Applied Microbiology cases

Salmonella enterica ssp typhi: Enteric (typhoid) fever
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A 22-year old man complains of continuous fever with chills since six days, headache, anorexia, malaise, abdominal discomfort and mild diarrhea. On examination coated tongue and slightly enlarged liver were observed.

What is your diagnosis?
It could be a case of enteric fever. Differential diagnosis includes febrile conditions such as abdominal abscess, malaria, amebic hepatic abscesses, appendicitis, brucellosis, tuberculosis, dengue fever, and typhus.

What is the specimen collected and how is the condition diagnosed with the aid of laboratory?
If enteric fever is suspected, diagnosis can be made by blood and feces culture. Freshly passed feces is collected in a sterile wide mouthed container. The enrichment medium used is tetrathionate broth or selenite F broth. Direct gram stained smear or hanging drop preparation is not useful. Feces is inoculated on to MacConkey's agar,  low selective media such as XLD Agar or DCA or SS AGar and highly selective medium such as Wilson & Blair's Agar. The plates are incubated at 37oC overnight. Subculture is made from enrichment broth on to MacConkey's agar for subsequent overnight incubation. For blood culture, 5 ml of venous blood is drawn and inoculated into blood culture broth such as bile broth or brain heart infusion broth and incubated at 37oC. Subcultures are made from blood culture broth to MacConkey's agar and discarded if it fails to yield any growth even after one week of incubation. To prevent contamination during subcultures, biphasic medium may be used (Castaneda method). Salmonella can also be isolated from urine, bone marrow, rose spots, rectal swab and duodenal aspirate. Culture of bone marrow aspirate is 90% sensitive until at least 5 days after commencement of antibiotics. Stool culture alone yields a sensitivity of less than 50%, and urine culture alone is even less sensitive. Blood, duodenal aspirate, and stool culture results are positive for S. typhi in approximately 85-90% of patients with typhoid fever within the first week of illness. S. typhi has also been isolated from the cerebrospinal fluid, peritoneal fluid, mesenteric lymph nodes, resected intestine, pharynx, tonsils, abscess, and bone. Detection of Salmonella antigen in blood by latex agglutination and coagglutination can also be performed. Detection of antibody later in the course of disease is by widal test, indirect hemagglutination, indirect fluorescent Vi antibody, ELISA for IgM & IgG antibodies to S. typhi polysaccharide, as well as monoclonal antibodies against S. typhi flagellin.

What is your observation?
Feces culture and subculture from blood culture broth on MacConkey's agar yield pale (non-lactose fermenting), smooth, low convex, circular colonies. XLD Agar shows pink coloured colonies with black center. Black colonies with metallic sheen is seen on Wilson & Blair's agar. Gram stained smear of the colony shows  gram negative bacilli, hanging drop shows motile bacilli. Results of biochemical reactions include positive catalase test, negative oxidase test, negative indole test, negative urea hydrolysis, negative citrate utilization test, positive MR test and negative VP test. TSI agar shows alkaline slant/acid butt with a steak of H2S but no gas.

What is your identification and how would you confirm it?
The isolate is identified as Salmonella typhi (Salmonella enterica ssp typhi) and is confirmed by slide agglutination using factor 9 'O' antiserum and flagellar anti-d antiserum. Presence of capsule may hinder agglutination; in such situations the suspension may be boiled for 20 minutes and retested.

What is the pathogenesis of enteric fever?
Enteric fever is transmitted by feco-oral route. Following ingestion of contaminated food or water (containing at least 104 bacteria), Salmonella gain entry into the small intestine after a variable incubation period of 1-2 weeks. Bacteria attach themselves to epithelium and subsequently penetrate lamina propria and submucosa where they are engulfed by monocytes. Bacteria resist intracellular killing and multiply in the monocytes. They reach mesenteric lymph nodes, multiply there and reach blood stream via thoracic duct resulting in primary bacteremia. During this transient bacteremia bacteria are seeded in the liver, gall bladder, spleen, lymph node, bone marrow, where they continue to multiply. Following multiplication in large numbers, the bacteria spill in to bloodstream again resulting in secondary bacteremia and marks the onset of clinical disease. When bacteria are shed from the gall bladder along with the bile juice, they reach the small intestine again and infect the peyer's patches and lymphoid follicles of ileum leading to inflammation and ultimately ulceration.

What is the course of untreated illness and its complications?
Untreated typhoid fever normally lasts for 3-4 weeks. As the disease progresses, febrile patient becomes more toxic and anorexic with significant weight loss, conjunctivae are infected, abdominal distension is severe and the patient may descend into the typhoid state, which is characterized by apathy, confusion, and even psychosis. Necrotic Peyer's patches may cause bowel perforation and peritonitis. Overwhelming toxemia, myocarditis, or intestinal hemorrhage may cause death. If the patient survives to the fourth week, the fever, mental state, and abdominal distension slowly improve over a few days. Some survivors become asymptomatic S. typhi carriers.

What is the role of carriers in transmission of disease and how are they detected?
Carriers are important source of disease as they disseminate the bacteria in the community. Approximately 2-4% of patients become carriers while some people become carriers after inapparent infection. Those who shed bacteria for a duration less than a year are called temporary carriers and those who shed for more than a year are called chronic carriers. Development of carrier stage is common in females and in elderly patients. A carrier may be fecal carrier or urinary carrier depending on the site of bacterial persistence. It is important to detect carrier state in food handlers as well as in community. Bacterial shedding is infrequent, hence repeated cultures may be required to recover Salmonella from urine, bile or feces. Carriers in a community may be traced by 'sewer-swab' technique. Demonstration of Vi antibodies has been used to detect carrier state. Pefloxacin is now considered an effective antibiotic in eradicating carrier state. If antibiotic treatment fails to eradicate the hepatobiliary carriage, the gallbladder should be resected. Cholecystectomy is not always successful in eradicating the carrier state because of persisting hepatic infection.

How is this condition treated?
Because of plasmid mediated development of resistance, antibiotics such as chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole are no longer used. Drugs of choice are either fluoroquinolones (ciprofloxacin, ofloxacin, pefloxacin) or third generation cephalosporins (cefotaxime, ceftraxone, ceftazidime). Unfortunately, resistance to both these groups are emerging. Antibiotics must be prescribed only on the basis of antibiotic susceptibility testing. Approximately 5-10% of patients treated with antibiotics experience relapse of typhoid fever after initial recovery. Relapses typically occur approximately 1 week after therapy is discontinued. After discharge, patients should be monitored for relapse or complications for 3 months after treatment has commenced.

How can this disease be prevented?
Since typhoid is spread mainly be feco-oral route and is more common in areas of poor hygiene, the disease can be controlled to some extent by general sanitary measures such as safe cooking and eating habits, proper disposal of excreta etc. For details on immunoprophylaxis, read the notes.

 

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