Enterobacteriaceae Notes

Family Characters (General Properties)

Members of the family Enterobacteriaceae should have the following properties:

• They are gram-negative, aerobes and facultative anaerobic bacilli
• Nonfastidious can grow in ordinary media like nutrient agar.
• Ferment glucose, and reduce nitrate to nitrite
• Oxidase test negative and catalase positive (except Shigella dysenteriae type-1)
• They are generally motile, except Shigella and Klebsiella.
• Natural habitat: Most of them are commensals in human intestine, called coliform bacilli

Read And Learn More: Micro Biology And Immunology Notes

Escherichia Coli

• coli is the most important species encountered as human pathogen.
• It is also the commonest aerobe to be harbored in the gut of humans and animals.
• After excreted in feces, it remains viable only for some days in the environment.
• Hence detection of fecal E. coli (thermotolerant E. coli that survives at 44°C) is taken as an indicator of recent contamination of drinking water with human or animal feces.

Clinical Manifestations of E. coli Infection

• Urinary tract infection (UTI): Caused by uropathogenic E. coli (UPEC) (described later)
• Diarrhea: Caused by six types diarrheagenic E. coli (described later)
• Other syndromes: Abdominal infections: E. coli is the most common cause of both primary bacterial peritonitis (occurs spontaneously) and secondary bacterial peritonitis (occurs secondary to intestinal perforation.
• It also causes visceral abscesses, such as hepatic abscess.
• Pneumonia (especially in hospitalized patients: ventilator associated pneumonia)
• Meningitis (especially neonatal meningitis)
• Wound and soft tissue infection.
• Osteomyelitis
• Endovascular infection and bacteremia.

Urinary Tract Infection (UTI)

• E. coli (uropathogenic E. coli or UPEC) is the single most common pathogen, accounting for 85–95% of all cases of UTI.
• UPEC serotypes O1, O2, O4, O6, O7 and O75 are responsible for most UTIs.

Route of Spread

• Ascending route: After colonizing the periurethral area, E. coli ascends the urinary tract to reach bladder and later to kidney.
• Descending route: Hematogenous seeding of E. coli into kidneys result in pyelonephritis.

Types of UTI

• Depending on the site involved, there are two types of UTI: Lower and upper UTI

Predisposing Factors that Promote UTI

• Females: Due to short urethra and close proximity to anus.
• Pregnancy: Physiological obstruction in urinary tract due to growing fetus may lead to prolonged stasis of urine and asymtpomatic bacteriuria.
• Others: Presence of urinary catheters, urinary stones or prostate enlargement.

Laboratory Diagnosis of UPEC

Specimen collection:

• Clean voided midstream urine: It is the most common specimen for UTI.
• Suprapubic aspiration is the most ideal specimen (for coma or infants).
• In catheterized patients: Collected from the catheter tube and not from the bag.
• Transport: Stored in refrigerator or stored by adding boric acid
• Direct examination: Screening tests done are as follows:
• Wet mount examination is done to demonstrate the pus cells in urine.
• Pyuria of > 8 pus cells/mm3 or 4 lakh pus cells excreted in urine/hour is taken as significant.
• Others: Leukocyte esterase test, Nitrate reduction test (Griess test), Catalase test Gram staining of urine is not a reliable indicator as low bacterial count in urine, pus cells rapidly deteriorate and may not be seen well.
• It may be limited to pyelonephritis and invasive UTI cases and a count of ≥ 1 bacteria/oil immersion filed is taken as significant.

Culture:
Culture Media: Urine is inoculated onto:

• MacConkey agar and blood agar combination or CLED (cysteine lactose electrolyte deficient) agar.

Kass concept of significant bacteriuria:

• A count of ≥ 105 colony forming units (CFU)/mL of urine is considered assignificant – indicates infection (referred as ‘significant bacteriuria’ developed by Kass)
• Low count of ≤ 104 CFU/mL is due to commensal bacteria (due to contamination during voiding) and is of no significance. However, low counts may be significant in following conditions:
• Patient on antibiotic treatment or on diuretic drugs
• Infection with some gram-positive bacteria like S .aureus, and Candida.
• Pyelonephritis and acute urethral syndrome
• Sample taken by suprapubic aspiration
• Quantitative culture is done to count the number of colonies. This is done by:
• Semi quantitative method, such as standardized loop technique Quantitative method, such as pour plate method.
• Antibody coated bacteria test is used to differentiate upper and lower UTI.

Diarrhea (Diarrheagenic E. coli)

1. Enteropathogenic E. coli (EPEC)

• EPEC frequently cause infantile diarrhea (outbreaks) and rarely sporadic diarrhea in adults.
• It is nontoxigenic and noninvasive

Mechanism of diarrhea:

• Adhesion to intestinal mucosa, mediated by plasmid coded bundle-forming pili
• A/E lesions (attaching and effacing lesions) on the intestinal epithelium.

2. Enterotoxigenic E. coli (ETEC)

• ETEC is the most common cause of traveler’s diarrhea causing 25–75% of cases:
• It causes acute watery diarrhea in infants and adults.
• Common serotypes associated are: O6, O8, O15, O25, O27, O153, O159, etc.
• It is toxigenic but not invasive

Pathogenesis:

• Attachment to intestinal mucosa is mediated by CFA (Colonization Factor Ag)
• Toxins: (i) heat labile toxin or LT (↑ cAMP), (ii) heat stable toxin or ST (↑ cGMP)
• Diagnosis is done by detection of toxins by in vitro and in vivo methods.

3. Enteroinvasive E. coli (EIEC)

• Common serotypes associated with EIEC are O28, O112, O114, O124, O136, O152, etc.

Pathogenesis: EIEC is not toxigenic but invasive. The epithelial cell invasion is mediated by a plasmid coded antigen called virulence marker antigen (VMA).

• EIEC is biochemically, genetically and pathogenically closely related to Shigella.
• Manifestations include ulceration of bowel, dysentery (resembling shigellosis).

Diagnosis:

• Detection of VMA by ELISA
• HeLa cell invasion assay
• Sereny test (inoculation into guinea pig eyes produces conjunctivitis)
• EIEC are biochemically atypical being nonmotile and lactose nonfermenters.

4. Enterohemorrhagic E. coli (EHEC)

• Serotypes associated with EHEC are:
  • O157: H7 (most common serotype)
  • Other serotypes are rare (O26: H11, O6, O55, O91, O103, O111 and O113).
• EHEC is usually transmitted by contaminated food, i.e. lettuce, spinach, sprouts and undercooked ground beef.
• Prevalent in industrialized countries (other Diarrheagenic E. coli are common in developing regions)
• Low infective dose (< 102 CFU) of EHEC can also initiate the infection.
• Pathogenesis: EHEC secretes a toxin called verocytotoxin or Shiga-like toxin (refer table for its mechanism)
• Manifestations: VT has predilection for endothelial cells causing capillary microangiopathy which leads to:

• • HC (hemorrhagic colitis): Manifests as gross bloody diarrhea, abdominal pain and fecal leukocytosis but no fever.
  • Hemorrhagic uremic syndrome (HUS): Injury to small vessels of the kidney and brain can lead to bloody diarrhea, thrombocytopenia, renal failure and encephalopathy but
    without fever. It is more common in children.

Diagnosis:

• Sorbitol MacConkey agar: EHEC in contrast to other E. coli, does not ferment sorbitol Toxin detection:
• Cytotoxicity in Vero cell lines (gold standard method)
• Fecal toxin antigen detection by ELISA or rapid tests
• PCR can be used to detect gene coding for VT.
• 5. Enteroaggregative E. coli (EAEC)
• It is so named because it adheres to HEp-2 cells in a stacked-brick fashion
• Most strains are ‘O’ untypeable but ‘H’ typeable

Pathogenesis:

• Colonization mediated by aggregative adhesion fimbriae I (regulated by aggR gene)
• Produce EAST 1 toxin (enteroaggregative heat stable enterotoxin 1)
• Manifestations: Persistent and acute diarrhea are common; in developing countries
• E. coli O104: H4 is an enteroaggregative strain that has caused major outbreaks in Germany
• in 2011. Peculiarity is, it produces Shiga toxin and can cause HUS.
• 6. Diffusely Adherent E. coli (DAEC)
• It is characterized by Ability to adhere to HEp-2 cells in a diffuse pattern
• Expresses diffuse adherence fimbriae which contribute to the pathogenesis.

Klebsiella

• Nonmotile, lactose fermenter
• Capsulated, produce mucoid colonies
• K. pneumoniae: Urease positive. It causes pneumonia, UTI, abdominal, wound and surgical site infection
• K. ozaenae: It causes ozaenae (foul-smelling nasal discharge)/atrophic rhinitis
• K. rhinoscleromatis: It causes rhinoscleroma.

Proteus

• Motile, Nonlactose fermenter
• Tribe character: PPA test +ve (phenylalanine deaminase test +ve)
• Both P. mirabilis and P. vulgaris are H2S and Urease positive
• Indole test: Positive for P. vulgaris, negative for P. mirabilis
• Pleomorphism: Gram-negative, appear in various forms—coccobacilli, bacillary and in filamentous forms.
• Odor: Produce characteristic putrid ‘fishy’ or ‘seminal’ odor in cultures.
• Swarming-Proteus has an ability to swarm (or spread) on solid media.
• Organism which swarm: Proteus, Clostridium tetani, V. parahemolyticus, and Serratia.
• Diene’s phenomenon: It is done to know the relatedness between different strains.
• Proteus forms struvite stone in bladder in alkaline urine.
• Nonmotile strains of Proteus (Basis of Weil felix test): OX 19, OX 2 (P. vulgaris), OX K (P.mirabalis) forms the basis of Weil Felix test.

Shigella

• Shigella species are the agent of bacillary dysentery.
• Four species has been recognized: S. dysenteriae, S. flexneri, S. boydii, S. sonnei.
• Most hardier: S. sonnei
• Most common species: In world: S. sonnei, In India:S. flexneri

Pathogenicity

Transmission:

• ingestion through contaminated fingers (MC), food, and water or flies and
• rarely by homosexuals

Infective dose:

• Shigella has a low Infective dose (10 to 100 bacilli). Others with low infective dose
• include EHEC, Entamoeba histolytica and Giardia.
• Bacilli enter the mucosa via M cells.
• Invasion: Mediated by a large virulence plasmid
• Direct cell to cell spread: This occurs by inducing actin polymerization of host cells.

Exotoxins:

• Shigella enterotoxin (ShET 1 and 2)- found essentially in S. flexneri
• Shiga toxin is a cytotoxin, produced by S. dysenteriae type 1.
• Endotoxin induces intestinal inflammation and ulcerations.

Complications:

• Intestinal complications, such as toxic megacolon, perforations and rectal prolapse.
• Metabolic complications, such as hypoglycemia, hyponatremia, and dehydration.
• Ekiri syndrome or toxic encephalopathy is a metabolic complication of shigellosis.
• Postinfectious phase: Patients expressing HLA-B27 develop autoimmune reactions, such as reactive arthritis, ocular inflammation, and urethritis months after S. flexneri infection (3% of cases).
• HUS and HC is produced by S. dysenteriae Type 1 (due to producing Shiga toxin, hence called Shiga bacillus)
• Rarely, it causes bacteremia, meningitis, pneumonia, vaginitis and keratoconjunctivitis.

Laboratory Diagnosis

• Specimen: mucus flakes of stool
• Culture Media: (Common media for both Shigella and Salmonella):
• Transport media: Sach’s buffered glycerol saline
• Selective media: DCA (deoxycholate citrate agar), XLD, SS Agar
• Enrichment Broth: Gram-Negative broth, selenite F broth, tetrathionate broth.
• Important biochemical properties: Nonmotile, Nonlactose fermenter except: S. sonnei (Late lactose fermenter)
• Catalase +ve except: S. dysenteriae type 1 Mannitol fermenting except: S. dysenteriae.

Treatment

• Because of the prompt transmissibility, every case of shigellosis should be treated with antibiotics, in addition to ORS.
• Ciprofloxacin is the drug of choice.
• Alternative drugs: Ceftriaxone, azithromycin, pivmecillinam, and some fifth-generation quinolones.
• Duration of treatment is about 3 days except for:
• S. dysenteriae type 1 infection 5 days Infections in immunocompromised patients 7–10 days.

Serratia
S. marcescens produces characteristic red nondiffusible pigment called prodigiosin.

• It is a saprophyte found in water, soil and food.
• It may grow in sputum after collection and makes the sputum red (due to pigment production).
• This condition is known as ‘pseudohemoptysis’.
• It is increasingly reported in various nosocomial infections. The hospital strains are often nonpigmented and multidrug resistant (produce AmpC β-lactamases).

Yersinia Pestis 
Yersinia pestis is the agent of plague, a zoonosis, transmitted from rodents by rat flea.

Epidemiology of Plague

• Plague is one of the greatest killer known to mankind. Africa accounts for maximum cases (97%).
• Plague Pandemics: There were three pandemics reported, each with a different biotype of Y.pestis.
• First pandemic (in AD541) associated with biotype Medievalis.
• Second pandemic (in 14th century) was called black death: with biotype Antiqua
• Third pandemic (1884): It mainly affected India and China: with biotype Orientalis.

Recent outbreaks of plague in India 1994 (Surat epidemic), 2002 (Shimla outbreak) and 2004 (bubonic plague in Uttaranchal) Four potential endemic foci are there in India at present which include:

• Region near Kolar,
• Beed-Latur belt in Maharashtra
• Rohru in Himachal Pradesh
• Dangud village,
• Uttaranchal.
• Epidemiological Factors
• Reservoir: Wild rodents, such as gerbils (Tatera indica), field mice, and forest bandicoot
• Source of infection are infected wild rodents, rat fleas and cases of pneumonic plague.
• Vector: Rat flea is the commonest vector of plague.
• Xenopsylla cheopis—the most efficient vector (found in north India)
• Xenopsylla astia (less efficient, found in south India).
• Human flea (Pulex irritans) may rarely serve as vector.
• Plague cycles: Plague exists in two natural cyclesDomestic cycle: Occurs between humans, rat fleas and rodents.
• Wild or sylvatic cycle occurs in nature between wild rodents independent of human.
• Mode of transmission: Human plague is frequently contracted from:

Blocked flea:

• The bacilli multiply and block the proventriculus of flea.
• The flea regurgitates the blood mixed bacteria into the bite, thus transmitting the infection.
• A partially blocked flea is more dangerous than a completely blocked flea as it survives longer inside burrows, may be up to 4 years in certain species.
• Extrinsic incubation period is the interval between the flea acquiring infection through blood meal and becoming a blocked flea; which is usually about two weeks.
• Cheopis index (No. of X. cheopis per rat) of > 1- Indicates Plague outbreak is likely to occur
• Seasonality: Plague is seasonal in north India (September to May) and throughout the year in South India.

Clinical Types of Human Plague

• Bubonic plague: It is the most common type, transmitted by the bite of an infected rat flea.
• Incubation period is about 2–7 days.
• Buboes: Enlarged regional lymph nodes are called buboes (MC site inguinal LN)
• It cannot spread from person to person as the bacilli are locked up in buboes.
• Pneumonic plague: Results from inhalation of bacilli in droplets expelled from patients/animals with pneumonic plague.
• Incubation period is short, about 1–3 days
• Clinical feature: Respiratory symptoms (cough or hemoptysis, dyspnea, and chest pain)
• Though rare (< 1%), it is highly infectious and highly fatal.
• Agent of bioterrorism-Aerosolized Y. pestis is a possible source of bioterrorism attack.
• Septicemic plague: Occurs secondary to spread of bubonic or pneumonic plague. Incubation period is about 2–7 days.
• Massive involvement of blood vessels results in hemorrhages in the skin and mucosa, hence the name black death.

Laboratory Diagnosis

• Specimen: Pus aspirated from bubo, Sputum and blood.

Direct Microscopy:

• Gram staining: Reveals pus cells and gram-negative oval coccobacilli with rounded ends surrounded by capsule.
• Wayson stain (or methylene blue staining): Demonstrates bipolar or safety pin appearance.

Culture:

• Y. pestis grows best at 27°C but the capsule develops best at 37°C.
• Blood agar: Colonies are nonhemolytic and dark brown pigmented MacConkey agar: NLF colonies
• Stalactite growth seen on nutrient broth with oil or ghee floated on top.
• F1 Antigen detection by direct immunofluorescence test, ELISA Antibodies to F-1 antigen may be detected by CFT, ELISA
• PCR can be done targeting gene coding F1 antigen, pesticin gene.

Treatment

• Streptomycin was given in the past, now Gentamicin is recommended for treatment.
• β-lactams and macrolides are generally not recommended as the response is poor.
• Chemoprophylaxis should be given to all contacts of pneumonic plague. Levofloxacin followed by doxycycline is DOC.

Vaccine

• WHO recommends using vaccine only for prevention of outbreak and not for general use.
• Formalin killed vaccine (Sokhey’s modification of original Haffkine vaccine):
• It is prepared in Haffkine institute, Mumbai.
• It is given subcutaneously, two doses 4 weeks apart and a booster after 6 months.
• Protection is short lasting (< 6 months).
• It is not protective against pneumonic plague and has considerable side effects, C/I in < 6 month of age.
• Live attenuated vaccine: Based on strain EV76, used in Soviet Union; causes side effects.

Yersiniosis

• Yersiniosis refers to Yesinia infection other than Y. pestis i.e.Y. pseudotuberculosis and Y. enterocolitica.

Clinical Manifestations

• Overall, Y. enterocolitica is more frequently reported clinically than Y. pseudotuberculosis.
• Self limited gastroenteritis (diarrhea with or without blood) occurs in younger children.
• Intestinal complications occur in older children, characterized by:
• Terminal ileitis (mostly in Y. enterocolitica)
• Mesenteric adenitis (mostly in Y. pseudotuberculosis)
• Pseudoappendicitis (by both species, appendectomy is not indicated)
• Postinfective phenomena (in adults): Occurs commonly with Y. enterocolitica as a result of autoimmune activity. Manifestations include:
• Reactive arthritis (people with in HLA-B 27)
• Erythema nodosum and Graves’ disease (independently of HLA-B 27)
• Super antigen: Some strains of Y. pseudotuberculosis express a super antigen mitogen which has caused scarlet-like fever in Russia and Japan (called Izumi-fever) and also linked to the
• pathogenesis of Kawasaki’s disease.

Laboratory Diagnosis

• Y. enterocolitica and Y. pseudotuberculosis can be differentiated from Y. pestis:
• Differential motility: They are motile at 22°C (but not at 37°C)
• Cold enrichment: Growth improves on refrigeration (4°C)
• Urease positive

Tests to differentiate Y. enterocolitica from Y. pseudotuberculosis:

•  Sugar fermentation
• Ornithine decarboxylase and VP test positive only for Y. enterocolitica.

Salmonella

Classification and Nomenclature

Salmonella is antigenically complex. There are several classifications proposed so far.

1. Clinical classification of Salmonella into Typhoidal and Nontyphoidal groups:

• Typhoidal Salmonella, e.g. S. typhi and S. paratyphi—They are restricted to human hosts, cause enteric fever
• Nontyphoidal Salmonella or NTSI: The remaining serotypes can colonize the intestine of a broad range of animals. They cause food-borne gastroenteritis and septicemia in man.

2. Antigenic classification (Kauffmann-White scheme): It is based on the somatic (O) and flagellar (H) antigens which can be detected by agglutination with antisera.

Serogroups: Based on O antigen, salmonelleae are classified into 67 serogroups:

• Serogroup 2 (S. paratyphi A): Contains O antigen type 2
• Serogroup 4 (S. paratyphi B): Contains O antigen type 4
• Serogroup 9 (S. typhi): Contains O antigen type 9
• Serotypes: Each serogroup is further typed into > 2500 serotypes based on flagellar Ag.

3. Molecular classification- According to molecular classification:

• Genus Salmonella consists of two species: Salmonella enterica and S. bongori.
• Most of the pathogenic serotypes are placed under subspecies enterica.
• Nomenclature of S. typhi is Salmonella species enterica, subspecies enterica, serotype Typhi.

Antigenic Structure

• Salmonellae possess three important antigens:
• Somatic antigen (O) and Flagellar antigen (H): Present in all serotypes/species

• Surface envelope antigen (Vi): Found in some serotypes/species

Vi Antigen

• Vi antigen is a surface polysaccharide envelope or capsular antigen covering the O antigen. The naming is due to the belief that Vi antigen is related to virulence.
• When Vi antigen is present, it renders the bacilli inagglutinable with the O antiserum.
• As Vi antigen is poorly immunogenic and antibody titers are low, not helpful in the diagnosis of cases. Hence the Vi antigen is not employed in the Widal test.
• However, the complete absence of the Vi antibody in a proven case of typhoid fever indicates poor prognosis.
• Vi antibody usually disappears early in convalescence, but if persists, indicates the development of the carrier state.
• Phage typing of S. typhi can be done by using Vi specific bacteriophages.
• Vi antigens can also be used for vaccination.

Pathogenesis

• Mode of transmission by oral route, MC through contaminated food or water.
• Infective dose of Salmonella: Minimum 103–106 bacilli are needed to initiate the infection.
• Risk factors that promote transmission include the conditions that decrease:
• Stomach acidity (an age of < 1 year age, antacid ingestion, or achlorhydria Intestinal integrity (inflammatory bowel disease, prior GIT surgery or on antibiotics)
• Entry: Salmonella enter through epithelial cells (M cells) lining the intestinal mucosa.
• The an uptake is called bacteria-mediated endocytosis, favored by bacterial type III secretion system
• Following entry, the bacilli remain inside vacuoles in the cytoplasm.
• Entry into macrophages: Salmonellae containing vacuoles cross the epithelial layer to reach submucosa, where they are carried by macrophage to bloodstream.
• Primary bacteremia occurs and then Spread organs, such as liver, spleen, lymph nodes and bone marrow
• Massive secondary bacteremia occurs from the seeded organs, which leads to onset of clinical disease.

Clinical Manifestations (Enteric Fever)

• Incubation period is about 10–14 days. Enteric fever is a misnomer as the manifestations are more extraintestinal than intestinal. Various manifestations include:
• Fever (step ladder pattern type of prolonged continuous fever)
• Other symptoms: Headache, chills, cough, sweating, myalgia and arthralgia Rashes (called rose spots) – seen in 30% of patients at the end of the first week.
• Early intestinal manifestations such as abdominal pain, nausea, vomiting and anorexia.
• Late intestinal manifestations: GI bleeding and intestinal perforation (3-4 weeks).
• Important signs include hepatosplenomegaly, epistaxis and relative bradycardia.
• Neurologic manifestations occur rarely which include meningitis, cerebellar ataxia and neuropsychiatric symptoms (described as ‘muttering delirium’ or ‘coma vigil’)
• such as paranoid psychosis and delirium.

Epidemiology

• Host: Humans are the only natural hosts for typhoidal Salmonellae.
• Mode of transmission: It is by ingestion of contaminated water and food.
• Rarely homosexual and laboratory acquired transmissions have been reported.
• Prevalence: Worldwide, an estimated 27 million cases of enteric fever with 2–6 lakh deaths occur annually Incidence is:
• Highest (>100 cases per 100,000 population per year) in south central and southeast Asia
• Medium (10–100 cases per 100,000) in the rest of Asia, Africa, Latin America.
• Low (<10 cases per 100,000) in other parts of the world.
• Locality and age distribution seen in enteric fever is:
• More common in urban than rural areas
• More common among young children and adolescents than in adults.
• Most important risk factor: Poor sanitation and lack of access to clean drinking water.
• S. typhi to S. paratyphi A ratio is 4:1. However, S. paratyphi A appears to be increasing, especially in India; may be due to increased vaccination for S. typhi.
• Carriage: Up to 10% of untreated patients become carriers.

Carriers of are of two types:

• Fecal carriers (more common): Multiply in the gallbladder and are excreted in feces.
• Urinary carriers: Multiplication takes place in kidneys and bacilli are excreted in urine.
• Duration of shedding: Carriers continue to shed bacilli in feces and urine for:
• Convalescent carriers: Three weeks to three months (after clinical cure)
• Temporary carriers: Three months to one year
• Chronic carriers: for more than 1 year

Laboratory Diagnosis of Enteric Fever

Widal Test

• Widal test is the investigation of choice second week and third week.
• As antibodies appear only after the end of the first week, it is not preferred in first week of illness.
• Principle: It is an agglutination test where H and O antibodies are detected in the patient’s sera by using H and O Ag.
• Antigens used: Four antigens are used:
• O antigens of S. typhi (TO), H antigens of S. typhi (TH), S. paratyphi A (AH) and S. paratyphi B (BH)
• (Paratyphoid O Ag are not used as they cross-react with the S. typhi O Ag due to sharing of factor 12)
• Strains used for Ag preparation: S. typhi 901 ‘O’ and ‘H’ strains and lab strains for S. paratyphi A, and B.

Results:

• O agglutination appears as compact granular chalky clumps (disc-like pattern), with clear supernatant fluid.
• H agglutination appears as large loose fluffy cotton-woolly clumps, with clear supernatant fluid.
• If agglutination does not occur, button formation occurs due to deposition of antigens and the supernatant fluid remains hazy.
• Antibody Titer: The highest dilution of sera, at which agglutination occurs.

Interpretation:

• Significant titer in most of the places in India is taken as: H agglutinin titer > 200 and O agglutinin titer > 100
• Low titers should be ignored and considered as baseline titers in endemic areas (due to prior exposure, people will always have some base line antibodies)
• O Ab appear early and disappear early and indicate recent infection. H Ab appear late and disappear late.
• O Ab are nonspecific. They are raised in all, i.e. S. typhi, S. paratyphi A and B H Ab are specific.
• TH, AH and BH antibodies are raised in S. typhi, S. paratyphi A and B infections respectively.
• False positive Widal test may occur due to:
• Anamnestic response: It refers to a transient rise of titer due to unrelated infections (malaria, dengue) in persons who have had prior infection or immunization.
• If bacterial antigen suspensions are not free from fimbriae
• Persons with inapparent infection or prior immunization (with TAB vaccine)

A four-fold rise in antibody titer in paired sera at 1 week interval is more meaningful than a single high titer:

• After 1 week if titer rises-indicates true infection
• After 1 week if titer falls- indicates anamnestic responses

False negative Widal test may occur in:

• Early stage (1st week of illness), Late stage (after fourth week) and in carriers Patients on antibiotics
• Due to prozone phenomena (antibody excess): This can be obviated by serial dilution of sera.

Other Antibody Detection Tests

• Typhidot test: 50 kDa OMP (outer membrane protein) antigen is used; it uses a dot ELISA format to detect both IgM and IgG separately after 2-3 days of infection
• IDLTubex test: O9 antigen is used, detects only IgM antibodies against S. typhi by a semiquantitative colorimetric method
• IgM dip stick test and ELISA detect anti-LPS IgM antibodies
• Dot blot assay: Flagellar antigen is used, detects only IgG antibodies.
• Demonstration of Serum Antigens
• Antigens of typhoidal Salmonellae are consistently present in the blood (early course of the disease), and in urine (late phase). Antigen detection: by-ELISA and Coagglutination test.

Molecular Methods

• PCR based methods (e.g. nested PCR): To detect and differentiate typhoidal salmonellae by targeting various genes, such as flagellin gene, Iro B and fliC gene.

Other Nonspecific Methods

• WBC count: Neutropenia is seen in 15–25% of cases. Leukocytosis is more common among children, during early phase.
• Liver function tests moderately deranged Muscle enzyme levels moderately elevated.

Detection of Carriers

• Culture: By stool and bile culture (detects fecal carriers) and urine culture (detects urinary carriers)
• Detection of Vi antibodies: It is done by tube agglutination test by using S. typhi suspension
• carrying Vi antigen (Bhatnagar strains). Even a titer of 1:10 is also considered as significant.
• Isolation of Salmonellae from sewage: It is carried out to trace the carriers in the communities.
• It can be done by:
• Sewer–swab technique
• Filtration using millipore membranes.

Treatment of Enteric Fever

Vaccines for Typhoid Fever

Parenteral TAB Vaccine

• It is heat-killed whole cell typhoid/paratyphoid A and B; no longer in use due to its side effects.

Parenteral Vi Polysaccharide Vaccine

• It is composed of Vi capsular polysaccharide antigen derived from S. typhi strain Ty2.
• Dosage: Single dose containing 25 μg of Vi antigen is given IM or subcutaneously.
• Vaccine confers protection for 2 years.
• Vi antigen elicits T independent IgG antibody response, booster given @ 2 years.
• Age: It is given only after 2 years of age.

Typhoral (Oral Live Attenuated S. Typhi Ty21a Vaccine)

• Typhoral is a stable live attenuated mutant of S.Typhi strain Ty21a, which lacks the enzyme
• UDP-galactose-4-epimerase (Gal E mutant).
• On ingestion, it multiplies for sometime, initiates the immune response but self destructs (dies
• of its own after 4–5 cell divisions, due to lack of enzyme) and therefore cannot induce any pathogenesis.
• It is indicated only after 6 years of age.
• Lyophilized form of the vaccine is available as enteric coated capsules.
• It is given orally before food, on alternate days- 1, 3, 5 and/or 7 (total of three or four doses).
• No antibiotics should be given during this period.
• Protective immunity starts after 7 days of the last dose and lasts for 4 years.
• Boosters are recommended every 3 years in endemic areas and every year for travelers.