Salmonella

Infections
an update
Dr.T.V.Rao MD

Dr.T.V.Rao MD

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Salmonella
• Causes Infections in Humans and vertebrates, • Enteric Fever ( Typhoid fever ) • Gastroenteritis • Septicemias, • Carrier state a concern
Dr.T.V.Rao MD 2

Salmonella
• A Very complex group • Contains more > 2,000 spp • Typed on the basis of Serotyping, and species typing • Divided into two groups 1 Enteric fever group 2 Food poisoning group – Septicemias.
Dr.T.V.Rao MD 3

Key points
• There are more than 2000 different antigenic types of Salmonella; those pathogenic to man are serotypes of S. enterica. • Most serotypes of S. enterica cause foodborne gastroenteritis and have animal reservoirs. • S. enterica serotypes Typhi and Paratyphi cause typhoid fever.

Dr.T.V.Rao MD

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Enteric Fever Typhoid Fever
• Caused by Salmonella typhi, and other Groups called as Paratyphoid A, B, C
• Salmonella typhi - Causes Typhoid • Salmonella Paratyphi A,B,C Causes Paratyphoid fevers. • Food Poison group • Spread from Animals – Humans • Causes Gastroenteritis – Septicemias, Localized Infection
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Typhoid fevers are prevalent in many regions in the World

Typhoid Mary Most Dangerous Woman in America

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Typhoid Mary
• A famous example is “Typhoid” Mary Mallon, who was a food handler responsible for infecting at least 78 people, killing 5. These highly infectious carriers pose a great risk to public health.

Typhoid Mary
• "Typhoid Mary," real name Mary Mallon, worked as a cook in New York City in the early 1900s. Public health pioneer Sara Josephine Baker, MD, PhD tracked her down after discovering that she was the common link among many people who had become ill from typhoid fever She was traced to typhoid outbreaks a second time so she was put in prison again where she lived until she died.

Morphology of Salmonella
• Gram negative bacilli • 1-3 / 0.5 microns, • Motile by peritrichous flagella
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S.typhi with Flagella

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Bacteriology –Typhoid fever
• The Genus Salmonella belong to Enterobacteriaceae • Facultative anaerobe • Gram negative bacilli • Distinguished from other bacteria by Biochemical and
antigen structure
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Different types of Salmonella
I - enterica II - salamae IIIa -arizonae IIIb -diarizonae IV - houtenae V - bongori VI - indica

Cultural Characters
• • • • Aerobic / Facultatively anaerobic Grows on simple media – Nutrient agar, Temp 15 – 41ºc / 37º c Colonies appear as large 2 -3 mm, circular, low convex,

• On MacConkey medium appear Colorless ( NLF )
Selective Medium - Wilson Blair Bismuth sulphide medium. Produce Jet black colonies H2 S produced by Salmonella typhi
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Enrichment Medium

Liquid Medium

• Selenite F medium • Tetrathionate broth • Above medium are used for isolation of Salmonella from contaminated specimens • Particularly stool specimens..
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Identifying Enteric Organisms
• • • • • • • • Isolates which are Non lactose fermenting Motile, Indole positive Urease negative Ferment Glucose,Mannitol,Maltose Do not ferment Lactose, Sucrose Typhoid bacilli are anaerogenic Some of the Paratyphoid form acid and gas Further identification done by slide agglutination tests
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Biochemical Characters
• • • • • • • • • • Glucose ,Mannitol ,Maltose produce A/G Salmonella typhi do not produce gas Lactose/Salicin/sucrose not fermented. Indole – Methyl Red + VP Citrate + Urea – H2S – produced by Salmonella typhi Paratyphi A do not produce H2S
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Resistance of Salmonella

• 55º c – 1 hour • 60º c – 15 MT • Boiling ,Chlorination, Pasteurization Destroy the Bacilli.
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Antigenic structure of Salmonella
Two sets of antigens • Detection by serotyping • 1 Somatic or 0 Antigens contain long chain polysaccharides ( LPS ) comprises of heat stable polysaccharide commonly.

• 2 Flagellar or H Antigens are strongly immunogenic and induces antibody formation rapidly and in high titers following infection or immunization. The flagellar antigen is of a dual nature, occurring in one of the two phases.

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Salmonella

Antigenic Structure
• • • • H – Flagellar antigens O – Somatic antigen, Vi – Surface antigen in some species only H antigens also called flagellar antigens, heat labile protein, • Boiling destroys antigenicity • When mixed with Antiserum produces agglutination and fluffy clumps are produced • H antigens are strongly immunogenic Induces antibodies rapidly,
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Antigens – Salmonella ( cont )
• O Antigens • • • • Forms integral part of Cell wall, Like Endotoxin 0 Antigens unaffected by boiling. When mixed with antiserum produce chalky clumps are formed, take more time reaction, at high temp 50º – 55º c • O antigens are less immunogenic. than H antigens
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Antigen (Vi) – Salmonella ( contd )
• Vi antigens • Many strains in S.typhi covers the O antigensprevents agglutination. • Resembles like K antigens • Destroyed after boiling at 60º c / 1 hour. • Vi a polysaccharide • Acts as virulence factor, protects the bacilli against Phagocytosis and activity of Complement • Poorly immunogenic • Low titer of antibodies are produced, Not diagnostic
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Classification of Salmonella • Classified on the basis of Kauffmann-White Scheme • Structure of 0 and H antigens are taken into consideration, • More than 2000 species characterized.
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Kauffmann – White scheme
• Serotype 0 antigens H antigens Phase 1 2 d 1,2 a b 1,2 I 1,7 g m 1,2
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1.Typhi 9,12,(Vi) 2 Paratyphi A 1,2.12 3 Paratyphi B 1,4,5,12 4 Typhimuruim 1,4,5,12 5 Enteritidis 1,9,12

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Antigenic Variation in Salmonella • May be phenotypic / Genotypic • H to O = loss of Flagella May be phase variation from I to II V to W variation S to R variation
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Pathogenicity
• Salmonella are definite parasites to humans. • Eg S.typhi. • S.paratyphi A, B ,C • Other groups Salmonella • The important clinical syndromes 1. Enteric fever, Septicemias, gastroenteritis.
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Enteric Fever: S. typhi
• • • • • Ileocecal penetration intraluminal multiplication mononuclear response (macrophages) Salmonella remains alive 2nd week - lymphoid hyperplasia (mesenteric lymph nodes) • back to bowel

Enteric Fever Typhoid

• Typhoid – caused by S.typhi • Paratyphoid Caused by Paratyphi A,B,C • Typhoid --- Like Typhus • Infective dose ID50 / 107,
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Fever
• All the events coincides with Fever and other signs of clinical illness • From Gall bladder further invasion occurs in intestines • Involvement of peyr’s patches, gut lymphoid tissue • Lead to inflammatory reaction, and infiltration with monocular cells • Leads to Necrosis, Sloughing and formation of chacterstic typhoid ulcers

Rashes in Typhoid
• May present with rash, rose spots 2 -4 mm in diameter raised discrete irregular blanching pink maculae's found in front of chest • Appear in crops of upto a dozen at a time • Fade after 3 – 4 days

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Events in a Typical typhoid Fever

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Pathology and Pathogenesis
• Bacilli enter through ingestion, • Bacilli attach to Microvilli,ileal mucosa, penetrate to Lamina propria and sub mucosa • Phagocytosis by Polymorphs and Macrophages • Enters the mesenteric lymph nodes • Enter the thoracic duct – Blood stream

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Infective Dose
• For human infections, the number of bacteria that must be swallowed in order to cause infection is uncertain and varies with the serotype. In most of these the median infective dose for most serotypes, including Typhi, has varied from 106 to 109 viable organisms. However, investigation of outbreaks suggests that in natural infection the infective dose might be fewer than 1000 viable organisms.
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Pathology and Pathogenesis
• Bacteremia Spread to Liver, Gall bladder, Spleen, Bone marrow, Lymph nodes, Lungs, Multiply in kidneys

Once again spill into Blood stream Causes clinical illness.
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Pathology and Pathogenesis
• Multiply abundantly in Gall bladder, • Bile rich source of Bacteria • Spill into Intestine, infects payers patches, Lymph follicles • Inflammation – Undergo necrosis, Slough off • Typhoid ulcers • Typhoid ulcers can cause perforation and hemorrhage • Duration of Illness 3 – 4 weeks • Incubation 7 -14, ( 3-56 days )
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What is Enteric Fever Typhoid Fever
• Enteric fever is caused by strains of S. Typhi or S. Paratyphi A, B or C; although S. Paratyphi B, which gene sequence analysis suggests is a variant of S. Java, is more likely to cause nontyphoidal diarrhoea. The clinical features tend to be more severe with S. Typhi (typhoid fever). After penetration of the ileal mucosa the organisms pass via the lymphatic's to the mesenteric lymph nodes, whence after a period of multiplication they invade the bloodstream via the thoracic duct.
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Progress in Enteric Fever
• The liver, gall bladder, spleen, kidney and bone marrow become infected during this primary bacteraemic phase in the first 7-10 days of the incubation period. After multiplication in these organs, bacilli pass into the blood, causing a second and heavier bacteraemia, the onset of which approximately coincides with that of fever and other signs of clinical illness.
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Progress in Enteric Fever
• From the gall bladder, a further invasion of the intestine results. Peyer's patches and other gut lymphoid tissues become involved in an inflammatory reaction, and infiltration with mononuclear cells, followed by necrosis, sloughing and the formation of characteristic typhoid ulcers occurs.
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Immunity in Typhoid
• Typhoid bacilli are Intracellular pathogens • Cell mediated immunity is crucial
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Diarrhea Nausea Vomiting Stomach pain Headache Fever Onset 12-72 hours after infection

Clinical manifestation
• • • • • • • • Head ache, malise,anorexia ,coated tongue Abdominal discomfort, Constipation / Diarrhea Step ladder type fever, Relative bradycardia, A soft palpable spleen Hepatomegaly Rose spots appear
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Events in a Typical typhoid Fever

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Complications of Enteric fever
• • • • • • • • Intestinal perforation, Hemorrhage, Circulatory collapse. Bronchitis Bronchopneumonia, Meningitis, Cholecystitis, Arthritis,Periostitis / Nephritis, Osteomyletis,
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Relapses in Typhoid Fever
• Apparent recovery can be followed by relapse in 5-10% of untreated cases. Relapse is usually shorter and of milder character than the initial illness, but can be severe and may be fatal. Severe intestinal haemorrhage and intestinal perforation are serious complications that can occur at any stage of the illness.
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Other complications
• Causes relapses in particular to patients treated with chloramphenicol. • S.paratyphi produce septicemias.
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Typhoid carriers
• Salmonella enterica causes approximately 16 million cases of typhoid fever worldwide, killing around 500,000 per year. One in thirty of the survivors, however, become carriers. In carriers the bacteria remain hidden inside cells and the gall bladder, causing new infections as they are shed from an apparently healthy host.

Carrier Stage in Typhoid Fever
• Most people infected with salmonella continue to excrete the organism in their stools for days or weeks after complete clinical recovery, but eventual clearance of the bacteria from the body is usual. A few patients continue to excrete the salmonellae for prolonged periods. The term chronic carrier is reserved for those who excrete salmonellae for a year or more.
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Carrier Stage in Typhoid Fever
• Chronic carriage can follow symptomatic illness or may be the only manifestation of infection. It can occur with any serotype, but is a particularly important feature of enteric fever: up to 5% of convalescents from typhoid and a smaller number of those who have recovered from paratyphoid fever become chronic carriers, many for a lifetime.
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Carrier Stage in Typhoid Fever
• The bacilli are most commonly present in the gall bladder, less often in the urinary tract, and are shed in faeces and sometimes in urine. The long duration of the carrier state enables the enteric fever bacilli to survive in the community in non-epidemic times and to persist in small and relatively isolated communities.
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Epidemiology
• Developed countries - Controlled. • Water supply/ Sanitation /Economically poor. • S.typhi and S.paratyphi are prevalent in India • Previously Typhi are more common Paratyphoid A on raise. • Age 5 – 20 years, Sanitation
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Epidemiology
• Sanitation has great role • Source an active patient or a Carrier shed the Bacilli. • Who are carriers. Convalescent carrier 3 weeks to 3 months Temporary carrier 3 months to 1 year Chronic carrier > 1 year, Women attain more carrier stage
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Epidemiology (Contd)
• • • • • • Bacilli persist in the Gall bladder and kidney Food handlers spread the infection Cooks great role S.typhi and S.paratyphi in humans S.para B in Animals, Typhoid spread through Water, Milk, Food HIV patients potentially susceptible for Typhoid disease.
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Bacteriological Diagnosis of Typhoid Fever
• Selective media, such as Deoxycholatecitrate agar or xylose-lysine Deoxycholate agar, are used for the isolation of salmonella bacteria from faeces. Fluid enrichment media, such as Tetrathionate or selenite broth, are also useful to detect small numbers of salmonellae in faeces, foods or environmental samples.
Dr.T.V.Rao MD 54

Bacteriological Diagnosis of Typhoid Fever
• Suspicious colonies from the culture plates are tested directly for the presence of Salmonella somatic (O) antigens by slide agglutination and subcultured to peptone water for the determination of flagellar (H) antigen structure and further biochemical analysis.
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Bacteriological Diagnosis of Typhoid Fever
• A presumptive diagnosis of salmonellosis can often be made within 24 h of the receipt of a specimen, although confirmation may take another day, and formal identification of the serotype takes several more days. A negative report must await the result of enrichment cultures - at least 48 h.
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How we Diagnose Typhoid Fever
• Diagnosis is made by any blood, bone marrow or stool cultures and with the Widal test (demonstration of salmonella antibodies against antigens O-somatic and H-flagellar ). In epidemics and less wealthy countries, after excluding malaria, dysentery or pneumonia, a therapeutic trial time with chloramphenicol is generally undertaken while awaiting the results of Widal test and cultures of the blood and stool.
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Laboratory Diagnosis of Typhoid Fever
• • • • • • 1 Isolation of Bacilli. A Gold standard 2 Diagnosis for presence of Antibodies, Positive Blood culture – A gold standard Isolation from Feces and Urine ? Detection of Antibodies Inconclusive. Newer methods
Detection of antigen in Blood and Urine
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Blood Culture
1 st week Positive in 90 % 2 nd week Positive in 75 % 3 rd week Positive in 60 % > 3 weeks positive in 25 % Draw 5 – 10 cc of Blood by venipuncture. ADD to 50 -100 ml of Bile broth. Incubate at 37 c /Subculture in MacConkey At regular intervals
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Blood Cultures in Typhoid Fevers
• Bacteremia occurs early in the disease • Blood Cultures are positive in 1st week in 90% 2nd week in 75% 3rd week in 60% 4th week and later in 25%
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Castaneda’s method of Blood Culture
• Double medium used Solid/Liquid medium in the same Bottle. • Bottle contains Bile broth/agar slant, • For subculture the bottle is merely tilted. • A subculture into MacConkey at regular intervals, • Reduces the chances of contamination • Increases the chances of isolation.
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Salmonella on Mac Conkey's agar

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Salmonella on XLD agar

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Clot culture
• Clot cultures are more productive in yielding better results in isolation. • A blood after clotting, the clot is lysed with Streptokinase ,but expensive to perform in developing countries.
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Bactec and Radiometric based methods are in recent use
• Bactek methods in isolation of Salmonella is a rapid and sensitive method in early diagnosis of Enteric fever. • Many Microbiology Diagnostic Laboratories are upgrading to Bactek methods
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Biochemical Characters
• • • • • Non Lactose fermenter, Motile Indole – MR + VP - Citrate + Ferment Glu/Mal/Man Do not ferment Lactose/Sucrose

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Slide agglutination tests
• In slide agglutination tests a known serum and unknown culture isolate is mixed, clumping occurs within few minutes • Commercial sera are available for detection of A, B,C1,C2,D, and E.
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Culturing other Specimens
• Feces Enrichment in Tetrathionate broth and Selenite broth • Culturing in MacConkey/DCA/Wilson Blair medium – Large black colonies. • Urine Culture – positive in 25 % • Other samples Bone Marrow,Bile,CSF/Sputum
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Serology
• • • • WIDAL Test – Tube agglutination test. Detects O and H antibodies Diagnosis of Typhoid and Paratyphoid Testing for H agglutinins in Dryers tubes, a narrow tube floccules at the bottom • Testing for O agglutinins in Felix tubes, Chalky • Incubated at 37º c overnight
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Widal Test
• In 1896 Widal A professor of pathology and internal medicine at the University of Paris (1911–29), he developed a procedure for diagnosing typhoid fever based on the fact that antibodies in the blood of an infected individual cause the bacteria to bind together into clumps (the Widal reaction).
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Widal test
• S.typhi O and H tubes • Paratyphi A/B H agglutinins only • Common antigens O in all Factor sharing 12 • Significance • I st week negative. • Titers raise in 2nd week Raise of titers diagnostic
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Diagnosis of Enteric Fever Widal test
• Serum agglutinins raise abruptly during the 2nd or 3rd week • The Widal test detects antibodies against O and H antigens • Two serum specimens obtained at intervals of 7 – 10 days to read the raise of antibodies. • Serial dilutions on unknown sera are tested against the antigens for respective Salmonella • False positives and False negative limits the utility of the test • The interpretative criteria when single serum specimens are tested vary • Cross reactions limits the specificity

Widal Test
• Single test not diagnostic. • Paired samples tests • Diagnostic. O > 1 in 80 H > 1in 160 H agglutinins appear first False positives in Unapparent infection, Immunization Previously infected
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Widal test
• Anamnestic response previous infection and responding to unrelated infection • Other Diagnostic tests CIE and ELISA Detection of Circulating antigens Co agglutination test.
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Limitation of Widal Test
• The Widal test is time consuming and often times when diagnosis is reached it is too late to start an antibiotic regimen. • In spite of several limitation many Physicians depend on Widal Test
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False Positive and Negative Reactions with WIDAL Test • The Widal test should be interpreted in the light of baseline titers in a healthy local population. This is especially important when there is a high local prevalence of non-typhoid salmonellosis. The Widal test may be falsely positive in patients who have had previous vaccination or infection with S typhi.
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False Positive and Negative Reactions with WIDAL Test
• Widal titers have also been reported in association with the dysgammaglobulinaemia of chronic active hepatitis and other autoimmune diseases.64 '8 '9 False negative results may be associated with early treatment, with "hidden organisms" in bone and joints, and with relapses of typhoid fever. Occasionally the infecting strains are poorly immunogenic.
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Diagnosis of Carriers and Environments
• Fecal carriers by isolation from specimens. or Bile aspirated. • Sewer swabs • Bacteriophage typing
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Prophylaxis
• TAB vaccine S.typhi 1,000 millions S Paratyphi A,B 750 millions. Injected subcutaneously 0.5 ml at 4 – 6 weeks. Live Oral Vaccine Typhoral Mutant S.typhi strain Ty 2 1a Lacking enzyme UDP galctose 4 epimerase 10 to9 Viable bacilli Given orally 1 – 3 – 5 days
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Key points
• Antibiotics have no place in the management of salmonella gastroenteritis unless invasive complications are suspected. • Clean water, sanitation and hygienic handling of foodstuffs are the keys to prevention.
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Prevention
• Vi Polysaccharide vaccine – Administered subcutaneously or intramuscular – Confers protection seven days after injection – Approximately 50% efficacy after three years • Ty 21 vaccine – Live attenuated strain of S. typhi – Administered orally in capsule form – Also available in liquid form which can be taken by children as young as two years of age

Vaccines
• An Inject able vaccine Typhium Vi • Contains purified Vi polysaccharide antigen from S.typhi strain Ty2 • A single dose, subcutaneous route • Given to children > 5 years • Immunity lasts for 2- 3 years. • Follow a booster
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Treatment
• Chloramphenicol 1948 /1970 resistance. • Other Important drugs Ampicillin Amoxicillin, Furazolidine Cotromoxazole Chloramphenical resistance /Mexico Kerala
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Antimicrobial Therapy in Typhoid
• With prompt antibiotic therapy, more than 99% of the people with typhoid fever are cured, although convalescence may last several months. The antibiotic chloramphenicol Some Trade Names CHLOROMYCETIN is used worldwide, but increasing resistance to it has prompted the use of other antibiotics BACTRIM SEPTRAN or ciprofloxacin

Other Drugs
• Fluroquinolones Ciprofloxacillin, Pefloxacillin Ofloxacillin Ceftazidime Ceftriaxone / Cefotoxaime
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Coalition against Typhoid
• Since May 2011, the Coalition against Typhoid (CaT) has featured monthly articles in the WHO’s Global Immunization Newsletters (GIN). The articles, written by CaT members from around the world, highlight important work being done to accelerate adoption of typhoid vaccines.

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Food Poisoning
• The laboratory diagnosis of bacterial food poisoning depends on isolation of the causal organism from samples of faeces or suspected foodstuffs. The more common food-poisoning serotypes, such as Enteritidis or Typhimuruim, may be characterized more fully by phage typing and antibiotic resistance typing (see above). Strains can be differentiated further by plasmid and pulsed-field gel electrophoresis typing so that the isolates from patients may be matched with those from the infected food and from a suspected animal source.
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Salmonella Gastroenteritis
• • • • • • • • Zoonotic disease S.enteritidis S.typhimurium S.halder S. agana S.indiana Contaminated poultry, Meat Milk, Milk products. Enters the shells of the Intact eggs – Chicken feed, and Fecal droppings.
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Nontyphoidal Salmonella
• • • • General Incubation: 6 hrs-10 days; Duration: 2-7 days Infective Dose = usually millions to billions of cells Transmission occurs via contaminated food and water Reservoir: a) multiple animal reservoirs b) mainly from poultry and eggs (80% cases from eggs) c) fresh produce and exotic pets are also a source of contamination (> 90% of reptile stool contain salmonella bacterium); small turtles ban. General Symptoms: diarrhea with fever, abdominal cramps, nausea and sometimes vomiting
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Mechanism of Pathogenicity Gastroenteritis
• ingestion • absorbed to brush border of epithelial cells of small intestine and colon • migrate to lamina propria, Ileocecal • multiply in lymphoid follicles • Reticuloendothelial hyperplasia and hypertrophy

Nontyphoidal Salmonella: Gastroenteritis
• Incubation: 8-48 hrs ; Duration: 3-7 days for diarrhea & 72 hrs. for fever • Inoculum: large • Limited to GI tract • Symptoms include: diarrhea, nausea, abdominal cramps and fevers of 100.5-102.2ºF. Also accompanied by loose, bloody stool; Pseudo appendicitis (rare) • Stool culture will remain positive for 4-5 weeks • < 1% will become carriers
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Nontyphoidal Salmonella: Bacteremia and Endovascular Infections
• 5%

develop septicemia; 5-10% of septicemia patients develop localized infections • Endocarditis: Salmonella often infect vascular sites; preexisting heart valve disease risk factor • Arteritis: Elderly patients with a history of back/chest + prolonged fever or abdominal pain proceeding gastroenteritis are particularly at risk. - Both are rare, but can cause complications that may lead to death
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Salmonella Gastroenteritis
• • • • Can occur as cross infection 24 hours Manifest with Diarrhea, omitting Abdominal pain mucous and blood in stools • Last for 2 – 4 days • Some times may lead to septicemias
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Diagnosis and Treatment
• Isolation by culturing • Rarely need antibiotics. • More frequent in Developed nations.
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Salmonella septicemias
• S.cholera suis • Deep abscess, Endocarditis • Isolation from Blood and Pus. • Chloramphenicol highly effective
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 Don’t eat raw or undercooked food  Cross-contamination of foods should be avoided  Do not prepare food or pour water if you are infected with the bacteria

Wash hands, kitchen surfaces, and utensils with soap and water after they have come in contact with raw meat or poultry

Wash hands after contact with animal feces Avoid direct/indirect contact between reptiles and infants

Simple hand hygiene and washing can reduce several cases of Typhoid

• The Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing World

• Email • doctortvrao@gmail.com

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