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

<p>Abstract</p> <p>Background</p> <p>The region around Chandigarh in India has witnessed a resurgence of cholera. However, isolation of <it>V. cholerae </it>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.</p> <p>Methods</p> <p><it>V. cholerae </it>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 <it>ctxA, ctxB, tcpA, toxT </it>and <it>toxR </it>genes and AFLP with six primer combinations was performed on 52 isolates (13 clinical, 34 environmental and 5 reference strains).</p> <p>Results</p> <p>All clinical and 3 environmental isolates belonged to serogroup O1 and remaining 31 environmental <it>V. cholerae </it>were nonO1-nonO139. Serogroup O1 isolates were <it>ctxA, tcpA </it>(ElTor), <it>ctxB </it>(Classical), <it>toxR </it>and <it>toxT </it>positive. NonO1-nonO139 isolates possessed <it>toxR</it>, but lacked <it>ctxA </it>and <it>ctxB</it>; only one isolate was positive for <it>toxT </it>and <it>tcpA</it>. Using AFLP, 2.08% of the <it>V. cholerae </it>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 <it>V. cholerae </it>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.</p> <p>Conclusion</p> <p>In a unique study from freshwater environs of a cholera-endemic area in India over a narrow time frame, environmental <it>V. cholerae </it>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.</p

Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa

The genomic characterization of Salmonella Paratyphi A from an outbreak of enteric fever in Vadodara, India

Salmonella enterica Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) are the causative agents of enteric fever, a systemic human disease with a burden of 300 000 cases per year in India. The majority of enteric fever cases are associated with S. Typhi, resulting in a paucity of data regarding S. Paratyphi A, specifically with respect to genomic surveillance and antimicrobial resistance (AMR). Here, we exploited whole-genome sequencing (WGS) to identify S. Paratyphi A genotypes and AMR determinants associated with an outbreak of S. Paratyphi A in Vadodara, India, from December 2018 to December 2019. In total 117 S. Paratyphi A were isolated and genome sequenced, most were genotype 2.4.2 (72.6 % of all cases), which is the globally dominant genotype. The remainder were genotype 2.3 (25.6 %), while only two isolates belonged to genotype 2.4.1. A single base-pair mutation in gyrA, associated with reduced susceptibility to fluoroquinolones, was present in all of the outbreak isolates; with 74.35 % of isolates having a S83F substitution and the remainder having an S83Y substitution. Our surveillance study suggests that S. Paratyphi A is an emergent pathogen in South Asia, which may become increasingly relevant with the introduction of Vi conjugate vaccines

Dipstick Test for Rapid Diagnosis of Shigella dysenteriae 1 in Bacterial Cultures and Its Potential Use on Stool Samples

International audienceBACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×10⁶ CFU/ml and 4.9×10⁶ CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6-99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8-99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1%) and 99.7% (95% CI:98-100%). CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys

Impact of a package of diagnostic tools, clinical algorithm, and training and communication on outpatient acute fever case management in low- and middle-income countries: protocol for a randomized controlled trial.

BACKGROUND: The management of acute febrile illnesses places a heavy burden on clinical services in many low- and middle-income countries (LMICs). Bacterial and viral aetiologies of acute fevers are often clinically indistinguishable and, in the absence of diagnostic tests, the 'just-in-case' use of antibiotics by many health workers has become common practice, which has an impact on drug-resistant infections. Our study aims to answer the following question: in patients with undifferentiated febrile illness presenting to outpatient clinics/peripheral health centres in LMICs, can we demonstrate an improvement in clinical outcomes and reduce unnecessary antibiotic prescription over current practice by using a combination of simple, accurate diagnostic tests, clinical algorithms, and training and communication (intervention package)? METHODS: We designed a randomized, controlled clinical trial to evaluate the impact of our intervention package on clinical outcomes and antibiotic prescription rates in acute febrile illnesses. Available, point-of-care, pathogen-specific and non-pathogen specific (host markers), rapid diagnostic tests (RDTs) included in the intervention package were selected based on pre-defined criteria. Nine clinical study sites in six countries (Burkina Faso, Ghana, India, Myanmar, Nepal and Uganda), which represent heterogeneous outpatient care settings, were selected. We considered the expected seasonal variations in the incidence of acute febrile illnesses across all the sites by ensuring a recruitment period of 12 months. A master protocol was developed and adapted for country-specific ethical submissions. Diagnostic algorithms and choice of RDTs acknowledged current data on aetiologies of acute febrile illnesses in each country. We included a qualitative evaluation of drivers and/or deterrents of uptake of new diagnostics and antibiotic use for acute febrile illnesses. Sample size estimations were based on historical site data of antibiotic prescription practices for malarial and non-malarial acute fevers. Overall, 9 semi-independent studies will enrol a minimum of 21,876 patients and an aggregate data meta-analysis will be conducted on completion. DISCUSSION: This study is expected to generate vital evidence needed to inform policy decisions on the role of rapid diagnostic tests in the clinical management of acute febrile illnesses, with a view to controlling the rise of antimicrobial resistance in LMICs. TRIAL REGISTRATION: Clinicaltrials.gov NCT04081051 . Registered on 6 September 2019. Protocol version 1.4 dated 20 December 2019

Studies of water damage in polyester glass laminates

Results are presented of an investigation into the combined effects of hot water and static uniaxial tensile stress on chopped strand mat reinforced polyester resins. Transport of water has been studied by measuring permeation rates, and the damage has been categorised by optical microscopy. Changes in flexural properties are reported. The resistance to crack propagation of polyester resins in the presence of water has also been studied. Isophthalic-acid based polyester resin/chopped strand mat laminates were prepared by the “contact moulding process”. Rates of permeation of water at a series of temperatures (35-80C) were measured by a gravimetric method for up to 2000 hours. The flow was Fickian in most cases, but there were circumstances where the transport became non-Fickian. The non-Fickian behaviour was observed at higher temperatures on prolonged exposures. A specially designed apparatus was used to stress laminates uniaxially in tension and to measure water permeation rates through the stressed samples. The effect of introducing external stress ([not greater than] 25% of the ultimate tensile strength) was to increase the rate of permeation. The progressive changes in the structures, as observed by microscopy took the form of (i) flat plate like circular disc cracks in the resins and resin rich areas of laminates, (ii) surface cracks in the resin casts and gel coats of laminates and (iv) partial failure of glass monofilaments by buckling. In most cases the damage was confined to regions very near the exposed surface. However, straining to 0.4% elongation led to the onset of debonding in transverse fibre bundles throughout the cross-section. Water damage was accelerated and modified by the presence of external stress. The laminates retained their flexural strength and modulus well. Laminates stressed to 0.3% strain at 80C in water for nearly three months retained 80% of their original strength. No catastrophic failure was observed. Single edge notched polyester resin specimens were exposed to water at three different temperatures for up to three months. The resistance to crack propagation as measured by the critical stress intensity factor was lowered at all exposure temperatures. An examination of the fractured surfaces revealed that no stable crack growth had occurred

Shigellosis: Epidemiology in India

Shigellosis is one of the major causes of diarrhoea in India. The accurate estimates of morbidity and mortality due to shigellosis are lacking, though it is endemic in the country and has been reported to cause many outbreaks. The limited information available indicates Shigella to be an important food- borne pathogen in India. S. flexneri is the most common species, S. sonnei and non-agglutinable Shigellae seem to be steadily surfacing, while S. dysenteriae has temporarily disappeared from the northern and eastern regions. Antibiotic-resistant strains of different Shigella species and serotypes have emerged all over the world. Especially important is the global emergence of multidrug resistant Shigellae, notably the increasing resistance to third generation cephalosporins and fluoroquinolones, and also azithromycin. This calls for a continuous and strong surveillance of antibiotic resistance across the country for periodic updation of the local antibiograms. The prevention of shigellosis is desirable as it will substantially reduce the morbidity associated with diarrhoea in the country. Public health measures like provision of safe water and adequate sanitation are of immense importance to reduce the burden of shigellosis, however, the provision of resources to develop such an infrastructure in India is a complex issue and will take time to resolve. Thus, the scientific thrust should be focused towards development of a safe and affordable multivalent vaccine. this review is focused upon the epidemiology, disease burden and the therapeutic challenges of shigellosis in Indian perspective