V. M. Vaidya

Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR

Aim: To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence‐associated genes.

Comparison of PCR, Immunofluorescence Assay, and Pathogen Isolation for Diagnosis of Q Fever in Humans with Spontaneous Abortions

ABSTRACT Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of Q fever in humans. We tested a total of 368 samples (placental bits, genital swabs, fecal swabs, and urine and serum samples) collected from women (n = 74) with spontaneous abortions for C. burnetii by a PCR assay targeting IS1111, the repetitive transposon-like region of C. burnetii (trans-PCR); real-time PCR; an indirect immunofluorescence assay (IFA); and the isolation of the pathogen. The IFA showed seropositivity for 25.68% of the women with spontaneous abortions, whereas trans-PCR and real-time PCR each detected the pathogen in 21.62% of cases. Overall, 25.68% of the subjects were positive by one or more assays. Real-time PCR showed a slightly higher level of sensitivity than trans-PCR. With the IFA as the reference, the two PCR assays showed a higher level of sensitivity (84.21%) than pathogen isolation (26.31%), while both the PCR assays and pathogen isolation were specific (100%). The detection of high numbers of C. burnetii cells in clinical samples and the frequent association of the pathogen with cases of spontaneous abortions observed in this study revealed that Q fever remains underdiagnosed and that the prevalence in India is underestimated.

Prevalence of Q fever in domestic animals with reproductive disorders.

The occurrence of Coxiella burnetii in animals with reproductive disorders was studied. A total of 920 samples (genital and faecal swabs, milk, urine and serum) were collected from cows (88), buffaloes (33), sheep (43) and goats (53) with a history of reproductive disorders and screened for C. burnetii by a PCR assay targeting the repetitive transposon-like region of C. burnetii (trans-PCR), real-time PCR, indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and isolation method. The overall prevalence of Q fever in animals with the history of reproductive disorders turned out to be 13.82%. The species-wise prevalence of Q fever among animals was observed to be 12.78% in cattle, 16.66% in buffaloes, 11.04% in sheep and 6.13% in goats. In comparison to IFA, the highest sensitivity (85.18%) was shown by both PCR assays followed by ELISA (74.07%) and isolation method (14.81%) whereas, isolation method was the most specific (100%) followed by both PCR assays (99.47%) and ELISA (98.44%). The high excretion rate of pathogen particularly in milk observed in the study posses a potential public health threat from infected animals.

Use of a phospholipase-C assay, in vivo pathogenicity assays and PCR in assessing the virulence of Listeria spp.

Listeria spp. was isolated from 19.8% of animals with a history of reproductive disorders. A total of 333 faecal, genital swab and blood samples from 111 animals (cattle, buffaloes, sheep and goats) were subjected to PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap) and pathogenicity testing by the phosphatidylinositol phospholipase-C (PI-PLC) assay, and by mouse and chick embryo inoculation. One isolate of Listeria ivanovii recovered from a genital swab from a sheep was found to be pathogenic. Virulence assessment was then carried out on two L. ivanovii and 29 Listeria monocytogenes isolates from various sources using these assays. Haemolytic L. monocytogenes isolates lacking the plcA gene and PI-PLC activity were deemed non-pathogenic when assessed by mouse and chick embryo inoculation tests, in spite of having the hlyA gene. The results suggested that the PI-PLC and PCR assays are reliable in vitro alternatives to in vivo pathogenicity tests for L. monocytogenes.

Street Vended Foods and Associated Environment in Two Major Cities of India: Microbial Safety Concern

Majority of the people in the cities liked to have street food even with the compromising their health. Therefore, the present study was carried out to find out the microbiological profile/safety of street vended foods and associated environmental samples collected from Mumbai and Delhi. A total of 166 samples of foods of animal origin (124) and associated environmental samples (42) were processed for aerobic plate count, enumeration of Escherichia coli, Staphylococcus spp., sulphite reducing Clostridia (SRC) and for detection of Salmonella spp. and Listeria monocytogenes, while water samples tested for most probable number. Swab samples showed low to marginal APC for Delhi while marginal to high in Mumbai. Notably, E. coli and Staphylococcus spp. count is higher in table and cloth swabs. Overall, 42.4%, 69.6% and 51.5% swab samples were positive for E. coli, Staphylococcus spp. and SRC, respectively. APC for raw chicken was 5.00 ± 0.17 & 5.45 ± 0.05 log10cfu g-1 for Mumbai and Delhi, respectively while that of 4.13 ± 0.18 and 4.53 ± 0.10 log cfu g-1 for raw egg and raw milk from Delhi and 4.00 log cfu g-1 for raw egg from Mumbai. Salad and chutney samples showed marginal to high APC (3.5 to 4.7 log cfu g-1) with the presence of E. coli and Staphylococcus spp. in majority of the samples in both the cities. Overall, 38.75, 51.25 and 35.0% raw foods; 52.0, 36.0 and 8.0% milk products and 22.72, 31.81 and 13.63% cooked food samples were positive for E. coli, Staphylococcus spp. and SRC, respectively. Salmonella spp. was present in only one chutney sample while L. monocytogenes was absent in all. Cooked chicken from Mumbai had 1.89 ± 0.56 log cfu g-1 with mean E. coli and Staphylococcal count is