A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species

Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

Risk factors for mortality in patients with leptospirosis during an epidemic in northern Kerala.

BACKGROUND: Epidemic leptospirosis is increasingly being reported from northern Kerala during the monsoon months. We investigated the risk factors for mortality during the 2002 epidemic. METHODS: Three hundred and forty patients suspected to have leptospirosis during the epidemic were studied by clinical examination, laboratory investigations and Leptospira serology (microscopic agglutination test). Two hundred and eighty-two seropositive cases were analysed for the clinical and laboratory profile, and risk factors for mortality using univariate and logistic regression analysis. RESULTS: Of the 282 seropositive cases, 58.9% were men. No significant association with occupational risk factors was seen; 62.9% had wounds on the feet. The majority had Weil syndrome with hepatic (69.8%) and renal (56.3%) involvement. Thrombocytopenia (65.8%) was common. Transient hyperglycaemia was observed in 10.3% of cases. Pulmonary haemorrhage (4.7%) and meningism (4.3%) were less common. Jaundice occurred in 46% of cases in the first week. The mortality rate was 6.03%. Hyperkalaemia (OR= 27.3), meningism (OR= 10.6), oliguria (OR=8.2), haemoptysis (OR= 5.4), bilirubin > 15 mg/dl (OR= 5.4), disorientation (OR=5), tachycardia (OR=4.1) and muscle tenderness (p=0.03) were the predictors of high mortality in univariate analysis. Only involvement of the lung and central nervous system were significant predictors of death in logistic regression. CONCLUSIONS: Leptospirosis is no more a mere occupational hazard in Kerala. Early occurrence of complications such as hepatitis mandates caution in the primary care setting. Lung and central nervous system involvement are significant predictors of mortality.

Evaluation of A newly developed DOT-ELISA kit for the diagnosis of human brucellosis.

A new dot-ELISA kit for detection of Brucella antibodies in human sera was developed and compared with that of serum agglutination test, Rose Bengal plate test, rapid slide agglutination and Coomb's antiglobulin test. Following testing of 120 human sera from suspected patients of occupational risk, 25 gave positive reaction in Rose Bengal plate test, 25 in rapid slide agglutination test, 26 in serum agglutination test, 27 in Coomb's antiglobulin test and 28 in dot-ELISA kit. Dot-ELISA kit picked up more positive than any other Serological test, indicating its superiority over the other laboratory tests for the diagnosis of brucellosis.

Comparative evaluation of protective antigen produced from Bacillus anthracis & Escherichia coli.

BACKGROUND & OBJECTIVE: Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples. METHODS: Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA. The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant. Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies. RESULTS: The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B. anthracis, B. anthracis pYS5 and recombinant Esch. coli, respectively. Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins. Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction. INTERPRETATION & CONCLUSION: The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.

Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis.

Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B. anthracis. Five clones reactive to this protein were stabilized and preserved. These MoAbs could detect nanogram levels of PA when tested in ELISA. In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B. anthracis and with other organisms. These MoAbs could detect PA from 22 confirmed clinical isolates of B. anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.

Use of restriction endonucleases for differentiation of pathogenic & saprophytic leptospires.

BACKGROUND & OBJECTIVES: PCR has been reported for amplification of the 482 bp genus specific region in 23S rRNA gene of Leptospira species. The sequence of this region was analyzed for specific restriction sites that could have yielded digestion products expected to provide differentiating banding profile for the pathogenic and the saprophytic group of leptospires. METHODS: Sixteen standard serovars of pathogenic group, two standard serovars of saprophytic group, 12 Leptospira isolates recovered from hospitalized patients with fever and jaundice or pyrexia of unknown origin and 23 isolates from different water sources were studied. Conventional tests, PCR methods and restriction digestion were used for confirming the identity of these isolates. RESULTS: All 12 isolates from patients and 1 from tap water source were identified as pathogenic and 22 isolates from water sources as saprophytic by the conventional tests and PCR. Of the 5 restriction endonuclease enzymes, viz, Apa I, Ban II, Hae III, Pst I and Sin I analyzed for digestion of PCR amplified 482 bp product, Apa I, Ban II, Pst I and Sin I provided fragments of different sizes providing distinct patterns for saprophytic and pathogenic leptospires. INTERPRETATION & CONCLUSION: The identity of a strain to genus Leptospira could be confirmed by PCR amplification of 482 bp region of 23S rRNA and with further restriction digestion a clear distinction into pathogenic or saprophytic group was achieved with the use of any of these 4 restriction enzymes.

Generation and characterization of monoclonal antibodies to adenovirus.

Monoclonal antibodies (MoAbs) were generated following immunization of Balb/C mice with adenovirus type 5 grown in Hep2 cell line. Six clones reactive to hexon antigen of the virus were stabilized, of which 4 had mu-heavy chain specificity and 2 were of gamma-heavy chain type. Three of the clones (ADV-1, ADV-3 and ADV-5) had a high ELISA reactivity to the hexon antigen but exhibited differential specificity to the adenovirus types tested. In Western blotting, ADV-1 and ADV-3 reacted with all the adenovirus types tested (types 3,4,5,7 and 8) with reactions at 116 kDa region (hexon antigen), in addition, ADV-3 also had reactivity at 80 kDa region, the penton antigen. Reactivity to these adenoviral types by the 2 MoAbs was demonstrable by dot ELISA. ADV-5 had a type specific reaction only to adenovirus type 5 in dot ELISA with specificity in the hexon region in Western blotting. The reactivity of these 3 clones was not observed to the normal Hep2 tissue culture antigens and to the 3 enteroviruses tested (polio, coxsackie A9 and echo 4).

Use of locally isolated saprophytic Leptospira strain for serological testing of human leptospirosis.

A saprophytic Leptospira isolate recovered from tap water was utilized for serological testing. One hundred-twenty Serum samples comprising 55 cases from PUO/febrile jaundice and 65 samples from apparently healthy individuals were tested by MAT and HA using this environmental saprophytic strain and the results compared with that of Leptospira biflexa semaranga patoc, the standard saprophytic strain commonly employed for sero-diagnosis of leptospirosis. The MAT data showed 96.4 per cent correlation between the two strains. Similarly, the HA results were matching to the extent of 94.5 per cent. Results, therefore, suggest that local saprophytic Leptospira strain may serve as a substitute to serovar patoc for serodiagnosis of leptospirosis.