An Assessment of a Public Health Initiative of Homeopathy for Primary Teething.

BACKGROUND: During primary teething, children suffer from running nose, mild fever, diarrhoea and other mild irritations and inflammations. A public health programme, 'Homoeopathy for the Healthy Child', was undertaken on a pilot basis focusing on promotion of healthy teething by provision of home-based care through six pre-identified homeopathic medicines for complaints commonly observed during primary teething. This article assesses the feasibility of this programme and reports the impact of this initiative on teething profile in children and episodes of diarrhoea and upper respiratory tract infection (URTI). MATERIALS AND METHODS: Accredited Social Health Activists (ASHAs) were trained in child care and usage of a kit comprising six medicines, namely Calcarea phosphoricum 6X (CP), Ferrum phosphoricum 3X, Magnesium phosphoricum 6X, Belladonna 30C, Chamomilla 30C and Podophyllum 30C. Calcarea phosphoricum was given regularly to each participating child from 6 months to 1 year of age. Home-based care for diarrhoea, URTI and mild fever was provided by ASHAs using the other five medicines in the kit. Dentition pattern and diarrhoea/URTI episodes were recorded over a period of the next 12 months. RESULTS: Eleven thousand four-hundred and twenty-six children were followed up regularly. Amongst those who enrolled at 6-7 months, a larger proportion of children were approaching expected teething in successive months as compared with children enrolled at 12 months, thus indicating that teething delays, if any, were overcome during this period. Incidence of diarrhoea and URTI showed decrease in the months after enrolment. Children responded favourably to the medicines given by ASHAs at the time of diarrhoea/URTI episodes, and ASHAs expressed satisfaction with the programme. CONCLUSION: An approach with regular use of CP and home-based care with homeopathy through health workers for common problems in teething children is acceptable to the community and enhances outreach of services to the public at large. Observations in terms of the healthy teething period may be further validated through studies of homeopathy with suitable comparator group.

Evaluation of Multiplex PCR for Rapid Diagnosis of Female Genital Tuberculosis.

BACKGROUND: Female genital tract tuberculosis (FGTB) is a very common disease in developing countries. Rapid and specific diagnosis is of paramount importance. PURPOSE: To evaluate Multiplex PCR using MPB 64 and IS6110 primers directed against M. tuberculosis for the diagnosis of FGTB and to compare the different methods available for diagnosis like histopathology, smear microscopy and TB culture. MATERIALS AND METHODS: Multiplex PCR was performed on endometrial biopsy samples of 21FGTB confirmed cases, 49 clinically suspected FGTB cases and 25 Non TB (control group) patients. RESULTS: : Multiplex PCR had sensitivity of 95.23% for confirmed cases and specificity of 100% for confirmed FGTB cases. In 49 clinically diagnosed, but unconfirmed FGTB cases multiplex PCR was positive in 61.22% cases. The overall sensitivity of microscopy, culture, Histopathology and multiplex PCR were 1.42%, 8.57%, 21.42%, 72.85% and specificity was 100%, 100%, 100% and 100% respectively. CONCLUSION: Multiplex PCR using MPB 64 and IS6110 primers has a high sensitivity and specificity in diagnosis of FGTB.

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.

BACKGROUND & OBJECTIVES: There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. METHODS: Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥ 0.25 µg/ml), SFM2 (≥ 4 µg/ml) and SFM3 (≥ 32 µg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥ 0.25 µg/ml) and SDM2 (≥ 4 µg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. RESULTS: Mutations were observed in gyrA Ser [83]→Leu, Asp [87]→Asn/Gly, Val [196]→Ala and in parC Phe [93]→Val, Ser [80]→Ile, Asp [101]→Glu and Asp [110]→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance ( p0 <0.05); while tolC and acrR expression levels did not. INTERPRETATION & CONCLUSIONS: Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val [196]→Ala in gyrA in clinical isolates and Phe [93]→Val, Asp [101]→Glu, Asp [110]→Glu and in parC in majority of laboratory-grown mutants.

Characterization of Shiga-toxigenic Escherichia coli isolated from cases of diarrhoea & haemolytic uremic syndrome in north India.

BACKGROUND & OBJECTIVES: Shiga toxin producing Escherichia coli (STEC) is an important zoonotic foodborne pathogen, capable of causing haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). As data from India on human infections caused by STEC are limited, this study was carried out for hospital based surveillance for STEC as a causative agent of diarrhoea, bloody diarrhoea and HUS at a tertiary care centre and to study the virulence gene profile and strain relatedness by multi locus variable tandem repeat analysis (MLVA). METHODS: A total of 600 stool samples were studied. Stool samples of every fifth patient presenting with non-bloody diarrhoea, all cases of bloody diarrhoea and diarrhoea associated HUS (D+HUS) were collected from October 2009 to September 2011. Stool samples were cultured for STEC and characterization of STEC was done by serogrouping, virulence genes analysis, and MLVA typing. RESULTS: STEC were isolated as a sole pathogen from 11 stool samples [5 of 290 (1.7%) non-blood diarrhoea and 5 of 300 (1.6%) blood diarrhoea cases]. STEC was also isolated from one fatal case of HUS who was an eight month old child. Only six of 11 isolates were positive for stx2 gene, whereas stx1 was present in all 11 isolates. Only one isolate was positive for eae. Other adhesion genes present were iha in five isolates, followed by toxB and efa1 in two each and saa gene in one, isolate. Among the plasmid encoded genes, espP, hly and etpD were each present in one isolate each. In the MLVA typing, diverse profiles were obtained except two untypeable isolates from different patients shared the same MLVA profile. Both these isolates were not epidemiologically linked. INTERPRETATION & CONCLUSIONS: This study demonstrated that STEC could be a causative agent of diarrhoea, bloody diarrhoea and sporadic HUS. However, further work needs to be done to study and explore the prevalence of these organisms in the food chain in this region.

An epidemiological and environmental study of Shiga toxin-producing Escherichia coli in India.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of worldwide importance, but a shortage of data exists for STEC isolation from India. Therefore, an epidemiological and environmental study that covers a large geographic area in north India was conducted. Ruminant stool samples (n=650) were collected from 59 dairies. Meat samples (n=450) were collected from local abattoirs and the main slaughterhouse of the region. Additionally, 600 human cases of diarrhea and hemolytic uremic syndrome were screened for STEC. Isolates were characterized for the virulence gene profiles and for the serogroups and were submitted to molecular typing by the multilocus variable-number tandem-repeat analysis (MLVA). Overall, 12.3% of animal stool samples and 6.3% of mutton samples (n=160) were positive for STEC. Additionally, STEC were isolated from 1.7% and 1.6% of watery (n=290) and bloody (n=310) stool specimens, respectively. Animal stool isolates were significantly more prevalent in hilly areas (p<0.05) than in plain areas. Polymerase chain reaction demonstrated the presence of stx1, stx2, hly, espP, saa, toxB, and iha genes in 117 (83.5%), 94 (67.1%), 77 (55%), 33 (23%), 62 (44.2%), 29 (20.7%), and 51 (36%) of the isolates, respectively. Five new serogroups (O55, O33, O173, O165, and O136) are being reported for the first time from India. Four isolates from serogroup O103 were found in mutton and stool specimens of cattle and humans (n=160). One isolate from serogroup O104 was isolated from a mutton sample. MLVA suggested the potential transmission of STEC from contaminated meat and bovine sources. This study confirms the frequent contamination of mutton samples (24%), whereas chicken and pork samples were negative for STEC. This study demonstrates the presence of STEC that carry a large repertoire of virulence genes and the potential transmission of STEC from contaminated mutton and animal stools in north India.

Prevalence of multidrug resistance in Mycobacterium tuberculosis isolates from HIV seropositive and seronegative patients with pulmonary tuberculosis in north India.

BACKGROUND: Multidrug resistant (MDR) and extensively-drug resistant (XDR) tuberculosis (TB) are a serious threat to the national TB control programs of developing countries, and the situation is further worsened by the human immunodeficiency virus (HIV) pandemic. The literature regarding MDR/XDR-TB is, however, scanty from most parts of India. We carried out this study to assess the prevalence of MDR/XDR-TB in new and previously treated cases of pulmonary TB and in HIV seropositive and seronegative patients. METHODS: Sputum and blood specimens were obtained from 2100 patients suspected of pulmonary tuberculosis and subjected to sputum microscopy and culture for TB, and HIV serology at our tertiary care centre in north India. The culture positive Mycobacterium tuberculosis isolates were subjected to drug susceptibility testing (DST) for first line anti-tuberculosis drugs, and the MDR isolates were further subjected to second line DST. Various parameters of the patients' were analyzed viz. clinical presentation, radiology, previous treatment history, demographic and socioeconomic data and microbiology results. RESULTS: Of the 2100 patients, sputum specimens of 256 were smear positive for acid-fast bacilli (AFB), 271 (12.9%) grew Mycobacterium spp., and M. tuberculosis was isolated in 219 (10.42%). Of the 219 patients infected with M. tuberculosis, 20.1% (44/219) were found to be seropositive for HIV. Overall, MDR-TB was observed in 17.4% (39/219) isolates. There were 121 newly diagnosed and 98 previously treated patients, of which MDR-TB was found to be associated with 9.9% (12/121) and 27.6% (27/98) cases respectively. There was significantly higher association of MDR-TB (12/44, 27.3%) with HIV seropositive patients as compared to HIV seronegative patients (27/175, 15.4%) after controlling previous treatment status, age, and sex (odd's ratio, 2.3 [95% CI, 1.000-5.350]; p-value, 0.05). No XDR-TB was found among the MDR-TB isolates. CONCLUSION: The present study demonstrated a high prevalence of drug resistance amongst pulmonary TB isolates of M. tuberculosis from north India as compared to the WHO estimates for India in 2010, though this could possibly be attributed to the clustering of more serious or referred cases at our tertiary care centre. The prevalence of MDR-TB in HIV seropositive patients was significantly higher than seronegative individuals. The study emphasizes the need to monitor the trends of drug resistance in TB in various populations in order to timely implement appropriate interventions to curb the menace of MDR-TB.

Evaluation of in-house loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of M. tuberculosis in pulmonary specimens.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) assay has come forward as a rapid, cost-effective molecular technique for diagnosis of tuberculosis (TB) in developing countries. This study evaluated Mycobacterium tuberculosis-specific in-house LAMP assay targeting 16s rRNA and compared it with other conventional tests and nucleic acid amplification assay (IS6110 PCR). METHODS: A total of 133 sputum specimens (103 from suspected pulmonary TB cases and 30 from non-TB controls) were subjected to conventional tests, IS6110 PCR and 16s rRNA LAMP assay. RESULTS: Of the 103 patients, the maximum number of cases were found to be positive by LAMP assay, that is, in 87 (84.5%) patients, followed by culture positive in 78 (75.7%), IS6110 PCR in 74 (71.8%), and smear positive in 70 (67.9%) patients. Of the 83 smear positive and/or culture positive cases, LAMP detected 77 (92.77%) cases, and was found to be superior to IS6110 PCR, which could detect 69 (83.1%) cases; a concordance of 0.6 was obtained between the two tests using kappa statistics. CONCLUSION: Overall, LAMP was simple and efficacious for early diagnosis of smear positive, culture positive cases as well as for confirmation of smear negative, culture negative cases, and was found to be superior to IS6110 PCR.

Cephalosporin-resistant Shigella flexneri over 9 years (2001-09) in India.

OBJECTIVES: To determine the pattern and antimicrobial resistance genes of cephalosporin resistance in Shigella flexneri and Shigella dysenteriae over 9 years. METHODS: Isolates of Shigella (S. flexneri, n = 119 and S. dysenteriae, n = 24) were tested for resistance to ceftriaxone and cefepime by disc diffusion, for MIC by Etest and for extended-spectrum ß-lactamase (ESBL) and AmpC production. The presence of antimicrobial resistance genes was investigated by PCR using specific primers for bla(TEM), bla(OXA-1), bla(CTX-M-15), bla(SHV) and bla(CMY-2) for all the isolates. RESULTS: Twenty (16.8%) S. flexneri isolates were resistant/intermediately susceptible to ceftriaxone/cefepime, while all S. dysenteriae were susceptible. In S. flexneri isolates, the MIC(50) values of ceftriaxone and cefepime were found to be 0.032 and 0.125 mg/L, respectively, while their MIC(90) values were 12 and 8 mg/L, respectively. The MIC(50) and MIC(90) for S. dysenteriae were below 1 mg/L for ceftriaxone; however, for cefepime the MIC(90) was found to be 4 mg/L. Of the 20 resistant/intermediately susceptible S. flexneri isolates, 9 were positive for ESBL production and 4 for AmpC production by phenotypic tests. All 20 isolates were found to be positive for bla(TEM), 10 for bla(CTX-M-15), 8 for bla(OXA) and 7 for bla(CMY-2); none was positive for bla(SHV). CONCLUSIONS: We report a high level of cephalosporin resistance with high MICs and ESBL- and AmpC-mediated antibiotic resistance in Shigella from north India.

Development and evaluation of a multiplex polymerase chain reaction for the detection of Mycobacterium tuberculosis from pulmonary specimens.

BACKGROUND: The diagnosis of pulmonary tuberculosis is still a major challenge. Using a polymerase chain reaction (PCR), one can detect Mycobacterium tuberculosis in clinical samples within a few hours. However, single gene targets may result in false negativity due to the absence of target DNA in some M. tuberculosis isolates. The objective of this study was to develop and evaluate a multiplex PCR (M-PCR) using IS6110 and devR primers for the detection of M. tuberculosis in sputum samples. METHODS: Sputum samples were collected from: (1) 200 confirmed cases of tuberculosis; (2) 100 suspected cases of tuberculosis diagnosed on the basis of clinical and radiological findings; (3) 200 non-tubercular patients suffering from respiratory diseases other than tuberculosis, in whom tuberculosis had been excluded. All 500 sputum samples were subjected to PCR using IS6110 primers, and M-PCR using IS6110 and devR primers; results were compared with conventional techniques. RESULTS: It was found that M-PCR was 97.5% successful in detecting the presence of tuberculosis in the confirmed tuberculosis group as compared to 84.5% by IS6110-based PCR. In the suspected tuberculosis group, M-PCR could detect 45% of cases as compared to 40% by IS6110-based PCR. Overall, the specificities of both the PCR and M-PCR were found to be 96.5%. CONCLUSIONS: This study demonstrated that the M-PCR assay is more sensitive than the IS6110-based PCR for the detection of M. tuberculosis in sputum specimens and could be applied in situations of highly suspected tuberculosis when all others tests including IS6110 PCR are negative.

Mycoplasma genitalium: an emerging sexually transmitted pathogen.

Mycoplasma genitalium is a member of genital mycoplasmas, which is emerging as an important causative agent of sexually transmitted infections both in males and females. The advent of polymerase chain reaction and other molecular methods have made studies on M. genitalium more feasible, which is otherwise a difficult organism to isolate. Besides Chlamydia trachomatis, M. genitalium is now an important and established cause of non gonococcal urethritis (NGU) in men, more so in persistent and recurrent NGU. Multiple studies have also shown a positive association of M. genitalium with mucopurulent cervicitis and vaginal discharge in females as well. The evidences for M. genitalium pelvic inflammatory diseases and infertility are quite convincing and indicate that this organism has potential to cause ascending infection. Lack of clear association with M. genitalium has been reported for bacterial vaginosis and adverse pregnancy outcomes. Diagnosis of M. genitalium infections is performed exclusively using nucleic acid amplification tests (NAATs), owing to poor or slow growth of bacterium in culture. Although there are no guidelines available regarding treatment, macrolide group of antimicrobials appear to be more effective than tetracyclines. The present review provides an overview of the epidemiology, pathogenesis, clinical presentation and management of sexually transmitted infections due to M. genitalium.

Molecular epidemiology of Vibrio cholerae causing outbreaks & sporadic cholera in northern India.

BACKGROUND & OBJECTIVES: Several outbreaks of cholera have been reported in Chandigarh region during a span of seven years from 2002-2008. The genetic characteristics of Vibrio cholerae isolates obtained during these outbreaks have not been adequately studied. The aim of this study was to do molecular typing of V. cholerae isolated from the sporadic and outbreak cases by pulsed-field gel electrophoresis (PFGE), Rep-PCR and ribotyping. METHODS: Fifty representative isolates of V. cholerae from outbreak as well as sporadic cases were subjected to molecular typing by PFGE, 173 isolates (163 clinical and 10 environmental) were typed by rep-PCR and ribotyping. Ribotyping was done by determination of rRNA restriction pattern of BglI restriction digestion and hybridization with 7.2 kb rRNA probe of pKK3535 plasmid using DIG DNA labelling and detection kit. Universal VC1 primer was used for rep-PCR. RESULTS: PFGE generated 15 pulsotypes, of which four matched the published pulsotypes and there were 11 new pulsotypes. PFGE was the most discriminatory method that could differentiate between isolates belonging to single ribotype. Pulsotype P1 corresponding to known pulsotype H1 was the major pulsotype till 2003. Pulsotype P3 corresponding to known pulsotype L emerged in 2004. The 2007 outbreaks in Punjab and Haryana were caused by P5 though P1 and P3 were isolated from the sporadic cases from the same region. The 2008 outbreak was caused by pulsotypes P6 and P7. Ribotype IV was the most predominant followed by RIII. This ribotype was not isolated after 2003 and ribotype IV became the most predominant 2004 onwards. Of the two unknown ribotypes (UNI and UN2), UNI was more common (27 isolates). Rep-PCR was the least discriminatory and divided all clinical isolates into four major profiles. The dendrogram analysis of PFGE revealed similarity of some clinical isolates with environmental isolates indicating the genetic relatedness. INTERPRETATION & CONCLUSION: Our findings showed that Rep-PCR was least discriminatory method. Ribotyping was a reliable and reproducible method. Ribotype IV was predominant ribotype followed by RIII. A total of 15 pulsotypes were generated and 11 of these were not reported earlier. Genetic relatedness was shown by clinical and environmental isolates which needs to be confirmed in future studies.

High occurrence of blaCMY-1 AmpC lactamase producing Escherichia coli in cases of complicated urinary tract infection (UTI) from a tertiary health care centre in north India.

AmpC beta lactamase producing Gram-negative bacteria have emerged worldwide. It is important to distinguish plasmid mediated AmpC ß lactamases from chromosomally mediated enzymes for surveillance, epidemiology and hospital infection control as plasmid mediated genes can spread to other organisms. Occurrence of blaCMY-1 AmpC ß-lactamase, a plasmid mediated cephamycinase was studied in 100 consecutive isolates of Escherichia coli from cases of complicated urinary tract infection (UTI). Screening for AmpC production was done by modified Hodge test, three dimensional test and AmpC disk test. All isolates showing a positive result by 2 out of 3 tests were then tested for blaCMY-1 gene by PCR. Fifty nine isolates were positive for AmpC ß lactamase production, 56.6 per cent were positive by PCR. Eight out of 13 isolates which were negative by EDTA disk method were positive by PCR, whereas none of the isolates negative by 3D and modified Hodge test was positive by PCR. Among admitted patients urinary catheterisation was the major risk factor followed by obstructive uropathy, three patients developed urosepsis. High occurrence of blaCMY-1 AmpC ß-lactamase warrants health care workers to endorse good hospital practices.

Does multiplex polymerase chain reaction increase the diagnostic percentage in osteoarticular tuberculosis? A prospective evaluation of 80 cases.

PURPOSE: Multiplex Polymerase Chain Reaction (MPCR) is a technique in which two or more gene targets are amplified in a single reaction. This has increased sensitivity of diagnosis as a single gene target may be absent in some Mycobacterium tuberculosis strains. METHODS: MPCR using two target genes specific for Mycobacterium tuberculosis, that is, IS6110 and MPB 64, ZN staining and Mycobacterial culture were performed on synovial fluid/pus samples of 80 (three confirmed, 77 suspected) patients of osteoarticular tuberculosis and 25 non tuberculosis patients. RESULTS: MPCR had a sensitivity of 100% in confirmed cases and 81.8% in clinically suspected cases. AFB was positive in one patient and Mycobacterial culture was positive in three patients. MPCR also had 100% specificity; MPB64 was positive in five patients in which IS6110 was negative whereas IS6110 was positive in two patients in which MPB64 was negative. CONCLUSIONS: MPCR is a sensitive and specific method for diagnosis of paucibacilliary conditions such as osteoarticular tuberculosis.

Genetic polymorphisms among Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in northern India.

Restriction fragment length polymorphism (RFLP) based on IS6110 is considered the gold standard for Mycobacterium tuberculosis molecular typing. It is useful to discriminate among M. tuberculosis strains, investigate outbreaks and distinguish between reactivation and re-infection. We studied polymorphisms among M. tuberculosis isolates from northern India using RFLP to determine the presence of a correlation between IS6110 based fingerprints and drug resistance and to look for relapse and transmission among patients and their contacts. RFLP patterns of PvuII digested genomic DNA of 100 M. tuberculosis isolates were analyzed using southern blotting with a 245 bp IS6110 probe. Drug sensitivity testing (DST) was conducted for rifampicin (40 microg/ml), isoniazid (1 microg/ml), ethambutol (2 microg/ml) and streptomycin (4 microg/ml) using the proportion method. A high degree of polymorphism was seen among the M. tuberculosis isolates and the number of IS6110 copies varied from 0 to 14, with a predominance of isolates with 11 bands. Seventy-five isolates had a high number of bands, 9 had an intermediate number, 6 isolates had a low number and 10 isolates had no bands. No correlation between IS6110 band numbers and RFLP banding patterns was found with drug resistance or for any particular geographical area, although clustering was seen amongst MDR-TB cases. No cases of relapses or transmissions were seen.

Is routine medical examination of food handlers enough to ensure food safety in hospitals?

Nosocomial food outbreaks due to infected food handlers is primarily due to inadequate knowledge and faulty practices of food handlers during diarrhoeal episodes. The aim of this study was to assess: 1) prevalence of enteropathogen infection among food handlers working in our hospital during 2007 to 2011 and 2) adequacy of precautions taken by them during gastroenteritis episodes. Stool samples submitted by food handlers during 2007 to 2011 were examined for the presence of enteropathogens by standard methodology. For the second part of the study, a questionnaire regarding practices during episodes of diarrhoea in food handlers or their family members was handed out to willing participants. During the years 2007, 2008, 2010 and 2011 respectively, 3.9%, 9.8%, 5.1% and 9.4% food handlers were found infected with enteropathogens. The most common parasite detected was Entamoeba histolytica. Bacterial enteropathogens prevalence was very low during these years. There was high awareness (78.8%) among the food handlers regarding routine testing of faeces. Only 64.7% knew that it was important to report for purpose of treatment and leave. While 9.4% had suffered from diarrhoeal episodes in between intervals of annual microbiological testing, only 4.7% took appropriate treatment and availed medical leave. A regular training programme on food safety should be established and emphasis should be laid on mandatory reporting and stool testing of kitchen personnel as well as abstaining from work till they are medically fit.

Amplified fragment length polymorphism of clinical and environmental Vibrio cholerae from a freshwater environment in a cholera-endemic area, India.

BACKGROUND: The region around Chandigarh in India has witnessed a resurgence of cholera. However, isolation of V. cholerae O1 from the environment is infrequent. Therefore, to study whether environmental nonO1-nonO139 isolates, which are native to the aquatic ecosystem, act as precursors for pathogenic O1 strains, their virulence potential and evolutionary relatedness was checked. METHODS: V. cholerae was isolated from clinical cases of cholera and from water and plankton samples collected from freshwater bodies and cholera-affected areas. PCR analysis for the ctxA, ctxB, tcpA, toxT and toxR genes and AFLP with six primer combinations was performed on 52 isolates (13 clinical, 34 environmental and 5 reference strains). RESULTS: All clinical and 3 environmental isolates belonged to serogroup O1 and remaining 31 environmental V. cholerae were nonO1-nonO139. Serogroup O1 isolates were ctxA, tcpA (ElTor), ctxB (Classical), toxR and toxT positive. NonO1-nonO139 isolates possessed toxR, but lacked ctxA and ctxB; only one isolate was positive for toxT and tcpA. Using AFLP, 2.08% of the V. cholerae genome was interrogated. Dendrogram analysis showed one large heterogeneous clade (n = 41), with two compact and distinct subclades (1a and 1b), and six small mono-phyletic groups. Although V. cholerae O1 isolates formed a distinct compact subclade, they were not clonal. A clinical O1 strain clustered with the nonO1-nonO139 isolates; one strain exhibited 70% similarity to the Classical control strain, and all O1 strains possessed an ElTor variant-specific fragment identified with primer ECMT. Few nonO1-nonO139 isolates from widely separated geographical locations intermingled together. Three environmental O1 isolates exhibited similar profiles to clinical O1 isolates. CONCLUSION: In a unique study from freshwater environs of a cholera-endemic area in India over a narrow time frame, environmental V. cholerae population was found to be highly heterogeneous, diverse and devoid of major virulence genes. O1 and nonO1-nonO139 isolates showed distinct lineages. Clinical isolates were not clonal but were closely related, indicating accumulation of genetic differences over a short time span. Though, environment plays an important role in the spread of cholera, the possibility of an origin of pathogenic O1 strains from environmental nonO1-nonO139 strains seems to be remote in our region.

Characterization of low molecular weight antimicrobial peptide from human female reproductive tract.

BACKGROUND & OBJECTIVES: The mechanisms that protect female upper genital tract from ascending infection by microbes present in vagina are only partially understood. It is expected that epithelial cells in mucosal surfaces and their secretions directly interfere with microbial colonization and invasion. This study was aimed to demonstrate the expression of 2 kDa antimicrobial peptide which was identified and purified from female genital tract tissues using chromatographic techniques. METHODS: Low molecular weight proteins were isolated from human female reproductive tract tissues obtained from premenopausal women. Antimicrobial activity of these LMW proteins was assessed against different reproductive tract pathogens viz., Neisseria gonorrhoeae, Group B streptococcus, Gardnerella vaginalis, Escherechia coli and Candida albicans. The expression of these peptides were also documented in reproductive tract tissues with the help of hyperimmune sera raised against the rabbits. The purified peptide was characterized by N-terminal sequencing. RESULTS: Immunohistochemical and immunofluorescence studies demonstrated that 2 kDa peptide was expressed in the stratified squamous epithelial cells of the ectocervix while it was absent in columnar epithelial cells of upper genital tract. Upregulation of the expression of this peptide was observed in patients of chronic non-specific cervicitis and acute on chronic cervicitis. This purified antimicrobial peptide also showed broad spectrum antimicrobial activity against different reproductive tract pathogens. INTERPRETATION & CONCLUSIONS: Considering the emerging bacterial resistance against conventional antibiotics, isolation and understanding of the expression of antimicrobial peptides from female reproductive tissue extracts may provide some leads towards the development of strategies for the treatment of reproductive tract infections.

Fecal contamination of drinking water supplies in and around Chandigarh and correlation with acute gastroenteritis.

The study evaluated drinking water from localities in and around Chandigarh for fecal coliforms, V. cholerae and Enterotoxigenic E. coli and correlate with occurrence of acute gastroenteritis occurring from the same region. Drinking water sample were collected from various sources from the defined area. Samples were tested for fecal coliforms and E. coli count by multiple tube method and pathogens by membrane filtration technique. E. coli were screened for heat labile toxin (LT) by the reverse passive agglutination method and heat stable toxin (ST) by ELISA. Stool samples from cases of acute gastroenteritis from the same region and time were collected and processed for V. cholerae, Enterotoxigenic E coli (ETEC) and others like Salmonella, Shigella and Aeromonas spp. Of 364 water samples examined, 116 (31.8%) samples were contaminated with fecal coliforms (58.5% rural, 33.4% semi-urban and 11.1% from urban areas). E. coli were grown from 58 samples. Ninety-two isolates of E. coli were tested for enterotoxins of which 8 and 24 were positive for LT and ST respectively. V. cholerae were isolated from 2 samples during the outbreak investigation. Stored water samples showed a significantly higher level of contamination and most of Enterotoxigenic E. coli were isolated from stored water samples. A total of 780 acute gastroenteritis cases occurred; 445 from semi-urban, 265 rural and 70 from urban areas. Out of 189 stool samples submitted, ETEC were the commonest (30%) followed by V. cholerae (19%), Shigellae (8.4%), Salmonellae (2.1%) and Aeromonas (2.6%). ST-ETEC (40/57) were commoner than LT- ETEC(17/57). In the present study, high levels of contamination of drinking water supplies (32.1%) correlated well with cases of acute gastroenteritis. Majority of cases of acute gastroenteritis occurred in the semi-urban area corresponding with high level of contamination (33.4%/). The highest level of water contamination was seen in rural areas (58.5%) but the number of acute gastroenteritis cases were lesser (33.9%) as ponds were infrequently used for drinking purpose. Safer household water storage and treatment is recommended to prevent acute gastroenteritis, together with point-of-use water quality monitoring.

Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms.

OBJECTIVES: This study was carried out to elucidate the role of reduced antibiotic penetration in the resistance of Staphylococcus aureus and Staphylococcus epidermidis biofilms to different antibiotics. METHODS: The biofilms of S. aureus ATCC 29213 and S. epidermidis ATCC 35984 were grown on black, polycarbonate membranes (diameter, 13 mm; pore size, 0.4 microm) placed on tryptic soy agar plates at 37 degrees C for 48 h. The penetration of oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin through the biofilms was determined by measuring the diameter of zones of growth inhibition (of S. aureus ATCC 25923, a quality control strain) on Mueller-Hinton agar plates following diffusion of each antibiotic from an overlying antibiotic disc through the biofilm to the agar medium versus the respective control assemblies. RESULTS: The penetration of oxacillin and cefotaxime (beta-lactams) and vancomycin (a glycopeptide) was significantly reduced through S. aureus and S. epidermidis biofilms whereas that of amikacin (an aminoglycoside) and ciprofloxacin (a fluoroquinolone) was unaffected. CONCLUSIONS: The results of this study indicate that the role of reduced antibiotic penetration in the drug resistance of S. aureus and S. epidermidis biofilms may vary with the antibiotic being used.

Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India.

BACKGROUND & OBJECTIVES: Extended spectrum beta lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. The enzymes are predominantly plasmid mediated and are derived from broad-spectrum beta lactamase TEM-1, TEM-2 or SHV-1 by a limited number of mutations. This study was undertaken to characterize ESBL producers among Escherichia coli and Klebsiella pneumoniae by PCR-RFLP, which were initially screened by phenotypic method. METHODS: A total of 100 isolates of each species (E. coli and K. pneumoniae) were screened for ESBL production. PCR analysis for ß-lactamase genes of the family TEM and SHV was also carried out. PCR products of TEM and SHV genes were subjected to digest with three different restriction enzymes. The digested products were run on 1.5 per cent agarose gel, stained and examined for DNA bands. RESULTS: PCR carried out on plasmid DNA alone detected 30 per cent ESBL positive isolates using TEM primer and 38 per cent using SHV primer, whereas PCR for both plasmid and chromosomal DNA showed 56 per cent positivity for TEM and 60 per cent positivity for SHV. INTERPRETATION & CONCLUSION: RFLP yielded homogeneous band pattern, suggesting that there may be a point source or a common evolutionary origin for all the ESBL isolates.