Laboratory Diagnosis of Enteric Fever

 The term “Enteric fever" collectively refers to typhoid and paratyphoid fever, produced by Salmonella Typhi and S. Paratyphi A, B and C.  The word ‘Typhoid’ means typhus like that comes from Greek word tûphos meaning hazy, describing state of mind those infected.  The Genus Salmonella belongs to Enterobacteriaceae.  Facultative anaerobe, Gram negative, non capsulated, non sporing bacilli motile with peritrichous flagella.  Distinguished from other bacteria by biochemical reactions and antigen structure.  Human is the only natural host.  Worldwide 28 million cases occur annually with an estimation of 2 lakh deaths.  Clinical classification- • Typhoidal • Non-typhoidal  Antigenic classification (Kauffmann-White Scheme)- • Serogroup- based on O antigen, 67 serogroups. • Serotype- based on H antigen, >2500 serotypes.  Molecular classification- • On DNA hybridization studies. • 2 species- S.enterica and S.bongori Serogroup Serotype name O Ag H Ag Phase I Phase II 2 S.Paratyphi A 1,2,12 a [1,5] 4 S.Paratyphi B 1,4,[5],12 b 1,2 S.Typhimurium 1,4,[5],12 i 1,2 S.Agona 1,4,12 f,g,s - S.Paratyphi C 6,7 c 1,5 S.Cholerasuis 6,7 c 1,5 S.Muenchen 6,8 d 1,2 S.Newport 6,8,20 e,h 1,2 S.Typhi 9,12 d - S.Enteritidis 1,9,12 g,m [1,7] S.Gallinarum 1,9,12 - - S.Dublin 1,9,12 g,p - S.Anatum 3,10,[15],[34] e,h 1,6 7 8 9 3,10 It possessi. Somatic antigen (O) ii. Flagellar antigen (H) iii. Surface envelope antigen (Vi)  Somatic antigen (O)- Flagellar antigen (H)- Part of cell wall lipopolysaccharide. Made up of protein flagellin. Heat stable, alcohol stable, formaldehyde labile Heat labile, alcohol labile, formaldehyde stable Less immunogenic. Appears and disappear early. More immunogenic. Appears and disappear late. When react with antibody forms compact, granular, chalky clumps. When react with antibody forms large, loose, fluffy clumps.  Vi antigen- • Surface polysaccharide capsular antigen. • It renders bacilli inagglutinable with O antisera. • Usually disappear early in convalescence. If persist indicates carrier state.  Fever (step ladder pattern), headache, chills, cough, sweating, myalgia, arthralgia.  Abdominal  Rose pain, nausea, vomiting, anorexia. spot- faint blanching salmon coloured maculopapular rash on trunk and chest.  Complications Neurologic GI bleed, intestinal perforation. manifestation occur rarely include meningitis and neuropsychiatric symptoms.  Depends on duration of illness Duration of illness Specimen and tests 1st week • Culture of blood, bone marrow aspirate and duodenal aspirate. 2nd & 3rd week • Serum – i. Ab detection by Widal ii. Ag detection • Stool and urine culture 4th week • Stool and urine culture Carriers • Stool and urine culture • Serum for Ab to Vi Ag • Sewage culture- indirect way  Culture and identification  Demonstration of serum antibodies  Demonstration of serum antigens  Molecular  Non methods specific methods  Blood culture-  Ideal method for diagnosis in first week.  Positive in approx. -90 % of cases in first week. -75 % of cases in second week -60 % of cases in third week -25 % of cases thereafter until fever subsides  Rapidly become negative on treatment. Culture media-  Monophasic medium- Bile broth 50-100 ml.  Castaneda’s biphasic medium- agar slope and bile broth 50-100 ml.  10-20 ml fresh blood is inoculated into culture bottle containing 50 – 100 ml of BHI broth.  LIQUOID (Sodium Polyanethol Sulphate) is added as anticoagulant. It also counteracts bactericidal action of blood.  Incubation at 37ᵒ C.  Serial subculture is done after 24 hr, 3rd day, 5th day and 7th day.  Subculture • is done on MacConkey agar- colonies are round (1-3 mm size), translucent and non lactose fermenting. • Blood agar- non hemolytic , grey- white, moist ,circular discs with smooth convex surface and entire edge.  With biphasic medium subcultures can be made just by tilting the bottles so that the broth runs over the agar slope.  If colonies appear over the agar slope, it is used for further identification.  Culture is declared negative only after 10 days of incubation.  Automated culture systems like BACTEC and Bact/Alert can be used as alternative.      Alternative to blood culture. 5 ml blood is withdrawn into a sterile test tube and allowed to clot. Clot is broken up with glass rod & added to a bottle of bile broth. Streptokinase (100 U/ml) is incorporated in broth. Advantage - 1) Higher yield than blood culture. 2) Sample of serum is obtained.  Highest yield in third week.  Valuable in patient on antibiotic (blood culture negative).  Repeated samples are to be tested (intermittent shedding).  Collect in sterile container & transport without delay.  Rectal swabs can be taken but not better.  0.033 M sodium or potassium phosphate buffer and glycerol is added if delay.  Barium enema doesn’t compromise the recovery of enteric pathogens.  Wet mount preparation- large number of pus cells, erythrocytes and macrophages.  Culture- to inhibit commensals, fecal specimen is inoculated simultaneously into enrichment broth and selective media.  Enrichment broth- • Selenite F broth • Tetrathionate broth • Gram-negative broth Selective media- • Low selective- MacConkey agar • High selective- i. DCA (Leifson’s Deoxycholate citrate agar)- NLF pale colonies with black center. ii. XLD (Taylor’s Xylose lysine deoxycholate) agar - initial yellow colonies(xylose ferm.) - later red colonies (lysine decarboxylation) with black centre. iii. HE (Hektoen enteric) agar - More inhibitory to coliforms. - Blue-green colonies with black centre. iv. SS (salmonella shigella ) agar-High selectivity of SS agar permit use of heavy inoculum. -Colorless colonies with black centre. v. Wilson Blair brilliant green Bismuth sulfite agar- Highly selective medium - S.Typhi- jet black colony with metallic sheen. - S.Paratyphi- green colour colony. URINE CULTURE –  Salmonella  Becomes are shed in urine infrequently. positive in 2 -3 week, highest yield in 4th week.  More commonly used for carrier detection.  Centrifuged and cultured on same media as for stool. OTHER  SPECIMEN - Bone marrow- 55-90% sensitive, useful when blood culture is negative especially when patient is on antibiotics.  Duodenal aspirate- if both blood and BM culture come negative .  Combination of blood, BM and intestinal secretions show >90% sensitivity.  Biopsy from rose spots, pus from suppurative lesions, CSF and autopsy specimens can also be taken.  Culture smear – gram negative (2-4 × 0.6 µm), non sporing and non capsulated bacilli.  Motility – motile with peritrichous flagella. TEST S.Typhi S.Paratyphi Catalase Positive Positive Oxidase Negative Negative Motility Positive Positive Indole Negative Negative Methyl Red Positive Positive VP Negative Negative Citrate Negative Negative Urease Negative Negative H2S Positive Negative PA Negative Negative Gelatin hydrolysis Negative Negative TEST S.Typhi S. Paratyphi 1.Glucose Positive (no gas) Positive (with gas) 2.Lactose Negative Negative 3.Sucrose Negative Negative 4.Mannitol Positive Positive 5.Sorbitol Positive Positive 6.Dulcitol Negative Positive 7.D-Xylose Positive Negative 8.L- rhamnose Negative Positive 9.L- arabinose Negative Positive 10.Melibiose Positive Positive 11.Raffinose Negative Negative Sugar fermentation tests- TEST S.Typhi S.Paratyphi Lysine Decarboxylase Positive Negative Ornithine Decarboxylase Negative Positive Arginine decarboxylase Negative Negative DNAse Negative Negative ONPG Negative Negative  Discovered by Fernand Widal in 1896.  Oldest and most widely used serological test.  Principle- Agglutinins against “O” (somatic) and “H” (flagellar) antigens of Salmonella group of organisms are detected by employing killed suspension of appropriate organisms.  If homologous antibody is present in patient’s serum, it will react with respective antigen in the reagent and gives visible clumping on test card and agglutination in tube.  ‘O’ and ‘H’ antigens of S.Typhi and ‘H’ antigen of S.Paratyphi A and B are used.  Paratyphoid ‘O’ antigen is not employed as they cross react with S.Typhi ‘O’ antigen due to sharing of factor 12.  Strain of bacteria used must be motile, smooth form of nonfimbriated organism in the specific phase of flagellar antigen.  S.Typhi 901 ‘O’ and ‘H’ strains and laboratory maintained strains of S.Paratyphi A and B are used.  For O antigen S.Typhi 901 O strain is cultured on 1:800 phenol agar. H Growth is emulsified in small volume of saline. Mixed with 20 times its volume of absolute alcohol Heated at 40-50ᵒ C for 30 minutes. Centrifuged and deposit is resuspended in saline Chloroform is added as a preservative suspension of bacteria is prepared by adding 0.1 % formalin to a 24 hrs. broth culture or saline suspension of an agar culture.  Slide test (Qualitative)  Slide test (Semi-quantitative)  Standard tube test method • Drop method • Double dilution method Place one drop of Positive control on one reaction circles of the slide. Mix contents of each circle uniformly over the entire circle with separate mixing sticks. Rock the slide, gently back and forth and observe for agglutination macroscopically within one minute. Place one drop of isotonic saline on the next reaction circle. (Negative Control). Add one drop of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four reaction circles respectively. Place one drop of the patient serum to be tested onto the remaining four reaction circles Add one drop of Widal Test antigen suspension ‘H’ to the first two reaction circles.  RESULT: • Agglutination – positive • No agglutination- negative  Add 1 drop of antigen suspension to each serum aliquot.  Mix thoroughly and rotate slide gently.  Observe reaction after a minute.  Agglutinaton – positive. Serum Antigen Corresponding titer 80 µl 1 drop 1:20 40 µl 1 drop 1:40 20 µl 1 drop 1:80 10 µl 1 drop 1:160 5 µl 1 drop 1:320  Tube test is performed by two methods- i. Drop method ii. Double dilution method  Two types of tubes are used: i. Dreyer’s tube (narrow tube with conical bottom) for H agglutination and ii. Felix tube (short round-bottomed tube) for O agglutination.  Now a days 3× 0.5 ml Kahn tubes are used for both types of agglutination. Tubes 1 2 3 4 5 Serum 1 in 10 10 5 2 1 0 Saline 0 5 8 9 10 Antigen (TO/TH/AH/BH) 15 15 15 15 15 Total 25 25 25 25 25 Final dilution of serum 1:25 1:50 1:125 1:250 control  Tubes are incubated in water bath (37ᵒC) for 2 hours.  Then incubated at 8ᵒ-12ᵒ C × 24 hrs.  Examine for agglutination. 0.5 ml  0.5 ml of corresponding antigen is added  Final dil. 1/20 1/40 1/80 1/160 1/320 control  Tubes are incubated in water bath (37ᵒC) for 2 hours.  Then incubated at 8ᵒ-12ᵒ C × 24 hrs. Take 4 sets of 8 test tubes and label them 1 to 8 for O, H, AH and BH antibody detection. Pipette 1.9 ml of isotonic saline into the tube No.1 of all sets. To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline. To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested and mix well. Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well. Transfer 1.0 ml of the diluted sample from tube no.2 to tube no.3 and mix well. Continue this serial dilution till tube no.7 in each set. Discard 1.0 ml of the diluted serum from tube No.7 of each set. Tube No.8 in all the sets, serves as a saline control. Now the dilution of the serum sample achieved in each set is as follows: Tube No. : 1 2 3 4 5 6 7 8 (control) Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280. To all the tubes (1 to 8) of each set add one drop of the respective WIDAL TEST antigen suspension (O, H, AH and BH) from the reagent vials and mix well. Cover the tubes and incubate at 37° C overnight (approximately 18 hours). O agglutination- compact granular chalky clumps with clear supernatant fluid. H agglutination- large loose fluffy cotton woolly clumps with clear supernatant fluid.  No agglutination- button formation due to deposition of antigens and hazy supernatant fluid.  Titer: highest dilution of sera at which agglutination occur is taken as antibody titer.  Significant  Any titer- titer is not significant, in endemic countries people have some base line antibodies.  Cut off values varies from place to place depending on endemicity of the disease.  Significant titer in our setting is 80. Widal test result Suggestive of Rise of TO and TH antibody Enteric fever due to S.typhi Rise of TO and AH antibody Enteric fever due to S. Paratyphi A Rise of TO and BH antibody Enteric fever due to S. Paratyphi B Rise of only TO antibody Recent infection due to any serotype S.typhi or S. Paratyphi A /B Rise of only TH antibody Convalescent stage/ Anamnestic response Rise of all TH, AH, BH antibody Post TAB vaccination  Widal test is time consuming (to first antibody titer) so too late to start an antibiotic regimen.  It should be interpreted in the light of baseline titers in a healthy local population.  False positive- • Anamnestic response- transient rise of titer due to unrelated infections (malaria, dengue etc.) in persons who have had prior enteric fever. • If bacterial antigen suspension not free from fimbriae. • Person with prior immunization (TAB vaccine).  Fourfold rise in antibody titer in paired sera at 1 week interval is done to rule out false positivity.  False negative- • Early / late stage • Carriers • Patients on antibiotics. • Severe hypoproteinemia. • Prozone phenomenon (antibody excess)- can be obviated by serial dilution of sera.  Variations also exist between laboratories in performance and reading the result.  Typhidot • test- It detects and separately identify IgM and IgG antibodies against outer membrane protein (OMP) of S.Typhi. • 50kD OMP antigen impregnated on nitrocellulose membrane.  IDL Tubex test- • It is a semiquantitative colorimetric test. • O9 antigen is used. • Detects only IgM antibodies.  IgM dipstick test and ELISA - detects anti-LPS IgM antibodies.  Dot blot assay- detects IgG antibodies against flagellar antigen.  ELISA  Coagglutination test - Demonstration of circulating Ag • COWAN Ι strain of Staph. aureus is used. • It contain protein A, which is stabilised with formaldehyde & coated with S.Typhi antibody. • This suspension (1%) give visible agglutination with patient’s serum. • Positive only in first week of disease.  CIEP  PCR detecting flagellin gene, iroB and fliC gene NON SPECIFIC METHODS  Neutropenia, leucocytosis.  Raised Liver function test  Raised muscle enzyme  Important for epidemiological & public health purpose.  Isolate bacilli from bile, feces & urine.  Repeated samples are tested because of varying intensity & shedding of bacilli.  Cholagogue purgative increase isolation chance.  Urinary carrier signify abnormality in urinary tract.  Detection of Vi antibodies- • Tube agglutination test • Bacterial suspension – - should be prepared from a selected strain that respond only to Vi agglutinin (Bhatnagar strain). - It should not acted on by the H & O antibodies. - Should NOT be used after 2 month (loses sensitiveness) - Titer of 1:10 is also considered significant. - Confirmation done by culture.  Sewer swab technique.  Sewage filtration through millipore membranes.  Phenotypic • • • • methods- Phage typing- National phage typing center- Lady Hardinge Medical College, New Delhi - By using specific bacteriophage called Vi phage II. - Most widespread phage types are E1 and A. - Now obsolete. Bacteriocin typing- Based on production of bacteriocin. Biotyping Antibiogram typing  Genotypic • methods- Plasmid typing-Based on numbers and molecular weight of plasmids. • Chromosomal based- Insertion sequence 200 typing - Pulse field gel electrophoresis - Ribotyping - PCR based methods