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serovar Typhi
Prof. Mochammad Hatta, MD, Ph.D, Clin Micro (Cons) Dept Microbiology, Molecular Biology and Immunology Laboratory, 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, rodshaped 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.
SALMONELLOSIS
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.
It is self-limiting, treatment is by water & salts replacement. Antibiotics are not usually needed.
TYPHOID FEVER
Typhoid fever is the most serious salmonella infection with significant morbidity & mortality. Caused by salmonella typhi & paratyphi. Incubation period is 1-2 weeks. Salmonella has somatic (O antigen) & flagellar H antigen. The O antigen is more specific for serologic testing.
FREQUENCY
An estimated 15-30 million cases of typhoid fever occur globally each 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. Incidence in Sudan is not exactly known, but estimated as 50 per 100,000 people/year.
PRECIPITATING FACTORS
Defects in cellular-mediated immunity (AIDS, Transplant patients & malignancy). Defects in phagocytic function (malaria, histoplasmosis & schistosomiasis). Splenectomy or functional asplenia (sickle cell dis) Low stomach PH ( patients on anti-ulcer drug). Prolonged use of antibiotics (altered gut flora). Injured gut barrier (bowel disease or surgery).
Dehydration is the most common complication of typhoid fever, but serious intestinal & extraintestinal complications may occur.
PATHOPHYSIOLOGY
After ingestion salmonella must survive the stomach acidic PH & colonize small intestine. Salmonella then attach to & penetrate the gut mucosa resulting in diarrhea from direct mucosal damage & by action of exotoxins. Another portal of entry is invasion of lymphoid tissue in the GIT (peyer patches) & multiplication within macrophages leading to bacteremia.
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
Mochammad Hatta
100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 Weeks
% Positive
Mochammad Hatta
DIFFERENTIAL DIAGNOSES
Cryptosporidiosis Campylobacter infection Cyclospora 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.
Laboratory diagnosis
Typhoid fever
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
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
Mochammad Hatta
MDR PCR product S.typhi Vietnam and Indonesian isolated Vietnam Indonesia
Mochammad Hatta
Mochammad Hatta
PCR for the detection of S. typhi specific DNA in blood, stool and urine samples from patients with suspected typhoid fever .
Patient group
No (%) of patients with the following result Blood Faeces Urine Neg Pos Neg Pos Neg
Pos
Mochammad Hatta
Mochammad Hatta
Salmonella bacteria on MacConkey agar Lactose-positive bacteria show pink colonies (upper left) Lactosenegative bacteria have colorless colonies (lower right)
Mochammad Hatta
Mochammad Hatta
Black colonies of
Mochammad Hatta
Mochammad Hatta
Mochammad Hatta
Control Test
Specificity (%) 94 92 95 93 98 95
38 (70.4) / 54 0 (0) / 2
32 (86.5) / 37 2 (7.7) / 26
Mochammad Hatta
No. positive (%) / total 85 (47.5) / 179 73 (65.2) / 112 4 (66.6) / 6 8 (13.1) / 61 0 (0) / 64 0 (0) / 259 2 (1) / 194
No. positive (%) / Total 30 (76.9) / 39 32 (82.1) / 39 38 (97.4) / 39 2 (4.3) / 47 36 (76.6) / 47 39 (83.0) / 47
8 15 29 6 13 27
Mochammad Hatta
Mochammad Hatta
80% sensitivity compared with blood culture PPV (92%) and NPV (64%) somewhat lower than that of culture Same day result Easy to perform High stability of components
Mochammad Hatta
Mochammad Hatta
Conjugate pad
Detection strip
Sink
Add 5l serum
Sample well
5 seconds
15 seconds
45 seconds
> 60 seconds
Mochammad Hatta
What is S.typhi ?
A bacteria Causes typhoid fever that affects 16 million people annually and causes 600,000 fatalities Has evolved the ability to spread from the intestine to the deeper tissues of humans, including the liver, spleen, and bone marrow
Resistant to many drugs Closely related to Salmonella typhimurium (also already sequenced), classified under the same species as Salmonella typhi Difference is that S. typhi causes typhoid fever and can only infect humans, whereas S. typhimurium causes food poisoning and can affect almost all animals
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 and sequencing 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 a emerging problem in treating infectious diseases Salmonella typhi is one example of MDR microorganism It is resistance to fluoroquinolones, the most effective antimicrobials for the treatment of typhoid fever
So..?
Since salmonella typhi is an example of an emerging MDR microorganism, studying this genome can contribute to the understanding of how such microorganisms adapt rapidly to new environmental changes that are presented by modern human society.
Some Statistics
Chromosome sequence is 4,809,037 bp in length 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 deletions and insertions, resulting in MDR Found the plasmid in Salmonella typhi that encodes resistances to all of the first-line drugs used for the treatment of typhoid fever Many other genes responsible for resistance in drugs were indentified
Side Note
Plasmid: A piece of symbiotic DNA, mostly in bacteria but also in yeast, not forming part of the normal chromosome DNA of the cell and capable of replicating independently of it. Plasmids carry a signal situated at their replication origin dictating how many copies are to be made, and this number can be artificially increased.
Results Cont
Salmonella typhis genome gives us hints as to why it only infects humans Because it has 204 pseudogenes. Working versions of these genes were discarded during typhis evolution for its current habitat in humans Vs. typhimurium only has about 40 pseudogenes
Results Cont
Both typhi and typhimurium have hundreds of genes that are different. This is very surprising because these two organisms are classified as a single species
CLINICAL PICTURE
Symptoms begin with sudden onset of highgrade 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 systemic complications 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.
LOCAL COMPLICATIONS
Intestinal hemorrhage
Intestinal perforation Paralytic ileus
SYSTEMIC COMPLICATIONS
Endocarditis Arteritis & arterial emboli Cholecystitis
TREATMENT
Medical care include rehydration, antipyretics & antibiotics. Drugs of choice are Ceftriaxone & ciprofloxacin but Cotrimoxazole & Chloramphenicol are still used 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 Notification of the case to the infection control nurse in the hospital. Trace source of infection.
continue breastfeeding infants & young children and give ORS & light diet for other patients in the first 48 hours.
PREVENTION
Education on hygiene practices like hand washing after toilet use & avoidance of eating in non hygienic restaurants.
PROGNOSIS
With early diagnosis and prompt treatment most 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.
TRANSMISSION
Infection follows ingestion of contaminated food or water. Meat, poultry, eggs & diary products are frequent sources.
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.
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 increasing problem 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 and affordable 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 Polymerase Chain 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 immunoglobulin M 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 in typhoid 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).