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Background: Emergence of atypical enteropathogenic Escherichia coli (EPEC) and hybrid E. coli (harboring genes of more than one DEC pathotypes) strains have complicated the issue of growing antibiotic resistance in diarrhoeagenic Escherichia coli (DEC). This ongoing evolution occurs in nature predominantly via horizontal gene transfers involving the mobile genetic elements like integrons notably class 1 integron. This study was undertaken to determine the virulence pattern and antibiotic resistance among the circulating DEC strains in a tertiary care center in south of India. Methods: Diarrhoeal stool specimens were obtained from 120 children (< 5 years) and 100 adults (> 18 years), subjected to culture and isolation of diarrhoeal pathogens. Conventional PCR was performed to detect 10 virulence and 27 antimicrobial resistance (AMR) genes among the E. coli isolated. Results: DEC infection was observed in 45 (37.5%) children and 18 (18%) adults, among which [18 (40%), 10 (10%)] atypical EPEC was most commonly detected followed by [6 (13.3%), 4 (4%)] ETEC, [5 (11.1%) 2 (2%)] EAEC, [(3 (6.6%), 0 (0%)] EIEC, [3 (6.6%), 0 (0%] typical EPEC, and [4 (8.8%), 1 (1%)] STEC, and no NTEC and CDEC was detected. DEC co-infection in 3 (6.6%) children, and 1(1%) adult and sole hybrid DEC infection in 3 (6.6%) children was detected. The distribution of sulphonamide resistance genes (sulI, sulII, and sulIII were 83.3 and 21%, 60.41 and 42.1%, and 12.5 and 26.3%, respectively) and class 1 integron (int1) genes (41.6 and 26.31%) was higher in DEC strains isolated from children and adults, respectively. Other AMR genes detected were qnrS, qnrB, aac(6')Ib-cr, dhfr1, aadB, aac(3)-IV, tetA, tetB, tetD, catI, blaCTX, blaSHV, and blaTEM. None harbored qnrA, qnrC, qepA, tetE, tetC, tetY, ermA, mcr1, int2, and int3 genes. Conclusions: Atypical EPEC was a primary etiological agent of diarrhea in children and adults among the DEC pathotypes. Detection of high numbers of AMR genes and class 1 integron genes indicate the importance of mobile genetic elements in spreading of multidrug resistance genes among these strains.

Background & objectives: Scrub typhus (ST), an important zoonosis caused by Orientia tsutsugamushi, is now prevalent throughout India. While demonstration of IgM antibody by Indirect Immunofluorescence Assay (IFA) is the gold standard serological test, IgM ELISA is an alternative. Demonstration of O. tsutsugamushi DNA in the blood or eschar confirms infection in the early febrile period. Methods: Scrub typhus nested PCR (n-PCR) for 56 kDa, 47 kDa and groEL genes and ST IgM ELISA were performed for 210 clinically suspected ST patients. As healthy controls, 70 voluntary blood donors were included. Statistical analysis was performed for laboratory parameters using Fisher exact test/chi-square test. Ninety-five PCR products of n-PCR positive samples were purified and submitted for gene sequencing. Results: PCR was positive for one or more gene targets in 75.71% of IgM ELISA positive patients and 10% of antibody negative patients. All voluntary blood donors were negative for both antibodies and DNA. Gene sequences of 95 n-PCR positive products confirmed the presence of Orientia tsutsugamushi DNA in the samples and NCBI database accession numbers MG601875 to MG601969 were obtained. Interpretation & conclusion: Compared to IgM ELISA, sensitivity of three PCRs was 30, 51.43 and 61.43% for 56 kDa, 47 kDa and groEL targets, respectively. Since IgM ELISA positivity can persist up to one year, PCR confirms ST diagnosis in the acute phase of the illness, in the presence of IgM and even before IgM appears. Inclusion of all three genes - 56 kDa, 47 kDa and groEL, instead of a single 56 kDa target, identifies and confirms maximum number of ST patients.

Background & objectives: Rickettsial diseases are important re-emerging infections that mostly go unnoticed or are misdiagnosed. Though few case reports of Indian tick typhus have been reported in Indian literature in the past 10 yr, prevalence surveys are few and far between. The objective of this research was to study the seroprevalence of spotted fever (SF) group rickettsiosis and its coinfection with scrub typhus (ST) in Puducherry region of south India, as these two diseases may show similar clinical presentations. Methods: During 2012-2015, paired sera of 320 febrile patients were examined for Rickettsia conorii IgM/IgG by ELISA and OX19 and OX2 agglutinins by Weil-Felix test. Additionally, patients were screened for ST IgM ELISA. Statistical analysis was performed for clinical and laboratory parameters in children and adults using Fisher's exact test and chi-square test with Yates correction. Results: Out of 320 patients, 142 (44.38%) had R. conorii IgM and/or IgG antibodies. Only IgM was present in 72 (22.5%) patients, while 36 patients were positive for IgG only and 34 were positive for both IgG and IgM. A total of 68 patients (21.25%) showed only OX19 and/or OX2 antibodies (titres ≥ 1 : 80). SF and ST coinfection was observed in 47 cases (14.69%). Interpretation & conclusion: Seroprevalence of SF in Puducherry was found to be quite high (44.38%). ST and SF coinfection was observed in 34.50% of the SG IgG positive patients, however, this require further evaluation by PCR to rule out cross-reaction or false positivity. At present ELISA seems to be an affordable alternative to highly subjective and technically demanding immunofluorescence assay (IFA) for serodiagnosis of SF.

Five IFA positive (including two qPCR positive) and 13 IFA negative patients had a history of domestic animal contact such as cattle, sheep, goat, dog or their residence situated near the abattoir [Table 1]. Laboratory definition and interpretation of acute QF[9] based on gold standard IFA is as follows: (i) IgM phase II + IgG phase II antibodies → acute QF; (ii) IgG phase II with or without IgG phase I → resolved past QF; and (iii) other combination viz., IgM/IgG phase II and/or IgM/IgG phase I → inconclusive, PCR to be performed. [...]the present study showed presence of QF in and around Puducherry. Authors thank the Indian Council of Medical Research (ICMR), New Delhi, for ad-hoc task force research project to the third author (SS) (30/3/41/2008/ECD-II), and to the Chairman, Vice-Chancellor, and Dean (Research and Allied Health Sciences) of Mahatma Gandhi Medical College and Research Institute, Puducherry, for providing financial assistance from Sri Balaji Vidyapeeth University Faculty Research Fund.

Background & objectives: Seroprevalence of Q fever (QF) caused by Coxiella burnetii has been reported from different parts of India. Usually serological/molecular tests are employed for detection of infection. The present study was undertaken to verify the validity of three different QF phase II IgM ELISA kits for acute QF diagnosis by comparing with the gold standard indirect fluorescent antibody assay (IFA). Methods: Fifty eight serum samples collected from 42 patients (26 patients provided acute sample only and 16 both acute and convalescent samples) which were examined by all three commercial kits, were cross-checked with QF Phase II IgM IFA for confirmation. Results: Eleven patients were positive for C. burnetii antibodies by IFA in acute and/or convalescent serum samples. Taking IFA as a reference, percentages of sensitivity, specificity, positive predictive value and negative predictive value for Virion-Serion/Vircell/NovaTec were 36.36, 61.29, 25.00, 73.08; 81.82, 35.48, 31.03, 84.62 and 100, 25.81, 32.35, 100 per cent, respectively. Interpretation & conclusions: The three different ELISA kits exhibited poor agreement amongst them and unacceptable level of false positivity. IFA remains to be the only option for diagnosing acute QF. Discrepancy between the clinical findings and IFA/ELISA results needs confirmation by C. burnetii DNA detection in real-time polymerase chain reaction.

In the course of our Indian Council of Medical Research project on coxiellosis in Puducherry and Tamil Nadu, 5.64% goat, 1.85% sheep, 1.06% buffaloes, and 0.97% cattle were positive for antibodies by enzyme linked immunosorbent assay kit (IDEXX, Liebefeld, Switzerland). In this preliminary study, we have proceeded to look for DNA in those antibody positive specimens employing an imported commercial polymerase chain reaction (PCR) kit. Blood samples were collected during slaughtering. All 15 blood samples of antibody positive ruminants and three antibody negative samples were subjected to conventional Trans-PCR assay with a commercial PCR kit (Genekam Biotechnology AG, Duisburg, Germany). An in-house Trans-PCR was included in the study for comparison. A total of 15 antibody positive and three antibody-negative serum samples belonging to 11 goat, 4 sheep, 1 cattle, and 2 buffaloes were tested in duplicate for the presence of DNA by the commercial agar gel PCR kit and an in-house Trans-PCR. Only one buffalo serum sample was positive for with a band at 243 bp in in-house Trans-PCR. Seropositivity for need not necessarily translate into infectivity status of the animal. Conversely, seronegative ruminants can shed . Rapid disintegration of DNA during the storage period is an important impediment in QF-PCR research. This is the first time the performance of this commercial PCR kit is being validated in India. Commercial PCR kit, Genekam did not identify any positive sample, probably because it targeted a larger amplicon of 687 bp.

Microbial biofilms pose a public health problem for persons requiring indwelling medical devices, as micro-organisms in biofilms are difficult to treat with antimicrobial agents. Thus the present study includes biofilm formation and antibiotic resistance pattern of uropathogens in hospitalised patients with catheter associated urinary tract infections (UTI). This prospective analysis included 100 urine samples from catheterised patients with symptoms of UTI over a period of six months. Following identification, all isolates were subjected to antibiotic sensitivity using modified Kirby- Bauer disc diffusion method. Detection of biofilms was done by tube adherence method and Congo red agar method. E.coli was found to be the most frequently isolated uropathogen 70%, followed by Klebsiella pneumoniae 16%, Pseudomonas aeruginosa 4%, Acinetobacter spp 2%, coagulase negative Staphylococci 6% and Enterococci Spp 2%. In the current study 60% of strains were in vitro positive for biofilm production. Biofilm positive isolates showed 93.3%, 83.3%, 73.3% and 80% resistance to nalidixic acid, ampicillin, cephotaxime and cotrimoxazole, respectively, compared to 70%, 60%, 35%, 60% resistance showed by biofilm non-producers for the respective antibiotics. Approximately 80% of the biofilm producing strains showed multidrug resistant phenotype To conclude E.coli was the most frequent isolate, of which 63% were biofilm producers. The antibiotic susceptibility pattern in the present study showed quinolones were the least active drug against uropathogens. The uropathogens showed the highest sensitivity to carbapenems. The next best alternatives were aminoglycosides. Significant correlation between biofilm production and multi-drug resistance was observed in our study.

Background and Aim: Diagnosis of query fever (QF) is mostly done on the basis of serological/molecular tests, due to the stringent requirement of biosafety level-3 containment facilities for isolating Coxiella burnetii in culture. QF is an important zoonosis and is considered to be an occupational hazard to livestock handlers. This report describes our study on the serological as well as molecular evidence of QF in animal handlers from Puducherry and surrounding Tamil Nadu, from where, to the best of our knowledge, no such reports are available so far. Materials and Methods: Seventy-five animal handlers were recruited, comprising veterinarians, slaughterhouse workers, butchers, and animal attendants of various government veterinary clinics from Puducherry and surrounding areas of Tamil Nadu state. QF serology was performed to identify Phase I and Phase II immunoglobulin G antibodies to C. burnetii. Nested polymerase chain reaction (N-PCR) was carried out to detect C. burnetii DNA in buffy coat samples by targeting IS1111 gene element. N-PCR-positive samples were sequenced and phylogenetic analysis was performed using MEGA software version 10.0. Results: A total of 21 animal handlers (28.1%) were positive for either serology or PCR. PCR alone was positive in 10 (13.4%), only serology was positive in 8 (10.7%), and both serology and PCR were positive in three samples (4.0%). GenBank accession numbers were obtained for 13 N-PCR-positive samples (MG548608-MG548620). Six of our study sequences showed close similarity with the reference isolates from Bengaluru, Colombia, Brazil, France, and Iran. Conclusion: A significant percentage of QF positivity in animal handlers of this part of South India, Puducherry, warrants a prospective study with follow-up of a large number of this occupational group.

Our objective is to study the seroprevalence of toxoplasmosis in the voluntary blood donors of Puducherry and surrounding districts of Tamil Nadu. A total of 275 healthy blood donors were screened for the presence of IgM and IgG antibodies to Toxoplasma gondii by ELISA test. Donor samples positive for IgM and/or IgG antibodies to T. gondii were subjected to IgG avidity ELISA. While, 54 out of 275 donors had IgG antibodies (19.66%), only one donor had IgM (0.36%) along with IgG. Among 54 IgG positive donors, only two had low avidity (3.7%), indicating recent exposure to the protozoa. Feasibility and cost effectiveness studies should be conducted throughout India to decide regarding screening of blood donors for toxoplasmosis.