Paban Kumar Dash

Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases.

Loop mediated isothermal amplification (LAMP) is a powerful innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases. The whole procedure is very simple and rapid wherein the amplification can be completed in less than 1 h under isothermal conditions employing a set of six specially designed primers spanning eight distinct sequences of a target gene, by incubating all the reagents in a single tube. Gene amplification products can be detected by agarose gel electrophoresis as well as by real‐time monitoring in an inexpensive turbidimeter. Gene copy number can also be quantified with the help of a standard curve generated from different concentrations of gene copy number plotted against time of positivity with the help of a real‐time turbidimeter. Alternatively, gene amplification can be visualised by the naked eye either as turbidity or in the form of a colour change when SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. LAMP does not require a thermal cycler and can be performed simply with a heating block and/or water bath. Considering the advantages of rapid amplification, simple operation and easy detection, LAMP has potential applications for clinical diagnosis as well as surveillance of infectious diseases in developing countries without requiring sophisticated equipment or skilled personnel. Copyright

Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay.

ABSTRACT The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3′ noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.

Rapid and Real-Time Detection of Chikungunya Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Assay

ABSTRACT The standardization and validation of a one-step, single-tube, accelerated, quantitative reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene for the rapid and real-time detection of Chikungunya virus (CHIKV) are reported. A linear relationship between the amount of template and time of positivity value over a range of 2 × 108 to 2 × 102 copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The comparative evaluation of the RT-LAMP assay with acute-phase patient serum samples demonstrated exceptionally higher sensitivity by correctly identifying 21 additional positive borderline cases that were missed by conventional RT-PCR (P < 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient serum samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2 × 108 to 2 × 101 copies. In addition, the field applicability of the RT-LAMP assay was also demonstrated by standardizing SYBR Green I-based RT-LAMP wherein the amplification was carried out in a water bath at 63°C for 60 min, which was followed by monitoring gene amplification with the naked eye through color changes. These findings demonstrated that the RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute-phase serum samples without requiring any sophisticated equipment and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries.

Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956–2007

BackgroundDengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV-1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001–2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis.ResultsThe region was found to be AT rich with no insertion or deletion. Majority of the nucleotide substitutions were silent, except 3 non-conservative amino acid changes (I → T, A → T and L → S at amino acid positions 59,114 and 155 respectively) in the Indian DENV-1 sequences, sequenced in this study. Except two 1997–98 Delhi isolates, which group in genotype I; all other Indian isolates group in genotype III. All Indian genotype III DENV-1 exhibited diversity among them, giving rise to at least 4 distinct lineages (India 1–4) showing proximity to isolates from diverse geographic locations.ConclusionThe extensive phylogenetic analysis revealed consistent existence of multiple lineages of DENV-1 genotype III during the last 5 decades in India.

Reemergence of dengue virus type-3 (subtype-III) in India: Implications for increased incidence of DHF & DSS

BackgroundDengue virus infection has recently taken endemic proportion in India implicating all the four known dengue serotypes. There was a major dengue outbreak in northern India including Delhi in October- December, 2003 and again in 2004. We have carried out a detailed investigation of the 2004 outbreak by Serosurveillance, RT-PCR, nested PCR, virus isolation and genotyping. We also report the molecular epidemiological investigation of these outbreaks.ResultsThe serological investigation of 162 suspected serum samples using an in-house dengue dipstick ELISA revealed 11%-IgM, 51%-IgG and 38%-both IgM and IgG antibody positivity. The RT-PCR analysis revealed presence of dengue RNA in 17 samples. Further subtyping and genotyping by nested PCR and nucleotide sequencing of C-prM gene junction revealed the association of subtype III of dengue virus type 3 in the outbreak.ConclusionThe sudden shifting and dominance of the dengue virus serotype-3 (subtype III) replacing the earlier circulating serotype-2 (subtype IV) is a point of major concern and may be attributed to increased incidence of DHF and DSS in India.

Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus.

The recent resurgence of Chikungunya virus (CHIKV) in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, no approved licensed vaccine is currently available. In the present study, a Vero cell adapted purified formalin inactivated prototype vaccine candidate was prepared using a current Indian strain implicated with the explosive epidemic during 2006. The bulk preparation of the vaccine candidate was undertaken in microcarrier based spinner culture using cytodex-1 in virus production serum free medium. The inactivation of the virus was accomplished through standard formalin inactivation protocol. The mice were immunized subcutaneously with alhydrogel gel formulation of inactivated virus preparation. The assessment of both humoral and cell-mediated immune response was accomplished through ELISA, plaque reduction neutralization test (PRNT), microcytotoxicity assay and cytokine production assay. The results revealed that formalin inactivated vaccine candidate induced both high titered ELISA (1:51,200) and plaque reduction neutralizing antibodies (1:6400) with peak antibody titer being observed during 6 -- 8 weeks of post-vaccination. In the absence of suitable murine challenge model, the protective efficacy was established by both in vitro and in vivo neutralization tests. Further assessment of cellular immunity through in vitro stimulation of spleenocytes from immunized mice revealed augmentation of high levels of both pro- and anti-inflammatory cytokines, indicating a mixed balance of Th1 and Th2 response. These findings suggest that the formalin inactivated Chikungunya vaccine candidate reported in this study has very good immunogenic potential to neutralize the virus infectivity by augmenting both humoral and cell-mediated immune response.

Japanese Encephalitis Outbreak, India, 2005

An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3.

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid and Real-Time Detection of Japanese Encephalitis Virus

ABSTRACT The standardization and validation of a one-step, single-tube accelerated quantitative reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay is reported for rapid and real-time detection of Japanese encephalitis virus (JEV). The RT-LAMP assay reported in this study is very simple and rapid; the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of six primers targeting the E gene of JEV. The RT-LAMP assay demonstrated exceptionally higher sensitivity compared to that of RT-PCR, with a detection limit of 0.1 PFU. The specificities of the selected primer sets were established by cross-reactivity studies with other closely related members of the JEV serocomplex as well as by evaluation of healthy human volunteers. The comparative evaluation of the RT-LAMP assay for clinical diagnosis with a limited number of patient cerebrospinal fluid samples revealed 85% concordance with conventional RT-PCR, with a sensitivity and a specificity of 100% and 86%, respectively. The concentration of virus in most of the clinical samples was 102 to 105 PFU/ml, as determined from the standard curve based on the time of positivity in the samples. In addition, the monitoring of gene amplification can also be visualized with the naked eye by using SYBR green I fluorescent dye. Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool for the rapid and real-time detection of JEV not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.

RNA interference mediated inhibition of Chikungunya virus replication in mammalian cells

Chikungunya has emerged as one of the most important arboviral infection of public health significance. Recently several parts of Indian Ocean islands and India witnessed explosive, unprecedented epidemic. So far, there is no effective antiviral or licensed vaccine available against Chikungunya infection. RNA interference mediated inhibition of viral replication has emerged as a promising antiviral strategy. In this study, we examined the effectiveness of small interfering RNAs (siRNAs) against the inhibition of Chikungunya virus replication in Vero cells. Two siRNAs against the conserved regions of nsP3 and E1 genes of Chikungunya virus were designed. The siRNA activity was assessed by detecting both the infectious virus and its genome. The results indicated a reduction of virus titer up to 99.6% in siRNA transfected cells compared to control. The viral inhibition was most significant at 24h (99%), followed by 48 h (65%) post infection. These results were also supported by the quantitative RT-PCR assay revealing similar reduction in Chikungunya viral genomic RNA. The siRNAs used had no effect on the expression of house keeping gene indicating non-interference in cellular mechanism. The specific and marked reduction in viral replication against rapidly replicating Chikungunya virus achieved in this study offers a potential new therapeutic approach. This is the first report demonstrating the effectiveness of siRNA against in vitro replication of Chikungunya virus.

Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

BackgroundDengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported.ResultsAn optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever) and alphavirus (Chikungunya). The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation vis a vis conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only.ConclusionThese findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.