A sensitive & specific multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei & Brucella species.

Background & objectives: Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella species are potential biowarfare agents. Classical bacteriological methods for their identification are cumbersome, time consuming and of potential risk to the handler. Methods: We describe a sensitive and specific multiplex polymerase chain reaction (mPCR) assay involving novel primers sets for the simultaneous detection of B. anthracis, Y. pestis, B. pseudomallei and Brucella species. An additional non-competitive internal amplification control (IAC) was also included. Results: The mPCR was found to be specific when tested against closely related organisms. The sensitivity of the assay in spiked blood samples was 50 colony forming units (cfus)/25 μl reaction, for the detection of B. anthracis, Y. pestis and Brucella species; and 150 cfus/25 μl reaction, for B. pseudomallei. The assay proved useful in correctly and promptly identifing the clinical isolates of the targeted agents recovered from patients, compared to the gold standard culture methods. Interpretation & conclusion: The assay described in this study showed promise to be useful in application as a routine detection cum diagnostic method for these pathogens.

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.

Dna probes for identification of leptospires and disease diagnosis.

A newly identified 1 kb DNA fragment amplified by PCR using (AG)8T inter-simple sequence repeats (ISSR) primer and a 631 bp segment of 16S rRNA ribosomal gene amplified by PCR using reported primers were labeled with a alpha32P dCTP for use as DNA probes. These probes were hybridized with DNA extracted from 19 standard pathogenic serovars, 3 standard saprophytic serovars, 33 pathogenic isolates (12 from patients, 1 from a tapwater source, and 20 from rodents), and 22 saprophytic isolates from environmental sources. The pathogen-specific 16S rRNA DNA probe specifically hybridized all 33 standard pathogenic serovars, to 13 pathogenic isolates. Similarly, the saprophyte specific 1 kb ISSR DNA probe specifically hybridized the 3 standard saprophytic serovars and the 22 saprophytic Leptospira isolates. The sensitivity of the 1 kb labeled saprophytic Leptospira specific DNA probe was 1.95 ng, and for the 16S rRNA pathogen specific probe 3.90 ng. The 16S rRNA gene segment DNA probe could also identify the leptospiremic stage in mice or guinea pigs infected experimentally with the pathogenic serovars australis, autumnalis or icterohaemorrhagiae. DNA probes therefore, owing to their high specificity and sensitivity, appear useful for easy, rapid, and reliable differentiation of pathogenic Leptospira strains and also hold promise for direct identification of organisms in blood samples to diagnose leptopsirosis.

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India.

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.

Characterization and utilization of monoclonal antibodies reactive to Yersinia pseudotuberculosis.

The 3 murine monoclonal antibodies, Yps1, Yps2 and Yps3 reactive to Y. pseudotuberculosis can be stabilized and all were found to be of IgG type. Monoclonal antibody, Yps1, recognized a glycoprotein antigen of the organism with reactivity at the 55-75 kDa region, while Yps2 and Yps3 recognized protein antigens of Y. pseudotuberculosis 65 kDa and 26-28 kDa molecular weight regions, respectively. The specificity of monoclonal antibodies was tested using dot ELISA and Western blotting with whole cell organisms or whole cell sonicated soluble antigens of different Yersinia species, Salmonella typhi, Klebsiella pnemoniae, Streptococcus abortus-equi and Escherichia coli. Monoclonal antibody, Yps1 exhibited cross-reactivity with soluble antigens and whole cell preparations of Y. pestis. Yps2 cross-reacted to soluble antigens of all the tested bacteria. Reactivity of monoclonal antibody, Yps3 was restricted to Y. pseudotuberculosis and Y. pestis with soluble antigen preparations. No reaction was observed with Yps2 and Yps3 to whole cell organism preparations from tested bacteria including Y. pseudotuberculosis. The co-agglutination reagent prepared by sensitizing staphylococcal cells with Yps1 monoclonal antibody produced a positive agglutination with all the 4 Y. pseudotuberculosis isolates and the 3 Y. pestis strains tested. Sandwich dot ELISA using monospecific antisera as a capture antibody and a monoclonal antibody, and Yps3 as a revealing antibody had a high level of specificity in detecting Y. pseudotuberculosis antigens.

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.