Salmonella Typhi 2012

7/28/2019 Salmonella Typhi 2012

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Salmonella enterica serovar Typhi

Prof. Mochammad Hatta, MD, Ph.D, Clin Micro (Cons)

Dept Microbiology, Molecular Biology and ImmunologyLaboratory, Fac.Medicine, Hasanuddin University,

Makasssar, Indonesia



INTRODUCTION

Discovered in 1880 & named after Daniel

Salmon, the pathologist who first isolated the

organism from porcine intestine.

Salmonella is a motile, gram-negative, rod-

shaped bacteria, which is a leading cause of

bacterial food-borne diseases.

Of the 2000 strains recognized, human

infection are caused mainly by 5 serotypes,

typhi, paratyphi, typhimurium, choleraesuis &

enteritidis.



Salmonella typically produces 3 distinct

syndromes: food poisoning, typhoid fever &

asymptomatic carrier state.

Salmonella gastroenteritis manifest as vomiting

& diarrhea within 6-48 hours after ingestion of food

or drink contaminated with bacteria.

SALMONELLOSIS

It is self-limiting, treatment is by water & salts

replacement. Antibiotics are not usually needed.



TYPHOID FEVER

Caused by salmonella typhi & paratyphi.

Incubation period is 1-2 weeks.

Typhoid fever is the most serious salmonella

infection with significant morbidity & mortality.

Salmonella has somatic (O antigen) & flagellar H

antigen. The O antigen is more specific for

serologic testing.



An estimated 15-30 million cases of typhoid

fever occur globally each year.

FREQUENCY

Incidence in Sudan is not exactly known, but

estimated as 50 per 100,000 people/year.

The disease is endemic in many developing

countries in Asia, Central America & Africa.

Outbreak of typhoid fever have been reported

recently from Eastern Europe.



PRECIPITATING FACTORS

Defects in cellular-mediated immunity (AIDS,

Transplant patients & malignancy).

Defects in phagocytic function (malaria,

histoplasmosis & schistosomiasis).

Low stomach PH ( patients on anti-ulcer drug).

Prolonged use of antibiotics (altered gut flora).

Injured gut barrier (bowel disease or surgery).

Splenectomy or functional asplenia (sickle cell dis)



MORTALITY & MORBIDITY

Whereas mortality caused by typhoid fever israre in western countries, it is associated with

significant mortality & morbidity in tropical

countries (10-30%).

Dehydration is the most common complication

of typhoid fever, but serious intestinal & extra-

intestinal complications may occur.

Infection with nontyphoidal salmonella produces

self-limiting gastroenteritis and food poisoning.



PATHOPHYSIOLOGY

After ingestion salmonella must survive the

stomach acidic PH & colonize small intestine.

Another portal of entry is invasion of lymphoidtissue in the GIT (peyer patches) & multiplication

within macrophages leading to bacteremia.

Salmonella then attach to & penetrate the gutmucosa resulting in diarrhea from direct

mucosal damage & by action of exotoxins.



Mochammad Hatta

• Viability : death point : 56o C

• In soil survival for 6 weeks

• Pathogenesis : infection by ingestion --- small intestinal via

lymphatics --- mesenteric glands -- multiplication --- blood via

thoracic duct --- bacteriaemic phase ( 1 - 10 days) : infection

liver, gall bladder, spleen, kidney & bone marrow.

Gall bladder --- invasion lymphoid tissue -- Peyer’s patches

& lymphoid follicles -- acute inflammatory reactions --- ulcer

haemorrhage -- perforation & necrosis



Mochammad Hatta

POSITIVE CULTURE AND IgM ANTIBODY RESPONS IN TYPHOID

FEVER

010

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8

Weeks

% P

o s i t i v e

BloodFaeces

Urine

IgM



DIFFERENTIAL DIAGNOSES

Cryptosporidiosis

Cyclospora

Campylobacter infection

Listeria monocytogenes

Escherichia Coli infection

Shigellosis



LAB FINDINGS

Salmonella can be grown from blood or bone

marrow in the 1st week, from stool in the 2nd

week & from urine in the 3rd week.

Special media are needed for transport & for

culture.

leukopenia is typical but WBC may be normal.

Widal test is not diagnostic, titer > 1:320 or 4

fold increase in titer support the diagnosis.



Typhoid feverLaboratory diagnosis

Polymerase Chain Reaction (PCR)

Culture of blood or bone marrow

80% during first week

Culture of urine or stool

in presence of characteristic clinical picture

Serology test

antibody test against somatic (O) or

flagellar (H) antigen



Mochammad Hatta

Deteksi Salmonella typhi dengan Nested PCR



Mochammad Hatta

Typhoid fever

(nested)

ST1 : 5’-ACT GCT AAA ACC ACT ACT-3’

ST2 : 5’-TTA ACG CAG TAA AGA GAG-3’

ST3 : 5’-AGA TGG TAE TGG CGT TGC TC-3’

ST4 : 5’-TGG AGA CTT CGG TCG CGT AG-3’

(M. Hatta & Henk L Smits. American J.Tropical Medicine & Hygeine, 2007)



Mochammad Hatta

Hasil nested PCR S.typhi dari penderitademam tifoid



Mochammad Hatta

MDR PCR product S.typhi Vietnam and Indonesian isolated

Vietnam Indonesia



Mochammad Hatta

MDR PCR product S.typhi Vietnam isolated

941 bp

819

639

310



Mochammad Hatta

PCR for the detection of S. typh i specific DNA in blood, stool and urine

samples from patients with suspected typhoid fever .

No (%) of patients with the following result

Patient group Blood Faeces Urine Neg Pos Neg Pos Neg Pos

Culture positive 1 (1) 71 (99) 16 (67) 8 (33) 22 (38) 36 (62)

Culture negative 21 (45) 26 (55) 7 (41) 10 (59) 11 (28) 28 (72)

Non-typhoid patients 12 (100) 0 (0) 2 (100) 0 (0) 10 (100) 0 (0)

(INCO-DC EC Research project Report, 2002)



Mochammad Hatta

CONSTRAINTS OF PCR TECHNIQUE 1. Quite expensive

2. Need special equipment

3. Need high skill and laboratory

4. Sophisticated



Mochammad Hatta

Salmonellabacteria on

MacConkey agar

Lactose-positivebacteria showpink colonies

(upper left)

Lactose-negative

bacteria have

colorlesscolonies (lowerright)



Mochammad Hatta

Biochemical reactions for identification of S.typhi by the API 20E procedure



Mochammad Hatta

Black colonies of Salmonella

typhi aftergrowth onbismuth

sulfite agar



Mochammad Hatta

Dipstick for Typhoid Fever

Procedure Add 5µl serum to 250µl detection reagent Incubate dipstick for 3 hours

Rinse with tap water Read by visual inspection

Result

(Mochammad Hatta, et al. American J. TropicalMedicine & Hygiene, 2002)



Mochammad Hatta



Mochammad Hatta

Dipstick for Typhoid Fever

Procedure Add 5µl serum to 250µl detection reagent Incubate dipstick for 3 hours Rinse with tap water

Read by visual inspection

ResultPatients with

clinical

suspicion of

typhoid fever from Makassar,

Indonesia

Control

Test

Typhoid Fever Dipstick



Mochammad Hatta

Typhoid Fever Dipstick CTD, Ho Chi Minh City, Viet Nam

Comparison of tests

Test Sensitivity (%) Specificity (%)

IgM ELISA 1:400IgG ELISA 1:1.600

IgA ELISA 1:200

756852

949295

Widal O1:400

Widal H 1:2004760 9398

Dipstick 77 95



Mochammad Hatta

Typhoid Fever Dipstick Semarang, Indonesia

Patient group,culture result

Number positive (%) / total

Dr. Kariadi Hospital(bone marrow culture)

S. typhi positiveS. typhi negative

3 district hospitals(blood culture)

S. typhi positiveS. typhi negative

38 (70.4) / 540 (0) / 2

32 (86.5) / 372 (7.7) / 26



Mochammad Hatta

Typhoid Fever Dipstick Makassar, Indonesia

Patient group No. positive (%) /total

Suspects

Clinical diagnosis: typhoid S. typhi culture positive S. paratyphi culture positive

Culture negative

Clinical diagnosis: otherCulture negative

Hospital controlsSchool children

85 (47.5) / 17973 (65.2) / 1124 (66.6) / 6

8 (13.1) / 61

0 (0) / 64

0 (0) / 2592 (1) / 194

Typhoid Fever Dipstick



Mochammad Hatta

Typhoid Fever Dipstick Makassar, Indonesia

Follow-up

Sample DPO No. positive (%) /Total

S. typhi culture positiveFirstSecondThird

S.typhi culture negativeFirstSecondThird

81529

61327

30 (76.9) / 3932 (82.1) / 3938 (97.4) / 39

2 (4.3) / 4736 (76.6) / 4739 (83.0) / 47



Mochammad Hatta

Typhoid feverCulture and Dipstick

Assay

Culture

Dipstick

Sensitivity

65.9%

47.5%

Specificity

100%

95%

PPV

100%

92%

NPV

74%

65%

Dipstick: finger prick blood, same day result



Mochammad Hatta

Rapid test for typhoid fever

80% sensitivity compared with bloodculture

PPV (92%) and NPV (64%) somewhatlower than that of culture

Same day result

Easy to perform

High stability of components



Mochammad Hatta

DIPSTICK FOR DETECT IGM ANTIBODIES 1. Simple and rapid

2. Required no equipment3. Highly stable reagents

4. Low cost

5. Easy to applied in field



TYPHOID Lateral FlowPrinciple

Immunochromatographic strip assay

Test Control

Sample pad / Conjugate Detection strip Sink

blood cell pad

separation filter



TYPHOID Lateral FlowMethod

Add 5l serum

Add 130l sample fluid

Wait 10 minutes Read result Sample well

Control line

Test line



Mochammad Hatta

Typhoid Fever Latex Agglutination

5 seconds 15 seconds

45 seconds > 60 seconds



What is S.typhi ?

A bacteria

Causes typhoid fever that affects 16

million people annually and causes600,000 fatalities

Has evolved the ability to spread from theintestine to the deeper tissues of humans,including the liver, spleen, and bonemarrow



What is it? Cont…

Resistant to many drugs

Closely related to Salmonella typhimurium (also

already sequenced), classified under the samespecies as Salmonella typhi

Difference is that S. typhi causes typhoid feverand can only infect humans, whereas S.

typhimurium causes food poisoning and canaffect almost all animals



Where and When? Sequenced by the Sanger Institute

On November 7, 2001

Mochammad Hatta. How diversity flagella variants of S. Typhi strains in

Indonesia Archipelago?. Wellcome Trust Advanced Course: Molecular Basis of Bacterial Infection: Basic and Applied Research Approaches . Wellcome Trust Genome Campus, The Sanger Institute,Hinxton, Cambridge, United Kingdom (UK), 11-17 May 2008. page 12-13.



Why?

It affects millions of people andsequencing the genome can help us find a

way to block its transmission in humans,eradicating it altogether

Can help improve diagnostic tools and

vaccines



Why?

Multiple drug resistance (MDR) is aemerging problem in treating infectious

diseases Salmonella typhi is one example of MDR

microorganism

It is resistance to fluoroquinolones, themost effective antimicrobials for thetreatment of typhoid fever



So…..?

Since salmonella typhi is an example of anemerging MDR microorganism, studying

this genome can contribute to theunderstanding of how suchmicroorganisms adapt rapidly to new

environmental changes that are presentedby modern human society.



Some Statistics

Chromosome sequence is 4,809,037 bp inlength

C+G content of 52.09% 4,599 protein-coding genes (402 of these

are pseudogenes)



Side Note:

Pseudogene: once functional stretches of DNA that have been inactivated by

mutation



Some Results

The genome shows hundreds of deletionsand insertions, resulting in MDR

Found the plasmid in Salmonella typhi thatencodes resistances to all of the first-linedrugs used for the treatment of typhoidfever

Many other genes responsible forresistance in drugs were indentified



Side Note

Plasmid: A piece of symbiotic DNA, mostlyin bacteria but also in yeast, not forming

part of the normal chromosome DNA of the cell and capable of replicatingindependently of it. Plasmids carry asignal situated at their replication origin

dictating how many copies are to bemade, and this number can be artificiallyincreased.



Results Cont…

Salmonella typhi’s genome gives us hintsas to why it only infects humans

Because it has 204 pseudogenes. Workingversions of these genes were discardedduring typhi’s evolution for its currenthabitat in humans

Vs. typhimurium only has about 40pseudogenes



Results Cont…

Both typhi and typhimurium havehundreds of genes that are different. This

is very surprising because these twoorganisms are classified as a singlespecies



CLINICAL PICTURE

Symptoms begin with sudden onset of high-

grade fever, headache & dry cough.

Fever is swinging or may show step ladder

pattern & patient initially feel well & mobile.

Abdominal pain & toxicity follow soon & by

the end of 1st week spleen is palpable & pink,

discrete, skin rash appears over the trunk.

Constipation is more common than diarrhea

which is usually greenish in color (pea soup).



CLINICAL PICTURE/2

Abdominal tenderness & hepatomegaly occur

in 50% of patients.

The pulse is relatively slow in relation to fever (Paget sign).

The tongue is coated with free margins &

halitosis may be present.

The sweat of some patients smell like yeast.



CLINICAL PICTURE/3

The 3rd week of illness is the usual time for

complications in the untreated patients.

Local gut as well as systemiccomplications may occur.

Serious infections may progress rapidly to

drowsiness & coma which is usually fatal(coma vigil).

Mortality is unlikely after the 4th week &

patients may become carrier if not treated.



Intestinal hemorrhage

Paralytic ileus

Intestinal perforation

LOCAL COMPLICATIONS

Zenker degeneration of abdominal muscles



Endocarditis

Arteritis & arterial emboli

Cholecystitis

SYSTEMIC COMPLICATIONS

Osteomyelitis & septic arthritis

Hepatic & splenic abscesses

Pneumonia or empyema

Meningitis

Urinary tract infection



TREATMENT

Medical care include rehydration, antipyretics& antibiotics.

Drugs of choice are Ceftriaxone & ciprofloxacin

but Cotrimoxazole & Chloramphenicol are stillused in developing countries. Ampicillin kills

bacilli hiding in the bile & hence prevents or

reduce the carrier state.

Chronic resistant carrier state may necessitate

cholecystectomy. Surgical care may also be

needed in patients with intestinal complications.



NURSING CARE

Isolation & barrier nursing is indicated

Trace source of infection.

continue breastfeeding infants & young children

and give ORS & light diet for other patients in the

first 48 hours.

Notification of the case to the infection control

nurse in the hospital.



PREVENTION

Education on hygiene practices like hand

washing after toilet use & avoidance of eating in

non hygienic restaurants.

Antibiotic prophylaxis is not needed for

house-hold contacts.

Proper handling & refrigeration of food even

after cooking.

Salmonella TAB vaccine is available but

affectivity is low (50% claimed protection).



PROGNOSIS

With early diagnosis and prompt treatmentmost patients with typhoid fever will recover in

due time.

Fever & toxicity subsides within 72 hours of antibiotic treatment.

Mortality is > 50% in untreated severe typhoid

fever particularly in children & elderly.

Recrudescence is rare but chronic carrier

state is reported in 10% of patients.



Infection follows ingestion of contaminated

food or water. Meat, poultry, eggs & diary

products are frequent sources.

TRANSMISSION

Pets, domestic animals and infected human

are potential reservoirs. Person to person &

animal to human transmission is recognized.

In healthy humans a dose of about one million

bacteria is necessary to produce symptoms.

f



References

Mochammad Hatta, Mirjam Baker, Stella van Beer, Theresia H Abdoel, Henk L Smits. Risk factors for clnical typhoid fever in villages in Rural South Sulawesi, Indonesia. International Journal of Tropical Medicine . Vol 4 (3): 91-99, (2009)

Mochammad Hatta and Ratnawati. Enteric fever in endemic areas of Indonesia: an increasingproblem of resistance. J. Infection Developing Countries (JIDC ). Vol 2(4); 298-301 (2008)

Rob Pastoor, Mochammad Hatta, Theresia H. Abdoel, Henk L. Smits. Simple, rapid andaffordable point-of-care test for the serodiagnosis of typhoid fever. J. Diagnostic Microbiology and Infectious Disease . Vol 61:(2);129-134, Feb (2008).

Mochammad Hatta and Henk L Smits. Detection of Salmonella typhi by nested PolymeraseChain Reaction in blood, urine and stool samples. American J. Tropical Medicine Hygiene.vol: 76;139-143 (2007).

Theresia H. Abdoel, Rob Pastoor, Henk L. Smits, Mochammad Hatta, Laboratory evaluation of a simple and rapid latex agglutination assay for the serodiagnosis of typhoid fever. Transactions of the Royal Society of Tropical Medicine and Hygiene . vol. 101 (10); 1032-1038 (2007)

Mochammad Hatta, Marga D.A Goris, Evy Heerkens, George C Gussenhoven, Jairo Goosken,Henk L Smits. Simple dipstick assay for the detection of Salmonellla typhi -specific immunoglobulinM antibodies and the evolution of the immune response in patients with typhoid fever American J. Tropical Medicine and Hygiene . vol 66: no 4; 416-421 (2002).

Mochammad Hatta, Mubin Halim, Theresia Abdoel, Henk L. Smits. Antibody response intyphoid fever in endemic Indonesia and relevance of serology and culture to diagnosis.Southeast Asian Journal of Tropical Medicine and Public Health . vol 33: no 4; 182-191(2002)

Salmonella Typhi 2012

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