Rapid detection of carbapenem resistance among gram-negative organisms directly from positive blood culture bottles.

Background: Carbapenemase producing gram-negative bacteria (GNB) has become a huge problem in majority of tertiary care centers worldwide. They are associated with very high morbidity and mortality rates, especially when they cause invasive infections. Therefore, rapid detection of these organisms is very important for prompt and adequate antibiotic therapy as well as infection control. The aim of this study was rapid detection of carbapenemase genes and thereby likely carbapenem resistance, 24-48 hours in advance, directly from the positive-flagged blood culture bottles using CHROMagar and Xpert® Carba-R. Methods: Aspirate from positively flagged blood culture bottles was subjected to differential centrifuge. All gram-negative bacilli on gram stain from the deposit were processed in Xpert® Carba-R and inoculated on CHROMagar. The presence of genes and growth on CHROMagar was compared with carbapenem resistance on VITEK-2 Compact. Results: A total of 119 GNB isolates were processed. One or more of the carbapenemase genes were detected in 80 isolates. On comparison with VITEK-2 result, 92 samples showed concordance for carbapenem resistance 48 hours in advance. There was discordance in 21 isolates with 12 major errors and 09 minor errors. The sensitivity of direct Xpert® Carba-R test for rapid detection of carbapenem resistance, 48 hours in advance, was 81.42%. The sensitivity of direct CHROMagar test for accurate detection of carbapenem resistance, 24 hours in advance, was 92.06%. Conclusion: The ability to detect carbapenem resistance with very high accuracy, 48 hours in advance, helps in appropriate antibiotic therapy and implementation of effective infection control practices.

Detection of dengue virus serotypes by single-tube multiplex RT-PCR and multiplex real-time PCR assay.

Background: All four dengue serotypes cause infection, with one of them predominantly reported from a particular geographical region. Coinfection by more than one serotype is reported from hyperendemic regions. These coinfections are clinically more severe than infection with a single serotype. This study was carried out to detect the predominant dengue serotype and presence of coinfections. Methods: Acute-phase serum samples of patients suffering from dengue infection were collected. They were screened for the presence of IgM, IgG and NS1Ag by a rapid test. Conventional multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and multiplex real-time RT-PCR assays were carried out for detection and serotyping of the dengue virus. Results: A total of 196 samples were positive by the rapid card test. Of these, 139 were NS1Ag positive, 40 were positive only for IgM, 5 were positive only for IgG and 12 samples were positive for different combinations of antigen and antibodies. All four serotypes were detected in these samples by PCR. DENV-3 was found to be most common circulating serotype. A total of 22 cases were found to have coinfection with more than one dengue serotypes. Samples having only antibodies and no antigen on rapid card test were also positive for virus by PCR. Conclusion: Prevalence of dengue co-infections is increasing. Moreover, it is important to screen for dengue virus in those samples also which do not show NS1Ag on rapid tests and have either one or both the antibodies. Real-time multiplex RT-PCR is found to be more sensitive in detecting coinfection than conventional multiplex RT-PCR.

Whole genome sequence of colistin-resistant Escherichia coli from western India.

BACKGROUND: With virtually dried out new antibiotic discovery pipeline, emergence and spread of antimicrobial resistance is a cause for global concern. Colistin, a cyclic polypeptide antibiotic, often regarded as last resort for multi drug resistance gram-negative bacteria, is also rendered ineffective by horizontal transfer of resistance genes. Surveillance of colistin resistance in GNB is essential to ascertain molecular epidemiology. METHODS: Whole genome sequencing (WGS) of an unusual colistin resistant urinary isolate of Escherichia coli was performed using Illumina MiSeq platform using 2x250bp V2 chemistry by following the manufactures protocol (Illumina Inc. USA). Multiple web-based bio-informatic tools were utilized to ascertain antibiotic resistant genes. RESULTS: An approximate 5.4 Mb of genome of the urinary isolate AFMC_UC19 was sequenced successfully. Mobile colistin resistance gene (mcr) on the plasmid responsible for horizontal spread was absent in the isolate. CONCLUSION: Colistin resistance has been reported previously in Klebsiella pneumoniae and it is a rare occurrence in Escherichia coli in Indian setting. Although the isolate lack mcr mediated colistin resistance, emergence and spread of colistin resistant in gram-negative bacteria pose a threat.

Estimating the proportion and IgG antibody response to SARS-CoV-2 in individuals joining a central educational institute from different parts of India by Enzyme-linked immunosorbent assay (ELISA) based serology.

BACKGROUND: Antibody response to SARS-CoV may be estimated to give trends and patterns emerging in a population during an evolving epidemic. The novel coronavirus has opened a new chapter in the history of pandemics and understanding the disease epidemiology. METHODS: The study was a cross-sectional descriptive study. Institutional Ethical clearance and informed consent were taken for participation in the study. The study population included all personnel reporting to the institute for training courses, permanent posting or joining back from leave during the study period of 2 months (16 June to 16 August 2020). The sample size was calculated assuming the prevalence of COVID-19 to be 1% with the absolute precision of 0.5% and 5% level of significance, and finite correction for population size of 500, and the calculated sample size was 377. Inclusion criteria were all personnel reporting to the institute from different states and districts. Exclusion criteria-Any personnel reported for a short visit of lesser than 14 days. Demographic details and details of any likely exposure to a confirmed COVID-19 case were noted. A blood sample was collected, and serological tests were done using ErbaLisa COVID-19 IgG kit by Calbiotech, as per the manufacturer's instructions. RESULTS: Overall seropositivity of IgG COVID-19 antibodies was 7.5% (31/413) (95% CI: 5.3-10.4%). Study population (n = 413) comprised of an adult population in the age range of 21 years-53 years, and the mean age was 31.4 years (SD = 6.2 years). CONCLUSION: As the personnel joining the institute have come from various parts of the country the study provides an estimation of antibodies against COVID-19.

Direct antimicrobial susceptibility testing from positive blood culture bottles in laboratories lacking automated antimicrobial susceptibility testing systems.

BACKGROUND: Timely initiation of appropriate antimicrobial can improve the outcome in terms of reduced morbidity and mortality in addition to reduced health-care costs. Availability of early preliminary Antimicrobial Susceptibility Test (AST) report will be useful in directing antimicrobial therapy. The aim of the study was to correlate AST by disc diffusion method, directly from positively flagged blood culture bottles, with the AST by automated method. METHODS: A total of 144 aerobic blood culture bottles flagged positive by the automated blood culture system were processed. The bacteria were pelleted by two-step centrifugation of the broth from the bottle and used to make a smear for Gram stain as well as an inoculum for antimicrobial sensitivity testing by Kirby Bauer disc diffusion method. Automated identification and AST were also carried out. RESULTS: On direct staining, 94 samples showed gram-negative bacilli, 39 showed gram-positive cocci, and 11 showed yeasts or polymicrobial growth. In the case of gram-negative bacteria, there was 99% categorical agreement between direct sensitivity testing and automated sensitivity testing with 1% disagreement. Among the gram-positive cocci, there was 96% categorical agreement with 4% disagreement between the two methods. CONCLUSION: High degree of agreement between the two methods is promising and applicable to situations where automated sensitivity testing is not available. Even if the systems are available, this method would prove useful as an adjunct to standard AST reporting. This sensitivity report can be generated earlier than the conventional AST, enabling choice of appropriate antimicrobial.

Molecular characterization and phylogenetic analysis of Chikungunya virus from Delhi, India.

BACKGROUND: Chikungunya virus is an alpha virus with high similarity to Dengue and Zika viruses, both in transmission cycle and in clinical presentation. Chikungunya is a re-emerging mosquito-borne infection known to cause small to very large outbreaks/epidemics at frequent intervals. In 2016, India witnessed a large outbreak of Chikungunya infection affecting more than 58,000 people. This study was undertaken to look at the genotypic phylogeny to know the relatedness with previously reported strains. METHODS: During the 2016 outbreak, samples from all patients clinically suspected to have Chikungunya were collected and subjected to testing for IgM antibody by ELISA and viral RNA detection by RT-PCR. Sequencing of the E1 gene segment was done to create a phylogenetic tree comparison with other strains. RESULTS: Serum samples were collected from 142 patients of clinically suspected Chikungunya infection. Majority of the patients were in the age group of 31-50 years accounting for more than 35% of the total cases. Twenty eight samples were positive for IgM antibody. Thirty seven samples were positive for viral RNA by RT-PCR. Only 06 cases were positive by both tests. Mutations in the amino acids K211E, M269V and D284E in the E1 gene segment of the Chikungunya virus were observed in the seven strains that were sequenced. On phylogeny tree, all the strains were found to belong to the ECSA genotype. CONCLUSION: Actively searching for the potential epidemic causing mutations and reporting of novel mutations may help in better understanding and probably forecasting of future CHIKV outbreaks and its nature.

Molecular detection of blaNDM-1 (New Delhi metallobetalactamase-1) in nosocomial Enterobacteriaceae isolates by nested, multiplex polymerase chain reaction.

BACKGROUND: Carbapenems are considered "drugs of last resort" in many life-threatening infections. Advent of carbapenemases like KPC, OXA-48, VIM, IMP, and NDM have greatly affected the efficacy of these drugs, posing serious threat to global health and infection control. NDM bears special significance to the India subcontinent, labeled as place of origin and reservoir. NDM tends to escape detection by routine phenotypic methods, requiring molecular confirmation. This study utilizes nested, multiplex polymerase chain reaction (PCR) for reliable detection of blaNDM-1 in nosocomial Enterobacteriaceae isolates. METHODS: This study was conducted to detect prevalence of blaNDM-1, blaIMP, blaVIMand blaKPC genes by multiplex PCR among multidrug/carbapenem-resistant nosocomial Enterobacteriaceae isolates. From March 2013 to April 2014, 100 consecutive non-repeat isolates of Enterobacteriaceae from various inpatient clinical samples were analyzed. Imipenem-resistant isolates identified by Kirby Bauer disk diffusion method with Clinical and Laboratory Standards Institute guidelines were further subjected to nested, multiplex PCR to simultaneously detect blaNDM-1, blaIMP, blaVIMand blaKPC genes. RESULTS: Out of 100 isolates, 17 (17%) were found to be imipenem-resistant. blaNDM-1 was detected in all 17 isolates by nested, multiplex PCR. blaVIM was co-carried in 4 isolates while one isolate co-harbored blaIMP with blaNDM-1. Imipenem resistance and NDM-1 carriage was predominant amongst Klebsiella isolates. Maximum NDM-1 producers were isolated from the intensive care unit (70.6%). CONCLUSION: NDM-1 prevalence in nosocomial Enterobacteriaceae isolates in our hospital was found to be 17%. A nested, multiplex PCR was used for rapid detection of various carbapenemase genes with high sensitivity and specificity which is essential not only for favorable patient outcome but also for timely implementation of appropriate infection control practices to prevent further spread of such organisms.