Working with the laboratory

Working with the laboratory

IDSP training module for state and district surveillance officers

Module 6

Learning objectives (1/3)

• List L1 and L2 laboratories in the district L3 laboratories in the state L4 and L5 laboratories in the country

• Understand the need of L1 and L2 laboratories to arrange for logistical support

• Identify what action is to be taken be the technician for sample collection in response to the diagnosis made by the medical officer


• List tests to be performed in L1 and L2 laboratories

• Identify quality assurance processes within the laboratory network

• Understand bio-safety issues• Identify transport modalities of samples

to higher levels


• Understand training needs of laboratory personnel

• Keep track of the flow of samples• Draw a flow diagram for reporting of the

laboratory investigations

Role of laboratories in disease surveillance

• Early diagnosis of diseases under surveillance

• Epidemiological investigation• Rapid laboratory confirmation of

diagnosis• Implementation of effective control

measures

Factors influencing laboratory confirmation in surveillance

• Advance planning• Collection of appropriate and adequate

specimens• Correct packaging• Rapid transport• Ability of laboratory to accurately perform

tests • Bio-safety and decontamination procedure

Types of case definitions in use

Case definition

Criteria used Who does it

Syndromic Clinical pattern Paramedical personnel and members of community

Presumptive Typical history and clinical examination

Medical officers of primary and community health centres

Confirmed Clinical diagnosis by a medical officer and positive laboratory identification

Medical officer and Laboratory staff

More

specifi

city

Laboratory network for the Integrated Disease Surveillance

ProjectLaboratorie

sDescription

L1 • Peripheral laboratories and microscopic centres

L2 • District public health laboratory

L3 • Disease based state laboratories

L4 • Regional laboratories and quality control laboratories

L5 • Disease based reference laboratories

Risk groups, biosafety levels, practices and equipment

BSL Laboratory type Laboratory practices Safety equipment

P1 Basic teaching, research

Good microbiological techniques

NoneOpen bench work

P2 Primary health services; diagnostic services, research

Good microbiological techniques, protective clothing, biohazard sign

Open bench plus biological safety cabinet for potential aerosols

P3 Special diagnostic services, research

As BSL 2 plusspecial clothing,controlled access, directional airflow

Biological safety cabinet and/or other primary devices for all activities

P4 Dangerous pathogen units

As BSL 3 plus airlock entry, shower exit, special waste

Class III biological safety cabinet, positive pressure suits, double ended autoclave(through the wall) and filtered air

Method of laboratory surveillance

• Routine passive surveillance Selected diseases

• Outbreak situations

Conditions under regular surveillance

Type of disease Disease

Vector borne diseases •Malaria

Water borne diseases •Diarrhea (Cholera)

•Typhoid

Respiratory diseases •Tuberculosis

Vaccine preventable diseases

•Measles

Disease under eradication •Polio

Other conditions •Road traffic injuries

International commitment •Plague

Unusual syndromes •Meningo-encephalitis•Respiratory distress•Hemorrhagic fever

Other conditions under surveillanceType of surveillance

Categories Conditions

Sentinel surveillance

•STDs •HIV/HBV/HCV

•Other conditions

•Water quality

•Outdoor air quality

Regular surveys •Non communicable disease risk factors

•Anthropometry

•Physical activity•Blood pressure

•Tobacco, blood pressure

•Nutrition

•Blindness

Additional state priorities •Up to five diseases

Diagnosis of malaria

• Laboratory criteria for diagnosis Detection and identification malaria parasite

microscopically

• Sample collection for microscopy Thick and thin blood smear

• Time of collection During fever or 2-3 hours after peak of

temperature Before patient receives anti-malarial

L1 L2 L3• Sample

collection• Smear

preparation• Microscopy and

reporting

• Same as L1

• Quality control for L1


Laboratory tasks at each level for the diagnosis of malaria

Diagnosis of cholera

• Laboratory criteria for diagnosis Isolation of Vibrio cholera O1 or O139 from

stools in any patient with diarrhea

• Sample collection Transfer a portion of specimen to a cotton

wool swab Insert it in alkaline-buffered salt solution If stool specimen could not be collected take

a rectal swab and insert it in the above solution

Laboratory tasks at each level for the diagnosis of cholera

L1 L2 L3•Stool sample collection•Transport to L2

•Stool sample microscopy•Culture•Biochemical and serotyping•Transport to L3 for sensitivity

•Drug sensitivity and phage typing•Quality control for L2•Training

Diagnosis of typhoid

• Laboratory criteria for diagnosis Serology – Widal or Typhi-dot test positive Isolation of S.typhi from blood, stool or other

clinical specimen

• Sample collection Blood / stool

L1 L2 L3• Typhi-dot test• Blood and stool

collection for culture

• Transport to L2

• Widal test• Typhi-dot• Blood and

stool culture

• Quality assurance for L1


• Special tests

• Training

Laboratory tasks at each level for the diagnosis of typhoid

Tuberculosis

• Laboratory criteria for diagnosis Demonstration of alcohol-acid fast bacilli in at

least two of the three sputum smears or culture positive for Mycobacterium tuberculosis

• Sample collection for microscopy Three specimens

• One spot specimen • One early morning specimen (preferably the next day)• One spot specimen when the early morning specimen

is being submitted for examination.

L1 L2 L3• Sputum collection• Smear

preparation• Microscopy and

reporting

• Same as L1


• Transport to L3 for culture

• Culture and sensitivity testing

• Quality control

Laboratory tasks at each level for the diagnosis of tuberculosis

Measles

• Laboratory criteria for diagnosis Presence of measles virus specific IgM antibodies At least four fold increase in antibody titre in paired

samples Isolation of measles virus

• Sample collection Serology

• An acute phase serum specimen (3-5ml of whole blood) be soon after onset of clinical symptoms but not later than 7 days

Virus isolation• Urine collected within 5 days of rash onset (1-3days best). • Do not freeze

L1 L2 L3• Collection of

blood and urine samples

• Transport to L3

• Same as L1 • Virus culture in designated labs.•Serology (?)

Laboratory tasks at each level for the diagnosis of measles

Polio

• Laboratory criteria for diagnosis Isolation of wild polio virus from stool

Laboratory tasks at each level for the diagnosis of polio

L1 L2 L3• Sample collection and transport to designated laboratories as per National Polio Surveillance Programme (NPSP) guidelines

• Sample collection and transport to designated laboratories as per NPSP guidelines

• Virus culture in designated laboratories

One or more of the above

Laboratory criteria for dengue

• Isolation of Dengue virus from serum, plasma, leucocytes or autopsy samples

• Demonstration of Dengue virus specific IgM antibodies or four fold or more rise in reciprocal IgG antibody titre

• Demonstration of dengue antigen in autopsy tissue by Immunochemistry or immunoflourescence or in serum samples by EIA

• Detection of viral genomic sequences in autopsy tissue, serum or CSF by PCR

Sample Period of collection

Storage for 24 to 48 hours

Transport

•Serum •5 days after onset

•+ 4oC •L2

•Plasma (Citrated blood)

•Within 5 days of onset

•+ 4oC •L3•CSF •Within 5 days

of onset

•Autopsy •(Brain, lung, liver)

•In the event of death

Sample collection for the laboratory diagnosis of Dengue

L1 L2 L3• Collection of blood

for serology and virus isolation

• Transport to L2

• Serology by ELISA or rapid methods

• Transport to L3 for culture

• Culture to be performed in a designated laboratories (which needs to be defined as a disease specific L3 or L4 / L5 laboratories)

• Serology by IgM ELISA and rapid tests


Laboratory tasks at each level for the diagnosis of Dengue

Laboratory criteria for the diagnosis of Japanese encephalitis

• Demonstration of Japanese encephalitis virus specific IgM antibodies

• Detection/isolation of antigen/virus• Demonstration of viral antigen in the

autopsied brain tissue by the fluorescent antibody test

Sample Period of Collection

Storage for 24 to 48 hours

Transport

•Serum •Within 6 days of onset

•+4C •L3•In cold chain

•CSF •Within 6 days of onset

•Autopsy (brain, lung, liver)

•In the event of death

Sample collection for the laboratory diagnosis of Japanese encephalitis

L1 L2 L3

• Collection of blood for serology and culture

• Transport to L3

• Same as L1 • Serology to be performed in a designated labs. (which needs to be defined as a disease specific L3 or L4 / L5 labs. due to the problem of availability of kits)

Laboratory tasks at each level for the diagnosis of Japanese

encephalitis

Laboratory criteria for the diagnosis of plague

• Gram staining on smear taken from bubo, blood or lung aspirate

• Detection of Y. pestis F1* antigen by direct fluorescent antibody testing or by other standardized antigen detection method

• Isolation from a clinical specimen• A significant (equal or more than 4-fold)

change in antibody titre to the F1 antigen in paired serum specimens

* Fraction 1. Glycoprotein from the capsule. Elisa technique

L1 L2 L3

• Assist in sample collection

• Staining and microscopy

• Transport sample to L3 laboratory

• No reporting(Wait confirmation)

• Culture, serology and confirmation to be performed in a designated L4/L5 laboratories

Laboratory tasks at each level for the diagnosis of plague

Leptospirosis

• Laboratory criteria for diagnosis Isolation from blood or other clinical materials by

culture Positive serology, preferably Microscopic Agglutination

Test (MAT) using a panel of Leptospira strains

• Sample collection Blood

• During first week of illness collect, second sample to be collected after about a week

Urine• Urine should be collected after second week of illness

and transported immediately in sterile container

L1 L2 L3

•Collection of blood and urine•Transport to L2

•Serology by latex agglutination/ IgM ELISA•DGM•Transport samples to L3 labs for culture

•Culture• MAT and serovar identification

Laboratory tasks at each level for the diagnosis of leptospirosis

Laboratory tests for water samples

• Most Probable Number (MPN) method for coliform bacteria

• H2S strip method for fecal contamination assessment

L1 L2 L3

• Collection of samples

• Rapid test- (H2S strip)

• Collection of samples

• Rapid test- (H2S strip)

• MPN test

• Same as L2• Quality control

for L2

Laboratory tasks at each level for the assessment of water quality

Functions of L1 laboratory technicians

• Collection of samples for investigations• Perform the laboratory tests assigned to L1 labs

Microscopy for malaria Microscopy for tuberculosis Typhi-dot test for typhoid fever H2S test for water quality

• Transport relevant sample to L2 laboratories for culture and serological investigations

• Assist Rapid Response Teams in sample collection

• Participate in External Quality Assurance conducted by L2 laboratories

Functions of L2 laboratory technicians

• Perform all tests performed by L1 laboratories• External Quality Assurance for L1 laboratories• Perform the tests assigned to L2 laboratories

Culture and sensitivity for cholera Serological test for typhoid, Dengue, Leptospirosis MPN test for water quality

• Transport relevant samples to L3 laboratories• Transport 5% of tested samples to L3 for testing

and quality assurance• Reporting test results to L1 laboratories for

samples received from L1 laboratories• Reporting tests result weekly to district

Quality assurance

Internal quality control

(Continuous, concurrent control of

laboratory work)

External quality

assessment(Retrospective and

periodic assessment)

Quality assurance

= +

Internal quality control

• Test request and specimen collection• Test processing

Temperature Reagent Maintenance of equipment

• Reporting and using test results

External quality assessment

• Within the state IDSP system L1 by L2 L2 by L3

• Through external agency External quality assurance scheme for

selected tests

Syndrome Action

•Fever •Blood smear for all patients

•Acute flaccid paralysis •2 stool samples at interval of 24 hours transported to the medical officer of the primary health centre in reverse cold chain

•Fever with rash, altered sensorium or bleeding

•Refer to the medical officer of the primary health centre for specific laboratory action •Fever more than 14 days

•Cough < or > 3 weeks

•Loose watery stools

•Acute jaundice

•Unusual syndromes

Action to be taken by the multi-purpose worker in the field

When to collect sample

•Single case of probable dengue •First 10 cases in outbreak situations

What specimens to be collected

•5ml of blood for serology•5ml of blood in citrate for virus isolation (If recommended by rapid response team)

Processing at the CHC by the technician

•Serum separation

Storage •Serum and blood in refrigerator. •If delay in transportation, store in –20C

Transportation •As quickly as possible within 24 hours in reverse cold chain to the district laboratory

Laboratory investigations by the PHC/CHC medical officer /laboratory technician

for Dengue

Processing at district / medical college / sentinel laboratories

•Serology - IgM Elisa / rapid test•Platelet count for hospitalized patients

Storage •–20C

Transportation •1st and 2nd serum and blood sample sent to state / reference laboratory

Processing at state / national laboratories

•Virus isolation and antigen detection•HAI and neutralization to detect rise in antibodies•Quality control of the IgM Elisa of the district

Laboratory investigations by the district and state laboratories for

Dengue


•Single case of probable Japanese encephalitis•First 10 cases in outbreak situations


•5ml blood for serology•CSF in hospitalized cases: Serology and virus isolation


•Serum separation

Storage •Serum and CSF in refrigerator. •If delay in transportation, store in –20C

Transportation •As quickly as possible within 24 hours in reverse cold chain to the state reference laboratory

Laboratory investigations by the PHC/CHC medical officer /laboratory technician for Japanese encephalitis /fever with

altered consciousness


•NIL

Storage •–20C

Transportation •CSF and serum sent to state / reference laboratory


•IgM Elisa for CSF and serum•HAI / neutralization for detection of rise in antibody titres.•Virus isolation and antigen detection in CSF


Japanese encephalitis


•Single case of fever


•Blood smear


•Staining and microscopy

Storage for quality assurance

•All positive •10% negative

Transportation •NIL

Laboratory investigations by the PHC/CHC medical officer /laboratory technician

for malaria or fever


•As in primary health care centre for cases seen at the district hospital

Storage • As in primary health care



•NIL


malaria


•Case of probable cholera •First 10 cases in outbreak situations


•Fresh stools or rectal swab in Cary–Blair medium


•NIL

Storage •In refrigerator

Transportation •As soon as possible •No need of cold chain if within 24 hours

Laboratory investigations by the PHC/CHC medical officer /laboratory technician for

cholera /loose watery diarrhea

Processing at district

•Culture, identification and sensitivity

Storage • Positive isolates at + 4oC

Transportation •Sealed stab culture of positive isolates to state reference laboratory

Processing at state laboratory

•Confirmation of serotype / phage typing•Antibiotic sensitivity•Quality assurance


cholera


•One case of probable typhoid•First 10 cases in outbreak situations


•5ml blood in citrate•5ml blood for serology (2 samples at one week interval if the first sample is negative and if requested by the district laboratory)


•Serum separation•Typhi dot test

Storage •In refrigerator (Serology)

Transportation •1st and 2nd serum sample and blood sample to be sent to the district laboratory

Laboratory investigations by the PHC/CHC medical officer /laboratory

technician for typhoid /fever > 7 days


•Serology - Widal in paired sera if first is negative•Blood, stool and bone marrow culture, identification and sensitivity

Storage •At + 4oC

Transportation •10% of positive and negative specimens to be sent to state for quality assurance


•Blood culture•Identification•Sensitivity


typhoid


•During outbreaks only •First 10 cases only


• 5ml blood for serology


•Serum separation

Storage •At - 20C deep freezer

Transportation •In reverse cold chain to the state/reference laboratory


technician for hepatitis/ acute jaundice


•NIL

Storage •At - 20oC

Transportation •Reverse cold chain to the state / reference laboratory


•IgM Elisa for HAV and HEV


hepatitis


•During outbreaks only •First 10 cases only


•5ml blood for serology•30 ml urine for virus isolation (If required by the rapid response team)


•Serum separation


Transportation •Immediately to the district laboratory within 24 hours, with reverse cold chain

Laboratory investigations by the PHC/CHC medical officer /laboratory technician for measles / fever with

rash


•Measles IgM Elisa

Storage •- 20oC

Transportation •10% of positive, all negative and urine samples to be sent to the state / reference laboratory


•Urine virus isolation•Antigen detection•Quality assurance of the positives•Test of negative for rubella


measles


•All probable cases of tuberculosis


• 3 sputum specimens •Spot/early morning/spot


•Smear staining and microscopy

Storage for quality assurance

•10% of positives •All negatives

Transportation •Sputum to the state laboratory for culture sensitivity testing


tuberculosis /cough > 3 weeks


•Smear, microscopy

Storage •10% of positives and all negatives to be kept for quality assurance



•For quality assurance: Blinded samples sent to districts


tuberculosis


•A single case of acute flaccid paralysis


•2 stools specimens at 24 hour interval


•NIL


Transportation •With 24 hours •Reverse cold chain•National polio laboratory


acute flaccid paralysis


•NIL

Storage •NIL

Transportation •Reverse cold chain

Processing at national polio laboratories (ONLY)

•Virus isolation•Identification•Quality assurance by the reference laboratory and WHO


acute flaccid paralysis


•Only at the voluntary counseling and testing sites or blood transfusion centres•Testing as per the recommendations of the National AIDS Control Organization (NACO)

Storage •- 20oC

Transportation •All positive specimens to the state laboratory

Processing at the state processing / national reference laboratory

•Confirmatory tests (Western blot)


HIV/HBV


•From probable cases•Samples to be collected by the rapid response team


•Aspirate from the bubo•Sputum from pneumonic plague cases •5 ml blood sample for serology


•NIL

Storage •NiL

Transportation •Immediately to the state/ national reference laboratory with P3 facilty


technician for plague


•At the medical college level only•Smear, microscopy of aspirate / sputum for bacilli

Storage •+ 4oC

Transportation •All samples by reverse cold chain in reverse cold chain to the nearest reference laboratory as specified by the rapid response team


•Isolation of bacteria by culture•Antigen detection•Direct fluorescent antibody testing of smears (for anti-F1 antibody)•PCR test


plague


•From probable cases


•5ml blood for serology


•Serum separation

Storage •At +4C

Transportation •Immediately by reverse cold chain to the district


technician for leptospirosis


•Rapid agglutination kit

Storage •+ 4oC

Transportation •To the state


•Microscopic Agglutination Test (MAT) for identification of serovars


leptospirosis


•When surveillance is conducted


•Blood sample

Transportation •To designated laboratories

Testing site •District laboratories•Medical college laboratories •Identified laboratories

Test to be done

•Blood sugar, serum cholesterol, triglycerides

Laboratory investigations by the health workers and medical officer of

the PHC for non communicable diseases

Laboratory data management

• Recoding Details of specimens received Tracking of samples Results of tests performed

• Analysis and interpretation of tests• Timely and accurate communication of

results

Information to be recorded on each specimen/ accompanied with each

specimen• Name, age, sex• Address in detail• Reporting unit referring the sample• Syndromic diagnosis• Date of onset of illness• Nature of sample, date of collection, date of

receipt and condition of sample• Investigation requested• Whether convalescent specimen or not

ID no Name and address of patient

Age Sex Prov. Diag.

Lab tests ordered

Lab results

Date sent to L2

Result from L2

Date of result

Sample laboratory register

The L form

• Weekly reports from laboratories to the district surveillance officer Prepared on the basis of the laboratory

register Filled by nodal person in the laboratory Sent every Saturday of each week

• Zero/NIL reporting• Electronic link between

District public health laboratory District surveillance unit

Points to remember (1/2)

• Categorization of labs - List of L1 and L2 labs in the districts & List of Disease wise L3 labs in the state

• List of tests that can be done at L1 and L2 labs

• List of diseases that can be confirmed only by L3 labs

Points to remember (2/2)

• Sourcing the consumables required by the labs • Samples that have to be collected for specific

disease• Bio Safety and waste management • Quality assurance

Working with the laboratory

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