ISSN: 1755 -6783 International indexed journal 2019 final.pdf · SP2000- 19 ©Annals of Tropical...

Special Issue May 2019 Vol. 19

ISSN: 1755-6783

International indexed journal

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©Annals of Tropical Medicine & Public Health-Special Issues

Table of Content May 2019 Vol. 19

Title Page Comparing the techniques of laminectomy SP2000-19

Development of X-ray application in cancer

treatment and the use of medical imaging techniques

SP2001-19

Correlation of FNAC lymph node cytology with CD4

count in HIV seropositive adults

SP2002-19

RnBeads 2.0: Comprehensive analysis of DNA

methylation data

SP2003-19

Safety disposal of electrophoresis gels and PCR

contaminate with ethidium bromide and alternative

methods

SP2004-19

In-vitro chick chorioallantoic membrane study of

chitosan capped 5-fluorouracil conjugated gold

nanoparticles

SP2005-19

Dental ergonomics: Awareness and practices among

dental undergraduates in Saudi Arabia

SP2006-19

Application: Coating of nanoparticles to minimizing

harmful environmental pollution by chemical

fungicide

SP2007-19

Genetic detection of hepatitis B virus by PCR

technique among children under 7 years in Hilla city,

Iraq

SP2008-19

Comparison of APGAR Score in neonates born after

elective spinal versus general anaesthesia

SP2009-19

Frequency of norovirus in Egyptian children with

gastroenteritis and confection with rotavirus

SP2010-19

A descriptive cross-sectional survey among

International Islamic University Malaysia’s student

on e-waste generation and public health problem in

Malaysia

SP2011-19

Resistance of Trypanosoma congolense forest

isolates to isometamedium chloride and diminazene

aceturate in West Democratic Republic of Congo

SP2012-19

Hemophagocytic lymphohistiocytosis in a Nigerian

child: A review of the literature

SP2013-19

Changing trends of lower respiratory tract

pathogens: A five year study

SP2014-19

Pathogens in milk and product diary: Study in Europe SPe168-19

i

©Annals of Tropical Medicine & Public Health-Special Issues

Salih (2019): Laminectomy techniques May 2019 Vol.19

SP2000-19 ©Annals of Tropical Medicine & Public Health

COMPARING THE TECHNIQUES OF LAMINECTOMY

ABDULNASER ABDULQADER SALIH1

1. DEPARTMENT OF NEUROLOGICAL SURGERY IN TIKRIT UNIVERSITY COLLAGE OF

MEDICINE

Email: [email protected]

Abstract

CL - shortcut for Classical Laminectomy - is the standard lumbar spine stenosis (LSS) treatment and

succeeds in (60: 87 %) of patients. Conversely, it is related with high difficulty rates such as back pain, spinal

instability and muscular atrophy. The goal in our study is to compare the clinical results of Transspinous-Split

Laminectomy TSSL, which reduces paraspinal muscle destruction, and protects the alignment of vertebrates. The

data consists of 50 patients which divided to group A with 25 patients who treatment via transspinous split

laminectomy and group B with 25 patients who treatment via classic laminectomy. The two groups were

administered by the same surgeon and the process technique is chosen randomly. The sample does not contain

pregnant patients and does not have any infectious diseases, malignant infections, previous treatment of spinal

fractures or disc herniation, and they have previous injury in the treatment of iatrogenic, spondylosis, spondylosis or

radiation-documented instability. The incision length and amount of hemorrhage were matched to group A and

group B. The length of the incision was shorter in group A. Avoided circumference dissecting muscles in group A

reduces the amount of hemorrhage. Paraspinal muscle atrophy (PMA) rating was lower in group A. The Minimal

muscle atrophy is scheduled during dissection of minimal muscular atrophy in TSSL technique. The reduction of the

values of postoperative Oswestry pain grade of patients compared to another group clinically verifies that adequate

decompression has been alleviated. The smaller the size of the incision, the less bleeding during the operation and

the injury of the paraspinal muscles results in less pain after the surgery and thus recovering faster. The minimally

invasive metod (TSSL) is a good alternative to the conventional of the traditional spinal laminectomy.

Key words: laminectomy, TSSL, back pain, lumbar spine stenosis, Oswestry pain grade

How to cite this article: Salih AA (2019): Comparing of techniques of laminectomy, Ann

Trop Med & Pub Health-Special issue; 19: SP2000-19

1. Introduction

Laminectomy or the decompression surgery removes the skeletal roof covering the spinal cord and nerves to provide

more gaps for them to move easily. Stenosis or narrowing of the spinal canal can produce chronic pain, numbness,

tingling, and muscle faintness in the arms or legs as shown in figure 1(1).

mailto:[email protected]



Is the removal of the entire bone lamina, part of the enlarged facet joints, and the thick ligaments above the spinal cord

and nerves.

Laminectomy

Types of decompression surgery

Figure 1 Spinal stenosis is a narrowing of the spinal canal

The stenosis (narrowing) is frequently produced by osteoporosis associated with age, inflated joints, and thick

ligaments. Stress relief may be suggested if your symptoms do not recover with physical therapy or medications.

Surgery requires hospitalization from 1 to 3 days and the recovery period takes from 5 to 6 weeks (2).

Decompression can be achieved on the spine wherever starting from the spine of the neck (cervical) to the lower

back (lumbar). The technique is performed through a surgical incision in the back (posterior). The lamina is the bone

that forms the back of the spinal canal and makes the surface above the spinal cord. Eliminating the lamina and other

soft tissues gives more room for nerves and allows removal of bone tingling. Depending on how narrow (extent of

stenosis), there may be one vertebra (one level) or more (multi-level)(3).

Laminotomy Is the removal of a small part of the lamina and ligaments, frequently on one side. Using this technique the normal

support of the lamina is left in place, which reduces the chance of spinal instability after surgery. Sometimes, an

internal endoscope can be used, allowing for a slighter, less invasive incision.

Laminaplasty Is the extension of the spinal canal via by cutting the lamina on one side and fluctuation them open like a door. It is

used only in the cervical area.

In various situations, the spinal fusion can be merged at the same time to help stabilize the parts of the spine treated

with the laminectomy. Fusion uses a combination of bone bait, screws, and rods to connect two separate vertebrae

together in one new section of bone. Joint integration prevents spinal stenosis from repeating and can help eliminate

the pain of an unstable spine (4).

Foraminotomy Is the removal of the bone around the neural puncture - the space between the vertebrae where the nerve root emerges

from the spinal canal. This technique is used when the dissolution of the disk causes the collapse of the high puncture,

leading to a pinched nerve. This can be done using a laminectomy or laminotomy.



not improved with physical therapy or medication

diagnostic tests (MRI, CT, myelogram) that show stenosis in

the central canal or lateral recess

leg pain worse than back pain

difficulty walking or standing that affects your quality of life

The surgery of decompression spinal contraction is optional, except for occasional cases in the tail syndrome or a

rapidly developing neurological deficit. The doctor may advocate treatment options, but only you can decide if

surgery is right for you. The patients have to be sure of check out all the risks and benefits before making a decision

of surgery. The decompression does not treat spinal stenosis and does not remove arthritis. It only releases some of

the symptoms. Regrettably, symptoms may repeat with the continued degenerative process that leads to stenosis (5).

significant pain, weakness, or numbness in your leg or foot

It may be a candidate to decompression if you have



The orthopedic or neurologist surgeon can achieve spinal surgery. Numerous spine surgeons will concentrate in

multifaceted spinal surgery. Patient have to ask surgeon about training, particularly if patient’s condition is

complicated or you have undergone more than one spinal surgery (6).

CL - shortcut for Classical Laminectomy - is the standard lumbar spine stenosis (LSS) treatment and succeeds in

(60: 87 %) of patients. Conversely, it is related with high difficulty rates such as back pain, spinal instability and

muscular atrophy. The long hospital rest and recovery time, and high blood loss. The minimal invasive operation

techniques began to develop after the operation microscopes are used. In 2002, Shiraishi described the technique of

vertebral lamination of the cervical spine that protected the paraspinal muscles. Furthermore in 2005; it is improved

to be used in the lumbar spine by Kota Watanabe. After 10 years, relevant publications supported the effectiveness

of this surgical technique (7).

The goal in our study is to compare the clinical results of Transspinous-Split Laminectomy TSSL, which reduces

paraspinal muscle destruction, and protects the alignment of vertebrates.

2. Methods

The data consists of 50 patients which divided to group A with 25 patients who treatment via transspinous split

laminectomy and group B with 25 patients who treatment via classic laminectomy. The two groups were

administered by the same surgeon and the process technique is chosen randomly. The sample does not contain

pregnant patients and does not have any infectious diseases, malignant infections, previous treatment of spinal

fractures or disc herniation, and they have previous injury in the treatment of iatrogenic, spondylosis, spondylosis or

radiation-documented instability.

The mean age of group A patients was 55.9± 1.25 and for group B was 53.23± 2.13. The gender of sample was 26

female and 24 male. Group A have treatment by TSSL which performed of 10 male and 15 female patients. Group B

have treatment by CL which performed of 14 male and 9 female patients. the symptoms mean duration were 5.10

years. Patients for group A and B had pain at lower back and leg. Group A have 15 patients with numbness and

group B have 10 patients only. Group A have 8 patients had claudication and group B had 11 patients claudication

as shown in figure 2.



Figure 2 Postoperative early period CT image

Figure 3 Postoperative MRI and CT images

Patients were assessed by oswestry scale, narcoticscale, and preoperative. Muscle atrophy was measured prior to

preoperative and postoperative surgery by MIR images. Patients' responses about walking in the oswestry scale were

analyzed using the assumption process of I=0, II=1, III=3, IV=4, V=5.

May 2019 Vol. 9

Muscle atrophy had assessed by determining two dimensions of the muscles prior to preoperative and postoperative

neurosurgery using weighted T2 images at the intervertebral disc level. In multi-level decompression is considered

and the average values are estimated. The rate of muscular atrophy was calculated using the following formula

Precentage of Atrophy Rating = (1 − 𝑇𝑜𝑡𝑎𝑙 𝑝𝑜𝑠𝑡𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑣𝑒 𝑎𝑟𝑒𝑎

𝑇𝑜𝑡𝑎𝑙 𝑝𝑟𝑒𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑣𝑒 𝑎𝑟𝑒𝑎

) × 100

After the effects of general anesthesia, patients placed on the operating table in the prone position, with semi flex

waist. The level of spinous operations has been distinguished with a portable endoscopy arm c. The operating

microscope was used at all stages of the surgery. The skin was passed under the uncover spinous operations. The

Spinous processes operations were divided into two sections with osteotomi process applied by high speed motor

and retractor incision was used to enlarge the surgery area. The Spinous processes and the muscles surrounding the

medulla connect to them and keep it. Data were analyzed using a statistical program (SPSS 14) and P <0.05 for

statistical significance.

3. Results

Group A have 13 patients (52%) with one level of decompression and 12 patients (48%) with multilevel. Group B

have 11 patients (44%) with one level of decompression and 14 patients (56%) with multilevel of decompression.

The units of measurement were measured of lengths of incision in centimeter, operating times in hours, amount of

bleeding in (cc), hospital stay in day, oswestry patients scores and grade, pain scores, anesthesia scores, VAS scores,

walk distances and tumor paraspinal musclesatrophy (PMA) before and after surgery for each group as shown in

table 1

.




Incision Length 3.72 ± 1.10 4.32 ± 1.95

Blooding 235.32 ± 188.15 263.12 ± 191.35

Before 63.50% ± 13.21 58.51% ± 17.25

Oswestry grade

After 19.50% ± 13.89 42.34% ± 18.54

Before 6.65 ± 1.52 5.32 ± 1.82

Pain grade

After 0.95 ± 1.63 2.42 ± 1.92

Before 2.74 ± 0.27 4.78 ± 0.57

Narcotic grade

After 1.02 ± 0.57 1.13 ± 0.24

Before 9.12 ± 1.61 8.24 ± 1.95

VAS

After 2.24 ± 1.75 4.87 ± 1.11

Before 3.52 ± 1.75 3.41 ± 1.32

Distance of walk

After 0.80 ± 0.52 2.82 ± 1.15

PMA 9.56% ± 1.85 38.52% ± 13.89

Table 1 Statistics

Items Group A Group B

Time of operation 2.31 ± 0.15 2.11 ± 0.55

Days for stay at hospital 2.15 ± 1.10 2.65 ± 2.12

The incision length and amount of hemorrhage were matched to group A and group B. The length of the incision

was shorter in group A. Avoided circumference dissecting muscles in group A reduces the amount of hemorrhage.

Paraspinal muscle atrophy (PMA)rating was lower in group A. the incision lengths, amounts of bleeding and

degrees of muscular atrophy assessment using Mann Whitney were tested and statistically better results were found

in group A (P = 0.001). The Minimal muscle atrophyis scheduled during dissection of minimal muscular atrophy in

TSSL technique. The main cause of postoperative back pain is loss of muscle function, which is better maintained in

TSSL technique.



4. Discussion

The lumbar spine stenosis (LSS) is the greatest familiar degenerative disorder in the lumbar spine which affects

about 1 in every 1000 people over age 55. The spread of the disease increases with age. The standard of lumbar

spine stenosis (LSS) for surgical treatment is pressure relief. The technique of surgical used is the removal of the

traditional spinal laminectomy to remove the pressure from the neurotransmitter described by Mexter and Pierre

from 70 years (8).

Corresponding to the results of 7-10 years of surgery, 26% of patients were re-operated and 34% of patients

presented with low back pain and hip after operation. Standard laminectomy involves the removal of a wide

laminectomy and partial or complete facial removal (9).

While this classic surgery offers sufficient nerve decompression, the posterior column, which has a significant role

in stabilizing the spine, is absolutely detached with the process. Removing the spinous process and lamina process

causes loss of tissue, formation of dead space, skin contour and cosmetic surgery problems, simplifies local wound

complications such as infection, and causes increased neural root scar adhesions. This in turn may increase pain and

lead to a syndrome of failure again in the coming period (10).

Since there is no an intersegmental such as the other muscles of paraspinal, and it was isolated only by the medial

branch of the dorsal ramus. The multifidus muscle is most susceptible to injury during backbone surgery. Lateral

deflection of the muscle dimension during the operation and subsequent compression of the medial part of the dorsal

branch can lead to the removal of muscle adenoid surrounding the uterus and postoperative muscular atrophy(11).

Line with others researchers, in their analysis of 18 patients, showed [sub laminar chimney pressure (CSD)]

technique were a way that minimized muscle damage with good neurological results in 2006. Published, they

reported that stripping the surrounding muscles of the marrow of a vertebra led to remove muscular embolism and

revascularization. Muscle contraction disrupts the local blood flow and increases oxidation, inflammatory cell

infiltration and increase in the nuclear factor KB associated with COX-2 expression the progressive edema of

muscle fibers. Postoperative injury pain is minimized using the new CSD procedure, as it does not involve muscle

stripping and muscles of paraspinal. Importantly, with this technique and early recovery, short hospital stay, a

decrease in complications related to hospital survival such as obstructive and urinary tract infections were also

observed(12).

Shetty and others involved in their report using LSPSL technology pointed out that this method provides a suitable

surgical area. They also observed that postoperative recovery was faster with minimal ligaments in the middle of the

midline and muscle damage (13).

5. Conclusion

In our report, it was shown that the minimally invasive common technique provide adequate expansion of the spinal

canal diameter by postoperative management of MIR and CT.

May 2019 Vol. 9


Consistency, the reduction of the values of postoperative Oswestry pain grade of patients compared to another group

clinically verifies that adequate decompression has been alleviated. The smaller the size of the incision, the less

bleeding during the operation and the injury of the paraspinal muscles results in less pain after the surgery and thus

recovering faster

In the end of this report, as a minimally invasive method (TSSL) is a good alternative to the conventional of the

traditional spinal laminectomy. The disadvantage of this study is the low number of patients. At last, as a minimally

invasive technique; TSSL is a good alternative to conventional laminectomy. The problem of this study is that the

sample does not expand (only 50 patients). To reach a high resolution result, the sample could be increased to

include different situations and situations with different age groups.

6. References

1. Robot-Assisted Decompressive Laminectomy Planning Based on 3D Medical Image. Yu Sun, Zhongliang Jiang,. 1, s.l. :

IEEE, 2018 , Vol. 12.

2. Clinical Comparison Between Patients Operated for Unilateral Radiculopathy via a Contralateral ( Facet-Sparing ) and

Ipsilateral Side Approach. Ahmet OGRENCI, Orkun Koban. 12, s.l. : Sedat Dalbayrak, 2017, Vol. 22.

3. Clinical Outcomes of Patients over 75 Years of Age with Degenerative Spondylolisthesis Following Bilateral Decompression

via Unilateral Approach. Mustafa Kemal Ilik, Mustafa Golen,. 1, s.l. : Turkish neurosurgery, 2017, Vol. 2.

4. Feng Chang, Ting Zhang,.Comparison of the Minimally Invasive and Conventional Open Surgery Approach in the Treatment

of Lumbar Stenosis: A Systematic Review and a Meta-Analysis. Singapore : Annals of the Academy of Medicine, , 2017.

5. Decompression surgery for lumbar spinal canal stenosis in octogenarians; a single center experience of 121 consecutive

patients. Alexander Antoniadis, Nils Harry-Bert Ulrich. 2, s.l. : British journal of neurosurgery, 2017, Vol. 15.

6. Lumbar Microlaminectomy vs Traditional Laminectomy. Langevin, J M. s.l. : Journal of neurosurgery. Spine, 2017.

7. How safe is minimally invasive pedicle screw placement for treatment of thoracolumbar spine fractures? Timo Michael

Heintel, Stefan Dannigkeit,. s.l. : European Spine Journal, 2016.

8. Microsurgical unilateral laminotomy for decompression of lumbar spinal stenosis: long-term results and predictive factors.

Karsten Schöller, Thomas Steingrüber,. s.l. : Acta Neurochirurgica, 2016.

9. Comparative Analysis of Inpatient and Outpatient Interspinous Process Device Placement for Lumbar Spinal Stenosis. Alicia

Ortega, J. Manuel Sarmiento. 3, s.l. : Journal of neurological surgery, 2015, Vol. 2.

May 2019 Vol. 9


10. Comparing cost-effectiveness of X-Stop with minimally invasive decompression in lumbar spinal stenosis: a randomized

controlled trial. Greger Lønne, Lars G. Johnsen,. Spine : s.n., 2015.

11. Does Obesity Affect Outcomes After Decompressive Surgery for Lumbar Spinal Stenosis? A Multicenter, Observational,

Registry-Based Study. Charalampis Giannadakis, Ulf S. Nerland,. s.l. : World neurosurgery, 2015.

12. Minimally Invasive Surgery for Intradural extramedullary Spinal Tumors : A Comprehensive Review with Illustrative

Clinical Cases. Martin H Pham, Ki-eun Chang,. 2016.

13. Minimally invasive removal of thoracic and lumbar spinal tumors using a nonexpandable tubular retractor. André Nzokou,

A G Weil, Daniel Shedid. Spine : Journal of neurosurgery, 2013.

Abbas (2019): X-ray application May 2019 Vol. 19

©Annals of Tropical Medicine & Public Health SP2001-19

Development of X-ray applications in cancer treatment and the use of medical

imaging techniques

Marwa H. Abbas

Department of dentistry, Al-Esra'a University College, Iraq

Email: [email protected]

Summary:

Role of radiotherapy in palliative care of cancer patients Radiotherapy plays an important role in

palliative care of cancer patients. This type of therapy accounts for about 30 to 50% of all radiation

delivered in a medical ward. The goal is to improve the patient's quality of life by providing rapid relief

of symptoms which depends on each specific localization. The cause of the symptoms must be

carefully identified (agreement between radiological and clinical findings) in order to establish the

precise target for radiation. Radiation dose and fractionation should be adapted to the patient's general

status and expected duration of life in order to limit the duration of treatment and secondary effects.

Key words: X-ray, radiology, prognosis, bone disease

How to cite this article: Abbas MH (2019): Development of X-ray application in cancer treatment

and the use of medical imaging techniques, Ann Trop Med & Pub Health-Special Issue; 19:

SP2001-19

Introduction

Painful bone metastases (20% of palliative care radiation protocols) can be irradiated in one or more

sessions with even analgesic effect with different results for prevention of fractures and cord

compression. Cord compression is a therapeutic emergency. The chances of recovering a motor deficit

after radiotherapy depend mainly on the rate of installation and the degree of deficit at the time of

treatment.

Patients with multiple brain metastases can be treated with 20-30 Gy delivered in 5-10 fractions on the

entire encephalon. More aggressive and technically more complex treatment (metastasectomy with

postoperative radiation, radiosurgery) can be reserved for selected patients. Radiotherapy can also be

effective for palliative care of patients with thoracic gold pelvic cancer.

The irradiation plays a major role in taking palliative load of patients with non-cancer curable [1].

Overall, the external irradiations carried out for palliative purposes represent 30% to 50% of activity of

a radiotherapy department, bone metastases painful alone constituting 20% of this activity. The purpose




of these treatments is to relieve and prevent symptoms, improve the quality of life or even increase

survival in some case. Because of their potentially myelo-toxic effects they must be fully integrated

into the in charge of the patients, especially not to risk compromising chemotherapy active. Moreover,

"time being precious when life is short "[2], the total duration of radiotherapy (or spreading) must be

adapted to the general condition of the patient and overall prognosis of the condition to limit maximum

unnecessary travel for patients whose life expectancy is short. Overall, a patient in desperate state never

benefits from palliative irradiation.

Reminder: fractionation of the dose in radiotherapy, the dose unit is gray (Gy), expressing the amount

of energy absorbed by the tissues irradiated. Apart from palliative situations or certain total body

irradiation protocols before transplant marrow, irradiations are not delivered in single dose but

according to a fractionation that is classically 5 weekly sessions of 1.8 to 2 Gy each. This fractionation

of the dose increases the effect of radiotherapy on the tumor while reducing its effects on the tissues

healthy.

In a palliative situation, it is possible to increase size of the fractions so as to shorten the total duration

of the treatment (spreading) while getting faster the desired analgesic or decompressive effect. This is

possible to the extent that the total doses required are much lower than during curative radiation. This

last element, associated with reduced life expectancy patients, limits the risk of complications late

radiotherapy. Schematically, it is therefore possible to deliver an irradiation with a spreading even

shorter than:

The life expectancy of the patient is short;

The target volume does not contain healthy tissue at risk acute complications (small

intestine, brain ...);

The total dose delivered is low;

The size of the fields is reduced.

Bone metastases:

Lesions with fracture risk should benefit of a first surgical treatment. The irradiations of bone

metastases account for 20% of the activity of a radiotherapy service. Split doses or doses unique are

comparable in terms of analgesic response objective.

External radiotherapy:

Overview 50% of patients with breast cancer, lung or prostate will develop metastases bones at one

time or another from the evolution of their disease. Conversely, 80% of bone metastases come from

breast cancer, lung cancer or prostate. Other cancers more rarely involved are those of the kidney,

thyroid and bladder. Without exception (including kidney cancers), bone metastases are in multiple

rule. Treatment of bone metastases includes two shutters:



the general treatment of the cancerous disease (chemotherapy, hormone therapy) or its

consequences bones (bisphosphonates);

the local treatment of bone metastases (surgery, radiotherapy).

Radiation therapy is a palliative treatment with aim to improve the quality of life of patients. Indeed, it

has the potential of being able to regress lesions tumors developed in bones and soft tissues adjacent

and thereby to obtain an analgesic effect, prolong the ambulatory state and reduce the risk of fracture

and spinal cord compression.

Several conditions must be met before starting radiotherapy of bone metastases:

1. Establish that the identified bone lesion is a metastasis. In the majority of cases, the diagnosis is

obvious. However, there are some difficult cases especially when the metastasis is unique and it occurs

more than 2 years after primary treatment of the primary tumor. He is then essential to confirm the

metastatic nature of the lesion by a biopsy or a puncture at the fine needle.

2. Make sure there is no indication of osteosynthesis prior to irradiation. Bone consolidation requiring

several months to be effective, it is better to use a primary surgery when the fracture risks are high.

Various criteria are used to evaluate in practice the fracture risk.

3. Integrate radiotherapy into the therapeutic plan general of the cancerous disease. Preservation of

capital medulla is different depending on whether it is a myeloma multiple for which myelotoxic

chemotherapy is considered or for carcinoma prostate for which androgenosuppressive hormone

therapy will constitute the bulk of systemic treatment.

4. Look for other bone metastases (scintigraphy bone), with little or no symptoms, but it is desirable to

irradiate at the same time because potentially fracturing in a relatively short (lytic metastases of long

bones or acetabulum for example).

The mechanisms by which radiotherapy allows to obtain an analgesic effect are poorly known. Indeed,

there is no clear correlation between the analgesic effect obtained on the one hand and the

radiosensitivity of the tumor or the dose delivered on the other hand. When the analgesic effect is rapid,

occurring in the first days after irradiation, one can invoke a reduction or a stop of the secretion of

chemical mediators of pain. The effects analgesics obtained later, in the weeks following the

irradiation, are rather in favor of a reduction tumor volume or bone recalcification for very late effects.

The average life expectancy of patients with bone metastases was evaluated at 29.3 months for prostate

cancers, 22.6 months for breast cancer and 3.3 months for bronchopulmonary cancers. However, some

subgroups of patients have a better prognosis. This is particularly the case of patients with exclusively

bone metastases secondary to carcinoma mammary (45% survival at 5 years with survival average of

52 months) or prostatic carcinoma hormone-sensitive (survival 43 months). The main criteria

correlated with life expectancy are as follows:



number of metastatic sites;

duration of the free interval between the first treatment primary cancer and the emergence

of metastases;

general condition of the patient reflected by the assessment of

the performance index (WHO or Karnofsky scale) (KPS));

Expected sensitivity of the neoplastic disease to a systemic treatment.

Overall, radiotherapy of bone metastases provides an analgesic effect in 70 to 80% of case. This

analgesic effect is:

Complete in 30 to 60% of the cases according to the series;

Delayed compared to the start of treatment; we can estimate that 50% to 60% of patients are calmed

in 2 weeks and 20% in 1 month or more.

Durable when it is complete (more than 1 year at 70% of the patients totally calmed);

Influenced by the nature of the primary tumor: the effect analgesic is more frequently obtained, more

complete and more durable when it comes to localizations of myeloma multiple or metastases of breast

cancer or prostate only when it comes to cancer metastasis thyroid, bronchial or renal;

Not influenced by the anatomical site of metastasis bone. Obtaining an analgesic effect (especially if

it is complete and durable) after irradiation of a first metastasis is a criterion of effectiveness during

irradiation another bone metastatic location. By against, the analgesic effect is delayed compared to the

beginning irradiation and allopathic analgesic treatment must be maintained the first sessions.

Indications: choice of dose and fractionation in 1998, the American College of Radiology

recommended the use of 3 regimens: 20 Gy in 5 fractions, 30 Gy in 10 fractions or 35 Gy in 14

fractions [3]. It suggests taking more account of hope of patients' lives by focusing on treatments short

for patients whose life expectancy is short.

Even Nielsen, who defends the principle of irradiation as a single dose, has reservation for this type of

treatment for unselected patients [4]. Single dose irradiations bone metastases are indicated when the

objective is to obtain a rapid analgesic effect. It is indeed effective treatment of painful bone

metastases, particularly simple and not very constraining for the patient, finally easily achievable for

the services of radiotherapy and inexpensive. The doses usually delivered range from 6 to 10 Gy

depending on the size of the fields and the organs at risk contained in the target volume. It is the

treatment of choice of metastases of non-bearing bones, poor lytic metastases of the carrier bones and

metastases Bones occurring in Patients with Hope life very reduced. Fractional dose irradiations should

be favored in patients with a relatively long life expectancy long when the goal is to get in addition to

the effect analgesic, i.e. tumor decompression (infiltration of the medullary canal, the paravertebral

muscles, a hole of conjugation ...) is a bone reconstruction (Lytic metastasis of the bones bearing). The



irradiations carried out after bone healing surgery are also performed in split mode. The most protocols

employees deliver 30 Gy / 10 fractions or 20 Gy / 5 fractions.

Diffuse metastatic osteosis in treatment failure is good indications of irradiation hemoglobin when the

hemogram of the patient allows it. These irradiations are most often performed in single dose (6 Gy on

the upper hemi-body, 8 Gy on the lower hemi-body). Arcangeli [5] published an analysis of the dose

response relationship in the field of analgesic radiotherapy of metastases bone. In this study, high doses

appeared increase the response rates but the methodology employee was questionable. It was indeed a

retrospective study conducted in a single institution, with a significant bias due to the fact that high

doses were delivered to patients with a good prognosis. Ben-Josef [6] also found a dose-response

relationship after a compilatory study of 5 randomized trials published.

The short-term analgesic efficacy of radiotherapy bone metastasis is established but there are persistent

divergences on the level of dose to be delivered and on the fractionation use. Phase III studies already

carried out did not show superiority of a treatment regimen compared to others [7-10]. As a result, wide

varieties of protocols are used. The most common deliver 8 Gy in 1 fraction, 20 Gy in 5 fractions or 30

Gy in 10 fractions; depending on the choice in practice various criteria such as the habits of the

institution, the convictions of the responsible practitioner, the workload of the processing units and the

means financial services. The tests that have been published have concerned for patients with a life

expectancy short, of the order of 3 to 4 months, either did not take counts the life expectancy of

patients in the criteria inclusion. Most studies done on radiotherapy bone metastases have focused on

the analgesic effect obtained during the 3 months following the treatment. In total, almost all of these

tests have confirmed that a single fraction of 6 to 10 Gy offered in the short term a rate of clinical

responses similar to that obtained by fractional irradiations. On the other hand, it appears from these

studies that long-term results single-dose radiotherapy is not established since there is no reliable data

available. Attempts involving randomized patients also showed that the re-irradiations were more

frequent in primotravity patients by a single dose but they have not studied precisely the reasons of

these re-irradiations. He can the effect is earlier or more intense recurrences of the syndrome after

single dose irradiation. It is also possible, as suggested by Steenland [11], that the first treatment with a

single fraction has conditioned a higher rate of early re-irradiations and this independently of the

intensity of the pain. The results published are in favor of reducing the number of pathological fractures

after fractional irradiation compared to single dose irradiations.

After irradiation, cells shown to be tumors were replaced by calcifying fibrous tissue gradually. An

irradiated lytic metastasis would be able to fill itself by direct osteogenesis without prior chondrogenic

phase, whereas a pathological fracture would require going through a chondrogenesis phase to be able

to consolidate. Chondrogenesis would be sensitive to radiotherapy, unlike osteogenesis. For this

reason, radiotherapy would reduce the consolidation of pathological fractures while it would have no

effect on the healing of lytic lesions not fractured. Radiologically there are signs of remineralization

lytic bone metastases after radiotherapy in 65 to 85% of cases. The essential benefit is obtained after



early radiotherapy of lyric lesions vertebral, pelvic or other weight-supporting bones from the body. No

reduction in the number of spinal cord compression has not been highlighted.

Hemi-corporal irradiation:

These irradiations were mainly used in analgesic treatment of multiple bone locations myeloma, breast

and prostate cancer. This treatment is indicated when these cancers are linked with systemic treatment

and that the patient keeps a hemogram satisfactory (the granular elements must be greater than 1,800 /

mm 3 and platelets at 100,000 / mm 3).

The previous bone irradiations do not constitute a contraindication. The irradiated volumes are as a

rule: for the upper hemi-body, extended from the angle from the jaw to the umbilicus: a single dose of

6 Gy or fractional irradiation of 12-24 Gy / 4-8 fractions; for the lower hemi-body, extended from the

umbilicus up to the knees: a single dose of 8 Gy or an irradiation fractionated from 15-30 Gy / 5-10

fractions.

The "middle" hemi-body, extended from the cervical spine to the upper third of the femurs, is more

rarely irradiated because the risk of bone marrow hypoplasia is high. The analgesic results published

are satisfactory. Thus, we obtain 77% of answers, 21% of which were answered completely. The

analgesic effect is obtained quickly, in 48 hours in 50% of responders and in 1 week in 80% of them,

sometimes after a brief period exacerbation of pain syndrome due to edema radiation-induced. The

immediate tolerance to these irradiations is globally very good. Acute side effects are mostly the fact of

the superior hemi-corporal irradiations. They may cause nausea and vomiting, fever and hypotension

and frequently lead to the suggestion of establishment of a venous route (alkalization), a premedication

with atropine, anti-emetic and corticosteroids, or else a medical observation during a few hours with

blood pressure monitoring. Immediate tolerance of hemi-bodily radiation lower is excellent. They can

be realized outpatient and require no precaution special. However, a few days later diarrhea occurs.

Late complications are rare. Some pneumopathies have been reported. In fact, only one monitoring of

the hemogram is necessary. Irradiation on the other half-body or chemotherapy can be performed 3 to 4

weeks later if the hemogram permits.

Metabolic radiotherapy

Although theoretically out of the frame of this review, metabolic irradiation is also a treatment

effective analgesic bone metastases. Irradiations metabolites by Strontium® or Quadramet constitute a

good alternative to external radiotherapy in case of multiple pain metastases, especially if they are

osteoconductive and the patient is still in good condition. Strontium chloride 89 (Metastro transmitter β

pure) is indicated for metastases of prostate cancer while the Samarium 153-EDTMP (Quadramet,

transmitter β and γ) is indicated for any tumor metastatic bone. Metabolic radiotherapy proves effective

in management of prostatic painful bone metastases, with 60 to 65% analgesic effect and a reduction

taking analgesics as well as the development of new painful sites.



The average response time is 6 months (Sr 89), 4.5 month (Sm 153).

The effect is all the more clear and prolonged bone is moderate. This treatment has low hematological

toxicity (granular line and platelets). It can however to appear a discrete and transitory worsening of the

pains (10 to 20%). The time of appearance is variable: long for the Metastron (up to 1 month), faster

for Strontium (1 week). Metabolic radiotherapy can be renewed within a minimum of 12 weeks.

Medullary compression:

Spinal cord compressions are therapeutic emergencies and the degree of recovery from the motor

deficit depends mainly on the speed of installation of the deficit and early management of treatment.

Metastatic spinal cord compressions represent a therapeutic emergency. Spinal pain previous

neurological symptomatology in more than 90% of the cases.

Intramedullary metastases are exceptional (1% of cases). Almost all spinal cord compressions

metastatic (99%) corresponds to invasion tumor of epidural space by 3 mechanisms:

Hematogenous bone spread responsible for a epidural invasion of contiguity (85% of cases);

Invasion of the para-vertebral spaces, in particular from the lymph nodes, gaining then the epidural

space after invasion of the bone tissue or by infiltration of the conjugation hole (10 to 15% of case);

Hematogenous dissemination directly localizing in the vessels of the epidural space (1 to 5% of

cases).

Bone metastases reaching much more frequently the vertebral body as the pedicle or blades, it is the

anterior part of the epidural space which is the more often invaded. These earlier epidurites are

associated with more or less destruction extension of the vertebral body opposite. Direct compression

of the marrow by the tumor tissue is accompanied by hemodynamic disturbances that may at any time

cause irreversible acute ischemia.

The posterior surgical approach limits considerably the possibilities of excision of these previous

lesions and makes run the risk of intraoperative neurological aggravation by lesion of posterior

vascular elements while the anterior vessels are already deficient made of the tumor. Surgery is

reserved for patients with doubted diagnosis, spinal instability, a compressive lesion obvious and

extirpable or a compression in an already irradiated territory. Traditionally, this is of a posterior

laminectomy allowing decompression of the spinal canal or more rarely of a posterior stabilization

(even a reconstruction posterior vertebral). Mortality rates and morbidity is 3 to 14% and 5 to 30% [12,

13]. A Postoperative irradiation is recommended at the dose of 20-30 Gy / 5-10 fractions [14]. A

prospective study [15] involving 209 patients mainly affected by localizations of mammary origin,

bronchopulmonary, prostatic and myeloma and treated with external radiotherapy associated with

corticosteroids, emphasized the importance of early maturity of care about the chances of recovery

from walking. Functional recovery is significantly influenced by the slow appearance of deficit signs.



Thus, a study of 98 cases [16] showed that, if the delay in setting up the disorders was long, the rate of

recovery was high. In case of rapid installation of motor disorders, only 10% of patients were improved

by irradiation.

The same author describes in a non-randomized trial [17] 3 fractionation schemes in patients with bone

marrow compression (49% of patients non-ambulatory): 30 Gy / 10 fractions, 37.5 Gy / 15 fractions, 40

Gy / 20 fractions, splitting being function the workload of the processing machines.

An outpatient activity was included in 19 to 24% of case, without difference between the 3 splits,

leading the authors to adopt the most splitting short (30 Gy / 10 fractions).

For altered patients (index WHO = 2, reduced life expectancy), Maranzano used an irradiation

hypofractionated of 8 Gy in 1 fraction in at least 53 patients paraparetic, repeated at 1 week interval in

case of response or stability. He reported 67% improvement in pain and 63% reduction motor deficit

without any radiation myelopathy [18].

When spinal cord compression occurs in territory already irradiated, reirradiation is at risk significant

amounts of radiation myelitis [19, 20]. In theory, a surgical solution must be preferred. In practice,

intervention is only exceptionally its own risks and the general condition often poor patients. The

utility of a reirradiation must then to be discussed on a case-by-case basis according to cumulative dose

and expectancy of the patient's life [21].

Brain metastases:

Panencephalic irradiation is recommended for symptomatic patients with metastases multiple. A

radiosurgical association or radiosurgery can be offered to selected patients whose prognosis is more

favorable. The brain is the 4 e metastatic site after the skeleton, lungs and liver [22]. Twenty-five

percent of patientsmetastatic will develop brain localizations secondary.

The median survival is 4 months longer (median 5-6 months) in patients treated with irradiation and

corticosteroids versus 1 month for patients treated with corticosteroids only [23]. The Radiation

Therapy Oncology Group analyzed the factors influencing the duration and quality of life of patients

treated for brain metastases and identified 3 prognostic groups. Median survival the highest (7.1

months) was for patients with KPS> 70, age <65 years, primary tumor controlled and absent other

metastatic locations. The adverse group had a median survival of 2.3 months [24].

Currently, an irradiation of 30 Gy in 10 fractions on the whole brain is the most common pattern used

in patients with metastases multiple cerebral In case of significant alteration of the condition In general,

an irradiation of 20 Gy in 5 fractions can be proposed [25].

For patients with isolated brain metastases, the radio-surgical association (metastasectomy then

postoperative pan-encephalic irradiation of 30 Gy / 10 fractions) appears to increase the median of

survival in the German study of Schackert (13 months vs.8 months) in patients in good general



condition and whose disease systemic was controlled [26]. Other studies have reported comparable

results with obtaining a profit significant in terms of survival, local control cerebral and quality of life

[27, 28].

Radiosurgery is a radiotherapy technique under stereotactic conditions to deliver a high dose of

radiation in a single session in a small intracerebral target volume with high precision [29].

The results obtained with this technique are similar to those of surgery in selected patients [30]. The

selection criteria usually used are the following: general state conserved (KPS = 70), less than 4 brain

metastases on MRI [31], and size <30 mm.

Chest tumors:

Bronchopulmonary Cancers:

Compressive, painful or haemorrhagic symptoms to a thoracic tumor constitute good indications of

radiotherapy. Endobronchial brachytherapy high dose rate may be an alternative external radiotherapy

in case of bronchial obstruction or tracheal.

Symptoms related to pleuro-pericardial effusion or an aero-digestive fistula is not indications

radiotherapy.

Thoracic irradiations performed for palliative purposes concern a large number of patients. Indeed,

several situations can lead to this indication:

Bronchial cancers initially too advanced or occurring in patients who are in poor general

condition to benefit from a surgical or radiochemotherapeutic project for curative purposes;

Bronchial cancers recurring locally;

Intrathoracic metastases of other cancers.

Again, many tests have been performed to try to shorten the duration of external beam radiotherapy in

these patients whose life expectancy is short. The Medical Research Council conducted two

randomized trials [32, 33] comparing an irradiation delivering 17 Gy in 2 fractions and 1 week at

irradiation performed with a more conventional fractionation (30-39 Gy / 10-13 fractions). The

palliation of the main symptoms (including cough and hemoptysis) was identical in the different groups

treated both in terms response rate than duration of the palliation, without significant change in

survival. Conversely, a test Canadian randomized patient [34] comparing a single session of 10 Gy at

an irradiation delivering 20 Gy in 5 fractions and 1 week reported better palliative effect (cough, pain

thoracic, gene in daily activity and quality of global life) in the split arm. In addition, survival was also

significantly longer in the arm split (6 months vs. 4.2 months) for the subgroup patients in good general

and non-metastatic condition. Endoluminal brachytherapy with high dose rate is also an effective

palliative treatment, especially in the treatment of endotracheal recurrences or endobronchial infections

occurring in irradiated territory and hemoptysis or dyspnea. This method is to circulate for a few



minutes a source of high-dose iridium in the light bronchial, over the entire length of the tumor

segment.

Applications are most often performed under anesthesia general, at the rate of an application of 5 to 15

Gy weekly for 1 to 5 weeks depending on the dose already received by the patient, the oncological

situation and condition general of the patient. The retrospective study of MD Anderson Hospital [35]

involving 175 patients reported a rate 78% objective response and significant improvement median

survival of responder patients (7 months vs. 3 months). Comparable results have been reported by a

British prospective study [36].

A randomized British trial [37] compared external irradiation (30 Gy / 10 fractions) at one application

unique endobronchial brachytherapy delivering 15 Gy. The conclusions of this study were in favor of

external beam radiotherapy which allowed for palliation more frequent and lasting symptoms than

brachytherapy as well as a modest but statistically of patient survival, at the price however, a higher

rate of radiation-induced dysphagia.

Note that the hemoptysis rate was similar in both arms (7%). Currently, the majority of authors agree to

reserve very concentrated irradiations (1 or 2 sessions from 8 to 10 Gy) to patients in poor general

condition, including life expectancy is very low. In total, patients in good general condition treated for

a non-metastatic disease seem to benefit from external irradiation spread as regards the improvement of

symptoms as survival time. Conversely, schemas using one or two strong fractions seem more adapted

to the symptomatic management of patients whose general condition is impaired and the prognosis

unfavorable short term. Radiation-induced esophagitis is the main complication acute occurring from

doses of 30 to 40 Gy. Smooth diet associated with a local treatment with anesthetics contact (gel or

syrup of Xylocaine) is often effective for dysphagia minor. For radio-mucositis more severe, general

treatment by major analgesics and corticosteroids as well as the installation of a probe may be

necessary. These dysphagia are transient, regressing a few days to a few weeks depending on their

severity [32].

For asymptomatic patients at the thoracic level, Falk et al did not found any benefit of immediate

irradiation compared to an irradiation deferred [38]. Survival rates were similar and only 42% of

patients in the group with "delayed irradiation" finally received palliative irradiation. This study

confirms the primary role of chemotherapy for patients asymptomatic in good general condition.

Superior cellar syndrome A superior vena cava syndrome due to bronchial cancer small cells or

untreated malignant lymphoma is a preferred indication of chemotherapy.

Radiation therapy is indicated if the SCS is due either to non-small cell lung cancer, either to a small

cell lung cancer or lymphoma in a situation of recurrence or failure of chemotherapy. The placement of

an endocaval vascular prosthesis represents a fast and efficient way to lift compression and can be a

complement or alternative to radiotherapy.



Three-quarters of the superior vena cava compressions are due to bronchopulmonary cancers, of which

40% to small cell cancers. About 5% of bronchopulmonary carcinomas are complicated by Upper Cave

Syndrome (SCS). Lymphomas are responsible approximately 10% of SCS cases. The clinical

symptomatology depends to a large extent on the speed of cave obstruction, slow obstructions allowing

the development of a collateral circulation that partially drains blood flow and thereby reduces the

importance of symptoms related to obstruction of the vein cellar.

The prognosis is not favorable in the absence of treatment with a median survival of 6 weeks.

Treatment aggressive and responsive symptoms by at least partially restoring a flow in the vena cava

and to obtain an overall survival rate at 1 year estimated at 25%.

In the case of small cell lung carcinomas not pretreated, chemotherapy is currently advised in first

intention whenever possible. Indeed, the results of chemotherapy are comparable to those of

radiotherapy, while having the advantage to treat distant metastases early and avoid irradiation of a

large cardiopulmonary volume. The regression SCS is fast (7 to 10 days) and is obtained in 43 to 100%

of cases depending on the series. A randomized study comparing chemotherapy with or without

radiotherapy confirmed the absence of benefit of radiotherapy in this situation [39]. Radiotherapy is

however formally indicated when the SCS occurs in the part of a bronchial carcinoma to small cells

chemoresistant from the outset or in local recidivism. The same attitude is recommended in case of

SCS secondary to lymphoma. Unlike small cell cancers, radiotherapy plays a vital role in the initial

care SCS due to non-small bronchial cancers cells.

It has been proposed to start the irradiation with 3 fractions from 3 to 4 Gy in order to obtain a fast

decompressive effect, with resumption of radiotherapy according to a fractionation conventional [40].

In a small series, Egelmeers [41] reported a response rate of 76% in group of patients irradiated for

SCS due to carcinoma bronchial non-small cell, half of responders remaining under control until death.

The obstruction of the superior vena cava can also be treated by percutaneous insertion of a prosthesis

expansive metallic endocave (stent). So a Japanese study [42] compared the pose of a stent to one

mediastinal radiotherapy or chemotherapy (10 patients).

The clinical response between stent and radiotherapy was identical (78 and 80%), as the median

survival time (145 and 146 days). Previous treatment did not change median survival and clinical

response rate. Other studies [43] confirmed the good results of this technique which allows obtaining a

quick lifting of the obstacle and may be supplemented by subsequent radiotherapy. Esophageal cancers

The importance of initial dysphagia is a factor essential prognosis of oesophageal cancers. Lifting the

dysphagia is the main goal of the mediastinal irradiation, possibly associated with an endoscopic

désobstruction. Esophageal cancers remain of poor prognosis with a 5-year survival rate of about 5% at

all stages confused. The main symptom is dysphagia causing malnutrition, impairment of general

condition and loss weight. It is also frequently painful. Lift dysphagia is the primary goal of treatment

symptomatic. It has been shown that a dose of at least 50 Gy delivered in 25 fractions and 5 weeks



increased survival rates overall and free interval without dysphagia. The patients very dysphagic do not

benefit from an increase of the dose beyond 50 Gy and a désobstruction mechanical is necessary.

Thus the retrospective study of Caspers [44] showed a radiotherapy dose equivalent to 50 Gy / 25

fractions / 5 weeks improved dysphagia in 70.5% of case. In this study, 50% of patients maintained the

benefit of this irradiation until their death. Severity dysphagia before treatment influenced survival

overall. A randomized Indian study compared a treatment endoscopic (esophageal dilatation and / or

prosthesis oesophageal) followed by external irradiation by oesophageal prosthesis alone [45]. For the

group of patients without initial esophageal fistula, the 1-year survival was better in the combined arm

(17% vs. 2.3%). In case of fistula present before treatment, radiotherapy did not improve median

survival but the size of this subgroup was too small to conclude to the inefficiency of radiotherapy.

In case of tumor recurrence or progressive continuation responsible for dysphagia, an endoscopic

treatment should be considered. Esophageal endoluminal brachytherapy with high dose rate has also

been used in patients whose life expectancy was less than 3 months or already treated by external

irradiation. The technique was similar to that used in the endo-luminal bronchial carcinomas. A single

fraction of 12 to 15 Gy proved effective on dysphagia, allowing obtaining response rates of 42% to

90% and a free interval of dysphagia ranging from 3.3 to 5 months [46].

A dose escalation of 2 fractions of 8 Gy or 3 fractions of 6 Gy [47] improved dysphagia and local

control but at the cost of complications mainly stenoses and fistulas.

Pelvic tumors:

Digestive or urinary obstructions should be benefit of a derivation before irradiation. Loco-regional

evolution of pelvic tumors may result in various clinical presentations. Thus, tumors centro-pelvic are

rather the cause of compressions or invasion of the bladder, rectum or of the vagina while latero-pelvic

tumors associate more or less completely limb lymphedema inferior, ipsilateral radiculalgia and

uretero-hydronephrosis. In case of obstruction of the urinary tract (obstacle to level of the

ureterovesical junction or bladder neck), the installation of a suprapubic catheter or a ureteral catheter

is necessary before any irradiation [48].

For rectal cancers, it has been shown that irradiation exclusive allowed to reduce syndromes rectal in

50 to 75% of cases, rectorrhagia in 2/3 of cases and pelvic pain in near the half of the cases. However,

a dose effect has been obviously, a minimum dose of 45 Gy delivered in 4 to 5 weeks being necessary.

Below this dose, the chances of being effective on the symptoms are limited (<15%). These palliative

effects are unfortunately transient, less than 15% of patients remain relieved at 1 year [49]. In order to

reduce the total duration of treatment, hypofractionated irradiations have been proposed. For example,

Swedish teams have established that preoperative, 25 Gy delivered in 5 fractions and 1 week were

equivalent to 45 Gy delivered in 25 fractions and 5 weeks [50].



For bladder cancer, palliative radiotherapy significantly improves symptoms such as dysuria, bladder

irritation and hematuria. A randomized study from the Medical Research Council [51] compared 2

different fractions (21 Gy / 3 fractions / 2 weeks vs. 35 Gy / 10 fractions / 12 days) in the setting

symptomatic load of 500 cancer patients bladder. The symptoms were reduced in 50 - 53% at the end

of irradiation and in 64 to 71% of patients at 3 months. There was no difference in effectiveness or a

difference in survival (median of 7.5 months) between the 2 arms. Other concentrated irradiation

schemes have been successfully proposed, for example 17 Gy issued in 2 fractions and 3 days [52]. In

terms of palliation, this scheme resulted in a better reduction of hematuria and pains that more

conventional irradiation (45 Gy / 15 fractions) but at the price of a reduction of survival that proved

significantly more short in the hypofractionated arm (9.77 months vs. 14.47 months).

For prostate cancer, the conclusions are similar. Radiation therapy improves the symptoms associated

with invasion of pelvic structures [53]. In total, palliative radiation therapy for pelvic cancers is an

effective, highly concentrated radiation protocol (1 to 3 sessions of 7 to 10 Gy) being reserved for

patients in poor general condition whose life expectancy is short.

Various irradiations:

For haemostatic radiotherapy of uterine cancers, Metrorrhagia is frequently indicative of cervical

cancer. Present at diagnosis in 70 to 80% of cases, they can be abundant and even represent a

therapeutic emergency. Irradiation allows rapid tumor reduction and Hemostasis by occlusion vessels.

In a retrospective study of 20 patients with metrorrhagia since 6.25 months in average and worsening in

the 5 last days preceding the consultation, not giving in to a dressing vaginal compress, Biswalet a [54],

have reported of haemostasis in all patients after 3 irradiation sessions. These results were similar to

those found by Kraiphibul [55].

Pancreatic cancers:

Pancreatic cancer is rarely diagnosed at early stage surgery. A derivation is necessary in case of an

inoperable tumor responsible for jaundice compression. The method choice of extrahepatic biliary

obstruction is represented by the endoscopic placement of prosthesis. This technique allows lifting the

obstacle in 80 to 90% of cases [56]. The way transhepatic is used in case of failure of the endoscopic

approach. Surgery is also possible by bypass biliary or digestive. The results of these different methods

on jaundice regression are equivalent to short term [57].

Inoperable, locally advanced cancers of the pancreas can benefit from a concomitant association

radiotherapy and chemotherapy if the general condition of the patients allows it. Studies have shown

that this association could increase the quality and duration of survival (9 to 10 months vs.5 to 6

months) compared to a treatment by external radiotherapy alone [58-60] or chemotherapy only [61].

In patients in poor general condition, radiotherapy palliative is relatively little used because the doses

necessary to achieve a palliative effect are relatively elevated while organs located in the party upper



abdomen have poor tolerance to irradiation. So, in a randomized trial, local control was obtained in

only 20% of patients at 2 years after an exclusive irradiation of 60 Gy [62].

Splenomegaly:

In myeloproliferative syndromes (especially in chronic myeloid leukemias and splenomegaly myeloid),

splenomegaly sometimes monstrous, occupying a part of the abdomen and possibly of the pelvis often

sign aggravation of the hematological disease. Palliative irradiation low dose is effective, allowing a

reduction in symptoms (gravity, pain, fever, sweating, slimming) in 60% of cases, splenic volume in 42

to 81% of cases and transfusion needs in 32 to 60% of cases [63, 64]. The irradiation field includes the

entire spleen. The total dose is 5 to 10 Gy delivered per daily fraction of 0.5 Gy. The size of the fields

is adapted to the regression of splenic volume during irradiation.

Side effects of radiotherapy:

The acute side effects of radiotherapy are correlated with the size of the fractions and the total dose

delivered. The usual protocols used (6 to 8 Gy in 1 fraction, 20 Gy in 5 fractions, 30 Gy in 10 fractions)

are in generally very well tolerated. Nevertheless, side effects may occur:

A cutaneous erythema, especially frequent at the level of folds and zones tangent to the beams,

resolving in 2 at 3 weeks;

Nausea and vomiting during irradiation abdominoplasty, especially when part of the stomach is

included in the fields (utility of anti-HT3 issued before each meeting);

Diarrhea after abdominal or pelvic irradiation including a significant intestinal volume;

Dysphonia and dysphagia during irradiation cervical or thoracic, including all or one part of the

pharyngolaryngeal sector or the esophagus. The doses delivered relatively low and especially the

expectation reduced life of these patients limit the risks occurrence of late complications. Only myelitis

radiation may be a problem during re-irradiation vertebral (especially where there is a risk of fields) in

patients with an expectation of life of several months.

Conclusion:

Palliative radiation therapy is an important part of the activity of a radiotherapy service and each

treatment must be adapted for the patient to draw a real benefit. Thus, we will favor a hypofractionated

irradiation or as a single dose for patients in terminal phase, which will be delivered quickly and will

not be less effective; transport and manipulation will be limited in these frail patients and the costs

treatment will be reduced [65]. More splitting conventional or a dose increase may in certain situations

(patient in good general condition, illness poorly evolved) improve local control rates and overall

survival. Innovative techniques (radiotherapy conformational, radiosurgery, brachytherapy dose rate)

have their place in this type of irradiation.



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Sumana et al (2019): FNAC lymph node in HIV+ adults May 2019 vol. 19


Correlation of FNAC lymph node cytology with CD4 count in HIV seropositive adults”

Mukherjee Sumana1, Mukhopadhyay Keya

1 , Bhattacharya Pranab

1

1. Department of Pathology, School Of Tropical Medicine, Kolkata

Corresponding Author:

Sumana Mukherjee, BH-62, Sector-2, Salt Lake, Kolkata

Phone numbers +919830945575

E-mail: [email protected]

Abstract:

Context: Lymphoid tissues are common targets of HIV infection.FNAC is the initial investigation of choice in

these cases.

Aims: To evaluate the usefulness of FNAC in HIV positive lymphadenopathy in our center..

Methods and Material: FNAC was performed in 153 HIVpositive patients presenting with lymphadenopathy.

Smears were stained with Giemsa, ZN and PAS/Grocotts/PAP according to cytological findings.

Statistical analysis: The data was analysed using the T Test

Results: Tuberculous lymphadenitis was the most common diagnosis (44%).Smear positivity was found in 29%

cases. Necrotizing granulomas and smear positivity was significantly higher in cases with CD4 count<200.

Reactive hyperplasia was significantly higher in the CD4> 200 category.

Conclusions: FNAC is very useful and gives specific diagnosis in most cases of HIV lymphadenopathy. Lower

CD4 count significantly increases the smear positivity for AFB.

Key-words: FNAC, lymph node, HIV, CD4 count

How to cite this article: Sumana M, Keya M, Pranab B (2019): Correlation of FNAC lymph node cytology

with CD4 count in HIV seropositive adults, Ann Trop Med & Pub Health-Special issue; 19: 2002-19.

Key Messages: FNAC should be the chosen diagnostic method in HIV lymphadenopathy

because it avoids unnecessary biopsy, saves time, is cost effective, safer for the operator and

has yields mostly specific diagnosis.




Introduction:

Lymphoid tissues are commonly targeted in HIV infections [1]

. HIV positive individuals thus commonly present

with enlarged lymph nodes. The degree of lymphadenopathy may range from progressive generalized to

transient.The commonest infection is tuberculosis and extra pulmonary involvement is common [2]

. Occurrence

of extra pulmonary tuberculosis has increased specially in those who are severely immunodeficient [3]

.

FNAC is the initial investigation of choice in these cases. Though FNAC may not clearly demarcate all

pathologies, it is useful in diagnosis of specific infections and involves lesser risk to the performer than biopsies

[4].

We tried to note the FNAC findings of all HIV positive patients sent to our department with lymphadenopathy

and corelated it with CD4 counts. We aim to evaluate the usefulness of FNAC in HIV positive

lymphadenopathy in our center

.

Materials and Methods:

Subjects: This is a cross sectional observational study of HIV infected subjects diagnosed in an ICTC unit in a

tertiary medical center. FNAC was performed on patients presenting with lymphadenopathy.

Sample size: 153 HIV positive adults with lymphadenopathy.

Inclusion criteria: Subjects above 18 years, seropositive for HIV, lymph node size at least 1 cm.

Exclusion criteria: Retroperitoneal or non-palpable nodes, inadequate material.

Data collection: Data collected included age, sex, site of lymph node enlargement, whether on ART therapy,

clinical examination of nodes, CD4 cell count by flow cytometry and cytological features. All the smears from

the aspirates were stained with Giemsa stain and ZN stain. PAS / Grocotts / PAP stains and culture were used

depending on cytological findings.

The following categories were used to record cytological data. The groups were (1) reactive hyperplasia, (2)

necrotizing granulomatous with or without AFB, (3) necrotizing only with or without AFB, (4) other specific

diagnosis like histoplasma, Cryptococcus, suspected lymphoma or metastasis, (5) inconclusive.

Statistical analysis: The data was analysed using the T test. P-values were calculated. A p-value of < 0.005 was

considered significant.

Results:

There were 108 males (70.5%) and 45 females (29.5%). The age range was 20 to 58 years. The commonest site

was cervical (40%) followed by axillary (25%) and inguinal (5%). Some patients (30%) presented with multiple

site involvement, commonly cervical and axillary. Most nodes (55%) were discrete, non-tender. Matted nodes

constituted 25% cases, while abscess or sinus formation was seen in 20% cases (Table 1). 112 cases were on

ART, while 41 were ART naïve.



Reactive hyperplasia accounted for 35% of diagnosis. There were 29% cases positive for AFB (figure 1). Only

necrotizing pattern (figure 2) were noticed in 35% cases among the AFB positiveswhile most smear positives

showed necrotizing granulomas (figure 3). Necrotizing granulomatous comprised 36%. Tuberculosis was

diagnosed when there was AFB positivity irrespective of cytology and/or presence of caseation necrosis with

epithelioid granulomas. Tuberculosis was diagnosed in 44% cases. Fungal infection comprised of 2 cases,

proven by culture. Lymphomas comprised 3.2% cases. Out of the 5 cases of lymphoma, biopsy confirmed NHL

in 4 cases and 1 was florid reactive hyperplasia. Most lymphomas and fungal infections (figure 4) were in CD4

count < 200 group (Table 2).

The number of reactive hyperplasias was significantly higher in CD4 > 200 group (p=0.00). While necrotizing

granulomas and AFB positivity was significantly higher in CD4 < 200 group (p=0.0066). Similarly, only

necrosis with AFB was significantly higherin CD4 < 200 group. There was no significant difference among

cases with necrosis and no AFB in the 2 CD4 groups (p=0.00). Cases with granulomas but no necrosis or AFB

were considered inconclusive at FNAC.

Discussion:

Male predominance has been established in most studies5,6

and the higher age limit varies up to 65 years7,8,9

In

our study , however, we did not get any case above 60 years of age. Agravatet al8 showed incidence of

lymphadenopathy decreased with increasing age.

Cervical nodes were most commonin our study like Neelima et al and others 9,

10,

11

Satyanarayana et al 4 found

axillary nodes most commonly. Liatjos et al12

and Naser S et al13

had categorized cytological findings in our line.

Similar to our findings, most workers found tuberculous lymphadenitis as the most common

diagnosis.1,5,8,9,11

Chronic granulomatous lymphadenitis without caseous necrosis and no AFB or fungi, which we

categorized as inconclusive 6,14

on FNAC comprised 12 of our cases (7.8%) is in between the 19% recorded by

Satyanarayana et al4 and the single case reported by Neelima et al.

10

CD4 count < 200 is considered advanced stage while counts 200-500 and >500 are early and intermediate

stages.15

We considered two groups based on similar cut off value.

Our study corroborates with other workers 10

that fungal infections and lymphomas are common in the low CD4

categories. Kumar Guru et al 6 also found highest CD4 counts in reactive hyperplasias like our study. We found

that metastasis has also been reported by some corroborating with our findings.15

We may have missed many

opportunistic infections like viruses and toxoplasma in this cytomorphological study. Diagnostic accuracy could

be increased by using appropriate immunofluorescence kits.

Conclusion

FNAC is very useful and gives specific diagnosis in most cases of HIV lymphadenitis. Lower CD4 count

significantly increases smear positivity for AFB and fungi and may help in considering segregation of these

patients.



Source(s) of support: Nil

Presentation at a meeting: Not Applicable

Conflicting Interest (If present, give more details): Nil

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Table 1: Site distribution of lymph nodes and clinical features among the cases

Lynph node Number Percentage

Site

Cervical 61 40%

Axillary 38 25%

Inguinal 8 5%

Combined 46 30%

Clinical Features

Discrete 84 55%

Matted 38 25%

Abscess or Sinus 31 20%

Table 2: Correlation of cytological features of lymph nodes with CD4 count

Cytological Feature CD4 > 200 CD4 < 200

Reactive Hyperplasia 53 5

Necrotizing granulomatous without AFB 24 --

Necrotizing granulomatous with AFB 11 20

Necrotizing without AFB / fungal stain positivity 4 2

Necrotizing with AFB 2 11

Lymphoma 1 4

Metastasis 2 --

Histoplasma -- 1

Cryptococcus -- 1

Inconclusive 10 2

Total Cases 107 46



Figure 1: AFB positivity, ZN stain, x 1000

Figure 2: Necrotizing inflammation, Giemsa, x100

Figure 3: Granuloma, Giemsa, x400

Figure 4: Histoplasma, GMS stain, x 400

Lafta & Gangadhar (2019): RnBeads 2.0 May 2019 vol. 19


RnBeads 2.0: Comprehensive Analysis of DNA Methylation Data

Methaq Hadi lafta1*

, Lekshmi Gangadhar2

1*Lecturer in ministry of education, Iraq,ORCID ID: 0000-0002-9978-6829

2Department of Nanotechnology, Noorul Islam Center for Higher Education, India, ORCID

ID: 0000-0002-1627-1230

ABSTRACT

DNA methylation is a widely investigated epigenetic mark with important roles in development and disease. High-

throughput assays enable genome-scale DNA methylation analysis in large numbers of samples. Here, we describe a

new version of our RnBeads software – an R/Bioconductor package that implements start-to-finish analysis

workflows for Infinium microarrays and various types of bisulfite sequencing. RnBeads 2.0 (https://rnbeads.org/)

provides additional data types and analysis methods, new functionality for interpreting DNA methylation

differences, improved usability with a novel graphical user interface, and better use of computational resources. We

demonstrate RnBeads 2.0 in four re-runnable use cases focusing on cell differentiation and cancer.

Keywords: Bisulfite sequencing, Computational epigenetic, DNA methylation, Epigenome-wide

association studies, Epigenotyping microarrays

How to cite this article: Lefta MH, Gangadhar L (2019): RnBeads 2.0: Comprehensive analysis of DNA

methylation data, Ann Trop Med & Pub Heatlh-Special Issue; 19: 2003-19

BACKGROUND

DNA methylation at CpG dinucleotides is a widely studied epigenetic marker that is involved in the regulation of

cell state and relevant for a broad range of diseases. Changes in DNA methylation at promoters and enhancers have

been associated with cell differentiation, developmental processes, cancer development, and regulation of the

immune system. The vast majority of current assays for DNA methylation profiling use bisulfite treatment to

selectively convert unmethylated cytosines (including 5-formyl-cytosine and 5-carboxy-cytosine) into uracil (which

is subsequently replaced by thymine), while methylated cytosines (including 5-hydroxy-cytosine) remain

unconverted. Bisulfite conversion thus transforms DNA methylation information into DNA sequence information

that can be read by next-generation sequencing or DNA microarrays [1].

Whole-genome bisulfite sequencing (WGBS) constitutes the current gold standard for DNA methylation profiling,

given its genome-wide coverage and single-base pair resolution [2]. However, WGBS requires deep sequencing of

the entire genome (which is a significant cost factor), while shallow sequencing leads to poor sensitivity for

detecting small differences in DNA methylation [3]. Reduced representation bisulfite sequencing (RRBS) offers a

cost-effective alternative for profiling large sets of patient samples, by focusing the sequencing on a subset of the

genome enriched using restriction enzymes. RRBS is particularly useful for studying DNA methylation

https://rnbeads.org/



heterogeneity, which profits from deep sequencing coverage and from analyzing many samples using a sequencing-

based assay [4]. Target-capture bisulfite sequencing enables the analysis of a defined set of genomic regions in large

numbers of samples at low cost per sample, but with high setup cost. Finally, the microarray-based Infinium DNA

methylation assays, including the MethylationEPIC BeadChip (EPIC) and its predecessor, the

HumanMethylation450 Bead Chip (450k)-facilitate standardized, high-throughput DNA methylation profiling of a

pre-defined subset of CpGs in large sample cohorts [5].

These assays have enabled DNA methylation mapping for a large number of cell types and following the concept of

epigenome-wide association studies (EWAS), for various diseases with a suspected role of epigenetic regulation [6].

The resulting datasets typically comprise DNA methylation profiles as well as sample annotations such as tissue or

cell type, phenotypic data (donor age, sex, etc.) and sample grouping (case versus control, treated versus untreated,

etc.). The primary goal for the bioinformatic analysis of such datasets is to identify characteristic and reliable DNA

methylation patterns, and to associate them with relevant annotation data. The various software packages exist that

support individual steps of the DNA methylation analysis are summarized as a feature table in Additional file 1:

Table S1. Many users would benefit from an integrative analysis tool that provides extensive, easy-to-understand

analysis reports and requires minimal configuration and no detailed bioinformatic knowledge of the various analysis

steps.

METHODOLOGY

We have previously developed the RnBeads software package as a start-to-finish pipeline for DNA methylation

analysis in accordance with established standards and practices [7]. Initially released in 2012 and published in 2014,

RnBeads has become a popular and widely used tool for DNA methylation analysis (200–300 downloads per month

from Bioconductor) [8]. Here, we present a new, substantially extended version of RnBeads, providing an up-to-

date, user-friendly, feature-rich, and readily scalable workflow for the bioinformatic analysis of DNA methylation

datasets. The new RnBeads 2.0 software package addresses feedback and feature requests from the tool’s active user

community, implements new analysis methods, introduces a graphical user interface, and improves computational

efficiency. With these advances, RnBeads provides state-of-the-art support for DNA methylation data analysis in an

easy-to-use way, with high flexibility and performance.

RnBeads includes modules for data import, quality control, filtering and normalization (“preprocessing”), export of

processed data (“tracks and tables”), covariate inference (e.g., predicting epigenetic age and cell type heterogeneity

from DNA methylation data), exploratory analysis (e.g., dimension reduction, global distribution of DNA

methylation levels, hierarchical clustering), and differential DNA methylation analysis (Fig. 1). Each analysis

module generates an HTML report that combines method descriptions, results tables, and publication-grade plots.

These reports provide the user with a comprehensive and readily sharable summary of the dataset.



Fig. 1 Differential DNA methylation analysis

Overview of the RnBeads analysis workflow and new features added in RnBeads 2.0. Conceptual drawing of the

RnBeads workflow for DNA methylation analysis, listing key features (right) for each of the RnBeads analysis

modules (center), with newly added features indicated in bold red text. Tab, tabular (e.g., comma-separated) files;

idat, Infinium signal intensity files; geo, download from the GEO data repository

Of the various features that we introduced into RnBeads since the original publication in 2014, we specifically

highlight the following four areas:

1. New data types and cross-platform analysis: RnBeads now supports EPIC microarrays and enables

seamless data integration across different DNA methylation assays (e.g., EPIC, 450k, and 27k microarrays

as well as WGBS and RRBS), which facilitates DNA methylation meta-analyses that combine several data

sources into a single set of results.

2. Extended analysis and inference methods: We added new functionality for handling incomplete data and

missing values, for detecting genetic evidence of sample contamination or low data quality, for quantifying

DNA methylation heterogeneity, and for DNA methylation-based inference of phenotypic information. We

incorporated the LUMP algorithm, which estimates immune cell content of tumors and other heterogeneous

tissue samples, and epigenetic age prediction for both Infinium microarray and bisulfite sequencing data.

These predictions are useful not only for inferring missing donor annotations, but also for detecting

deviations indicative of accelerated aging or evidence of sample mix-ups. Additional new features include

the identification of genomic regions characterized by differential DNA methylation variability and

genomic region enrichment analysis using the LOLA tool.

3. New user-friendly interface: We provide a graphical user interface for RnBeads that facilitates the

configuration and execution of DNA methylation analyses. Together with RnBeads’ interactive and self-



explanatory HTML reports, this new interface makes RnBeads analyses more readily accessible for users

with limited R/Bioconductor knowledge.

4. Improved computational efficiency: Using parallelization and automatic distribution of RnBeads

analyses across a high-performance computing (HPC) cluster, we were able to process datasets comprising

hundreds of RRBS/WGBS profiles and thousands of microarray-based profiles in a single analysis run.

RESULTS AND DISCUSSION

To illustrate the practical utility of these new RnBeads features, we present four use cases: (i) DNA methylation in

human peripheral blood samples, (ii) cell type-specific DNA methylation in human hematopoiesis, (iii) DNA

methylation heterogeneity in cancer samples, and (iv) cross-platform DNA methylation analysis. Detailed re-

runnable versions of these analyses, including configurations and results, are available for visualization and

download from the RnBeads website (https://rnbeads.org/methylomes.html). These pre-configured analyses and pre-

calculated reports also provide a good starting point for learning about the use of RnBeads, thereby complementing

the tutorials provided on the RnBeads website (https://rnbeads.org/tutorial.html), and for configuring custom

analyses that integrate newly generated datasets with publicly available reference data.

Use case 1: Analyzing DNA methylation in a large cohort of peripheral blood samples

To illustrate the use of RnBeads for analyzing DNA methylation microarray data in a large cohort, we obtained

Infinium 450k profiles for peripheral blood samples of 732 healthy individuals. We also included reference profiles

for sorted blood cell types, in order to account for inter-individual differences in the frequency of different cell

types. First, we used RnBeads to infer donor age and sex for each sample, thereby filling in a handful of missing

annotations with imputed values, while also checking for potential sample mix-ups among those samples that have

donor age and sex documented as part of their annotation as in Fig. 2 a, b. Second, we performed reference-based

estimation of immune cell composition as implemented in RnBeads, and we found that the inferred immune cell

content as well as other annotations are indeed associated with important principal components of the DNA

methylation dataset as in Fig. 2 c, d. Our results emphasize the need to correct for these covariates when identifying

CpGs and genomic regions that are associated with the annotation(s) of primary interest. Third, we compared

chronological age with the fraction of CD4+ T cells inferred from the DNA methylation data using sorted blood cell

types as reference as in Fig. 2e and observed a negative correlation, consistent with the known age-related shift

toward myeloid (instead of lymphoid) hematopoiesis. In summary, this use case illustrates the prediction of age, sex,

and cell composition based on DNA methylation data, and it provides a framework for microarray-based

epigenome-wide association studies.

https://rnbeads.org/methylomes.html

https://rnbeads.org/tutorial.html



Fig. 2Infinium 450k profiles for peripheral blood samples

Analysis of a large DNA methylation dataset of blood samples profiled using Infinium 450k.

A) Scatterplot showing the correlation between epigenetic age predicted from DNA methylation and reported

chronological age for 729 healthy donors (three individuals were excluded because no chronological age

was reported).

B) Positioning of the samples in two-dimensional space for sex prediction

C) Statistical association between principal components (columns) and sample annotations (rows). Significant

associations with p values below 0.01 are marked by filled circles, while non-significant values are

represented as empty circles.

D) Principal component analysis for 792 blood-based DNA methylation profiles, comprising 732 peripheral

blood samples and 60 sorted blood cell populations, using the same principal components as in panel c.

Immune cell content was estimated using the LUMP algorithm. E Scatterplot showing the negative

correlation between chronological age and the estimated fraction of CD4+ T cells

Use case 2: Dissecting the DNA methylation landscape of human hematopoiesis

The efforts of the International Human Epigenome Consortium and its contributing projects have resulted in large

sets of publicly available WGBS data for various cell types. To demonstrate RnBeads’ ability to process such large

reference collections, we analyzed a DNA methylome dataset comprising 195 WGBS profiles and 26,238,599

unique CpG sites (after the preprocessing step) for various hematopoietic cell types as in Fig. 3a, which was

originally established by the BLUEPRINT project. Focusing on pre-defined genomic region sets including the

Ensemble Regulatory Build, we observed the expected distribution of DNA methylation, with high levels of DNA

methylation in genome-wide tiling regions, slightly lower levels at enhancers and transcription factor binding sites,

and much lower levels (and a bimodal distribution) of DNA methylation at gene promoters and transcription start

sites as shown in Fig. 3b. The DNA methylation profiles clustered according to cellular lineage (lymphoid vs.

myeloid cells), cell maturation stage (naive vs. effector/memory cells), and cell type as shown in Fig. 3c. Comparing

two myeloid cell types (monocytes and neutrophils), RnBeads identified decreased DNA methylation levels in



monocytes at a subset of putative regulatory regions as in Fig. 3d. LOLA analysis for enrichment of genomic region

sets (a new feature we introduced in RnBeads 2.0 to facilitate biological interpretation) identified characteristic

enrichment for cell type-specific regulatory regions (including monocyte-specific open chromatin and its associated

histone modifications) and for the binding sites of important hematopoietic transcription factors such as CEBPB and

SPI-1/PU.1. In summary, this use case demonstrates the scalability of RnBeads to large DNA methylation datasets

(which involves the distribution of analysis jobs across an HPC cluster for efficient parallelized calculation), region-

based analysis of DNA methylation, and biological interpretation by region set enrichment analysis.

Fig. 3 Unique CpG sitesfor various hematopoietic cell types

Genome-wide analysis of DNA methylation in hematopoietic cells profiled using WGBS.

A) Overview of cell types and sample numbers in the BLUEPRINT WGBS dataset (August 2016

release), which was analyzed with RnBeads.

B) Distribution of DNA methylation levels for different types of genomic region sets.

C) t-SNE dimension reduction based on Euclidean distances of mean DNA methylation in putative

regulatory regions.

Cell types are color-coded as in panel a. d Density scatter plots showing differential DNA methylation levels

between monocytes (N = 20) and neutrophils (N = 10). Point density is indicated by blue shading. The 0.1% of

regions in the most sparsely populated areas of the plot are shown as individual points. The 500 highest-ranking

hypomethylated regions in monocytes compared to neutrophils are indicated in purple. E Log-odds ratios of the

LOLA enrichment analysis for the 500 regions highlighted in panel d. The top 20 most enriched categories of the

LOLA Core and Extended databases are shown. Differently colored bars represent different types of genomic region

data (e.g., peaks for histone marks or transcription factor binding sites) as Mf, macrophage; GM, lymphoblastoid

cell; Mo, monocyte; REMC, Roadmap Epigenomics Mapping Consortium.

Use case 3: Quantifying DNA methylation heterogeneity in a childhood cancer cohort

Epigenetic heterogeneity has recently emerged as a key property of tumor samples [9]. To demonstrate the utility of

RnBeads for cancer research, we re-analyzed 188 recently published RRBS profiles of Ewing sarcoma tumors, cell



lines, and mesenchymal stem cells [10]. Ewing sarcoma is a pediatric bone cancer characterized by low genetic

heterogeneity but striking changes in the epigenome [11]. Data processing and quality control resulted in 2,217,186

unique CpG sites that were covered by at least five sequencing reads in more than 50% of the samples. Based on

these CpGs, we aggregated DNA methylation values in each sample across annotated genomic regions, including

putative regulatory elements defined in the Ensembl Regulatory Build [12]. Principal component analysis showed

the expected separation between tumors, cell lines, and mesenchymal stem cells, with higher sample-to-sample

heterogeneity among the tumors and cell lines compared to mesenchymal stem cells (Fig. 4a). We compared the

primary tumors with the cell lines using the differential DNA methylation module of RnBeads, and we found that

most of the differentially methylated regions were hypermethylated in the cell lines (Fig. 4b). We also observed

increased variance in the cell lines (Fig. 4c). LOLA analysis detected markedly different enrichments among

differentially methylated regions (DMRs) and differentially variable regions (DVRs), indicating that the two

measures provide complementary information about the DNA methylation landscape (Fig. 4d–f). Regions

hypermethylated in Ewing sarcoma cell lines were enriched for DNase hypersensitive sites in various healthy tissue

samples (Fig. 4d), consistent with the widespread hypermethylation and silencing of non-essential regulatory regions

in cell lines. In contrast, hypervariable regions were enriched for transcription factor binding and histone

modifications in cancer cell lines and embryonic stem cells (Fig. 4f), indicative of increased regulatory plasticity of

the Ewing sarcoma cell lines compared to the primary tumors. In summary, this use case describes the analysis of an

RRBS-based dataset (which benefits from region-based analysis due to fluctuations in single-CpG coverage), and it

demonstrates the utility of RRBS and RnBeads for investigating DNA methylation heterogeneity in tumor samples.

Fig. 4. Component analysis for investigating DNA methylation heterogeneity in tumor samples.

Dissection of DNA methylation heterogeneity in Ewing sarcoma samples profiled using RRBS is as follows.

A. Principal component analysis of an RRBS dataset for Ewing sarcoma tumors, cell lines, and mesenchymal

stem cells, based on DNA methylation values aggregated across Ensembl Regulatory Build regions.

B. Density scatter plot comparing the aggregated DNA methylation levels between Ewing sarcoma tumors

(N = 140) and Ewing sarcoma cell lines (N = 16). Marked in purple are the most highly ranking

differentially methylated regions up to an automatically selected rank cut off.



C. Density scatter plot comparing DNA methylation variance between Ewing sarcoma tumors and cell lines.

Significant differentially variable regions are marked in brown.

D. Enrichment (log-odds ratios) based on LOLA analysis for the differentially methylated regions shown in

panel b and in panel e. Differently colored bars represent different types of genomic region data.

E. Density scatter plot comparing the log-ratios between DNA methylation levels and variance in Ewing

sarcoma tumors and cell lines.

F. Enrichment (log-odds ratios) based on LOLA analysis for differentially variable regions shown in panel c

and in panel e. ESC, embryonic stem cell; CGIs, CpG islands.

Use case 4: Analyzing DNA methylation data across different assay platforms

Several generations of Infinium DNA methylation microarrays have been used over the years, and it can be

necessary to combine multiple datasets in an integrative analysis. RnBeads now provides dedicated methods for

cross-platform analysis, making it possible to combine RnBeads data objects across the different versions of the

Infinium microarray (27k, 450k, EPIC) and with bisulfite sequencing data (RRBS, WGBS). To demonstrate this

feature, we analyzed a benchmarking dataset comprising three different assay platforms: Infinium 450k microarrays,

Infinium EPIC microarrays, and WGBS [13]. All three datasets were loaded and preprocessed separately using

RnBeads, which resulted in data objects with 443,053 (450k), 801,716 (EPIC), and 25,918,426 (WGBS) unique

CpG sites, respectively. Applying the RnBeads method for combining datasets with the option of including only

CpGs covered by all three platforms, these objects were merged into a combined dataset comprising 408,621 shared

CpGs. This combined dataset was processed using the RnBeads analysis modules.

We observed differences in the global distribution of DNA methylation levels between assays (Fig. 5a).

Nevertheless, the principal component analysis showed that the biological differences between samples dominated

over the technical differences between platforms (Fig. 5b). Focusing specifically on the comparison between a

prostate cancer cell line (LNCaP) and prostate epithelial cells (PrECs), we observed the highest correlation between

replicates for the same assay in the same cell type (Pearson’s r = 0.9979, Fig. 5c). Nevertheless, the correlation

between different assays in the same cell type (Pearson’s r = 0.9655, Fig. 5d) was still high and much stronger than

the correlation between different cell types for the same assay (Pearson’s r = 0.6471, Fig. 5e). In summary, this use

case highlights the feasibility and practical utility of cross-platform analysis of DNA methylation using RnBeads\.



Fig. 5 Global distribution of DNA methylation levels

Cross-platform data integration using RnBeads is as follows.

A. Distribution of DNA methylation levels for the same samples profiled across different assay platforms.

B. Principal component analysis of the assay comparison dataset. Point shapes and colors depict assay

platforms and cell types, respectively.

C. Density scatter plots comparing replicates of prostate epithelial cells profiled using the EPIC assay (panel

c); prostate epithelial cells profiled using the EPIC assay and WGBS (panel d); and prostate epithelial cells

as well as a prostate cancer cell line profiled with the EPIC assay (panel e).

Point density is indicated by blue shading. The 0.1% of CpGs in the most sparsely populated areas of the plot are

shown as individual points. All plots are based on CpGs that were covered by all three assay platforms. Pearson

correlation coefficients are shown below each diagram as NAF, non-malignant tissue-associated fibroblasts; CAF,

cancer-associated fibroblasts; PrEC, prostate epithelial cells; LNCaP, prostate cancer cell line.

Comparison to other software tools for DNA methylation analysis

To assess the computational efficiency of RnBeads, we compared its performance to that of other software packages

for DNA methylation analysis, separately for DNA methylation microarray data, RRBS data, and WGBS data (see

the “Methods” section for details and Additional file 2: Table S2 for tool configurations) [14]. Given that the

different tools provide vastly different feature sets, we considered three scenarios: (i) data import only, (ii) core

modules and (iii) comprehensive analysis with more features activated (Additional file 3: Figure S1). RnBeads was

the only tool that supported both microarray-based and bisulfite sequencing-based analysis. For microarray-based

analysis, the low-level data processing packages minfi, methylumi, and wateRmelon were faster than ChAMP and

RnBeads (which need to prepare the dataset for their more extensive downstream analyses) [15]. Compared to

ChAMP, RnBeads was more memory-efficient and faster in the comprehensive setting. For bisulfite sequencing-

based analysis, RnBeads showed better performance than methylKit on the WGBS dataset in the core module

setting, but somewhat longer runtime and higher memory usage on the RRBS dataset. These differences can be

attributed to the reformatting into memory-efficient data structures that RnBeads performs during data import [16].

In summary, the runtime performance of RnBeads was similar to that of other tools with more limited functionality,



suggesting that the choice of the most suitable tool for DNA methylation analysis depends mainly on the desired

features and analysis modes. To assist with an informed selection, we thus surveyed a broad range of tools for DNA

methylation analysis and assembled a detailed feature table based on the tool documentations (Additional file 1:

Table S1). RnBeads emerged from this comparison as the software that implements the most comprehensive

workflow for analyzing DNA methylation data, while also providing a user-friendly interface and extensive options

for reporting and reproducibility.

CONCLUSION

RnBeads is an integrated software package for the analysis and interpretation of DNA methylation data. Due to its

modular design and optional graphical user interface, the software is well suited for both beginners and experts in

the field of DNA methylation analysis. Interactive reports provide a comprehensive overview of DNA methylation

datasets while also fostering reproducibility (by documenting parameter settings) and robustness (by making it easy

to evaluate different parameter settings). RnBeads 2.0 implements various new features that arose from

technological advances and valuable user feedback. For example, support for the Infinium EPIC assay and for cross-

platform data integration broaden the scope of RnBeads analyses; new and improved methods for the DNA

methylation-based prediction of age and sex are useful for large cohort studies; a graphical user interface makes

many RnBeads features more easily accessible; the analysis of DNA methylation variability adds a new dimension

to epigenome-wide association studies; and LOLA-based region set enrichment analysis facilitates the biological

interpretation of DNA methylation differences. RnBeads also establishes a convenient way of interfacing with

reference epigenome datasets and integrating them into custom analyses, as demonstrated by the integration of

sorted blood cell profiles in our first use case. We processed several such datasets and provide re-runnable analyses

on the website (https://rnbeads.org/methylomes.html). The presented use cases provide concrete examples for

RnBeads analysis of DNA methylation and illustrate some of the tool’s key features. Typical applications of

RnBeads include epigenome-wide association studies, reference epigenome analysis, investigation of cancer

heterogeneity, and epigenetic biomarker development.

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2. Makambi, K. (2003). Weighted inverse chi-square method for correlated significance tests. Journal of Applied

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Lafta & Shamran (2019): Electrophoresis gels and PCR contamination May 2019 vol. 19


Safety Disposal of Electrophoresis Gels and PCR contaminate with

Ethidium Bromide and alternative methods.

Methaq Hadi lafta¹ Amal Raqib Shamran²

Iraqi Education Ministry, Qadisiyah ¹

University of Babylon /collage of science for weman²

Abstract:

Electrophoresis gel is commonly used in molecular biology laboratories for the identification of nucleic acid

(DNA or RNA) and proteins. This gel will typically be agarose-based. This electrophoresis process utilized an

organic fluorescence dye or inorganic such as Silver (which is an EPA regulated material) to stain DNA or

proteins. Waste of the DNA identification process must be managed and disposed in a manner to protect public

health and the environment. Purpose: To ensure safe, prudent disposal as well as reduce the amount of

Hazardous Waste material. This can be accomplished by choosing less toxic materials and work practical

methods that minimize thquantity of waste generated as well as the toxicity of the waste material itself. In cases

where safer materials or work practices cannot be employed, waste collection methods and regulatory agency

requirements are to be followed. Background: There are a number of different protocols and alternative dyes

used in the preparation of electrophoresis gels. Gels can be cast with or without dyes. The DNA /proteins can

be stained by adding the dye to the sample before electrophoresis; the gel can be placed in a dye solution after

electrophoresis has been completed. Waste Management: Waste disposal requirements will vary on the dye

used and the methodology used to stain the cells.

The electrophoretic waste of the DNA or proteins product identification process must be managed and disposed

in a manner that is consistent with the commitment it has to protect public health and the environment. The

following provides Lab managers/generators with the proper procedures for managing and disposing

electrophoresis-associated gel wastes.

Key words: PCR, electrophoresis, ethidium bromide, contaminate, waste management

How to cite this article: Lafta MH, Shamran AR (2019): Safety Disposal of Electrophoresis Gels and PCR

contaminate with Ethidium Bromide and alternative methods, Ann Trop Med & Pub Health-Special Issue;

19: 2004-19.

MUTAGENIC DYES

Ethidium Bromide, Acridine Orange, SYBR Green I, SYBR Green II, SYBR Gold, GelStar. These dyes have

been determined to have mutagenic properties. The gel that have been cast with these dyes in them, unwanted

dye stock solutions, and all contaminated debris must be collected for disposal by the HWMU. Gels that have

undergone Electrophoresis and staining, and then have been destained.

Where all excess dye has been washed out the gel (the only dye left in the gel is a trace amount contained in

DNA /protein sample material) can be discarded in the trash. Contaminated “non-sharp” lab debris (e.g.,

gloves, towels, tubes, etc.) should be collected and disposed of the HWMU.

The buffer solutions that have been run through the approved filter should be checked under an appropriate light

source for complete removal of



the dyes, and if it passes (does not fluoresce), the liquid can be disposed of

down the drain with a copious amount of water as long as it contains no other materials that would cause it to be

regulated as a Hazardous Waste.

Waste Management of Materials for Disposal through the Hazardous Waste Management Unit.

What is hazardous waste?

■Chemicals (room 0114)

■'Risk waste': Blood, cells, viruses and bacteria, sharp or pointed objects (room 0116)

■Biological waste: Dead experimental animals, etc. (room 0116 / freezer room in the waste area)

■Radioactive waste (room 0115)

Chemicals

The safety data sheet will tell you whether your waste is to be classified as hazardous waste. In addition

it will provide information about the necessary protective equipment and about any protective devices that must

be used.

All waste must be packaged and labelled properly. If possible, use the original packaging if this is

suitable. Containers must be in good condition, and caps must be screwed on well. Avoid using packaging

material that can be confused with household products – for example coffee jars, soft-drink bottles and the like.

The university purchases plastic cans for hazardous waste, labels for marking waste, and polystyrene

boxes that can be used to protect glass bottles against impact. These are kept in the waste area in the basement.

If you can not find any available or have any other questions, please contact the HSE coordinator.

Each substance/substance compound must be clearly labelled with:

■contents (name of chemical and approximate composition). If the contents are not known, the

container must be labelled “Content not known".

■date

■name, department and telephone number

Chemical waste, used oil and batteries must be placed in Room 0114 in the basement of Domus

Medica.

Volatile substances must be place in the innermost part of the waste room with suction removal. The

waste must be put on the shelves, away from the floor.

Explosive chemicals must not be put in this room. Contact the HSE Coordinator for the disposal of

explosives.

The room must be kept locked at all times.



Hazardous waste is collected by Ragn Sells AS.

Risk- and biological waste

When handling 'risk waste', adequate protective equipment and, if relevant, protective devices must be

used. Read the safety data sheet for information.

'Risk waste' and biological waste can include:

■Biological material

■Sharps and cutting equipment

■Substances that may be carcinogenic/mutagenic

■Cytostatics etc.

All waste must be packaged and labelled properly. 'Risk waste' is packaged in yellow plastic containers

that are labelled with a completed declaration form for risk waste. Yellow plastic containers and declaration

forms are available in the waste department in the basement. If you can not find any available or have any other

questions, please contact the HSE coordinator.

Yellow toxic waste containers must be placed in the basement of Domus Medica, room number 0116.

The room must be kept locked at all times.

Risk- and biological waste is collected by Ragn Sells AS.

Radioactive waste

When handling radioactive waste, adequate protective equipment and if relevant protective devices

must be used, such as closed hoods. Please read the safety data sheet for more information. All radioactive waste

needs to be clearly marked with a label for radioactive waste. These are available in the waste department. If

you can not find any available or have any other questions, please contact the HSE coordinator.

Radioactive waste, which is also 'risk waste' and biological waste, must also be packed into yellow

plastic containers and be marked with a label for radioactive waste. This also applies to waste that has

previously been radioactive but that has become inactive. Please use bequerel as a unit for activity.

All radioactive waste, as well as waste that has previously been radioactive, must be logged when it is

placed in the waste room. The log is kept inside of room 0115. If you have any problems using the log you are

required to notify the HSE coordinator.

The waste must be placed in the basement of Domus Medica, Room 0115. The room must be kept

locked at all times.

Waste described in this document must be stored in the following manner:

■32P is kept in plexiglass shielding containers

■51Cr and 125I are kept in lead containers

■35S, 3H and 14C can be stored without shielding



Mutagenic or Toxic product of (PCR) and Contaminated “Non-Sharp” Lab Debris (e.g., gloves, pads ,

tubes, etc.) into a plastic container 5 gallon bucket ( optimal of volume of waste generated). This container

should have a plastic bag as an inner liner. The container must be closed at all times except when immediately

adding or removing wastes from the container. Contact the Hazardous Waste Management Unit (HWMU) if you

need a 5 gallon bucket to collect your waste. Labelled the container’s which waste constituents are present in the

pail (e.g., “Hazardous Waste - Ethidium Bromide Contaminated Gels, Gloves, Paper”).

Sharp items (e.g., needles, Pasteur pipettes, Tips, etc) are to be placed into the containers or 5-gallon

pails. See below for the proper means for disposing of contaminated sharps lab debris.

For a pickup. An empty replacement pail will be provided at the time of the collection if needed. Collection and

Disposal of Chemically Contaminated Sharps

Chemically contaminated sharps (needles, Pasteur pipettes, etc.) must be collected in an approved sharps

shelter (NOT RED – use the white/translucent or yellow). It must be labelled(Hazardous Waste – Chemically

Contaminated Sharp). Any biohazard labels should be removed or completely defaced.

■ DNA and PCR products

■Kits

■ Agaros seals

■Used running buffer

Protective Equipment

■Gloves (nitrile gloves when handling ethidium bromide)

■Laboratory coat

■Safety glasses

Procedure for disposal

■ All DNA, plasmids etc. must be handled as toxic waste.

■ All pipette tips, tubes etc. that have been used for DNA work must be disposed of in yellow toxic waste

containers.

■ Agaro seals with EtBr and SYBR-safe/SYBR-green etc. are disposed of in yellow toxic waste containers.

■Contaminated glass equipment must be rinsed well before being put ready for washing.

■Kits: waste must be handled in accordance with the HSE data sheet that comes with the kit.

■Small volumes of isopropanol and ethanol waste must be collected in Eppendorf tubes and disposed of in

yellow toxic waste containers. Larger volumes must be handled as hazardous waste.

■Used running buffer must be handled as an absorption medium. Used absorption media must be disposed of in

yellow toxic waste containers. Read the safety data sheet that comes with the absorption medium.

Examples of absorption media

■ Activated carbon, activated charcoal, C3014-500G, untreated, granular, 20-60 mesh



■Destaining bags, cat. no: 730-0997

■BondEX 50 Set (5), product code M740703

Handling GMO(Genetically Modified Organisms ) waste

■Waste from GMO must be handled as biological waste and regarded as toxic waste. This applies to both solid and liquid waste.

■Waste from GMO in class 3 and class 4 must be inactivated before it is placed in yellow toxic waste

containers.

■Inactivation takes place by autoclaving for one hour at 121 degrees, or by using Virkon 1-3% or 5%

hypochlorite.

Alternative Dyes

SYBR® Safe, GelRed, GelGreen, and EvaGreen. These dyes determined to be nonmutagenic dyes in

testing by independent licensed testing laboratories.All gels and contaminated “non-sharp” lab debris ( gloves,

towels, etc.) that are processed using this dye can be discarded in the trash.Spent running buffer solutions and

destaining solutions that contain the dyes can either be collected and disposed of through the HWMU or

collected and run through an approved filter device.

The buffer solutions that have been run through the approved filter should be checked under the

appropriate light source for complete removal of the dyes, and if it passes (does not fluoresce), the liquid can be

disposed of down the drain with a copious amount of water as long as no other materials are present that would

cause the material to be a (Hazardous Waste). The filters that have been used up and are no longer effective

must be disposed of through the HWMU.

Figure (1) show the right disposed of carcinogenic Lab waste



References:

Armour, Margaret-Ann. Hazardous Laboratory Chemicals Disposal Guide, 3rd Edition.

Ethidium Bromide MSDS: http:/msds.ehs.cornell.edu/msds/siri/files/cfg/cfggr.html

GelRed, GelGreen Safety Report:

http://www.biotium.com/product/product_info/Newproduct/GelStains.asp

EvaGreen Safety Report:

http://www.biotium.com/product/price_and_info.asp?item=31000&Sub_Section=09A

GelStar Product Protocol:

http://www.cambrex.com/Content/Documents/Bioscience/GelStar%2818111%29.pdf

GelRed, GelGreen Ames Testing: z

www.biotium.com/product/product_info/Safety_Report/GR%20&%20GG%20safety.pd

http;//juniordentist.com

http://www.biotium.com/product/product_info/Newproduct/GelStains.asp

http://www.biotium.com/product/price_and_info.asp?item=31000&Sub_Section=09A

http://www.cambrex.com/Content/Documents/Bioscience/GelStar%2818111%29.pdf

http://www.biotium.com/product/product_info/Safety_Report/GR%20%26%20GG%20safety.pd

Praseetha et al (2019): Study of chitosan May 2019 vol. 19


IN-VITRO CHICK CHORIOALLANTOIC MEMBRANE STUDY OF CHITOSAN

CAPPED 5-FLUOROURACIL CONJUGATED GOLD NANOPARTICLES

Akhila Rajan

1, P.K. Praseetha

2*, Methaq Hadi lafta

3, V.N. Ariharan

4, S. T. Gopu Kumar

5

1,2*Department of Nanotechnology, Noorul Islam Centre For Higher Education, Kumaracoil –

629180, India.

3Lecturer in Ministry of Education, Iraq.

4Inbiotics, William Hospital Campus, Nagercoil – 629001, India.

5Thampi Institute of Medical Educational and Research, William Hospital Campus,

Nagercoil – 629001, India.

E-mail:[email protected]

ABSTRACT

Introduction: Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4) -

linked D-Glucosamine (DE acetylated unit) and N-acetyl-D-Glucosamine (acetylated

unit). It is made by treating the chitin shells of shrimp and other crustaceans with an

alkaline substance, like sodium hydroxide. Chitosan has a number of commercial and

possible biomedical uses. Chitosan's properties also allow it to be used in transdermal

drug delivery.

Aim: The aim of the work is to deliver therapeutic compound to the desirable site in the

treatment of diseases.

Methods: Chitosan Nanoparticles (NPs) was prepared using the ionic deletion method. 5’

Fluorouracil (5’FU) loaded Chitosan nanoparticle (5FCN) was synthesized and tested

for its Angiogenic activity on Cell lines by a chick chorioallantoic membrane (CAM)

assay.

Conclusion: The angiogenic activity of 5-FU loaded chitosan nanoparticles provided a platform

for treating cancer with biopolymer nanomaterials. The combination of nanoparticles

with Chitosan in the form of Nano composite matrices provides the high surface area

required to achieve a high loading of enzymes, drugs, and a compatible micro-

environment to facilitate stability.

Keywords: 5-fluorouracil, chick chorioallantoic membrane, Chitosan, Gold Nano particle.

How to cite this article: Rajan A, Praseetha PK, et al (2019): In-vitro chick chorioallantoic

membrane study of chitosan capped 5-fluorouracil conjugated gold

nanoparticles, Ann Trop Med & Pub Health-Special issue; 19: Sp2005-19.




INTRODUCTION

Recent advancements in the synthesis of various nanomaterials of different sizes, shapes and functions have

established nanotechnology as an indispensable technology for agriculture 1. Materials which show unique

properties linked to their size (ranging from 1 to 1000 nm at least in one dimension) are considered

nanoparticles and deemed under nanotechnology. Nano materials have some high value properties like high

surface-to-volume ratio, more molecules/reactive groups on the surface; prefers Nano-encapsulation and are

independent of gravity 2.

The rapid expansion of nanotechnology promises to have great benefits for society. Nanomaterial is matter at

dimensions of roughly 1-100 nm, where a unique phenomenon offers novel applications. The major advantages

of nanoparticles as a delivery system are in controlling particle size, surface properties, and release of

pharmacologically active agents in order to achieve the site-specific action of the drug at the therapeutically

optimal rate and dose regimen. Although nanoparticles offer many advantages as drug carrier systems, there are

still many limitations to be solved such as poor oral bioavailability, instability in circulation, inadequate tissue

distribution, and toxicity 3.

Chitin is found in the exoskeleton of some anthropoid’s, insects, and some fungi. Commercial sources of chitin

are the shell wastes of crab, shrimp, lobster, etc. Chitosan is usually prepared by the DE acetylation of chitin.

The conditions used for DE acetylation will determine the average molecular weight (Mw) and degree of DE

acetylation (DD). The DD of typical commercial chitosan is usually between 70 and 95%, and the Mw between

10 and 1,000 kDa.

The use of nanoparticles in the biological field has increased due to the superior properties that they possess

when compared to their bulk counterparts such as enhanced permeability and retention and the ease with which

they are taken up by the cells as demonstrated by Y. Liu et al in the use of Nano vectors for gene delivery in the

case of low density carbon nanotubes 4. Apart from this, nanoparticles own a functional surface which gives the

nanoparticles the ability to bind, adsorb and carry other compounds thus, making them suitable for drug

delivery. Besides this, the nanoparticles also protect the drug from degradation, enable a prolonged release of

the drug, improve the bioavailability of the drug, reduce the toxic side effects of the drug and offer an

appropriate form for all routes of administration 5. Of these nanoparticles, noble metal nanoparticles are

preferred due to their optical properties, non-toxicity and biocompatibility8 when compared to the other metals.

Majorly, gold nanoparticles can convert light or radio frequency into heat, thus enabling the thermal ablation of

the targeted cancer cells 6. This type of phenomenon has been observed for gold nanoparticles. Hence, the

ability to combine drug delivery and photo thermal therapy on gold nanoparticle based delivery platforms prove

it to be a system that is capable of eliminating the cancer cells 7. One major drawback that arises while using

gold nanoparticles is its agglomeration during reduction from its metal salts. It has reported the importance of

the control of nanoparticle size as the increased particle size reduces the scope for their use in drug delivery

since it is well known that particles with size ranging from 10 to 50 nm are the ones that are easily taken up by

the cells. Thus, an effective way to prevent the aggregation of gold nanoparticles is mandatory. An effective

strategy for this would be the use of a stabilizing agent or surfactant. The most promising and environment

friendly stabilizing agents are enzymes 15 and polymers. The role of a stabilizing agent/surfactant is played by a

chitosan18. Chitosan is a biopolymer that is biocompatible, biodegradable, eco-friendly, non-toxic and has NH2



and OH groups which act as chelating sites for drugs (5-Fluorouracil in our case) and for other molecules.

Moreover, as the combination of nanoparticles with Chitosan in the form of Nano composite matrices provide

the high surface area required to achieve a high loading of enzymes, drugs, and a compatible micro-environment

to facilitate stability as Chitosan is suitable for use as a drug delivery carrier. There are a number of reports on

the use of gold nanoparticles and Chitosan separately for drug delivery 8. A Nano composite system consisting

of both gold and Chitosan will encompass the properties of both the moieties, which makes it a powerful choice

for use in the bio-compatible, targeted delivery and sustained release of 5-Fluorouracil (5-FU).

MATERIALS AND METHODS

The chicken chorioallantoic membrane (CAM) assay is a standard assay used to evaluate the angiogenic

potential of biomaterials. Angiogenesis is the formation of new blood capillaries from existing blood vessels for

supplying nutrients to the cells that are distant from existing blood vessels. Angiogenesis is a complex process

that is mediated by the endothelial cells that line blood vessels. Angiogenesis is a regulated process involving a

number of stimulators such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF),

angiopoietins, activators of integrins and inhibitors such as thrombospondin, angiostatin and endostatin.

5 Days Old Fertilized Hen Eggs, Pyruvic Acid (Cat No:107360, Sigma), NaCl (Cat No.RES0926S-A, Sigma),

Parafilm (#380030, Tarsons), DMSO (#PHR1309, Sigma), D-PBS (#TL1006, Himedia), 50 ml centrifuge tubes

(# 546043 TORSON), 1.5 ml centrifuge tubes (TORSON), 1 ml serological pipette (TORSON), 10 to 1000 ul

tips (TORSON).

Equipment’s used Sterilized Sharp Forcep, 18-gauge hypodermic needle (#72-567, Harward Apparatus),

Camera (Canon), Pipettes: 2-10μl, 10-100μl, and 100-1000μl. Assay controls Negative Control (0.9% NaCl),

Positive Control (1% w/v SDS).

The chickchorio-allantoic membrane (CAM) assay for screening the effect of test samples on angiogenesis was

performed. Fertilized white leghorn chicken eggs were collected from a local hatchery at day ‘5’ and checked

for the damage. The eggs were cleaned with 70 % ethanol and incubated under conditions of constant humidity

at 37°C. On the 3rd day of incubation a small hole was drilled at the narrow end and 2-3 ml of albumin was

withdrawn with 18-gauge hypodermic needle. The window was sealed with transparent tape and again

incubated. On the 7thday of incubation a small square window was opened in the shell and sterile gel foam

(3mm×3mm×1mm) piece was implanted on top of the membrane. The vehicle control group was impregnated

with sterile normal 0.9% NaCl, standard group was impregnated with 1% SDS and the test group was

impregnated with IC50 Concentration. The eggs returned to the incubator and they were incubated undisturbed

till 24hours. After 24hours of incubation the eggs were removed from the incubator and the CAM tissues

directly beneath each sponge was resected from control and treated CAM samples. The number of vessel branch

points contained in a squire region equal to the area of each sponge was counted and findings from 3 CAM

preparations were analyzed for each treatment group. The resulting angiogenesis index is the mean of new

branch points in each set of samples.

Chitosan Coated With Gold Nanoparticles



100 ml Solution (2 ml acetic acid + 98 ml water) + O.34 gm. Chitosan (0.34 %) + 2 ml gold solution (5nM) +

2ml sodium borohydrate solution (50 mm). Stirred for 3 hours (room temperature). PH of the solution brought

to pH 5.0 using NaOH. The Solution is centrifuged at 3500 RPM for 15 minutes to separate Chitosan coated

with Gold Nanoparticles. The sample is dried at 50OC overnight.

5-Fluorouracil Loaded Chitosan - Gold Nanoparticles

100 ml Solution (2 ml acetic acid + 98 ml water) + O.34 gm. Chitosan (0.34 %) + 3.8 mm 5-Fluorouracil + 1.4

mm sodium polyphosphate + 0.5 % v/v Tween 80+ 2 ml gold solution (5nM) + 2ml sodium borohydrate

solution (50 mm). Stirred for 3 hours (room temperature).PH of the solution brought to pH 5.0 using NaoH. The

Solution is centrifuged at 3500 RPM for 15 minutes to separate Chitosan coated with Gold Nanoparticles. The

sample is dried at 50OC overnight.The conjugated nanoparticle is characterized by FTIR, UV, and TG/DTA.

RESULT AND DISCUSSION

FTIR Spectrum of 5flurouracil Linked Gold Coated Chitosan

The FTIR spectrum of Chitosan coated gold nanocomposite exhibits an NH2 twisting peak at ~1566.50 cm-1,

stretching at ~ 715.07 cm-1, bending at 1404.11 cm-1, a peak of NH3 + at ~1661.48 cm-1. These peaks

correspond well to the peaks of pure Chitosan except for minor differences that establish the formation of the

nanocomposite. The splitting of the NH3 + and NH2 peak increases with increasing the amount of gold in the

nanocomposite. This is because of the neutralization of the protonated amine group. An enhancement in the

intensity of the NH2 peak on increasing the amount of gold is an indication of the increase in the percentage of

gold, incorporated into the nanocomposite. The FTIR spectrum of 5-FU encapsulated nanocomposite is shown

in figure 1. All these observations show the encapsulation of 5-FU to Chitosan coated gold nanocomposite.

3500 3000 2500 2000 1500 1000

Wavenumber cm-1

Figure 1: FTIR Spectrum of 5Flurouracil linked Gold coated Chitosan.

Tra

nsm

ittan

ce [%

]

85

75

80

90

95

3249

.86

2932

.69

1651

.48

1565

.86

1556

.50

1404

.11

1247

.67

1151

.00

1069

.92

1011

.13

92

1.2

4

89

1.7

2

75

1.0

7

68

8.9

5

64

5.9

7

61

2.7

8

58

8.4

8

56

9.6

4



19.51% 475.9Cel

76.80uV 531.5Cel

81.62uV

463.7Cel

61.76uV -342uV.s/mg

550.6Cel

62.41uV 52.27%

495.5Cel 503.1Cel 60.00uV 58.98uV

-181uV.s/mg

TG/DTACurve Of 5flurouracil Linked Gold Coated Chitosan

Thermal Analysis (TA) techniques study the properties of materials as they change with temperature. It provides

information on properties like thermal capacity, mass changes, enthalpy, coefficient of heat expansion etc. It is

widely used in solid state chemistry to study solid state reactions, thermal degradation reactions, and phase

diagrams. The weight loss curve indicates changes in sample structure, heat stability, and other

parameters.Differential Thermal Analysis (DTA) involves exposing the material under study and an inactive

reference to similar heat cycles. Any temperature distinction amongst test and reference is recorded. In this

procedure, the heat flow to the specimen and reference is kept constant instead of the temperature. The peak

intensities are observed at 475.9 cells, 531.5Cel, 550.6 Cel.

80.00

70.00 90.00

60.00

80.00

50.00

70.00

40.00

60.00

30.00

50.00

20.00

40.00

10.00

0.00

100.0

200.0

300.0

400.0 Temp Cel

500.0

600.0

700.0

30.00

800.0

Figure 2. TG/DTA Curve of 5Flurouracil linked Gold coated Chitosan.

UV Spectrum Of 5-Flurouracil Linked Gold Coated Chitosan

The UV-Vis spectra taken for the composite containing different weight percentages of gold is shown in figure

3. The UV-VIS spectra for the formation of gold nanoparticles was monitored and obtained. A single peak

corresponding to the surface plasmon resonance of gold was observed in the wavelength range 210-300 nm. A

peak in this range is generally attributed to the surface plasmon excitation of small spherical gold nanoparticles.

The damped nature of the peak is indicative of the small particle size. In small particles, the mean free path of

the electrons is reduced, which eventually leads to the peak dampening. The intensity of the peak increased with

concentration of gold, which implies that the amount of gold binding to Chitosan increased

.

TG

%



2.5

2.0

1.5

1.0

0.5

0.0

200 300 400 500 600 700

Wavelength (nm)

Figure 3. UV Spectrum of 5-Flurouracil linked Gold coated Chitosan.

Angiogenic Activity Of 5-Fluorouracil Loaded Chitosan And Gold By Chickchorio-

Allantoic Membrane (CAM) Assay

Sample A (C+G) and Sample B (C+F+G) were evaluated to check the Angiogenesis Activity on the Fertilized

Chick Eggs. The used Concentrations of the compounds and No of Branching Points resulted mentioned in the

Table 1.

Table 1. Details of Drug Treatment to respective Fertilized Eggs

Sl.No Test Compounds Concentration

1 Untreated NaCl (0.9%)

2 Standard Pyruvic Acid

3 Sample A (C+G) IC50 (uG)

4 Sample B (C+F+G) IC50 (uG)

Control

266

5-Flurouracil loaded chitosan linked Gold

Abso

rban

ce (a

.u)



Sample A (C+G)

Sample B (C+G+F

STD

Figure 4. Angiogenic activity of the drug under various samples.

CAM Study Of The Test Compounds Using Fertilized Hen Eggs

The Observations in Statistical data of CAM Study suggesting that on Fertilized Eggs, given Test Compound-A

(C+G)) showing a slight increase in the growth of nerve branches with the blood vessel density 26 after the

incubation of 24hours of the drug treatment. On the other hand, Test Compound-B (C+F+G)) showing a high

increase in the growth of nerve branches with the blood vessel density 43 after the incubation of 24hours of the

drug treatment compared to the Standard Drug showing 86% of Blood vessel Density. In this study, The

angiogenesis index in the chick CAM was High in the Sample B Compared to the Sample A. The networks in

CAM and the newly synthesized vessels participated actively in the circulating of blood cells. Taken together,

the observations in the present study suggest that the given Test Compounds exhibit strong angiogenic actions



and also may have the potential to be a useful activator of the large number of serious diseases characterized by

deregulated angiogenesis

.

Figure 5. The Angiogenic activity of Chitosan loaded gold nanoparticles.

Fertilized Hen Eggs is treated with Chitosan and gold nanocomposite under different samples, branching points

count and blood vessel density is calculated for different samples. The result is tabulated in table 2. The blood

vessel density is minimum at control and standard (86) and the blood vessel density increase in the sample B

(43.66666667).

Table 2. Details of Drug Treatment to different samples and counting of blood vessels

Concentration Unit: uG/mL Incubation: 24hrs

Test CONTROL STD Sample A Sample B

Concentration 0.9% NaCl 1% SDS IC 50 IC 50

No of Branching Points Count 1 76 168 102 121



Mean No of Branching Points 78.33333333 164.3333333 104.6666667 122

Blood Vessel Density 0 86 26.33333333 43.66666667

CONCLUSION

It was observed from the present investigation that, the angiogenic activity of 5-FU loaded chitosan

nanoparticles provided a platform for treating cancer with biopolymer nanomaterials. Chitosan is safe to use as a

carrier for the drug 5-FU, which was observed from the present investigation. This could contribute to the



discovery of a new method for the treatment of various cell lines that may overcome the difficulties in the

currently available procedures or therapies. The increased angiogenic activity was found even at lower

concentrations of the drug. Thus synthesized nanoparticles were more efficient in the biomedical applications in

cancer treatment for their high angiogenic activity.

ACKNOWLEDGEMENT

The authors are thankful to the chancellor, Noorul Islam Centre for Higher Education, Kumaracoil for

providing all facilities.

ETHICAL CLEARANCE

Ethical clearance is taken from advisory committee, Department of Nanotechnology, Noorul Islam

University.

SOURCE OF FUNDING

This work is a self funded project.

CONFLICT OF INTEREST

No conflicts of interest declared.

REFERENCES

1. Meredith PA, Elliott HL. Clinical pharmacokinetics of amlodipine. Clinical pharmacokinetics. 1992 Jan 1;22(1):22-31.

2. Yamamoto K, Hagino M, Kotaki H, Iga T. Quantitative determination of domperidone in rat plasma by high-

performance liquid chromatography with fluorescence detection. Journal of Chromatography B: Biomedical Sciences

and Applications. 1998 Dec 11;720(1-2):251-5.

3. Carmeliet P. Angiogenesis in health and disease. Nature medicine. 2003 Jun;9(6):653.

4. Ng YS, D'Amore PA. Therapeutic angiogenesis for cardiovascular disease. Trials. 2001 Dec;2(6):278.

5. Rajan A, Gopukumar ST, Praseetha PK. Journal of Global Trends in Pharmaceutical Sciences. 2013 Feb. 13-22.

6. Dutta J. Engineering of Polysaccharides via Nanotechnology. InMultifaceted Development and Application of

Biopolymers for Biology, Biomedicine and Nanotechnology 2013 Jun. 87-134.

7. Tong R, Cheng J. Anticancer polymeric nanomedicines. Journal of Macromolecular Science, Part C: Polymer Reviews.

2007 Aug 1;47(3):345-81.

8. Kim CK, Ghosh P, Pagliuca C, Zhu ZJ, Menichetti S, Rotello VM. Entrapment of hydrophobic drugs in nanoparticle

monolayers with efficient release into cancer cells. Journal of the American Chemical Society. 2009 Jan 9;131(4):1360-

1.

Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19


Dental Ergonomics: Awareness and practices among dental

undergraduates in Saudi Arabia

M Zakirulla1*

, Salem Almoammar1, Amerah Bedah Muraih

1, Amjad Ali Al-whlan

1, Ohood Saeed Al-merei

1, Rasha

Hussain Al-zahrani1, Abdulaziz Bedah Alshahrani

2

1. Department of Pediatric Dentistry & Orthodontic Sciences, College of Dentistry, King Khalid University,

Abha, Kingdom of Saudi Arabia.

2. Damman University, Damman, Kingdom of Saudi Arabia.

* Corresponding Author: Dr. M Zakirulla, Assistant Professor, Department of Pediatric Dentistry &

Orthodontic Sciences, College of Dentistry, King Khalid University, Abha, Kingdom of Saudi Arabia. E-mail:

[email protected].

ABSTRACT

Aim: The present study aims to assess knowledge, practice, and condition of work place regarding ergonomics

among dental students in Abha city, Saudi Arabia. Materials and Methods: A cross-sectional study was carried out

on the sample size of 156 dental students (Males 91 & Females 68) from 5th

, 6th

year and internship year of the BDS

program (Students involving clinical courses) in College of Dentistry, King Khalid University, Abha, Saudi Arabia.

The survey with close-ended, self-administered questionnaires that focused on various positions while working was

used as the data collection method. The survey data was collected and organized into Microsoft Excel spreadsheets

(Microsoft Inc., USA), and was statistically analyzed utilizing the Statistical Package for the Social Sciences version

20.0 software (IBM Inc., USA). The statistical test used here was the chi-square test and P values less than 0.05

were considered to be statistically significant (P < 0.05). Results: when students were asked about this question,

92% of students agreed with sitting dentistry, 5% practised standing dentistry, and 3% were not sure. Student’s

interest in learning and exercising the correct working posture: 93% of students said yes. Majority of students were

interested in learning the right working posture. When the students were asked about the importance of

properposture in dental clinics: 90 % of students said yes. Conclusions: Findings confirmed that there is a positive




awareness of ergonomics among dental students. There is no statistical difference between males and females in

knowledge and behaviour related to ergonomic principles among dental students.

Keywords: Ergonomics, dental students, Posture, Pain, Awareness, Saudi Arabia.

How to cite this article: Zakirulla M, Almoammar S , et al (2019): Dental Ergonomics: Awareness and

practices among dental undergraduates in Saudi Arabia, Ann Trop Med & Pub Health-Special Issue; 19:

2006-19.

INTRODUCTION

Ergonomics is derived from Greek words, “Ergo” meaning work and “Nomos” meaning natural laws or systems.

Ergonomics is an applied science which deals with devising ways and product designs to enhance efficiency and

safety atthe workplace. The goal of ergonomics is to reduce the risk of work‑related injury at workplaces.[1]

Ergonomics in dentistry is defined as a reduction in cognitive and physical stress, preventing occupational diseases,

thereby improving efficiency with better quality and greater comfort for both the practitioners and patients.[2]

Dentistry is a profession that generally produces muscular pain and soreness which are innocuous and slow to

appear. As a result, the symptomswere usually ignored until they become chronic.[3]

Improving worker productivity and occupational health and safety at workplaces are current global

concerns.[4]Musculoskeletal disorders (MSDs), also known as cumulative trauma disorders are injuries to muscles,

nerves, tendons, ligaments, joints, cartilage, and spinal discs. They often present as pains in the upper extremities,

neck, and back and shoulders, etc.[5] Poor posture at work, repetitive movements, ill‑structured job, poor

workstation design, and prolonged working time among others have been reported as risk factors for the

development of MSDs. MSDs of the upper limbs isone of the most frequent occupational problems among dental

health‑care workersand have psychological and social in addition to physical consequences. When they reach a

certain level of severity, they directly affect a person’s ability to work, causing absenteeism, and even early

retirements.[6] Dental students are also at high risk of developing these disorders,mainly owing to the deleterious

postural habits they acquired during professional training [7]. Garcia et al.[8] perceived high risk for the



development of musculoskeletal disorders in students in their final year of graduation. Dental students are also at

high risk of developing these disorders, mainly owing to the deleterious postural habits they acquired during

professional training. [6-8]

In literature, researchers have documented the varied prevalence of MSDs among dentists. Dental education can

play an important role intraining dental students, helping them to adoptadequate knowledge related to ergonomic

posture.[9]Occupational health programs and training activities are not being carriedout satisfactorily.[10] Injury

preventionand dental ergonomics education are still in its infancyin Saudi Arabia. With this in mind, this work aims

toanalyze knowledge, practice, and condition of workplace regarding ergonomics among dental students in Abha

city, Saudi Arabia.

MATERIALS AND METHODS

A cross-sectional study was carried out on the samplesize of 156dental students (Males 91 & Females 68)from 5

th,

6th

year and internship year of the BDS program (Students involving clinical courses) in College of Dentistry, King

Khalid University, Abha, Saudi Arabia. The inclusion criteria were 5th

, 6th

year and internship year of the BDS

program, whovolunteered to participate in the study. The survey with close‑ended, self‑administeredquestionnaires

that focused on various positions whileworking was used as the data collection method. Thequestionnaire was

prepared by us keeping in mind thedifferent faulty positions of students working in the dentalclinics.Written consent

was taken from the respondents,and ethical approval for performing the survey was obtained from the Scientific

Research committee of King Khalid University, College of Dentistry.The questions were designed such that they

evaluatedthe level of postural awareness by eliciting the details of the body posture of the dentist. The

questionnairewas formulated which comprised Variables included inthe study were age (only quantitative variable),

gender, classof research, knowledge, and practice of ergonomic postureand condition of work place. The other part

of the questionnaire comprised 12questions were prepared based on other studies.[11] Using the convenience

sampling, the questionnaireswere distributed to all the 185 male,and female dental students present andworking in

the student clinics of the College of Dentistry. All the students were aged between 20 years and 26 years. Among the

185 students, 159 (86%) responded.



A self-administered structured questionnaire was developed and tested among a convenience sample of 20dental

students, who were interviewed to gain feedback on the overall acceptability of the questionnaire in terms of length

and language clarity, according to their feedback the questions were corrected. Face validity was also assessed

before the start of the study. Both descriptive and analytical statistical measurements were used to describe the main

variables by SPSS 18 (IBM Corporation, Armonk, New York, USA) software. Chi‑square, ANOVA was used to

compare the qualitative and quantitative variables. The statistical significance of the coefficients in the statistical

analyses will be tested at 0.05 (< = 0.05) level.

RESULTS

Knowledge related to posture during working patient on a dental chair [Table 1]: when students were asked about

this question, their 92% of studentswere agreed with sitting dentistry, 5% were practised standing dentistry, and 3%

were not sure. Student’s interest in learning and practising thecorrect working posture:93% of students said yes.

Majority of students wereinterested in learning the right working posture. When the students were asked about the

importance of properposture in dental clinics: 90 % of students said yes.The necessity to teach correct working

posture in classes and need education regarding this:95% of studentsthought that it is necessary to teach correct

workingposture during the clinical classes, so that theywould be able to analyze themselves regarding

theproblem.Whenthey were asked the position of head and elbow while working:79% ofstudents agreed for the

straight posture of the head,and 64 % said elbow should be at shoulder level.Regarding students asked about the

lower back is supported by the back of the stool while treating a patient on dental chair: 83% of students saidyes,and

3 % of students said no. Correlation between incorrect sitting postureswill cause harm to the body and affect dental

practice in the long run: 94 % ofstudents agreed with the statement. 78% of student agreed that they experienced

neck, finger, shoulder, back, or any other muscular pain related to your work in the dental clinic [Figure 1].



Table 1: Distribution of dental students in the study according to gender and class, by number and percentage of

respondents in each group.

GENDER 5TH YEAR 6TH YEAR INTERN TOTAL P VALUE

Male 31 (34%) 28 (30%) 32 (36%) 91 (58%)

0.859 Female 26 (38%) 20 (29%) 22 (32%) 68 (42%)

Total 57 (37%) 48 (31%) 54 (32%) 156 (100%)

*P<0.05; n = Number; % = Percentage. Figure 1: Do you ever experienced neck, finger, shoulder, back, or any other muscular pain related to your work in the

dental clinic?



Table 2: Comparison of males and females dental students in terms of knowledge and attitude towards Ergonomics

QUESTIONS Males

(n)-91

% Females

(n)-68

% Total

159

% P value

Q1.Do you practice

Sitting dentistry 84 93 62 91 146 92

Standing dentistry 4 4 4 6 8 5 0.908

Both 3 3 2 3 5 3

Q2. Do you think that adopting the correctposture

in clinics is important?

0

Yes 81 89 63 93 144 90

No

Don’t know

10

0

11

0

5

0

7

0 15

0

10

0

0.437

Q3. Do you think incorrect workingposture will

cause harm to your body and affect your dental

practice in long run?

Yes 89 97 60 88 149 94

No

Don’t know

2

0

3

0

4

4

6

6 6

4

3

3

0.028*

Q4. Is your lower back supported by the back of the

stool?

Yes 76 84 57 84 133 83

No

Don’t know

12

3

13

3

10

1

15

1 22

4

14

3

0.747

Q5. Have you ever experienced neck, finger,

shoulder, back, or any other muscular pain related

to your work in the dental clinic?

Yes 64 70 60 88 124 78 0.026*

No

Don’t know

21

6

23

7

6

2

9

3 27

8

17

5

Q6. What is the position of your head while

working?

Straight 73 80 53 78 126 79 0.678

Bent forward and tilted 15 16 11 16 26 16

Bent forward 3 4 4 6 7 5

Q7. What is the position of your elbow while

working?

At the shoulder level 55 60 47 69 102 64

Above the shoulder level, 23 25 18 26 41 26 0.119

Below the shoulder level 13 15 3 5 16 10

Q8. What is your seat position when you are

working on the patient?

Straight, 79 87 59 87 138 87

tilted slightly forward 12 13 9 13 21 13 0.992

Q9. What correct working posturecalled as?

Ergonomics 86 95 67 98 153 96

Astronomics 3 3 1 1 4 3 0.354

Economics 2 2 0 0 2 1

Q10. Is it possible to include correct working

posture education in preclinical subject?

Yes 89 98 62 91 151 95

No

Don’t know

2

0

2

0

5

1

7

2

7

1

4

1

0.114

*P<0.05; n = Number; % = Percentage.



DISCUSSION

The right and timely application of the principles of ergonomicsat work places promotes the health, efficiency, and

well‑being ofthe workers.[12] However, the practicability of ergonomic principlesis a function of its awareness.

Against this background, thisstudy aimed at assessing the awareness and knowledge ofergonomics among dental

undergraduates in Saudi Arabia.

In the present study, a bulk of respondents (89% males; 93% females) agreed that adopting correct postures in

clinics is important a majority of respondents were aware of the fat that muscular pain is related to improper lower

back supported by the stool. Posture reflects the position that an individual maintains inspace via his bone–muscle-

skeletal system, according to a staticor dynamic balance.[13] By maintaining a good posture, thebody lowers its

energy expenditure, improves organ functioningand is protected against disturbances that could

undermineoccupational practice.[14] In our study, 98% of males and 91% of femalesstudents were interested in

learning the proper workingposture,and it should be included in the curriculum rightfrom the preclinical classes.

Learning the proper workingposture would help reduce pain and discomfort. Itwas found that the students were of

the opinion that learningthis would not only help reduce pain but also will behelpful in the long run for their future

clinical practice duringclinical postings as well as professional working places. There was no statistical significant

difference in knowledge between both males and females undergraduate dental students.

Majority of respondents agreed with the posture of head and seat backrestis straight while working on the

patient in the dental chair. Dental stool must fit correctly; it must offer neutral back, neck,and shoulder support for

optimal posture, be at the correctheight and tilt, and offer optimal arm and elbow support.[15] In another study, 60%

of students in their final year ofdental school had difficulty visualizing the oral cavity during aprocedure, because of

incorrect use of the dental stool backrest,which resulted in an inadequate working posture.[16] Garciaet al. likewise

found that 11.7% of dental students adopt amalaligned right-tilted posterior column, probably to improve

viewing.[17]

Dental professionals with poor applicationof principles of ergonomics have increased the risk for

thedevelopment of work‑musculoskeletal disorders (MSDs),which could adversely affect his performance on the

job, qualityof test result, and ultimately patient’s management and care.MSDs, also known as cumulative trauma

disorders or repetitivestress injuries,[18] are injuries to muscles, nerves, tendons,ligaments, joints, cartilage, and

spinal discs. They oftenpresent as pains in the upper extremities, neck, and back andshoulders, etc.[19] MSDs are an



increasing health problem inworkplaces, resulting in workers’ disability, loss of precioustime from work, and huge

economic and social costs.[20] Poorposture at work, repetitive movements, ill‑structured job,poor workstation

design, and prolonged working time amongothers have been reported as risk factors for the developmentof MSDs

among clinical laboratory workers.[20,21].

A poor understanding of ergonomic principlesamong dental students, a gap between thetheoretical discipline and its

clinical application will be unsuitable for the ergonomically correct dental working environment. In thissense, the

education systemmust allow students to apply their theoretical ergonomicsknowledge to their clinical practice. The

use of digitalimages reproducing ergonomic dental positioning proved to bea positive methodology in this aspect.

Early acquisition of ergonomicsknowledge among dental students can results in improved assimilation and

incorporation,preventing deleterious habit formation.[15, 22]

A key limitation of this research is that this study is questionnaire-based, knowledge and practice of

ergonomics may not be best assessed by this method. The sample size in this study is small,and furtherresearch with

a larger sample size and a more elaboratequestionnaire will help understand thedentists’ requirements. This will

enable the planningand execution of appropriate training programs toprevent ill health effects associated with

improper postures during dental practice.

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3. Kumar RV, Anuroopa P, Nalini MS, Savita S. Prevalence of musculoskeletal pain and assessment of ergonomics factor in

under graduate dental students: An observational study. J Health Sci Res 2014;5:1‑5.

4. Shikdar AA, Sawaqed NA. Ergonomics, and occupational health and safety in the oil industry: A managers’ response.

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5. Darragh AR, Harrison H, Kenny S. Effect of an ergonomics intervention on workstations of microscope workers. Am J

OccupTher 2008;62:61‑9.

6. de Carvalho MV, Soriano EP, de França Caldas A Jr., Campello RI, de Miranda HF, Cavalcanti FI, et al. Work‑related

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7. Rambhad C, Pande N, Radke U. Assessment of knowledge of ergonomics among preclinical undergraduate students: A

cross-sectional study. J IntClin Dent Res Organ 2018;10:65-70.

8. Garcia PP, Pinelli C, Derceli JR, Campos JA. Musculoskeletal disorders in upper limbs in dental students: Exposure level to

risk factors. Braz J Oral Sci 2012;11:148‑53.

9. Valachi B, Valachi K. Preventing musculoskeletal disorders in clinical dentistry: Strategies to address the mechanisms

leadingto musculosketal disorders. J Am Dent Assoc 2003;134:1604-12.

10. Sartorio F, Franchgnoni F, Ferriero G, Vercelli S, Odesclchi L, Augusti D, et al. Work related musculoskeletal disorders in

dentistryprofessionals. 2. Prevention, ergonomic strategies and therapeutic programs. G Ital Med Lav Ergon 2005;27:442-8.

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Communit Dent 2015;5:S107-11.

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13. Wilson EL, Madigan ML, Davidson BS, Nussbaum MA. Postural strategy changes with fatigue of the lumbar extensor

muscles. GaitPosture 2006: 23: 348–54.

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OccupSaf Ergon 2007;13:3-14.

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18. Kozak A, Schedlbauer G, Peters C, Nienhaus A. Self‑reported musculoskeletal disorders of the distal upper extremities and

the neck in German veterinarians: A cross-sectional study. PLoS One 2014;9:e89362.

19. Darragh AR, Harrison H, Kenny S. Effect of an ergonomicsintervention on workstations of microscope workers. Am

JOccupTher 2008;62:61‑9.

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ClinPathol2010;133:543‑8.

22. Rising DW, Bennett BC, Hursh K, Plesh O. Reports of body pain in a dental student population. J Am Dent Assoc 2005:

136: 81–6.

Khaleel (2019): Coating of nanoparticle May 2019 Vol. 19

Application: Coating of Nanoparticles to Minimizing Harmful Environmental Pollution by Chemical Fungicide

Rana Ibrahim Khaleel

1 *

1* College of Engineering,University of Samarra, Samarra, Iraq

[email protected]

Abstract

Field experiment carried out for evaluation using nano-silver solution to reduce environment

pollution by fungicides. Four concentrations of AgNPs, (100 ppm, 50 ppm, 25 ppm, and 12.5 ppm)

were prepared by diluting the original stock solution with distilled water. From the concentration

was ready used in coated orang fruit compared with fungicide and uncoated fruit as control

treatment. The fruits were kept in box carton (40x40x30cm) and incubated under room conditions

for 28 days to study the formation to Green mold and Black fungal and shelf life for fruit stored.

The result showed high infection recorded in uncoated fruit compared with other treatments with

values reached (86.66 and 91.1) for Green mold and Black rot respectively, while ppm recorded

completely inhibition infections (0%) for fungal decay index. Also coating fruit with 100 and 50

ppm recorded significant different reached (3.8 and 3.9%) from other concentrations and fungicide

that, recorded high weight loss with mean reached (13.66%) after four weeks from storage.

Nanotechnology applied could play a fundamental role for this purpose and research in packaging

fresh fruit and minimize environmental pollution by chemical fungicides.





Keywords: Silver-Nanoparticles, Potential Alternative, Orange, Fungicide.

How to cite this article: Khaleel RI (2019): Application: Coating of Nanoparticles to

Minimizing Harmful Environmental Pollution by Chemical Fungicide , Ann Trop Med & Pub Health-Special Issue; 19: 2007-19.

Introduction

Nanoscale materials have emerged as novel antimicrobial agents owing to their high surface area to

volume ratio and the unique chemical and physical properties, which increases their contact with

microbes and their ability to permeate cells (Nejad et al., 2014). Also, nanotechnology has amplified

the effectiveness of silver particles as antimicrobial agents. Nanoparticles such as silver is known to

attack a broad range of biological processes in microorganisms including cell membrane structure

and functions (Lamsal et al., 2012). Nanoparticles also inhibit the expression of proteins associated

with ATP production, although its specific antimicrobial mechanisms are still not completely

understood. The use of nano-sized particles as antimicrobial agents has become more common as

technological advances make their production more economical. One of the potential applications of

Nanoparticles

is to manage plant disease. Since silver displays multiple modes of inhibitory action to

microorganisms (Kamran et al., 2011), it may be used for controlling various plant pathogens in a

relatively safer way compared to synthetic fungicides. Until now, . Reducing the particle size of

materials is an efficient and reliable tool for improving their biocompatibility. In fact,

nanotechnology helps in overcoming the limitations of size and can change the outlook of the world

regarding science (Gavanji et al., 2013). Furthermore, nano-materials can be modified for better



efficiency to facilitate their applications in different fields such as bioscience and medicine. This

simple size comparison gives an idea of using nanoparticles as very small probes that would allow

the elucidation of the cellular machinery without introducing too much interference (Lamsal et al.,

2012). Understanding of biological processes on the nano scale level is a strong driving force

behind development of nanotechnology (Salata et al., 2004). Current study came to investigate in

activity of silver nanoparticles as alternative of fungicides to decrease environmental pollution.

2. Methodology

AgNPs Solation Preparation

AgNPs, WA-CV-WA13B (CV)were used in the experiment that classified and using in different

manufacturing processes. Four concentration (100 ppm, 50 ppm, 25 ppm, and 12.5 ppm) were

prepared by diluting the original stock solution with distilled water and stored at 4℃ until further

use.

Fruit collection of Fungal Pollination

Citrus fruit (orange) of medium size and in good physical conditions such as being insect and

disease and damage free were purchased from local market in Samarra, Iraq. The fruits were

visually inspected for any damage or disease during collection. They were brought to the

enlivenment lab and kept in a cool room at 4oC for 24hrs to be used the next day for the experiment.

Preparation Fruit for Inoculation



The current research in cooperated a modification of this range to selected 100, 50, 25 and

12.5ppm. Fruits (oranges) washed with tap water, air-dried and then sterilized by immersion in 70%

ethanol for 1 min. The fruits were then randomly divided into six equal groups (Four nano-treatment

+ two controls). Fruits were wounded at a depth of 5.0mm with a 1.25mm diameter needle at the

equator for all the treatments and inoculated by spraying with suspension of fungi. The treated fruit

were left for an hour after spraying to stabilize the spores on the wound.

Preparation Fruit of Coating Treatment

From the concentration were prepared previously the homogenous solutions (100, 50, 25 and

12.5/ppm) used in coated fruit using a clean and soft brush. Coated fruits were allowed to dry at

ambient temperature about 30 minutes, after that transfer to package it.

Packaging Treated Fruit

The fruits were kept in box carton (40x40x30cm) and incubated under room conditions (25ºC±2

and 65-75 % RH) for 28 days to study the formation of fungal rot and shelf life for fruit stored .

There were three replicate trials of five fruits per replicate using the Completely Randomized

Design (RBD).

Fungal decay

Fungal decay in stored fruit was evaluated after 21 days under storage conditions. The percentage

of infection was determined using scale of 1-5 (Scale modified from Tzortzakis, 2006) as shown in

Table 1.



Table .1 Percentage of infection fungal decay index

Percentage infection Remarks

0 Clean

1-5 Trace

5-15 Slight

15-30 Moderate

>30 High

Shelf Life Fruit

The percentage of shelf life fruit was calculated by determining the progressive reduction in fruit

weight during storage after a period of 21 days in relation to the original fresh weight at the

beginning of storage (Tune et al., 2007).

WL (%) = WL before storage - WL after storage/ WL before storage X 100 (Eq 1)

Statistical Analysis

The experiment was laid out in the Completely Randomized Design (CRD) with three replicate.

Data collected and analysed using analysis of variance (ANOVA). Significant differences between

mean values were determined using Duncan’s Multiple Range test (P≤ 0.05). Following ANOVA

statistical analysis, which were performed using SPSS version 19- 2012 (SPSS Inc., Chicago,

USA).

3. Result and Discussion



Fungal Decay Index

The statistical analysis (ANOVA) as reported in Table.2 shows statistical different between the

treated and untreated fruit. High infection recorded in uncoated fruit compared with other treatments

with values reached (86.66 and 91.1) for Green mold and Black rot respectively. Also not

statistical difference (P ≤0.05) between 25, 50 and 100 ppm treatments which exhibited

significantly decrease in disease severity trace degree to completely inhibition infections (0%),

compared with 12.5ppm and fungicide which recorded (6.66 and 11.10) fungal decay index .

Table. 2 Effect of coating fruit with different concentrations of Silver (12.5 to 100 ppm) on the

development of mycelium growth Penicillium digitatum and Aspergillus niger(colony

diameter/cms) on the orange surface during storage for 21 days at 25oC±2 and 65-75% RH.

Treatments (ppm)

Penicillium

digitatum

Aspergilius

Niger

**Infection

12.5 6.66 b 11.10

c Slight

25 0 a 2.22

b Trace

50 0 a 0 a Trace

1000 0a 0

a Trace

Water 86.66 c 91.10

d High

Fungicides 6.66 b 11.10

c Slight

* Alphabets different in the same columns shows significant difference at (P≤0.05) between concentrations and average was calculated from three replicates.

Result the current study comes a similar to previous studies that, nanoparticles with antimicrobial

properties can be employed for safer food packaging. Nanoparticles such as titanium dioxide, zinc

oxide and magnesium oxide, as well as a combination of them, once functionalized can be efficient

in killing micro-organisms and cheaper and safer instead of metal based nanoparticles (Barik et al.

2008; Cui et al. 2010). Finding study of Sasson et al (2007) reported that, Nanotechnology has the

potential for efficient delivery of chemical and biological pesticides using nanosized preparations or

nanomaterial based agrochemical formulations. The benefits of nano material based formulations



are the improvement of efficacy due to higher surface area, higher solubility, induction of systemic

activity due to smaller particle size and higher mobility and lower toxicity due to elimination of

organic solvents in comparison to conventionally used pesticides and their formulation. Also

findings Abdelmalek and Salaheldin ,(20116) are agreement with result current study that, Silver

nanoparticle, 150 ppm, showed potent antifungal activity against the isolated fungi from fresh fruit.

Also current result come out with study (Lucimeire et al., 2014) that, coatings chitosan

nanoparticles of 110 nm showed higher antimicrobial activity against mold and yeasts, and

mesophilic and psychrotrophic bacteria.than the other treatments. Silver nanoparticles may directly

attach to and penetrate the cell membrane to kill spores, although penetration of silver nanoparticles

into microbial cell membranes is not completely understood (Morones and Elechiguerra,2005).

Shelf life Fruit

Effect different coatings on percentage of weight loss were shown in Table.3.The result showed

difference between the effects of the coating treatments on weight loss at different concentrations.

Coating fruit with 100 and 50 ppm recorded significant different (3.8 and 3.9%) from other

concentrations and fungicide after four weeks from storage. Also statistical analysis (P≤ 0.05) did

not showed significant difference between concentration 25 and 12.5ppm ( 5.42 and 5.5) which

difference from fungicide that, recorded high weight loss with mean reached (13.66%) after four

weeks from storage.

Table.3 Effect of coating fruit using nano-silver solution on weight loss (%) that storage at 25±2oC

and 65- 75 % RH for four weeks(28S) days.



Treatments

W1 W2 W3 W4 Mean

100ppm 2.9 a 3.5

a 3.9

a 4.9

b 3.8

a

50ppm 3.5b 3.5

a 4

b 4.63

a 3.9

a

25ppm 5 c 5.5

cb 5.5

c 5.70

c 5.42

b

ppm 5 c 5

b 6

d 6

d 5.5

b

Uncoated fruit 10.3 e 11.9

d 13.7

e 18.76

e 13.66

c

*Alphabets different in the same column show significant difference using Duncan’s Multiple Range test (P≤ 0.05) and average was

calculated from three replicates.

The treatment of 100ppm of silver showed significant decrease in weight loss compared with

uncoated fruit and other treatments Table. 3 Active coating using nano-silver may be helped to

improve and modify the atmosphere around the fruits, thereby preventing extremely low levels of

O2 and/or high levels of CO2, which could produce anaerobic metabolism with possibility of off-

flavour generation (Serrano et al. 2008; Valero et al. 2006 ; Valverde et al. 2005). Result current

study recorded clear increase in weight loss ranged (5.7- 6%) with treated 25 and 12.5 ppm

respectably. Current finding in agreement with report each of (Bautisty et al. 2003; Ali et al .

2011) that, coating fruit by some materalis such as chitosan conc.2-5% with crude plant extract of

custard apple leaves, papaya leaves and papaya seeds 1000-3000 did not influence the percentage

weight loss during the storage fruit but there was inhibition of fruit decay percentage. Result

current study are agreement with finding (Li and Wang. 2006 ; Li et al. 2009) that, nano-packing

technology applied on fruit and vegetables exhibited better physico-chemical , sensory

physiological and preservation properties compared to normal packaging . Also study of (Lopez-

Rubio et al. 2006 ; Jimenez et al. 2004) that, Nano encapsulation of active compounds used in



edible coating control rates and conditions and thus protect the coated items from moisture, heat or

other extreme conditions while enhancing their stability and viability.

Figure .1 Effect of coating fruit using nano silver on weight loss (%) at 25±2

oC and 65- 75%

RH storage condition for 28 days.

The experimental data by (Arnon et al. 2015; Ouzounidou et al. 2013) confirmed that -Ag coatings

markedly reduced the weight loss in those kinnow mandarin treatments. Weight loss in fresh fruits

and vegetables has not only been associated with vapor pressure at various locations, but also occurs

through respiration phenomenon. Result current study confirmed finding (Zambrano-Zaragza et al.

2013) that, Nano-emulsions and Nanoparticles can improve barrier properties and increase

functionality of coating because submicronic systems allow for more efficient control of coating

properties with better distribution and homogeneity on the coated fruit skin. Also support study

20

18

16

14

12

10

8

6

4

2

0

W1 W2 W3 W4

100ppm 50ppm 25ppm 12.5ppm Uncoated

We

igh

t lo

ss



(Hazirah and Armylisas, 2016) that reported, Nano-particles are recognized as micro additives and

submicroscopic particles which enhance the physical properties of food packaging such as edible

films and coating.

Conclusion

Application of coating nano-materials as further proved the quality of fresh fruits by decline fungal

decay index and increasing lengthen fruit shelf life. Using of the silver nanoparticles as alternative

fungicides for crop protection is emerging technology in food packaging but still need more

studies to assesses nano-silver to avoid side negative and their possible negative and toxicological

effects on environmental and human health.

Acknowledgements

Acknowledgements and Reference heading should be left justified, bold, with the first letter

capitalized but have no numbers. Text below continues as normal.

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guava(Psidium guajava L.) shelif-life food research international, 51(2): 946-953.

Khamis et al (2019): Genetic detection of HBV May 2019 vol. 19


Genetic detection of hepatitis B virus by PCR technique among children under

7 years in Hilla city, Iraq

AMAL RAQIB SHAMRAN1, Tsahel H. AL.Dulaimi

1, Ammar S.Khamis

2 RULA DHAHIR ABDULMOHSIN

1

1. Collage of science for woman

2. Collage of pharmacy University of Babylon

Abstract

Hepatitis disease is mean the inflammatory and destruction of the liver. Its commonly caused by viral

infection, Hepatitis B Virus (HBV) is one of the main resone of liver destruction, it spread when people come in

contact with the blood, open sores, or body fluids of someone who has the (HBV), 3.5% of global population is

chronic infected with HBV although the incidence of HBV infections is decreasing owing to vaccination and to a

lesser extent. Hepatitis is a serious public health problem distressing many of people worldwide. Inadequate

data is available on this issue in Iraq. This study was carry out with the aim of determining the genetic

sequencing in human and risk factors of hepatitis Bvirus (HBV) among the general population and among blood

donors. Methods: Blood samples from volunteers; have been detected the hepatitis-gen by PCR ,we see that

the 48 out of 250 children patients ( 19.2% ) among 5months to 5 years , in this study population the biggest

percentage were 45.83 % in aged 3-5 years while the second percent was 31.25 % in aged month-1year,while

it was 12.5% in age 1-3 years and 10.41% in age 5-7 years, table (1) ,in this study we see that male were 22

out of 48 ,45% while the femal were 26 out of 48 ,55%.

Key words: HBV, PCR, hepatitis, genetic detection

How to cite this article: Shamran AR, Al-Dulaimi TH, et al (2019): Genetic detection of hepatitis B virus by

PCR technique among children under 7 years in Hilla city, Iraq, Ann Trop Med & Pub Health-Special Issue; 19:

2008-19

Introduction

Hepatitis generally is disease involving the liver as target for viruses and other disease agents .The infections

that occur in the liver is called Hepatitis. There are many variety agents in addition to the virus infections that

cause liver destruction such as bacteria, fungi, drugs (1). Viruses which responsible for liver diseases include

HBV, HCV, HDV. HBV was discovered in 1963 by Dr.Blumberg at the National Institute of Health (NIH), (2).

HBV infections were recognized and reported as main reasons of significant morbidity and mortality in the

world (3). WHO reported that 2 billion people are infected with HBV and 450 millions have chronic infections

and the others peoples have risk of infected by HBV and HDV, many factors seem to associated with fibrosis

,age, daily alcohol consumption , gender (4). Many studies have reported that the risk of infection of the HBV

has increased in the countries which have high level of sexual activities, drugs use, family history of HBV

infections, vertical transmission haemolysis and exchange organs and bloods (5). This study aims of

determining the genetic sequencing in human and risk factors of HBV in the general population and among

blood donors.

Material l and methods

In this study total 250 patients children blood samples were collected from Alnoor-hospital ,during the period

Nov2017-Mar2018; then analyzed by PCR technique .



HBV DNA Detection through PCR for detection of HBV, the specific purification of the gen was amplified by

PCR using specific forward & reverse primers.

Viral DNA Extraction : DNA of the virus was isolate from the blood samples by from patients by (Qiagen

kit)according to manufacture protocol .

PCR – test,Thermal Cycling, Place the tubes wich contain (DNA sample ,master mix ,10mM

ofsenseprimer(Macrogen,Korea),5-CCGAATTCGCCACCATGCATCCTGCTGCTATGCCTCATCT-3and 10mM of

antisense primer (Macrogen, Korea), and 5-CCC-GAATTCGCCACCATGCGAACCACTGAACAAATG-GCACT-3 .

Cycling Profile

94°C to 45 second, (denaturation)

94° to 1 minute

64°C to 45 seconds (annealing)

72°C to 72 second (elongation)

For 30 cycles,

72°C to 7 minutes

Agarose Gel Electrophoresis, for visualization of the amplification products, Manufacture organisation

advocate using a 4 % NuSieve® 3:1 Plus TB buffer precast gel, 4% NuSieve, three:1 agarose gel in 1X TB buffer

contain 0.5μg/ml EthB.

Results

Screening of Cases infected with HBV was 48 out of 250 children patients ( 19.2% ) among 5 months to 5

years, in this study population, the biggest percentage were 45.83 % in those aged 3-5 years while the second

percent was 31.25 % in those aged month-1year,while it was 12.5% in age 1-3 years and 10.41% in age 5-7

years, table (1) ,in this study we see that male were 22 out of 48 ,45% while the femal were 26 out of 48 ,

55%. There is no significant different of the gender in the patients, and the same number of patients who live

in the rural and the urban (24 patient 50%), table (2).

Table 1: age group of patient population group

No.

Age group /year

Tested subject

No.

%

1 1 month-1year 15 31.25

2 1-3years 6 12.5

3 3-5years 22 45.83

4 5-7 years 5 10.41



age group of hepatitis distribution

year1 month-1

years year- 1

years years-

years years-

Figure 1: age group of hepatitis patients

Table2: general characteristic of the study of the patients’ population

Gender Inhabit

Male Female Urban Rural

No. % No. % No. % No. %

22

45.38

26

54.16

25

50

25

50



Figure 2: positive result

Discussion

All though there have been massive advances in immunization for HBV infections over three decades, the

burden of HBV remains a public health concern around the world. 2 billion people are estimated to be

affected by HBV disease, which causes a broad range of liver sickness, including intense or fulminant hepatitis,

inert administration state, reactivation, relentless hepatitis, cirrhosis and hepatocellular carcinoma (HCC). An

estimated 420 million people around the world requires ceaseless HBV contaminations; 15%-40% of them may

die due liver disease complications or HCC [3]. Symptoms of HBV disease differs around the globe, around

45% of the overall people live in risky environment, including sub-Saharan Africa, the Pacific and particularly

Asia(6). A study by Ye et al (2006) reported that a small number of case become much less infected due to

perinatal transmission from mother to infants. A number of studies reported of similar findings. The

extraordinary sexual activities in this study may explain the reason for the risk among the subjects, although

there is no statistical association between them (P>0.05). This contradicts the study of Omer and

Thewaini(1979) which reported that the males were higher than females in ratio (1.6:1) in Iraqi people as well

as Al- Mashhadani (1998) stated that males were higher than female (2:1),while Sabri reported that it was

2.5:1 . The prevalence of the virus within the urban and rural areas were identical as reported by Boisier et al

(1996) which reported that the agriculture regions have tremendous large HBV infection of around 26% while

the city has 53% (7).

References

[1] G. B.Gaeta,C. Sardaro,G.Giusti et al., “Prevalence of anti-HCV antibodies in patients with chronic liver disease and its

relation-ship to HBV and HDV infections,” Infection,vol.18,no.5,pp. 277–279, 1990.

[2] D. Lavanchy, “Hepatitis B virus epidemiology, disease burden, treatment, arid current and emerging prevention and

control measures,” Journal of Viral Hepatitis, vol.11, no.2, pp.97–107, 2004.

[3] T. L. Wright, “Introduction to chronic hepatitis B infection,” American Journal of Gastroenterology, vol.101, no.1, pp.S1–

S6, 2006.



[4] A. Sangiovanni ,G.M.Prati, P. Fasanietal., “The natural history of compensated cirrhosis due to hepatitis C virus: a 17-

year cohort study of 214 patients,”Hepatology,vol.43,no.6,pp.1303– 1310, 2006.

[5] B. Tsatsralt-Od, M. Takahashi, T. Nishizawa, K. Endo, J. Inoue, and H. Okamoto, “High prevalence of dual or triple

infection of hepatitis B, C, and delta viruses among patients with chronic liver disease in Mongolia,” Journal of Medical

Virology,vol.77, no. 4, pp. 491–499, 2005.

6) Takako Utsumi et al (2014) Molecular epidemiology of hepatitis B virus in Asia World J Med Genet. May 27, 2014; 4(2):

19-26

7) P. Boisier et al (2009) reported that Hepatitis B virus infections in general population in Madagascar evidence for

different epidemiology pattern in urban and rural areas , VOL 117,issue 1 august (1996) 133-137

8) Ye F., Yue Y., Li S., Chen T., et al. (2006). Presence of HBsAg, HBcAg and HBV DNA in ovary and ovum of the patients

with chronic hepatitis B virus infection. Am. J. Obstet. Gynecol. 194:387-392.

9) Al-Mashhadani J.I., (1998). Seroepidemiological study on HBV and HCV infections among health care workers. Ph. D.

thesis. College of Medicine. Baghdad University.

Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19


COMPARISON OF APGAR SCORE IN NEONATES BORN AFTER ELECTIVE:

SPINAL VERSUS GENARAL ANAESTHESIA.

Idrees Jameel Khalaf1

1. Department of surgery Tikrit College of medicine Tikrit, Iraq

ABSTRACT:

BACKGROUND: ten point Apgar score has been used to compare the effect of spinal anesthesia versus general

anesthesia on Apgar score of neonates born by elective caesarean section.

STUDY DESIGN: Randomized prospective study.

SETTING: This study was conducted in the Department of pediatrics-Haweejha general Hospital/Kirkuk/Iraq., between

December2017toJune2018.

SUBJECTS AND METHODS: In this study was achieved on sixteen women bestowing for Elective lower segment

caesarean section. Thirty mothers were given general anaesthesia and other 30 mothers received spinal anaesthesia. The Apgar

score was recorded at 1 minute and 5 minute interim after each delivery.

RESULTS: Out of thirty mothers, who received general anaesthesia, 25 patients (82.3%) give birth to neonates having

Apgar score ≤ 6 at one minute after birth and the remaining 5 neonates (17.7%) had Apgar score of ≥7. On the other hand out of 30 mothers who received spinal anaesthesia only 10 mothers give birth to neonate having Apgar score ≤ 6 at one minute after birth, who improved at 5 minutes interval, and their Apgar score were ≥7.It had been found that those neonates who were born under G.A were ten folds more likely to have Apgar score less than or equivalent to 6 at first

minute compared to those with spinal anaesthesia, the odds ratio=10 and 96%assurance interval of the odds ratio (2.95-

35) and p=0.00025 which is highly significant, G.A had greater risk on newborn at the first minute.

KEY WORDS: Apgar score, Spinal Anesthesia, General Anesthesia, Neonate, Elective Caesarean Section.

How to cite this article: Khalaf IJ (2019): comparison of Apgar Score in neonates born after elective spinal

versus general anaethesia, Ann Trop Med & Pub Health-Special Issue; 19: 2009-19.

INTRODUCTION: An Apgar test is usually performed for the fetus twice: once at first minute after

birth, and again at 5 minutes after birth. Infrequently, if there are apprehensions about the baby

condition and the first two scores are low, less than 7, the scoring is also performed at 10, 15and 20

minutes after delivery (1)

. Five factors are used to estimate the baby’s condition and each factor is

scored on a scale of 0 to 2, with 2 being the best score for each: Activity and muscle tone, Pulse,

Grimace response, Appearance and Respiration. Scores obtainable are between0- 10, with 10 is highest

possible score. There are several factors that affect Apgar sore counting false positive and false



(3)

negative score(2)

.

The Apgar score was mostly designed as a research implement, enabling the grouping

ofinfantsaccordingtotheirconditionatbirth.In1953,VirginiaApgar,M.D.publishedherproposal

foranewmethodofevaluationofthenewborninfant.APGARscoreisaclinicalexperimentimplementedo

na newbornoneandfiveminutesafterbirth.Itisacompositemeasureofbreathingeffort,heartrate,

muscletone,reflexes,andskincolorandisanindicatorofthenewborn'sneedformedicalconsiderationpres

entlyafterbirth.Apgarscore(AS)isusuallyusedforvaluationofnewbornsinstantlyafter

birthandconsistsoffivevariablesviz.Respiratoryefforts,heartrate,color,muscletone,andreflex

irritability. It is being used as a standardized tool for expressing the physiologic condition of

newborn at birth and also to record fetal to neonatal transition. However, Apgar score has

mainconfineslikehavingalimitedtimeframeandcountingsubjectiveconstituents.Eachoftheseis given

a score of 0,1 and 2. Thescoreisconventionallyreportedat1and5minafterbirth,A score of 0-3 at 5

min is a suggestive criteria for asphyxia insult and is a predictor of neonatal mortality.

Newbornswithascoreof≥7are measured normal .

Apgar scoring is a research instrument rather than a criteria for clinical valuation on which

to baseadministrationchoiceorprognosis.Apgarscoreat1,5 min have allow specificity for asphyxia

and subsequently poor predictive value for long term neurological squeal, score can be falsely

lowinverypreterm,maternaldrugintake,CHD,andCNSdisfigurements,hencelowscorescannotbe

alwaysparalleledtoasphyxia.Thoughitisusefulinassesscardiopulmonarystatus,tellaboutneedof

resuscitation and its effectiveness. It is assigned every 5 min until 20 min or till 2 consecutive

score are 7 or greater. The neonatal revival program (NRP) guidelines state that “Apgar scores

should not be used to dictate appropriate resuscitative actions, nor should interventions for

depressedinfantsbedelayeduntilthe1-minuteassessment.”However, an Apgar score that remains 0

beyond 10 minutes of age may be useful in determining whether additional resuscitative efforts

are indicated. The outcome of anaesthesia either spinal or general depends upon the condition of

the mother and more importantly effects on newborn. APGAR score is the best parameter to

assess the direct condition of the baby.(4,5)

This is the reason to select this

topic.Theutmostimportantcauseoffetaldistressinanyanestheticmethodisthelesseninginthe amount

of O2 available to the fetus as a result of the reduction of uteroplacental blood flow. Motherly,

placental, and fetal factors play roles in such reduction. The effect of anesthetic drugs is direct or

through the changes in the mother (6)

.The Apgar scores are taken at 01and 05 minutes after

delivery. Of the two scores, the 05 minutes score is regarded as the better predictor of survival in

infancy in the long term. Whereas the 01 minute score definitely has the value for; assessing the

effectsofdifferentdrugsgiventothemotherduringtheCesareansection.Thismethodisevenmore

interesting because it is noninvasive, (7,8)

MATERIAL AND METHODS:

.



This study was carried out in Mansoura hospital from February 2017 to July 2018, and was

performed on 60 healthy full term mothers presenting for elective lower section caesarean section

,thirty mothers were given general anaesthesia and other sixteen mothers received Spinal

anaesthesia, the Apgar scores were recorded at 1minute and 5 minute interval after each delivery.

Total sixteen mothers were encompassed and a written consent was taken from each patient.

Method for general anaesthesia: History was taken for all patients in the suite which include age,

parity, duration of pregnancy and any maternal health history, anaesthesia interrelated obstetric

history, blood pressure measurement, and airway assessment. An18gauge intravenous catheter is

employed.

Method for general Anaesthesia: Before medication done with injection. Cimetidine 200mg i/v,

Injection Metoclopramide 10mg i/v 1 ± 2 hours prior to induction. Patients was pre oxygenated

for 3 ± 5 minutes, induction was done with, Injection Thiopentone 4mg/kg body weight i/v,

Injection Succinyl choline 1.5mg / kg i/v. After endotracheal intubation, 50% oxygen with nitrous

oxide and 0.5% halothane breathing was given each time. General anaesthesia was maintained

with non-depolarizing muscle.

Exclusion criteria: the following patients were excepted: Premature pregnancy <37weeks of

gestation, Liver, heart or kidney failure associated with gravidity, Uncontrolled metabolic

disorder (diabetes Mellitus, hypertension, thyrotoxicosis), And Multiple fetus gravidity.

Spinal anaesthesia: History was taken from patients particularly about back surgery, valvular

heart disease, hemorrhage tendency, pre-existing neurological deficits, blood pressure, pulse rate,

chest inspection was done and back examination for any deformity or back surgery at site o

injection, two 18 gauge intravenous catheters are employed, 500-1000ml of crystalloid solution

was preloaded then patient was placed in sitting position and space between 3rd

and 4th

lumbar

spine was identified and marked. After attractive all aseptic measure lumbar puncture was done

with22 gauge size spinal needle and hyperbaric Bupivacaine 0.5%, 2.5ml (12.5mg) was

administered. Directly after injection of Bupivacaine, patient was placed in supine position with

wedge under right hip for left uterine dislodgment. Monitoring was for pulse, N.I.B.P., oxygen

permeation. The following was recorded during every caesarean section under general or spinal

anaesthesia in different stages of birth: time of induction, time of incision to skin, time of incision

to uterus, time of delivering the baby. We contemplate that every fetus should deliver with less

than 10 minutes after induction of GA. In this study, Apgar score of all 60 neonates was recorded

at 1minute and 5 minutes after delivery. Birth weight was recorded and Apgar score of each baby

was compared with standard Apgar score chart.

Statistical analysis: By using statistical package for social sciences (SPSS) software for window

V.18.US , data of all cases were come in and analyzed; descriptive analytic statistic were

performed using appropriate statistical tests. Age, history was taken from patients specially about

back surgery, valvular heart disease, bleeding tendency, pre-existing neurological deficits, blood

pressure, pulse rate, chest examination was done and back examination for any deformity or back



surgery at site o injection, two 18 gauge arterial catheters are employed, 500-1000ml of

crystalloid solution was preloaded then patient was placed in sitting position and space between

3rd

and 4th

lumbar spine was identified and marked. After taking all aseptic measure lumbar

puncture was done with22 gauge size spinal needle and hyperbaric Bupivacaine 0.5%,

2.5ml(12.5mg) was administered. Immediately after injection of Bupivacaine , patient was

placed in supine and weight were expressed as (Mean± SD).

Student (t) test was used to compare these incessant variables and the mean of both groups. All

data were presented as tables, graphs or paragraph. In all statistical measures and tests P value set

at ≤0.05 to be consider as significant.

RESULTS:

Recording of Apgar score: In this study Apgar score of all 60 neonates were recorded by

neonatologist. Apgar scores were recorded at 1 minute and 5 minutes after delivery. Birth weight

of every baby was recorded. Apgar score of each baby was compared with the standard Apgar

score chart as shown below:

Apgar score (table I) of the two groups was compared by Statistical analysis performed with the

use of power calculation.

\ TABLE I: APGAR SCORE: (B= Body/ E=extremities/ C= Completely/ S= Spontaneous)

APGAR

Score

Heart

rate

Respiratory

effort

Reflex

irritability

Appearance

(color)

Muscle tone

0 Absent Absent No-response Blue or pale Flaccid

1 <100 Irregular Grimace B-pink / blue-E Good tone

2 >100 Good Cough/sneeze C-pink S-flexion

For all studied 60 mothers, the mean age of the mothers was (28.75±0.11), and the mean birth

weight of neonate was (3.1±0.16) of GA-group. While, the mean age of the mothers was

(27.91±3.12), and the mean birth weight of neonate was (31.7±0.18) of Spinal-group There was

no significant difference in age of the mothers nor had the birth weight of neonates been found in

between two groups.( P. value >0.05), table (2). Out of 30 patients who received general

anaesthesia, 25 patient (82.3%) give birth to neonate having apgar score ≤ 6 at one minute after birth and the remaining 5 neonates (17.7%) had apgar score of ≥7. At 5 minutes, twenty five neonates with low Apgar score at one minute were improved after resuscitation and showed

Apgar score of ≥7.

Table 2: Descriptive statistics of studied group.



Variables

anaesthesia Mother Age Neonates’ Birth weight

Mean ±SD(year) Ranger (year) Mean± SD(kg) Ranges (kg)

An

aes

thes

ia

GA group 28.75±0.11 18 - 37 3.1± 0.16 2.8 - 3.5

Spinal group 27.91±3.12 18 - 37 31.7 ± 0.18 2.8 - 3.5

All cases 28.6±3.75 18-37 3.07± 0.14 2.8 - 3.5

P .value 0.58 0.56

On the other hand out of 30 patients who received spinal anaesthesia, only 10 patients give birth

to neonate having Apgar score ≤ 6 at one minute after birth, who improved at 5 minute interval,

and their apgar score were ≥7. The Apgar score at 5 minutes of all 30 neonates, in spinal anaesthesia group, was ≥7.

It had been found that the infants who were born under GA were ten folds more likely to have

Apgar score less than or equal to 6 at one minute compared to those with spinal anaesthesia, the

odds ratio=10 and 96% confidence interval of the odds ratio (2.95-35) and p=0.00025 which is

highly significant, G.A had greater risk on infant at the one minute, table (3).

Table 3: Apgar score at one minute and type of anaesthesia.

Apgar score at 1min

Odd ratio

P. value Anaesthesia ≤6 ≥7 Total

GA-group Count 25 5 30

10

0.00025 %within anaesthesia 82.3% 17.7% 100%

Spinal-group Count 10 20 30

%within anaesthesia 35.5% 64.5% 100%

Total Count 35 25 60

%within anaesthesia 59.1% 40.9% 100%

DISCUSSION:

The Apgar score is a applied method of systemically assessing newborn infants directly after birth

to help identify those demanding resuscitation and to predict survival in neonatal period the 1 min.

Apgar score may signal the need for instant resuscitation, and the 5, 10, 15 and 20min.score may

indicate the probability of successfully revivifying an infant. Due to a number of factors a low

score may be including drugs given to the mother during labour, caesarean section under general

anaesthesia and immaturity(9)

. The major causes of maternal death in Pakistan are general

anesthesia. Despites the advances in anaesthetic techniques, monitoring facilities and availability

of different drugs, young women are still dying of anaesthesia related complication (10)

. In another



study observed that Apgar scores of neonates whose mothers conventional general anaesthesia

were lower than neonates whose mommies received spinal anaesthesia(11)

.

Delivery of baby by caesarean section has become gradually common, and both general and

spinal anaesthesia have certain advantages and disadvantages, but regional anaesthesia has

become the favored technique because general anaesthesia associated with higher maternal

mortality and foetal depression(12)

.

Apgar’s was between the first to report that babies delivered by Caesarean section under spinal

block were, in general, more vigorous at birth than those whose mothers had cyclopropane.

Numerous workers report a marginal enhancement in one-minute Apgar scores in infants

delivered by Caesarean section under epidural block (13,14,15,5)

,but others have found no

difference.(16,17)

.

Datta et al., observed that in absence of hypotension there is no change in Apgar scores or acid

base status with prolonged induction to delivery interval in spinal anaesthesia. Morgan describe

long skin incision to delivery time more than 8 minutes and uterine-incision-to delivery time more

than 180 seconds have been associated with foetal hypoxia and acidosis regardless of the type of

anaesthesia. The Apgar scores of neonates whose mothers received general anaesthesia were

lower than neonates whose mothers received spinal anaesthesia. (18)

.

A study other done that there is that there is statistically significant risk of fetal acidemia of

varying severity with the use of regional anaesthesia in women delivered by cesarean without

labor. Also, the umbilical artery blood PH values less than 7.10 were observed in 4% of fetuses,

among whom 1% had PH values less than 6.99. On the other hand no infant had PH values less

than 7.10 when general anaesthesia was used. They also concluded that the prevalence of low PH

values was significantly increased in those infants exposed to any of regional anesthetic

techniques compared to general anaesthesia (19,20,21)

.

In other studies for two groups of patients, one received general anaesthesia and other spinal

anaesthesia and found that no significant difference was seen in the mean 1 minute Apgar scores

in the two groups, however more neonates of the general anaesthesia group appeared depressed

soon after birth, needing free flow of oxygen and bag and mask ventilation(5)

. There are different

opinions about the ideal time at which the fetus should be delivered after induction of anaesthesia.

Barter was the first to emphasize that parturient woman should be prepped and draped before

induction of general anaesthesia(22)

. Numerous workers have mentioned that delivery is best

completed 6-8 minutes after induction of general anaesthesia as nitrous oxide could cause

neonatal depression by diffusion through the placenta (23,10)

.

A Study done at Abbasi Shaheed Hospital from March 2009 to July 2009, accomplish that there is

no significant difference between the effects of general anaesthesia and spinal anaesthesia on

Apgar score of neonates at 5 minutes interval, born after full term elective caesarean section of

healthy patients Present anaesthetic techniques, however limit the dose of intravenous agents such



that fetal depression is usually not clinically significant with general anaesthesia and

recommended that spinal anaesthesia is safe for caesarean section of healthy patients (24)

. The

result of this study was corresponding to our result i.e. there is significant difference only at one

minute after delivery. In the other study was done at Los Angeles, California, the result of this

study was that the neonates delivered with general anaesthesia had scored significantly lower on

some of the test items than neonates delivered by spinal anaesthesia at one minute after delivery (25).

The results were in the same trend with those reported by (Haider et al.,), who found that the

general anaesthesia of 25 patients give birth to neonates having Apgar score ≤ 6 at one minute after birth and the remaining 5 neonates had Apgar score of ≥7. On the other hand out of 30 mothers who received spinal anaesthesia only 10 mothers give birth to neonate having Apgar

score ≤ 6 at one minute after birth, who improved at 5 minutes interval, and their Apgar score

were ≥7. It had been found that the neonates who were born under G.A were ten folds more likely to have Apgar score less than or equal to 6 at first minute compared to those with spinal

anaesthesia (26)

.

CONCLUSION:

From this study it is recommended that spinal anaesthesia is safe for caesarean section of healthy

patient and more safe for newborn delivery than general anaesthesia at one minute valuation of

Apgar score & more comfortable for the mother. It is preferable to do caesarean section under

spinal anaesthesia, to avoid intravenous agents which cause foetal depression at one minute after

delivery. It can be further estimated by a large studies on Apgar scores in neonates following

both elective and substitute cesarean sections.

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edition: 223.

2. Klieyman Behrman ,Jenson Stanton .Nelson Text book of paediatrics : 18 edtion,2007;94:679.

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6. Petropoulos G, Siristatidis C, Salamalekis E, Creatsas G. Spinal and epidural versus general

anesthesia for elective Cesarean section at term: effect on the acid-base status of the mother

and newborn. Journal of Maternal-Fetal and Neonatal Medicine. 2003; 13(4):260–66.

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8. Khoury AD, Moretti ML, Barton JR. Fetal blood sampling in patients undergoing elective

cesarean section: a correlation with cord blood gas values obtained at delivery. Am J Obstet

Gynecol 1994; 171:679-84.

9. Klieyman Behrman ,Jenson Stanton .Nelson Text book of paediatrics : 18 edtion,2007;94:679.

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edi-torss. Care of high-risk neonate. 5th ed. Philadel-phia: W.B. Saunders; 2001. pp.45-61.

11. Sultana A. Effect of Type of Anaesthesia on neo-natal outcome. Annals 2004;9(2):552±6.

12. G.Edward Morgan, J .R.,Maged S .Mikhail. spinal Epidural &caudal block: clinical

Anaesthesiology , 2nd edition, alang medical book 1996 :902:300:291:301.

13. Apgar V, Holadady A, Jamesl S, Prince CE , Weisbrot IM .Comparison of regional and general

anesthesia in obstetrics. With special reference to transmission of cyclopropane across the

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14. Shniders M, Levinson G . Anaesthesia for obstetrics. Baltimore: Williams and Wilkins, 1979.

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scores indicate asphyxia? Lancet 1982;1:494-96.

16. Fisher J T, Mortola JP, Smith B , Fox GS, Weeks SK. Neonatal pattern of breathing

following caesarean section: epidural versus general anaesthesia. Anesthesiology 1983;

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Al-Shalah (2019): Pathogens in milk May 2019 Vol.19

©Annals of Tropical Medicine & Public Health SPe168-19

Pathogens in milk and product dairy: study in Europe

Zainab Musadaq Al-shalah1

1. Al-Qasim Green University, College of Food Science, Babylon, Iraq

Summary:

Milk and milk products contain natural microbial flora and / or addition to the origin of the diversity of

products placed on the French market. The origin of contaminations by pathogenic bacteria varies

according to the nature of the product and its mode of production and processing. The contamination of

milk and milk products by pathogenic germs can be of endogenous origin, and it then follows a

mammary excretion of the animal sick; it can also be of exogenous origin, it is then a direct contact

with infected herds or a contribution from the environment (water, personnel).

The whole process of processing and processing milk can slow down the multiplication of the germs

possibly present or, on the contrary, favor their development. For each of the main germs likely to be

found in these products, the authors describe aspects of physiology and of bacterial ecology, the

incidence in dairy products and consequences in public health. The most frequently mentioned germs

are

Mycobacteria, Brucella, Listeria monocytogenes, Staphylococcus aureus, Enterobacteria, including

Escherichia coli producing toxins and Salmonella. Currently, control of these pathogenic bacteria in

milk and derivatives requires the establishment of systems of control and surveillance that rely on

regulations now become European. The means of prevention must take into account the data now well-

known in the field of microbiology for milk and dairy products. More and more, the presence of

pathogenic bacteria in a food will have to be examined from the perspective of risk analysis for the

consumer with respect to these micro-organisms.

Keywords: Brucella , Contamination, Escherichia coli, Milk, Listeria monocytogenes ,

Mycobacteria , Prevention , Milk Products , Risk , Salmonella, Public Health, Staphylococcus aureus

How to cite this article: Al-Shalah ZM (2019): Pathogens in milk and product dairy:

study in Europe, Ann Trop Med & Pub Health; 19: SPe168-19

Salmonella:

Salmonella are Gram-negative bacteria of the type aerobic-anaerobic option belonging to the

family of Enterobacteriaceae and possessing all their characteristics biochemical. Provided

with peritrichous flagella, they are generally mobile but some serovars are motionless as S. Gallinarum

pullorum and others having lost their flagella. The genus Salmonella comprises two species :

Salmonella enterica and Salmonella bongori; the enterica species is itself subdivided in six subspecies



defined on the basis of characters biochemical and genotypic by hybridization results DNA / DNA

(42). The subspecies thus differentiated are enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb),

houtenae (IV) and indica (VI). The vast majority of isolated strains in humans and warm-blooded

animals belong to the subspecies enterica. Subspecies II, IIIa and IIIb are frequently found in the

commensal flora from the gut of cold-blooded animals but can be as isolated from the environment or

warm-blooded animals. The subspecies IV, VI and the bongori species are very rarely found and do not

seem to have any preferential habitat.

Each subspecies is subdivided into serovars on the base of somatic and flagella antigenic characters.

The combination of these antigenic factors makes it possible define a complete antigenic structure,

which according to the Kauffmann-White scheme characterizes a strain. The current nomenclature

grants to keep for each serovar the name that had been assigned for the first time to a strain of

identified antigenic formula; this denomination corresponds most often to an origin zoological or

geographical area for strains belonging to the first subspecies. Serovars from other subspecies are

designated only by their antigenic formula and the indication of the subspecies. To date, there are more

2,300 different serovars; new serovars are sometimes still isolated in France and recognized by the

Center National Reference Standard for Salmonella and Shigella (Institute Pasteur, Paris). Although

any isolated Salmonella is considered potentially pathogenic for humans, the determination of the

serovar makes it possible to better characterize the strain and restore it in its ecological context or

Epidemiological. Salmonella can multiply at temperatures between 5 ° C and 45 ° C with an optimum

at 35 ° C-37 ° C and at pH 4.5-9 with an optimum optimum between 6.4 and 7.5. Most salmonella can

develop in foods with water activity (Aw between 0.945 and 0.999). The potential oxidation reduction

may also be a determining factor in the growth of this microorganism.

Salmonella can be divided into three distinct groups:

- Those that are specifically adapted to humans and isolated exclusively in humans: this is the case of S.

Typhimurium, S. Paratyphi A and S. Sendai responsible for typhoid or paratyphoid fevers; serovars

specifically adapted to animal species, such as S. Gallinarum pullorum in poultry, S. Abortusovis in

sheep, S. Abortusequi in horses, S. Typhisuis and S. Choleraesuis in pigs;

- The other so-called ubiquitous serovars belonging to the third group and that can infect humans as

well which animal. They are most frequently isolated in the countries industrialized and are at the

origin of most salmonellosis human and animal. The gut of animals is the most important reservoir in

salmonella and contributes greatly to their spread in the environment where they can survive but

without multiply. The contamination of man can be done directly by contact or, most the intermediary

of contaminated food, a large number of food products being likely to be vectors. Many animal species

as well as humans can host the micro-organism in a non-apparent way as those healthy carriers

allowing even more easily this dissemination. The prevalence of contaminations by Salmonella in dairy

herds is variable according to countries and publications. In California, more than 72.7% of dairy cows

would show signs of salmonella infection, the observed serovars being S. Typhimurium, S. Dublin, S.



Montevideo, S. Newport and S. Anatum (85). Few data are published for countries European. In France,

according to data from the serotyping of the National Center for Veterinary Studies and (CNEVA) of

Paris, salmonella of origin accounted for 18% of strains ten years ago inventories of animal origin,

while currently they corresponding to 36% of this same population (9, 10, 11, 17). At the same time,

the severity of salmonella infections requires the use of anti-infective treatments, although antibiotic

resistance of the original strains cattle has also increased.

In France and also in other European countries, S. Typhimurium is the most frequently found serovar in

cattle. According to data from the inventory of Salmonella from CNEVA Paris (2014-2015), S.

Typhimurium represents 66.6% of all isolated strains of origin bovine, whereas ten years ago serovar

Dublin was still predominant (10, 57). Currently, this last serovar appears to be increasing again in

cattle (9, 10, 11). The infected farms constitute a potential reservoir of contamination of milk and

products derived from raw milk. However, it seems that the contamination occurs more frequently

from the middle outside, from the environment or by contact with animals infected at the time of

milking as intramammary. According to the Salmonella inventory data, 443 strains were identified in

dairy products during the 2014-2015, accounting for 3.6% of the total stem from food hygiene. This

figure appears to be low, corresponds to a sharp increase in number of strains from milk and milk

products, doubtless in connection with the Decree of 30 March 2014 which Mandatory search for

Salmonella in milkshakes consumption and milk-based products when they are on the market. Table I

summarizes the evolution of the number of isolated strains and their distribution in different categories

of dairy products. The results presented depend heavily on the establishment of guidelines and

monitoring or control plans in this sector. The recent data show that serovar Typhimurium is largely

predominant and accounts for more than 35% of isolated strains, two thirds of which come from raw

milk.

Table I: Evolution of the number of isolated strains of Salmonella in milk and dairy products

Products

analyzed

Raw milk 15 44 134 94 257

Powdered

milk 9 10 6 3 0

Cheese 1 19 60 47 156

Milk products 15 13 16 25 30

Total 40 86 216 169 443

isolated strains

in hygiene

alimentary

0.9 1.3 3.2 1.9 3.6



Is followed by serovar Montevideo with more than half of strains are isolated from cheeses (10, 63).

Although Salmonella is the leading cause of food poisoning in France, milk and products are rarely

responsible for salmonellosis Raw milk is rather infrequently contaminated and this contamination is

then most often of external origin. The pasteurized milk is usually free of all salmonella because they

are eliminated during the pasteurization. Incidents can only occur by recontamination after

pasteurization .Powders milk were responsible for several incidents; indeed, he has been shown that

some salmonella could withstand the freeze-drying heat treatment. The contamination of milk powders

may also have a external origin. Contamination of pasteurized cheeses can only be done after the

pasteurization step. The growth and survival of salmonella during the manufacturing and refining vary

according to different parameters, in particular, the acidification of the medium. A low pH variation

can reduce (pH 4.55) or contrary to promoting (pH: 4.95) the development of salmonella (68). Lactic

acid bacteria like certain Streptococcus or Lactobacillus that produce acids result in the elimination of

salmonella. During stages of manufacture and ripening, salmonella can also multiply according to the

ambient temperature. They can then persist until the moment of the market for these products.

However, for cheeses long ripening period (cooked pressed pasta), elaboration substances with

bactericidal action makes it possible to eliminate salmonella; other substances present in cheeses

marbling may also have an inhibitory action on the growth of certain strains: this is the case with

ferments according to their composition play an important role in the inhibition and elimination of

salmonella (30.) Salmonellosis can be classified in two major categories: those due to serovars strictly

humans responsible for typhoid syndromes or paratyphoid appearing in our regions as a case isolated

sporadic or restricted localized foci, and due to ubiquitous serovars of lower pathogenicity in healthy

adults, leading to clinical symptoms of food poisoning of favorable prognosis if the Host resistance is

not lowered. After a period incubation period usually from 12 to 36 hours, the table classic clinical is

that of a gastroenteritis that can be accompanied by fever, diarrhea, abdominal pain and vomiting.

Fragile populations such as subjects immunocompromised, infants and the elderly are more susceptible

to infection. The infectious dose needed to the onset of these symptoms is variable depending on the

samovar, the nature of the food and the host: it is usually estimated at 106 live bacteria but can be

above or below this value (50). During the year 2014 data from the Weekly Epidemiological Bulletin

identified 267 reported outbreaks of food-borne illness Collaborative Salmonella (TIAC) for 3,840

16.7 % were hospitalized .

Serovar Enteritidis was found in 65.5% of the outbreaks due to Salmonella. Salmonella TIACs are

more likely to occur family catering (60%) in a collective setting (35%). When the offending food

could be identified, it was the more often eggs or egg products (47.5%). Milk and dairy products were

involved in 5.5% of the cases during the year 2014; no case from these same products has declared

during the year 2015.

In France and other European countries, different types products have been involved in recent years in

episodes of food poisoning: this is raw milk, goat cheese, mozzarella, soft cow cheese, Cheddar,

Vacherin, Milk Powder, Cream and Sauce cream and ice cream. Of the milk powders, those intended

for infant feeding have recently been responsible for gastroenteritis in infants in France and in the



United Kingdom. The survey conducted by the National Network public health network and the "Salm-

net" European network enabled trace the origin of this contamination and to identify the serovar

Anatum. Such contamination by salmonella had already been responsible for TIAC in Austria, there is

a ten years and in Australia, twenty years ago. Raw milk cheeses were responsible for two community

outbreaks of salmonellosis in the seven occurred in France between 2016 and 2017; the first was due to

serovar Paratyphi B; the second, caused by serovar Dublin, covered both France and Switzerland (24,

25). In France, the surveillance of the TIAC is carried out jointly by the National Reference Centers of

Pasteur Institute of Paris, by the National Laboratory of (LNS) receiving the reports of diagnosed cases

retransmitted by the Departmental Directorates of Affairs health and social services (DDASS). The

National Health Network The public carries out epidemiological investigations in the event of declared

outbreaks. At the same time, surveillance of zoonoses is under the control of the Directorates of

Services veterinarians from each department and the centralization of data is carried out at the level of

the Directorate-General Food. Strengthening and better organization monitoring systems have allowed

for better census of cases in recent years.

A European network has recently been set up to collect all the data from the centers of national

reference of each country. He allowed to observe Isolation frequencies of some identical serovars for

each country and also to carry out surveys epidemiological data covering several European countries

(34(. 92/46 / EEC helped to increase the number of It also seems that the implementation of the

Directive of Salmonella research in dairy products and either therefore causing the apparent increase in

the incidence of Salmonella in these product categories. Apart from the role played by the supervisory

bodies and control of food poisoning, the best way to combat the increase in the number of

salmonellosis is to set up a number of prevention measures at all levels of production. It is already

announced by the World Health Organization. Health (WHO) in 1983 (A. Zuniga Estrada, L. Mota de

la Garza and A. Lopez Merino, personal communication), with several recommended control axes

since production until consumption. These preventive measures first of all relate to the farms, where

they must allow animals to better resist infection with reducing the spread of salmonella and

maintaining a good level of hygiene through basic rules. This requires the establishment of awareness-

raising actions breeders accompanied by corrective actions in case of animals infected. Procedures on

the respect of methods appropriate cleaning and disinfection procedures have been by WHO (70). If the

use of vaccination is commonly used in the United States of America, where 45% of cows’ dairy

farmers in California are probably protected (85), this practice is little used in France. Monitoring milk

collected with regular monitoring of milk tank and tank allows a first sort that requires, in case of a

positive result, a more thorough investigation the origin of contamination in the production of the milk.

Checks are regularly carried out in milk processing workshops, whether at the level of the

pasteurization or during the manufacture and refining of cheeses. Control of the manufacturing

parameters and ripening (hygrometry, temperature, pH) as well as the place of a hazard analysis critical

control system points (HACCP: risk analysis, critical points for their control) can reduce the risks of

contamination throughout the processing chain. Finally, the consumer must be informed of the

consequences non-compliance with storage or storage instructions some products. In the United States



of America, lately, a investigation by the Center for Disease Control and Prevention showed that most

toxi-infections were generated by the consumer himself.

Listeria monocytogenes:

The genus Listeria belongs to the phylogenetic branch of Clostridium as well as Staphylococcus,

Streptococcus, Lactococcus and Bacillus. On the basis of the DNA / DNA hybridization results and the

partial sequencing of 16S ribosomal RNA, the genus Listeria is currently divided into six species,

divided into two phylogenetic branches (55). The first includes Listeria (L) monocytogenes, L. ivanovii

(subsp. Ivanovii and subsp. iondoniensis), L. innocua, L. welshimeri, L. seeligeri. The second consists

of a single species Listeria bacteria are in the form of small bacilli of regular shape from 0.5 μm to 2

μm long and 0.4 μm to 0.5 μm in diameter, rounded at the ends and not forming neither capsule nor

spore. They are Gram positive, being able to appear in Gram stain, isolated, in V, in clusters and

sometimes even in chains.

Their growth is possible between 0 ° C and 45 ° C (temperature optimum temperature: 30 ° C-37 ° C),

for pH values between 4.5 and 9.6 , up to 10% NaCl and for A w of 0.92. Between 20 ° C and 25 ° C,

they are mobile thanks to flagella implantation is peritrich.

Listeria monocytogenes can be considered as an agent food pathogen "perfect" because it is ubiquitous,

very resistant to difficult conditions (temperature, Aw, pH ...) and especially she is able to grow at

temperatures of refrigeration of food. The virulence of the strains could besides be exalted by their low

development temperature (48).

Listeria bacteria are in the form of small bacilli of regular shape from 0.5 μm to 2 μm long and 0.4 μm

to 0.5 μm in diameter, rounded at the ends and not forming neither capsule nor spore. They are Gram

positive, being able to appear in Gram stain, isolated, in V, in clusters and sometimes even in chains.

Their growth is possible between 0 ° C and 45 ° C (temperature optimum temperature: 30 ° C-37 ° C),

for pH values between 4.5 and 9.6 , up to 10% NaCl and for A w of 0.92. Between 20 ° C and 25 ° C,

they are mobile thanks to flagella implantation is peritrich , Listeria monocytogenes can be considered

as an agent food pathogen "perfect" because it is ubiquitous, very resistant to difficult conditions

(temperature, Aw, pH ...) and especially she is able to grow at temperatures of refrigeration of food.

The virulence of the strains could besides be exalted by their low development temperature (48).

Three large reservoirs at Listeria are thus identified. All first a "human" reservoir and an "animal"

reservoir, since a large frequency of healthy carriers is generally observed (6). Finally, a tank

"Environment", namely soil, vegetation, surface, wastewater, silage or food from which the man can be

contaminated. However, in in the majority of cases, the number of L monocytogenes is low (less than a

hundred or a few hundred per gram) (83).

This high incidence in food is also at the origin some major Listeriosis epidemics, the contaminations

being mainly due to of animal origin and in particular to dairy products. Thus, in France, it is estimated

that on average between 1% and 9% of raw milk samples are contaminated but the concentration is



most often less than one bacterium / ml. Two routes of contamination are usually described

contamination by the cow (mastitis) is infrequent but the level of contamination is often high (1000 to

100,000 L. monocytogenes / ml in district milk), associated with an abnormal cell count without

clinical signs individuals ;- contamination by the environment is more frequent, but contamination

levels are also lower. The poor quality silage are therefore a source significant contamination since the

presence of L. monocytogenes in these multiplies by twenty the risk of contamination of the tank milk.

At the dairy, L monocytogenes is normally destroyed by efficient pasteurization. Contamination

possibly highlighted then result usually is a technological problem (insufficient pasteurization) or

contamination secondary.

During processing, it is estimated that 0.5% to 10% cheeses are contaminated with L. monocytogenes;

it's about mainly soft cheeses, however 75% contaminated cheeses have low levels of 1 to 100 L.

monocytogenes / g. However, some cheeses pasteurized soft can hold up to 106 / g. Contamination

may be limited to crust or extended to the dough. Indeed, Listeria generally follows the evolution of the

pH which is not homogeneous in cheese (the pH gradient can reach one or two units between the heart

and the crust). The development of Listeria is therefore more favorable under crust, thanks to the flores

of surfaces that alkalize the matrix. We can globally distinguish several types of behavior of L.

monocytogenes according to the matrix cheese (48). For some cheeses such as cheeses fresh, acidic

soft pasta (goat cheese ), Hard cheese ripening very long (parmesan, mozzarella), the matrix itself has

destructive properties. Other cheeses have inhibiting properties: pressed pastes some blues. Still others

allow growth of Listeria; these can reach levels of contamination sometimes very high in pasta soft

pasteurized (106 / g). Levels of contamination in soft pasta with raw milk are very variable, depending

on the contamination of the raw material and following the flora associated, since some flora have an

inhibitory action it can be added that L. monocytogenes has been isolated in other dairy products such

as unsweetened condensed milk, concentrated milk by ultra filtration, ice cream, or certain dried milks

(drying by the "spray" destroys L. monocytogenes, which survives but does not develop not).

Although listeriosis is a rare infection caused by virulent strains of L. monocytogenes, it has done much

talk about her in recent years because of the seriousness of the disease. This is, indeed, food-borne

infection most lethal (20% to 30% of cases). Thus, even diagnosed early, listeriosis high mortality

rates, on the one hand because the diagnosis is difficult to achieve, given the very long incubation times

variables (2-70 days) (6), on the other hand because treatments are quite random (development of

antibiotic resistance).

The infection can evolve into a mild form (episode febrile influenza type with sometimes meningeal

syndrome) but it can evolve into a more serious form like a meningitis or sepsis, leaving in 5% to 10%

of cases neurological squeal.

For example, 60% of patients during the Swiss epidemic do not have any predisposing condition). The

infectious dose is still unknown but no case human listeriosis has been linked to food containing less

than 100 L. monocytogenes / g or ml. This dose will be difficult to evaluate because it depends, among

other factors, the immune status of the host and the virulence of the strain. Since the early 1980s, the



number of listeriosis has tendency to increase, mainly in the industrialized. Thus, if we list fifty cases

per year in France until 1970, then about 150 cases per year until 1975, the 1980s were marked by a

significant increase in the number of listeriosis cases (824 cases in 2014, 522 in 2017).

This situation is mainly due to an increase in food chains and a restoration development collective, but

also to more epidemiological surveillance organized as well as an increase in life expectancy and

number of immunocompromised individuals. Establishment of listeriosis surveillance systems in many

industrialized countries has thus made it possible to estimate the incidence of this disease. This one is

also variable from one country to another because it probably depends on everything less in part, eating

habits. So, for the year 2013, the incidence is close to ten cases / 106 inhabitants in France, while it is

estimated at around two cases / 106 inhabitants in Switzerland or Canada and seven to eight cases / 106

inhabitants in the United States of America. In France, surveillance is carried out by the National

Center reference for phage typing and molecular typing Listeria (Institut Pasteur, Paris) and by the

LNS which receives the reports of cases diagnosed by a network of biologists in the field of regulation.

Staphylococcus aureus:

Staphylococcus and Micrococcus are two genera bacteria that have long been grouped together in the

family of Micrococcaceae. This family is going to be soon overhauled, because of the remoteness

phylogenetic of these two genera. The micrococci represent a heterogeneous group of the branch of

Actinomycetes, while staphylococci form a homogeneous group connected to the branch of

Clostridium (20). The bacteria of the genus Staphylococcus are Gram cocci positive, non-sporulating,

grouped into clusters, immobile, facultative anaerobes and possessing a catalase. Thirty three species

have already been identified by DNA / DNA hybridization and new species or subspecies are regularly

described (44). The basic criterion of their classification is the coagulase production. There are three

species to positive coagulase: Staphylococcus aureus, S. intermedius, S. hyicus, and thirty coagulase

negative species.

The presence of staphylococci in food represents a risk to human health, because some strains mainly

belonging to the species S. aureus produce enterotoxins whose ingestion causes toxiinfection

Staphylococcal food .

Milk and dairy products only become toxic if they are contaminated with enterotoxin producing strains

and if favorable conditions for multiplication important bacterial and toxinogenesis can be found met.

Staphylococcus aureus is part of the flora of the skin and mucous membranes of man and animal.

Parasite usually harmless, it can cause infections (skin abscess, mastitis). Contamination of milk may

occur by through healthy or infected carriers, or by the environment. In cattle, S. aureus is isolated in

nostrils. It is found in small skin lesions and in the sleeves of milking machines. Colonization of teats

can lead to infection of the udder. It can be estimated that 25% of dairy cows can be have breast

infections. During investigations carried out in France and the United Kingdom from 2006 to 2016, the

frequency of isolation of S. aureus in infected quarters has been 20% to 40% for unseen infections, and

15 % to 30% in clinical mastitis (76). S. aureus is the most common bacterium involved in latent



infections and chronic subclinical mastitis. Mastitis being difficult to eradicate, they represent the main

source of contamination of raw milks by S. aureus. Excretion of S. aureus in district milk varies from 0

to 1 0 4 - 1 0 5 bacteria / ml in case of subclinical mastitis and up to 1 0 8 bacteria / ml in case of

clinical mastitis. In the mixing milk, there are on average 102 to 103 S. aureus / ml. In humans, nasal

carriage concerns 20% to 50% of people. S. aureus is disseminated on the skin and hands of

temporarily or permanently. The man is considered as the main vector of contamination during the

manipulations intervening throughout the chain food. However in raw milk and milk cheeses raw,

human biotype strains remain a minority compared to strains of bovine biotype . The S. aureus strains

are not all toxinogenic. According to the numerous surveys carried out on this subject, and optimal

conditions of culture, in the laboratory, the percentage of toxigenic strains would be 30% to 60% in

sheep and goat strains, compared to only 4% in 10 % in cattle strains (21).

The temperature of milk storage and manufacturing of cheeses plays a vital role. Staphylococcus

aureus grows at temperatures included between 6 ° C and 46 ° C (optimum temperature: 37 ° C) at a

pH between 4 and 9.8 (optimum pH: 6 to 7). This species tolerate a high concentration of NaCl (up to

20%) and a Aw very reduced (0.83). Toxinogenesis is involved in conditions a little more restrictive

than those required for the growth (20).

Once formed, the enterotoxins are remarkably stable. They resist the irradiation, the enzymes

proteolytic and especially to heat. While the bacteria is destroyed during pasteurization of milk,

enterotoxins are only partially inactivated. They are not completely inactivated after 20 to 40 min at

120 ° C, according to a study with unpurified toxins at 100 ng / ml in phosphate buffer (90). More

importantly, they could form complexes with each other or with the food, preventing their detection

after heat treatment while their biological activity persists (82, 90).

Pasteurized milk is more favorable for the growth of S. aureus than raw milk because this

microorganism is a bad competitor in the presence of other bacterial flora. In the raw milk, the initial

number of S. aureus must be equal or greater than that of the concomitant flora to be able to multiply

sufficiently and produce enterotoxins .

The technological parameters of cheese making are usually favorable for the growth of S. aureus. This

occurs in the tank, then, if the pH does not drop Normally, it continues during pressing but usually not

beyond. The first twenty-four hours manufacturing therefore seem decisive. However, in case of lactic

flora, staphylococci can continue to multiply during the first weeks refining. Their number then

decreases gradually . Enterotoxins can be detected when the number of S. aureus reaches about 106 to

107 / g. In milk cheeses believed, this is exceptional because even when the level bacterial exceeds this

value, the surrounding conditions are not usually favorable for toxinogenesis. The type of cheese also

matters (temperature and pH gradient during manufacture, salt concentration, activity of the

antagonistic flora) (5, 21).

Staphylococcal food poisoning is characterized by violent and repetitive vomiting occurring 30 minutes

to 8 hours after ingestion. Disease is short lived but very challenging and spectacular complications are



sometimes observed in ingested toxin dose and sensitivity individual. Hospitalization is reported in

14% of case on average, but mortality is exceptional (22). In France, from 1988 to 1995, staphylococci

were incriminated in more than 300 households, or 10% of TIAC reported to the health authorities.

They occupy the second row for the number of outbreaks after salmonella and reached during this

period more than 7,000 patients of which 55% in schools (2 2, 4 3). Foods responsible for food

poisoning Staphylococci are varied. Milk and dairy products are suspected in 25% of the 300

households mentioned above (22). On the one Generally, dairy products are infrequently incriminated

in original food-borne illness bacteria: they accounted for 4% of total outbreaks, France, from 1988 to

1995 (22) and, in other countries, from 0.9% to 8.3 % of households by country (32). In contrast,

staphylococci are the most common bacteria more often implicated in the toxi-infections foodstuffs due

to dairy products in France as United Kingdom (22, 56).

European Directive 92/46 / EEC of 16 June 1992 fixes the health rules for the production and placing

on the market of raw milk, heat-treated milk and products based on milk (15). Staphylococci are

presented as germs evidence of lack of hygiene, but it must be remembered that in the case of raw milk

products, the main source of contamination is bovine mastitis (5). S. aureus must be enumerated in raw

cow's milk (m = 500), cheeses fresh (m = 10), soft cheeses and marbled pasteurized milk (m = 100),

raw milk and milk cheeses thermised (m = 1000). If they contain more than 10,000 colony forming

units (CFU) per gram of S. aureus, an enterotoxin test should be performed (culture toxins isolated

strains from cheeses or toxins extracted from cheeses). However, the implementation evidence of

enterotoxins remains technically difficult despite the existence of many detection kits . Prevention of

food-borne illness Staphylococci involves setting up a program action against bovine mastitis, the

maintenance of milk refrigeration temperature and strict compliance with the rules hygiene during

handling on the farm and at the dairy, in order to to limit the number of S. aureus present in the milk.

She also requires know-how, a monitoring of parameters technologies and a choice of lactic ferments

to inhibit the growth of S. aureus as much as possible during the manufacture of raw milk cheeses (5,

20, 61).

Finally, improvements are needed at the level of declaration and investigation of toxi-infections food.

Indeed, many homes are not reported and when they are, it is not always possible to evidence of the

causative bacteria or the food in question (43, 56). In addition, most food poisoning reported are very

poorly documented. Lack of information epidemiological data, the risk due to the presence of

Staphylococci in dairy products will remain difficult to assess.

Escherichia coli:

Escherichia (E) coli form a group of motile bacilli gold still, Gram-negative, of the family of

Enterobacteriaceae. They can multiply at temperatures between 4 ° C and 46 ° C, with an optimum

growth at 37 ° C and pH between 4.6 and 9.5. E. coli that causes diarrhea, acute gastritis or colitis of

man are referred to as E. coli pathogens. Differentiation criteria based on their serotype, their virulence

and their clinical consequences have classified these pathogenic strains into four groups:



Enteropathogenic E. coli distingites (EPEC), E. coli enterotoxigenic agents (ETEC), enteroinvasive E.

coli (EIEC) and enterohemorrhagic E. coli (EHEC).

EPECs are associated with epidemics of childhood diarrhea. They may, depending on the strain,

produce toxins or invade epithelial or intestinal cells. Clinically, the disease is characterized by fever,

vomiting, abdominal pain and severe diarrhea, accompanied by a large amount of mucus in the stool

and a little blood (49). ETECs are characterized by the production of one or two toxins, one

thermolabile (LT), the other thermostable (ST). Tea disease is characterized by watery diarrhea by

abdominal pain, discomfort and nausea. ETECs are also recognized agents of the traveler's diarrhea

(49). The infectious dose for ETEC is high: 1 0 8 - 1 0 1 0 (58).

EIECs are characterized by signs of toxemias with discomfort and fever. EIECs proliferate in tissues

epithelial cells until necrosis occurs (49). The infectious dose for this pathovar is also important: 1 0 6 -

1 0 8 (58). EHEC is responsible for hemorrhagic colitis, uremic hemolytic syndromes (HUS) and

purpura thrombocytopenia. E. coli 0157: H7 are the most often responsible for this hemorrhagic colitis.

These E. coli can produce two potent cytotoxins (VT toxins) .The infectious dose is not known with

certainty purpose it is weak (<10 / g). Escherichia coli are normal commensal of the intestine of man

and animals. It represents 80% of the flora aerobic intestinal. It is found in very large numbers in the

faecal faeces. From there, it spreads in nature: soil, waters. Its presence in the environment always

signifies a faecal contamination. A commensal bacterium, whatever its species, can acquire certain

pathogenicity factors through the contribution a new generic support (plasmid, bacteriophage,

transposons) or by the expression of genes previously silent, and thus become pathogenic (39).

Of the pathogenic bacteria that can end up in raw milk, some are usually at a very low level and are

unlikely to develop. Others are at appreciable levels and can multiply. It's the case, among others, from

E. coli that usually comes from the skin udders (77). This bacterium of fecal origin can survive on

soiled soil. Its implantation in the material milking is unusual. Some strains, fortunately rarely present,

when they are at a high level in the raw milk or in cheese, can produce gastroenteritis due to the

production of toxins. The transmission of enterohemorrhagic E. coli is essentially related to product

contamination food. Food contamination can occur during factory manufacture (followed by

multiplication possible during transport and storage), ie when meal preparation (kitchen staff). The

interpersonal contamination also exists by intermediate healthy carriers with a low dose infective.

Infectious diarrhea is a case sporadic or anademic episodes (TIAC, for example), even epidemic.

Specificities exist, on the one hand according to the regions and seasons of infection, but also

according to the age and socio-economic level of concerned and their way of (life(diets, consumption

of imported foods, fast and collective catering, travel) (39).

Seven biovars are currently identified in B. abortus, three in B. melitensis and five in B. suis (Table II).

The studies hybrid DNA / DNA and DNA / RNA compatible with the existence of a single species of

Brucella ovis and B. canis are the only two species naturally in rough phase. Bacteria of the genus

Brucella are small sticks, very frequently coccobacillary, aerobic and low glucidolytic. Their length

varies from 0.6 μm to 1.5 μm and their thickness 0.5 μm to 0.7 μm. Their morphology is very constant



except sometimes in older cultures. The Brucella are immobile and do not form spores. No flagella, pili

or capsule. Gram-negative bacteria, Brucella do not show bipolar staining and are resistant to

discolouration by weak acids (2, 16).

Many species animals (especially ruminants, but also suidae, carnivores, equidae and rodents) can be

naturally infected by various species of Brucella (Table II). Because of the frequency and severity of

contracted human cases directly or indirectly from the animal, the brucellosis is considered a major

zoonosis.

Table II: Brucella (species and biovars) (2,69)

Species biovar Morphology

colonies

Host

Preferential

Brucella abortus 1.2.3.4.5.6.9 s Cattle

B. melitensis 1.2.3 s Sheep, goats

B. am 1 s Pigs

2 s Pigs, hares

3 s pigs

4 s Rennes

5 s Wild rodents

B. neotomae - s Neotomes *

B. ovis - R Sheep

B. canis - R Dogs

S: naturally in smooth phase R: naturally in rough phase * Neotoma lepida (desert wood rat)

In the South, the prevalence of bovine infection is now very low (for example, in France prevalence of

livestock was 0.28% in 1995) (4). In these regions of southern Europe, ovine and caprine considerably

diminished over the last ten years because of strengthening of control measures, but the prevalence

remains significant (livestock prevalence rate sheep infected in France in 1995: 2.27%) (4), in

particular in mountain areas and around the Mediterranean. Brucellosis in these species largely

explains the human cases still observed in these areas. The most common vehicle of human infection

by ingestion is raw milk, or one of its drifts, especially the cream and, in many countries, fresh cheeses,

which sometimes the cheapest source of protein and no longer available (69). Milk and dairy products

of origin cattle, sheep or goats are most frequently incriminated, but the infection can also be

contracted atfrom dairy products derived from buffaloes, camelids or yaks. Brucellosis of small

ruminants is considered nevertheless as being responsible for the majority of the cases food-borne

humans. In France, for example, the consumption of goat cheese is the first cause of unproductive

human brucellosis (54%), absorption cow's milk accounted for only 16% of these cases (80, The

generalization of the pasteurization of the milk of origin intended for consumption or processing and

level of sanitary requirements allowing the marketing raw milk products explain this situation. The

eating habits, and especially the predilection of certain populations for raw milk and its derivatives



concur still largely to the maintenance of human brucellosis. The survival of Brucella in milk and dairy

products is related to many factors, including the type of product, the water, temperature, pH changes,

action the other bacteria present, and the duration and storage conditions of the product (38). The

results of some studies (11, 19, 65, 75, A. Zuniga Estrada, L. Mota Garza and A. Lopez Merino,

personal communication) are shown in Table III. Low concentration in medium liquid, Brucella is

easily destroyed by heat. So, the classic pasteurization, the ultra-high method temperature or a simple

prolonged boil (10 min) kill the Brucella contained in the milk (19).

Wirth Brucella, it is usually reduced in milk that has been kept for some days. Survival in ripened

fermented cheeses seems pretty short. We do not know the time of minimal fermentation necessary for

their total destruction, but it is conventionally estimated that three months are sufficient in fresh

cheeses, the survival of Brucella can be much longer, strictly lactic fermentation and of short duration

and desiccation favoring their survival. Alone past pasteurization of the milk or cream allows to

prevent the health hazard posed by these products human. Brucella are also sensitive to radiation

ionizing agents at commonly used doses, especially in colostrum. Prevention of food-borne human

brucellosis therefore goes first and foremost by the heat treatment of milk intended for direct

consumption or processing. The sale of raw milk and dairy products untreated heat must be, for its part,

closely monitored and limited to free holdings. These measures, even when accompanied by campaigns

information, are insufficient, however, to reduce significantly the incidence of human brucellosis,

numerous cases originating from direct contact with infected animals. Only countries that have

implemented animal brucellosis control programs were able to observe a consequent reduction in the

number of cases humans. The control and prevention of animal brucellosis requires the respect of a

general hygiene in the farms and the setting in place at regional level a restorative on sanitary and / or

medical measures. All of these measures cannot be really effective without a health education, training

and mobilization of professionals concerned. Finally, no measure of prophylaxis cannot be considered

without identification sustainable use of animals and flocks and strict control of their movements

(trade, transhumance).

Various general hygiene measures make it possible to limit the extension of the infection. At the farm

level, isolation animals at the time of parturition and the destruction of non-living products and foeto-

maternal appendages as well as disinfection of livestock premises are two measures essential in this

respect. Staff must also submit to a systematic disinfectant treatment of body parts and clothing that

may have been in contact with contaminated products. Given the relative inefficiency and the cost of

treatment antibiotic in livestock, elimination by slaughter of seropositive animals is one of the solutions

used in the fight against animal brucellosis. This solution implies, when setting up the program of fight,

a relatively low disease prevalence rate so that this plan is economically bearable. Such plans are

usually based on the systematic control of flocks (individual serology or regular ring-test on milk

tanker) and animal health measures positive or in clinical outbreaks, with slaughter positive animals or

total slaughter of herds infected.



Bacteria of the genus Mycobacterium belong to the family of Mycobacteriaceae which consists of

Actynomycetales with rudimentary pseudomyelium usually presents as small bacilli, immobile,

sometimes having bulging, cuneiform or branched (0.2-0.6 μm over 1.0-10 μm). They are characterized

by their ability to maintain color despite action combined with alcohol and diluted acids: they are acid-

resistant. The optimal temperature of mycobacteria ranges from approximately 28 ° C to 45 ° C. Within

the genus Mycobacterium, there are more than 40 species recognized, including M. tuberculosis, M.

leprae and several mycobacteria called "atypical". The classification taxonomy within the genus is

based on the form of colonies, pigmentation, growth characteristics bacterial and biochemical reactivity

(Tables IV and V).

The bacteria are divided into two main groups depending on whether their growth is slow or fast. In

practice, we consider that growth is slow when it takes more than seven days incubation to obtain

visible growth.

Table IV: Cultural characteristics of the main bacteria of the genus Mycobacterium

mycobacteria Pigmentatio

n

37

°

C

42

°

C

> 7

day

s

mycobacti

n

Pyr

.

Pn

b

Tc

h

pz

a

Tb

1

em

b

M. tuberculosis - + - + - - - + - - -

M. bovis - + - + - + - - + - -

M. africanum - + - + - + - V - V -

M.

paratuberculosi

s

- + - + + + V + v Nf +

M. avium - + + + v + + + nf v +

+: positive v: variable Pyr. : pyruvate TCH: hydrazide of thiophene carboxylic acid, 2 μg /

Tb1:thiosemicarbazone, 10 μg / ml ,,, - : negative nf: not done PNB: Paranitrobenzoate, 500 μg / pza :

pyrazinamide EMB: Ethambutol, 2 μg / ml

The bacteria belong to the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. microti)

are mandatory pathogens. Apart from M. tuberculosis and M. leprae, several species of mycobacteria

are pathogenic for humans under certain conditions. Some species like the M. avium complex, M.

kansasii, M. malmoense and M. xenopi are more likely to cause diseases than others. The majority of

potentially pathogenic atypical mycobacteria have a slow growth. From an economic point of view, the

main diseases Mycobacterial infections in animals are due to M. bovis and M. paratuberculosis.

Mycobacterium bovis can be transmitted from animals to humans and to provoke in the latter a disease

identical to tuberculosis due to M. tuberculosis. Other mycobacteria, such as M. avium, M. marinum,

M. farcinogenes and M. silvaticum may be responsible infections in animals of very different species.



M. bovis, agent of bovine tuberculosis, is able to infect humans and certain animal species, especially

goat, pork, dog and cat. From the point of view of veterinary medicine and public health, this germ is

the most important cause of diseases mycobacteria in animals.

The symptoms of the disease in cattle vary according to the organ reached. In case of pulmonary

infection, a dry cough appears and worsens as the disease develops. This cough is accompanied by

slimming. Infection of the uterus and / or mammary glands is responsible for a sterility and decreased

milk production. The disease tends to slow worsening. Cattle with tuberculosis are the main source of

M. bovis which is transmitted from cattle to humans mainly in two ways: by air (aerosols) and digestive

(consumption of raw milk infected). Man with pulmonary tuberculosis M. bovis is also a source of

infection for other subjects and possibly for cattle.

Table V: Biochemical characters of the main bacteria of the genus Mycobacterium

mycobacteria Nia. Nit. Cat.

20 °

C

Cat.

68 °

C

Urea 1 2 3 4 5 6

M. tuberculosis + + + - + + + +

M. bovis - - + - + + + +

M. africanum V V + - V + + +

M.

paratuberculosis

- - + + - + + +

M. avium - - + + - + + +

Nia. : niacin Cat. : catalase v: variable +: positive - : negative Nit. : nitrate reductase

Conclusion:

Other pathogenic microorganisms may be encountered in milk and milk products, among which

Yersinia enterocolitica, Campylobacter jejuni, Coxiella burnetii, Streptococcus agalactiae, Clostridium

botulinum, Bacillus cereus, toxin-producing molds and viruses. The presence and persistence of these

germs in milks and dairy products depend on their resistance to treatments raw milk (pasteurization,

acidification, curd heating, ripening conditions) and the initial level of contamination in raw

milk.Pasteurization treatments (72 ° C for 15 s) eliminate pathogenic bacteria in vegetative form, but

those in sporulated form resist ) B. cereus, C. botulinum) .

Campylobacter, very fragile germs, are found only in raw milks and insufficiently pasteurized milks

The number increased by 4-5 log instead of 3 log the foot-and-mouth disease virus is also susceptible

to pH drop. It is inactivated in 17 s at pH 6.7 at 72 ° C and in 55 s at pH 7.6 at 72 ° C .



The survival of these pathogenic microorganisms in the dairy products is also more or less dependent

on manufacturing process. So C. burnetii can be put in evident in butter and cooked pressed cheeses

after several weeks, while in the cheeses soft and acidic dairy products, germs are very fast inactivated

(33). The FMD virus also survives longer in cooked pressed cheeses than in soft cheeses. Studies from

milk milks cow naturally infected with fever virus foot-and-mouth disease products for the production

of cheddar and camembert cheese revealed that the foot-and-mouth disease virus survives in the

cheddar after 60 days of storage but not after 120 days . In the pie chart, the virus survives 21 days at 2

° C but not 35 days. As for poisoning due to C. botulinum, in cheese technology, the parameters

(acidification, Aw, salt content) do not allow cell growth vegetative growth of C. botulinum or spore

germination (12).

Reference

1. Alton G. G. (1985). - The epidemiology of Brucella melitensis in sheep and goats. Brucella

melitensis, a seminar of the CEC (J.M. Verger & M. Plommet, eds.). Martinus Nijhoff, Dordrecht,

Boston and Lancaster, 187-186.

2. Alton G.G.Jones L.M., Angus R.D. and Verger J.M. (1988). - Techniques for the brucellosis

laboratory. National Institute of Agricultural Research, Paris, 190 pp.

3. Anon. (1993). - Escherichia coli producers of verotoxins (E. coli 0157: H7). Summary of activities

of the Food and Health Quality Branch animal husbandry (1987-1993). MAPAQ publication,

September.

4. Anon. (1996). - Annual Statistical Survey 1995. Epidemiology and prophylaxis in ruminant culture,

Directorate General of Food, Ministry of Agriculture, fishing and food, Paris, 157 p.

5. Asperger H. (1994). - Staphylococcus aureus. In the importance of pathogenic microorganisms in

raw milk (Hahn, ed.) Monograph, Document No. 9405, International Dairy Federation, Brussels, 24-

42.

6. Astier-Dumas M. (1992). - Development: listeriosis. Med. And Nat., 28 (6), 332-333.7. Benet J.J. &

Thorel M.-F. (1992). - Prophylaxis of bovine and human tuberculosis a success? Sci. Vet. Med. comp.,

94, 175-186.

8. Bohnert M. (1995). - Survival and multiplication of Escherichia coli in food (ETEC diarrheal

pathovars, EPEC, EIEC, EHEC). Communication, Pasteur Institute of Lille, 13 pp.

9. Brisabois A., Frémy S., Guignard A., Moury F., Oudart C, Piquet C. & Pires Gomes C. (1995). -

Inventory of Salmonella, 1992-1993. National Center for Veterinary Studies and food, Maisons-Alfort,

142 pp.

10. Brisabois A., Frémy S., Moury F., Oudart C, Piquet C. & Pires Gomes C. (1996). - Inventory of

Salmonella, 1994-1995. National Center for Veterinary Studies and food, Maisons-Alfort, 136 pp.



11. Carrère L., Lafenêtre H., Quatrefages H. & Noronha F. (1960). - Duration of survival of Brucella in

cheese Roquefort. Bull. Acad. clothes. Fr., 33, 469-473.

12. Center for Continuing Education and Professional Development managers of the milk industry

(CEPIL) (1992). - The groups microbials of dairy interest. CEPIL, Paris, 567 pp.

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