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With increasing awareness regarding biorisk management worldwide, many biosafety laboratories are being setup in India. It is important for the facility users, project managers and the executing agencies to understand the process of validation and certification of such biosafety laboratories. There are some international guidelines available, but there are no national guidelines or reference standards available in India on certification and validation of biosafety laboratories. There is no accredited government/private agency available in India to undertake validation and certification of biosafety laboratories. Therefore, the reliance is mostly on indigenous experience, talent and expertise available, which is in short supply. This article elucidates the process of certification and validation of biosafety laboratories in a concise manner for the understanding of the concerned users and suggests the important parameters and criteria that should be considered and addressed during the laboratory certification and validation process.

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There is a global mandate even in countries with low resources to improve the accuracy of testing biomarkers in breast cancer viz. oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2neu) given their critical impact in the management of patients. The steps taken include compulsory participation in an external quality assurance (EQA) programme, centralized testing, and regular performance audits for laboratories. This review addresses the status of ER/PR and HER2neu testing in India and possible reasons for the delay in development of guidelines and mandate for testing in the country. The chief cause of erroneous ER and PR testing in India continues to be easily correctable issues such as fixation and antigen retrieval, while for HER2neu testing, it is the use of low-cost non-validated antibodies and interpretative errors. These deficiencies can however, be rectified by (i) distributing the accountability and responsibility to surgeons and oncologist, (ii) certification of centres for testing in oncology, and (iii) initiation of a national EQA system (EQAS) programme that will help with economical solutions and identifying the centres of excellence and instill a system for reprimand of poorly performing laboratories.

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Background & objectives: Lactobacilli species that are better adapted to vaginal environment of women may colonize better and offer protection against vaginal pathogenic bacteria. In this study, the distribution of common Lactobacillus species was investigated in pregnant women. Methods: Sixty seven pregnant women were included in the study and vaginal samples were collected for Gram staining. Women were classified as normal vaginal flora, intermediate flora and bacterial vaginosis (BV) based on Nugent's score. Vaginal samples were also collected for the identification of Lactobacillus spp. by multiplex polymerase chain reaction (PCR) profiling of 16S rDNA amplification method. Results: Lactobacillus crispatus (100%) was the most predominant Lactobacillus spp. present in pregnant women with normal flora, followed by L. iners (77%), L. jensenii (74%) and L. helveticus (60%). While, L. iners was commonly present across groups in women with normal, intermediate or BV flora, L. crispatus, L. jensenii and L. helveticus decreased significantly as the vaginal flora changed to intermediate and BV. In women with BV, except L. iners other species of lactobacilli was less frequently prevalent. Species such as L. rhamnosus, L. fermentum, L. paracasei and L. casei were not detected in any vaginal sample. Interpretation & conclusions: L. crispatus, L. jensinii and L. helveticus were predominant species in women with normal flora. L. crispatus alone or in combination with L. jensinii and L. helveticus may be evaluated for probiotic properties for the prevention and treatment of BV.

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Background & objectives: Pioglitazone was suspended for manufacture and sale by the Indian drug regulator in June 2013 due to its association with urinary bladder carcinoma, which was revoked within a short period (July 2013). The present questionnaire-based nationwide study was conducted to assess its impact on prescribing behaviour of physicians in India. Methods: Between December 2013 and March 2014, a validated questionnaire was administered to physicians practicing diabetes across 25 centres in India. Seven hundred and forty questionnaires fulfilling the minimum quality criteria were included in the final analysis. Results: Four hundred and sixteen (56.2%) physicians prescribed pioglitazone. Of these, 281 used it in less than the recommended dose of 15 mg/day. Most physicians (94.3%) were aware of recent regulatory events. However, only 333 (44.8%) changed their prescribing pattern. Seventeen of the 416 (4.1%) physicians who prescribed pioglitazone admitted having come across at least one type 2 diabetes mellitus patient (T2DM) who had urinary bladder carcinoma, and of these 13 said that it was in patients who took pioglitazone for a duration of more than two years. Only 7.8 per cent of physicians (n=58) categorically advocated banning pioglitazone, and the rest opined for its continuation or generating more evidence before decision could be taken regarding its use in T2DM. Interpretation & conclusions: Majority of the physicians though were aware of the regulatory changes with regard to pioglitazone, but their prescribing patterns were not changed for this drug. However, it was being used at lower than the recommended dose. There is a need for generating more evidence through improved pharmacovigilance activities and large-scale population-based prospective studies regarding the safety issues of pioglitazone, so as to make effectual risk-benefit analysis for its continual use in T2DM.

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Background & objectives: Cystic fibrosis (CF) is a life-limiting genetic condition resulting in chronic respiratory infections, pancreatic enzyme insufficiency and associated complications. This pilot study was undertaken to assess the glucose tolerance and insulin secretion and sensitivity among Indian children with CF. Methods: Children with CF under regular follow up at the Paediatric Pulmonology Clinic of a tertiary care hospital in New Delhi, India, were enrolled. Children who had a history of acute exacerbation or intake of systemic steroids within the last two weeks were excluded. Anthropometry, pulmonary function and disease severity (Shwachman) score were assessed. Fasting venous sample was drawn to assess glucose, insulin, haemoglobin and calcium. Oral glucose tolerance test was performed, and blood glucose and insulin were assessed at 30, 60, 90 and 120 min. Insulin secretion and sensitivity indices were calculated. Results: Twenty nine patients with a mean age of 11.2±4.1 yr were enrolled. Stunting, thinness, anaemia and hypocalcaemia were present in 31.0, 13.8, 37.0 and 48.3 per cent of the patients, respectively. Abnormal glucose tolerance (AGT) was present in 21.4 per cent. Insulin secretion was similar in individuals with AGT and normal glucose tolerance (NGT), but insulin sensitivity index was lower (0.12±0.02 vs 0.15±0.01, P<0.001) and homeostatic model assessment of insulin resistance higher [1.63 (0.53-1.76) vs 0.83 (0.28-4.43), P<0.05] in individuals with AGT compared to NGT. Interpretation & conclusions: AGT was observed in 21.4 per cent of children with CF. The CF patients with AGT had significantly lower insulin sensitivity compared to patients with NGT. Future multicentric studies with a large sample should be conducted to assess insulin secretion and sensitivity indices in CF patients compared to healthy controls.

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Background & objectives: With the use of early and appropriate use of antibiotics, outcomes have improved in the mother-infant dyads exposed to preterm pre-labour rupture of membranes (PPROM). This study was undertaken to evaluate immediate neonatal outcomes in infants born before 33 completed weeks of gestation to mothers with PPROM versus without PPROM. Methods: During the study period from January 2013 to December 2013, a total of 182 mother-infant dyads were prospectively included in the study. Among the enrolled, 69 were in the PPROM group and 113 in the control group (no PPROM). Mother-infant dyads in PPROM group were covered with antibiotics. The primary outcome was the combined adverse neonatal outcome consisting of sepsis, necrotizing enterocolitis >Stage II or pneumonia or oxygen at day 28 or cystic periventricular leucomalacia or mortality before discharge. Results: Baseline maternal and neonatal variables were comparable across the two groups, except for higher incidence of singletons, maternal pregnancy-induced hypertension (PIH) in the control group and higher proportion of males, complete steroid coverage and oligohydramnios in the PPROM group. The proportion of infants with combined adverse neonatal outcome was similar between the two groups [odds ratio (OR): 1.43; 95% confidence interval (CI): 0.77-2.6]. Both the groups were comparable for most other neonatal morbidities and outcomes, except screen-positive sepsis (OR: 3.7; 95% CI: 1.17-11.5) which was higher in PPROM group. Interpretation & conclusions: Mothers with PPROM and their newborns when treated with timely and appropriate antibiotics had neonatal outcomes similar to those not exposed to PPROM.

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Background & objectives: A high incidence of hearing impairment is reported from the village of Dhadkai in the State of Jammu and Kashmir, India. Prevalence of endogamy in this community suggested a common genetic basis for the disorder. A genetic study was undertaken to ascertain the basis for the high incidence of hearing impairment in this region. Methods: In a two-step approach to identify the causative mutation/s, a whole-genome-based linkage analysis of an extended family of 45 members was carried out, which included 23 affected and 22 unaffected members. Mutational analysis for the candidate deafness genes helped reveal causative mutations in the family. In addition, seven deafness-causing genes, Cx26, SLC26A4, CLDN14, TMPRSS3, TMC1, TMIE and USH1C, were analyzed in smaller families with hearing impairment. Results: In the 45-member extended family, the critical chromosomal region mapped to 2p24-p22.The c.2122C>T (p.R708X) mutation in OTOF in 2p24-p22was identified as being the causal change. Linkage to 2p24-p22 locus was not observed in a particular branch of this extended family. Analysis of seven known deafness-causing genes in this branch revealed a mutation, c.254T>A (p.V85D), in CLDN14. Among seven small families unrelated to the 45-member extended family, hearing loss was attributable to p.R708X in OTOF in three families and to p.V85D in CLDN14 in one family; a new mutation c.1668T>A (p.Y556X) SLC26A4 was identified in two families and the causative change could not be identified in one family. Interpretation & conclusions: This study suggested considerable genetic heterogeneity in the causation of hearing loss in Dhadkai. Recessive mutations were observed in at least three genes causing hearing loss: OTOF (p.R708X), SLC26A4 (p.Y556X) and CLDN14 (p.V85D). Mutation p.R708X appeared to be the major cause of hearing impairment in Dhadkai.

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Background & objectives: Acute myocardial infarction (AMI) is a major health concern in India. The aim of the study was to identify single nucleotide polymorphisms (SNPs) associated with AMI in patients using dedicated chip and validating the identified SNPs on custom-designed chips using high-throughput microarray analysis. Methods: In pilot phase, 48 AMI patients and 48 healthy controls were screened for SNPs using human CVD55K BeadChip with 48,472 SNP probes on Illumina high-throughput microarray platform. The identified SNPs were validated by genotyping additional 160 patients and 179 controls using custom-made Illumina VeraCode GoldenGate Genotyping Assay. Analysis was carried out using PLINK software. Results: From the pilot phase, 98 SNPs present on 94 genes were identified with increased risk of AMI (odds ratio of 1.84-8.85, P=0.04861-0.003337). Five of these SNPs demonstrated association with AMI in the validation phase (P=0.05). Among these, one SNP rs9978223 on interferon gamma receptor 2 [IFNGR2, interferon (IFN)-gamma transducer 1] gene showed a significant association (P=0.00021) with AMI below Bonferroni corrected P value (P=0.00061). IFNGR2 is the second subunit of the receptor for IFN-gamma, an important cytokine in inflammatory reactions. Interpretation & conclusions: The study identified an SNP rs9978223 on IFNGR2 gene, associated with increased risk in AMI patient from India.

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Background & objectives: Data on serial cervical length (CL) measurements in pregnancy at term to predict spontaneous labour onset are scarce and conflicting. This study was conducted to observe CL changes preceding spontaneous onset of labour, by serial transvaginal sonography (TVS) and transabdominal sonography (TAS), in nulliparous Indian women near term. Methods: Only nulliparous women with a singleton foetus in cephalic presentation and who confirmed their gestational age were recruited. Sonographic CL measurements were taken at weekly intervals from 36 wk gestation onwards by a single ultrasonologist. Transabdominal and transvaginal measurements were undertaken using the suitable transducer probes with the women in the supine position. Results: A total of 104 women with spontaneous onset of labour were evaluated. There was substantial variation in CL measurements, both by TVS and by TAS, from 36 to 40 wk gestation, although the two sets of measurements correlated closely. Mean CL changed significantly over the last three weeks before delivery. However, only one-third of the women showed CL change of >5 mm per week in the last three weeks. There was poor correlation between gestational age at delivery and the last measured CL, either by TVS or TAS. Length >3.1 mm, measured by TVS at 38 wk gestation, predicted post-dated pregnancy to a limited extent. Interpretation & conclusions: Inter-individual variations in CL and in CL changes were large. Thus, it was not practical to predict spontaneous onset of labour by sonographic CL measurement near term. Post-dated pregnancy may be predicted with limited success. Further studies should explore other parameters, in addition to CL.

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Background & objectives: Infectious complications have been reported to occur in up to 45 per cent of patients, following pancreatoduodenectomy (PD). The incidence of perioperative infectious and overall complications is higher in patients undergoing preoperative invasive endoscopic procedures. The aim of the study was to compare the role of a carbapenem administered as three-once daily perioperative doses on infectious complications in patients at high risk for these complications versus those at low risk. Methods: A retrospective study with some secondary data collected from records was carried out on the data from a prospectively maintained surgical database of patients undergoing PD for pancreatic and periampullary lesions at a tertiary referral care centre, between June 2011 and May 2013. Patients were divided into two groups for comparison based on whether they underwent at least one preoperative endoscopic interventional procedure before PD (high-risk - intervention and low-risk - no intervention). All patients were administered three-once daily doses of ertapenem (1 g). Results: A total of 135 patients in two groups were comparable in terms of demographic and nutritional, surgical and histopathological factors. No significant difference between the two groups in terms of the overall morbidity (38.7 vs 35.7%), infectious complications (9.7 vs 4.8%), mortality (2.2 vs 2.4%) and mean post-operative hospital stay (9.2 vs 8.9 days) was observed. Interpretation & conclusions: Perioperative three-day course of once-daily administered ertapenem resulted in a non-significant difference in infectious and overall complications in high-risk patients undergoing PD as compared to the low-risk group.

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Culture-negative bacteraemia has been an enigmatic entity with respect to its aetiological agents. In an attempt to actively identify those positive blood cultures that escape isolation and detection on routine workflow, an additional step of MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) based detection was carried out directly from the flagged blood culture bottles. Blood samples from 200 blood culture bottles that beeped positive with automated (BACTEC) system and showed no growth of organism on routine culture media, were subjected to analysis by MALDI-TOF MS. Forty seven of the 200 (23.5%) bacterial aetiology could be established by bottle-based method. Based on these results, growth on culture medium could be achieved for the isolates by providing special growth conditions to the fastidious organisms. Direct identification by MALDI-TOF MS from BACTEC-positive bottles provided an opportunity to isolate those fastidious organisms that failed to grow on routine culture medium by providing them with necessary alterations in growth environment.

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Background & objectives: Coxsackievirus B (CVB), a member of human Enterovirus group, is the most common cause of viral myocarditis. Coxsackievirus adenovirus receptor (CAR) is identified as a key determinant for the entry of CVB in the target cells. Thus, blockade of receptor by RNA interference (RNAi) may inhibit the entry and pathogenesis of CVB in cardiac cells. The present study was aimed to determine the effect of CAR small dsRNA (siRNA) on coxsackieviral load and CAR expression in coxsackievirus-infected cardiomyocytes. Methods: Transfection efficiency in rat cardiomyocytes (H9c2) was determined by the fluorescent microscopy and flow cytometry. CAR siRNA dose was optimized based on cell viability and relative CAR messenger RNA (mRNA) expression. Cardiomyocytes were transfected with CAR siRNA followed by infection with 100 multiplicity of infection of CVB, which were harvested after 24, 48 and 72 h post-infection (p.i.). RNA was extracted for relative CAR mRNA expression. Cells were freeze-thawed thrice for estimating coxsackieviral load. Results: The efficiency of transfection was optimized to be >80 per cent and CAR siRNA dose of 60 pmol was standardized. The knockdown of CAR by siRNA decreased its expression twice the expression in normal cardiomyocytes after 24 h p.i. of CVB. The treatment with CAR siRNA resulted in significant two log reduction of CVB load in cardiomyocytes infected with CVB at 24 h p.i. and retained till 72 h p.i. Interpretation & conclusions: The inhibition of CAR by siRNA was found to be effective against CVB in cardiomyocytes. However, this treatment strategy has to be evaluated in vivo to develop a new treatment strategy for patients suffering with viral myocarditis.

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Background & objectives: Human dirofilariosis is a well-recognized zoonosis caused by several species of the genus Dirofilaria. The disease is prevalent among canines and human beings in Kerala. The objective of the present study was to confirm the human Dirofilaria isolates by molecular characterization. Methods: The worms or segments obtained from human sources were subjected to diagnostic polymerase chain reaction (PCR) targeting the cytochrome oxidase subunit 1 (COI) and 5S ribosomal RNA (rRNA) genes of Dirofilaria repens. The amplicons were sequenced and analyzed. Results: The filariid nematodes recovered from ocular as well as subcutaneous tissue of human origin were identified as D. repens based on PCR targeting COI as well as 5S rRNA genes. The phylogenetic analysis of the COI gene nucleotide sequence obtained in the present study showed that D. repens shared the closest evolutionary relationship with D. honkongensis. Interpretation & conclusions: Molecular identification of D. repens isolated from human source assumes significance from the point of zoonotic threat of this mosquito-borne nematode. Phylogenetic analysis revealed a close evolutionary relationship with Asian isolate of D. honkongensis. Timely detection and treatment of infection in dogs, together with mosquito control, should be an integral part of the control strategy of this disease.

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