Sharma Vp

Evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of Influenza A viruses

n Abstractn n Backgroundn Influenza A viruses (IAVs) have always remain a serious concern for the global economy and public health. A rapid, specific and sensitive detection method is always needed to control the influenza in its early stages by timely intervention of therapy and early clinical management.n n n Objectivesn To develop RT-LAMP assays for detection of influenza A viruses, their further subtyping into seasonal (H1N1, H3N2) and novel pandemic H1N1 viruses and to evaluate clinical applicability of optimized RT-LAMP assays on patients’ samples.n n n Study designn In this study, we optimized RT-LAMP assay to detect IAVs by using primers against matrix gene and subtyping of IAVs was done by using primers against hemagglutinin gene. Optimized RT-LAMP assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step RT-PCR and real-time RT-PCR.n n n Resultsn RT-LAMP assays successfully detected and differentiated IAVs into H1N1, H3N2 and pdm09/H1N1 subtypes. One hundred and sixty seven clinical swab samples from influenza suspected patients were taken and tested with RT-LAMP assay, detecting 30 (17.9%) samples positive for Influenza A virus. Out of 30 samples, 21, 7 and 2 were found positive for pdm09/H1N1, H3N2 and seasonal H1 respectively. Conventional one-step RT-PCR detected a total of 27 (16.2%) samples for influenza A and further subtyping showed 20 and 7 samples positive for pdm09/H1N1 and H3N2 virus respectively whereas none was found positive for seasonal H1N1. RT-LAMP assay demonstrated higher sensitivity (93.8%) than conventional RT-PCR (84.4%) for influenza A viruses detection in clinical samples.n n n Conclusionsn RT-LAMP assay is rapid, sensitive, specific and cost effective method for detection of influenza A viruses than conventional one-step RT-PCR and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries.n n

Comparative Analysis of Molecular Methods for Detection of Influenza Viruses

Aims: Influenza is a serious threat to human population worldwide therefore continuous surveillance is required to update influenza seasonal vaccines. A rapid, sensitive, specific and cost effective diagnostic method will be much helpful for patient management in the present scenario. Present study is conceptualized for detection of influenza viruses by molecular methods and compare with ‘gold standard’ virus isolation. Study Design: Standard strains of Influenza virus were used to standardize the molecular diagnostic assays and results were then compared with virus isolation. Place and Duration of Study: Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India, between December 2015 and April 2016. Methodology: Standard strains of Influenza A and B virus were used for influenza virus isolation using virus culturing in MDCK (Madin-Darby Canine Kidney) cell line by following standard tissue culture procedure. Isolated viruses were detected by Hemagglutination assay (HA) and typed by Hemagglutination inhibition assay (HI). Conventional one step RT-PCR, Taqman real time RT-PCR and RT-LAMP (Reverse transcription loop mediated isothermal amplification) were standardized on RNA extracted from standard strains. Sensitivity and specificity of these molecular methods were Original Research Article Sharma and Kaushik; BMRJ, 17(3): 1-10, 2016; Article no.BMRJ.28858 2 compared with each other as well as with virus culture (gold standard). Results: Both influenza A and B virus strains were cultured in MDCK cells and produced cytopathic effect during virus culture. Conventional RT-PCR and real time RT-PCR detected both type of Influenza viruses. RT-LAMP also successfully detected and typed influenza viruses. RTLAMP proved to be more rapid than other two molecular assays. Conclusion: Molecular diagnostic methods are useful in detection and typing of Influenza viruses and these methods provide results in short period of time when compared with traditional virus culture methods. RT-LAMP is rapid, sensitive, specific and cost effective method for influenza virus detection and subtyping.

Emerging trends of Nipah virus: A review.

Since emergence of the Nipah virus (NiV) in 1998 from Malaysia, the NiV virus has reappeared on different occasions causing severe infections in human population associated with high rate of mortality. NiV has been placed along with Hendra virus in genus Henipavirus of family Paramyxoviridae. Fruit bats (Genus Pteropus) are known to be natural host and reservoir of NiV. During the outbreaks from Malaysia and Singapore, the roles of pigs as intermediate host were confirmed. The infection transmitted from bats to pigs and subsequently from pigs to humans. Severe encephalitis was reported in NiV infection often associated with neurological disorders. First NiV outbreak in India occurred in Siliguri district of West Bengal in 2001, where direct transmission of the NiV virus from bats‐to‐human and human‐to‐human was reported in contrast to the role of pigs in the Malaysian NiV outbreak. Regular NiV outbreaks have been reported from Bangladesh since 2001 to 2015. The latest outbreak of NiV has been recorded in May, 2018 from Kerala, India which resulted in the death of 17 individuals. Due to lack of vaccines and effective antivirals, Nipah encephalitis poses a great threat to public health. Routine surveillance studies in the infected areas can be useful in detecting early signs of infection and help in containment of these outbreaks.

Molecular Detection of Hepatitis C Virus (HCV) by Conventional One-step RT-PCR Coupled with Nested PCR

Aims: HCV causes both acute and chronic infections and can be easily transmitted through contaminated blood or other body fluids. The present study deals with the molecular detection of HCV with help of one-step RT-PCR assay followed by nested PCR and agarose gel electrophoresis. Study Design: RNA extracted from the confirmed positive samples of HCV was utilized for the standardization of the one-step RT-PCR assay and nested PCR assay for diagnosis of HCV. Place and Duration of Study: Centre for Biotechnology, Maharshi Dayanand University, Rohtak Haryana, India, during period of one year (January-December 2015). Methodology: HCV positive samples were obtained from Department of Medicine, Maulana Azad Medical College (MAMC), New Delhi, India. Published primers from most conserved regions of HCV were taken and these primers were able to amplify all the strains of HCV. One-step RT-PCR kits, Short Research Article Sharma et al.; IBRR, 7(2): 1-6, 2017; Article no.IBRR.34463 2 primers, extracted RNA from these positive samples were used for standardization of molecular diagnostic assays. The results were checked by 2% agarose gel electrophoresis. Results: Positive samples of HCV were detected by nested PCR. Positive samples showed sharp band of 405bp while there was no amplification in the negative control. Conclusion: Rapid tests have low sensitivity and specificity while molecular assays are rapid, sensitive and specific. Conventional one-step RT-PCR assay followed by nested PCR is rapid, specific, sensitive and it is also less costly than real-time RT-PCR. Cost of an assay is an important factor in controlling a disease in resource limited settings of developing countries.