Development and validation of LCMS method for determination of Ethyl Glucuronide (EtG) in urine.

Background: Ethyl glucuronide (EtG) is a phase II metabolite of ethanol and is an upcoming biomarker for recent alcohol intake. Monitoring of alcohol intake in case of alcohol-dependent syndrome is very useful for early intervention and preventing harmful effects. EtG has also been identified as a very useful marker in differentiating antemortem ingestion of alcohol from postmortem production of alcohol. This study was undertaken with an objective of developing a sensitive and specific method for determination of EtG in urine. Methods: Triple quadruple Liquid Chromatography (LC)-Mass Spectrometry (MS) with Electrospray Ionization (ESI) negative mode has been used for developing the multiple reaction monitoring method by using the Polaris 3 C 18-Ether analytical column. A simple sample preparation method was adopted using the Bond Elute Plexa PAX SPE cartridge. The developed method was also tested on actual urine samples from 15 individuals after consumption of 60 and 90 ml of whiskey at different time intervals. Results: A simple method was developed for determination of EtG in urine, with a sensitivity of 100 ppb and a recovery of 75%. Validation of the method on urine samples revealed that EtG could be detected for up to 18 h in individuals who ingested 60 ml of whiskey and up to 24 h in those who ingested 90 ml of whiskey. Conclusion: The simple method was developed for determination of EtG in urine and validated on actual urine samples. This method can now be used in aircraft accident investigation to differentiate postmortem production of alcohol, and the method is also a very useful tool to monitor Alcohol dependent Syndrome (ADS) cases.

Determination of serotonin metabolites in urine sample by liquid chromatography-mass spectrometry as biomarkers of recent alcohol intake: Implication for aircraft accident investigation.

BACKGROUND: Determination of ethanol levels in aircraft accident victims constitutes an important part of investigation. However, postmortem production of alcohol by microbial fermentation is known to interfere with the results. Distinguishing postmortem produced alcohols from antemortem ingested is very important in interpretation of results. Ratio of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA) metabolites of serotonin, has known to provide a convenient, rapid, and reliable solution as antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio after ingestion of alcohol (5-HTOL/5-HIAA = >15 pm/nm). METHODS: Triple quadruple (QQQ) liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization positive mode has been used for development of single tube multiple reaction monitoring (MRM) method for simultaneous quantification of 5-HTOL and 5-HIAA in urine. Deglucuronidation of 5-HTOL glucuronide in urine by beta-glucuronidase followed by simple sample preparation has been adopted. Examination of the ratio on urine samples from 15 individuals after consumption of 60 and 90 ml of whiskey has been carried out at different time interval. RESULTS: A single method for analysis of both the analytes was developed with sensitivity of 50 ppb and recovery of around 80-90%. Examination of the ratio on urine samples revealed that the ratio was >15 in all groups consuming 60 ml and 90-ml whiskey up to 12 h after alcohol ingestion. CONCLUSION: This is a unique highly sensitive single LC-MS method, which has been developed for simultaneous estimation of both 5-HTOL and 5-HIAA on same instrument for proving antemortem alcohol ingestion with high degree of sensitivity and specificity.

Study of acute hypoxia markers in healthy subjects: Utility in post-crash investigation.

BACKGROUND: Lactic acid is being routinely used as a marker of hypoxia in aircrash investigation. Since lactic acid estimation as a marker of hypoxia in postmortem samples for aircrash investigation is prone to many interfering factors, like the postmortem production and hemolysis. A study was carried out to evaluate other hypoxia markers other than lactic acid which could be later added as markers of hypoxia in postmortem investigations of aircraft accidents. METHODS: 25 healthy males of age 20-40 yrs volunteered participants were subjected to an simulated altitude of 15,000 ft for 30 min and the mean plasma concentration of Hypoxia Inducing Factor 1α (HIF 1α), Erythropoietin (EPO), Vascular Endothelial Growth Factor (VEGF) and lactic acid (LA) were analyzed from their venous blood sample collected at 4 intervals viz. Ground level pre exposure, 15,000 ft at 15 min, 15,000 ft at 30 min and Ground level 3 h post exposure. RESULTS: Statistical analysis revealed significant increase in mean plasma concentration of lactic acid, HIF-1α and EPO on exposure for duration of 15 min and 30 min at an altitude of 15,000 ft. CONCLUSION: Our study reveals that HIF-1α and EPO are sensitive to hypoxia exposure as compared to lactic acid and can be used in association with LA as hypoxia markers. However stability of these proteins in postmortem conditions needs to be studied and the potential for estimation of mRNA transcripts of HIF-1α and EPO, which would be stable in postmortem conditions, can be explored.

Clinical presentation, etiology, and survival in adult acute encephalitis syndrome in rural Central India.

BACKGROUND: Acute encephalitis syndrome (AES) is a constellation of symptoms that includes fever and altered mental status. Most cases are attributed to viral encephalitis (VE), occurring either in outbreaks or sporadically. We conducted hospital-based surveillance for sporadic adult-AES in rural Central India in order to describe its incidence, spatial and temporal distribution, clinical profile, etiology and predictors of mortality. METHODS: All consecutive hospital admissions during the study period were screened to identify adult-AES cases and were followed until 30-days of hospitalization. We estimated incidence by administrative sub-division of residence and described the temporal distribution of cases. We performed viral diagnostic studies on cerebrospinal fluid (CSF) samples to determine the etiology of AES. The diagnostic tests included RT-PCR (for enteroviruses, HSV 1 and 2), conventional PCR (for flaviviruses), CSF IgM capture ELISA (for Japanese encephalitis virus, dengue, West Nile virus, Varicella zoster virus, measles, and mumps). We compared demographic and clinical variables across etiologic subtypes and estimated predictors of 30-day mortality. RESULTS: A total of 183 AES cases were identified between January and October 2007, representing 2.38% of all admissions. The incidence of adult AES in the administrative subdivisions closest to the hospital was 16 per 100,000. Of the 183 cases, a non-viral etiology was confirmed in 31 (16.9%) and the remaining 152 were considered as VE suspects. Of the VE suspects, we could confirm a viral etiology in 31 cases: 17 (11.2%) enterovirus; 8 (5.2%) flavivirus; 3 (1.9%) Varicella zoster; 1 (0.6%) herpesvirus; and 2 (1.3%) mixed etiology); the etiology remained unknown in remaining 121 (79.6%) cases. 53 (36%) of the AES patients died; the case fatality proportion was similar in patients with a confirmed and unknown viral etiology (45.1 and 33.6% respectively). A requirement for assisted ventilation significantly increased mortality (HR 2.14 (95% CI 1.0-4.77)), while a high Glasgow coma score (HR 0.76 (95% CI 0.69-0.83)), and longer duration of hospitalization (HR 0.88 (95% CI 0.83-0.94)) were protective. CONCLUSION: This study is the first description of the etiology of adult-AES in India, and provides a framework for future surveillance programs in India.

Yeast expressed recombinant Hemagglutinin protein of novel H1N1 elicits neutralising antibodies in rabbits and mice.

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

Emergence of dengue virus type 4 (genotype I) in India.

Dengue is an emerging arboviral disease and currently poses the greatest arboviral threat to human health. In recent decades, there has been a substantial increase in dengue outbreaks in many parts of the world including India. We performed an in-depth investigation of a major dengue outbreak in Andhra Pradesh, southern India in 2007 by serology, virus isolation, RT-PCR and genotyping. The results revealed an unusual emergence of dengue virus type 4 (DENV-4) along with the prevailing DENV-3. Phylogenetic analysis based on complete envelope gene of 182 globally diverse DENV-4 isolates demonstrated the involvement of a unique clade of genotype I of DENV-4 in the outbreak. This study also demonstrated a clear shift in the dominant serotype from DENV-3 to DENV-4 in India. This is the first report regarding the molecular characterization of Indian isolates of DENV-4, which has the potential to be involved in future outbreaks.

Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against Chikungunya virus in vero cells.

The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented and explosive epidemics in India and the Indian Ocean islands after a gap of 32 years is a major public health concern. Currently, there is no specific therapy available to treat CHIKV infection. In the present study, the in vitro prophylactic and therapeutic effects of chloroquine on CHIKV replication in Vero cells were investigated. Inhibitory effects were observed when chloroquine was administered pre-infection, post-infection, and concurrent with infection, suggesting that chloroquine has prophylactic and therapeutic potential. The inhibitory effects were confirmed by performing a plaque reduction neutralization test (PRNT), real-time reverse transcriptase (RT)-PCR analysis of viral RNA levels, and cell viability assays. Chloroquine diminished CHIKV infection in a dose-dependent manner, with an effective concentration range of 5-20 microM. Concurrent addition of drug with virus, or treatment of cells prior to infection drastically reduced virus infectivity and viral genome copy number by >/=99.99%. The maximum inhibitory effect of chloroquine was observed within 1-3 hr post-infection (hpi), and treatment was ineffective once the virus successfully passed through the early stages of infection. The mechanism of inhibition of virus activity by chloroquine involved impaired endosomal-mediated virus entry during early stages of virus replication, most likely through the prevention of endocytosis and/or endosomal acidification, based on a comparative evaluation using ammonium chloride, a known lysosomotropic agent.

Development of a quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) for detection and quantitation of Chikungunya virus.

Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT-PCR assay was developed to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative RT-PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 10(2) to 10(10) copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT-PCR result with real-time RT-PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of CHIKV in acute-phase serum samples.

Chemokine profiling of Japanese encephalitis virus-infected mouse neuroblastoma cells by microarray and real-time RT-PCR: implication in neuropathogenesis.

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. JE virus (JEV) infection causes prominent neurological sequelae in approximately one-third of the survivors. In humans, the inflammatory response of CNS consequent to JEV induced viral encephalitis is mediated through chemokines released by various cells of CNS. In the present study, the chemokine profiles of mouse neuroblastoma cells (N2A) following JEV infection was analyzed by cDNA microarray followed by real-time RT-PCR. Eighty mRNA transcripts belonging to various functional classes exhibited significant alterations in gene expression. There was considerable induction of genes involved in apoptosis and anti-viral response. Modified levels of several transcripts involved in proinflammatory and anti-inflammatory processes exemplified the balance between opposing forces during JEV pathogenesis. Other genes displaying altered transcription included those associated with host translation, cellular metabolism, cell cycle, signal transduction, transcriptional regulation, protein trafficking, neurotransmitters, neuron maturation, protein modulators, ER stress and cytoskeletal proteins. The infection of neurons results in the synthesis of proinflammatory chemokines, which are early important mediators of leukocyte recruitment to sites of viral infection. Our results clearly suggest the implication of chemokines in neuropathogenesis of JEV infection leading to neurological sequelae. Pro- and anti-inflammatory agents targeted against chemokines such as CXCL10 may provide possible therapeutic modalities that can mitigate the morbidity associated with JEV infection of the CNS.

Appearance of E1: A226V mutant Chikungunya virus in Coastal Karnataka, India during 2008 outbreak.

Chikungunya has resurged in the form of unprecedented explosive epidemic in 2006 after a long gap in India affecting 1.39 million of persons. The disease continued for the next two consecutive years affecting 59,535 and 64,548 persons during 2007 and 2008 respectively. The 2008 outbreak being the second largest among these three years the information regarding the etiology and the mutations involved are useful for further control measures. Among the 2008 outbreaks the Coastal Karnataka accounts for the 46,510 persons. An in-depth investigation of Chikungunya epidemic of Coastal Karnataka, India, 2008 by serology, virus isolation, RT-PCR and genome sequencing revealed the presence and continued circulation of A226V mutant Chikungunya virus. The appearance of this mutant virus was found to be associated with higher prevalence of vector Aedes albopictus and the geographical proximity of coastal Karnataka with the adjoining Kerala state. This is the first report regarding the appearance of this mutation in Karnataka state of India. The present study identified the presence and association of A226V mutant virus with Chikungunya outbreak in India during 2008.

Oxidative damage and gene expression profile of antioxidant enzymes after T-2 toxin exposure in mice.

T-2 toxin is one of the most potent trichothecenes, and on exposure causes severe human and animal diseases. We investigated the dose- and time-dependent effect of T-2 toxin on certain biochemical variables, oxidative damage in terms of antioxidant enzyme activity, and gene expression profile in mice. Mice treated intraperitoneally with either 1 LD50 or 2 LD50 dose (5.61 and 11.22 mg/kg body weight, respectively) of T-2 toxin showed significant alterations in hepatic alanine amino transferase, aspartate amino transferase, and lactate dehydrogenase. Significant changes in hepatic lipid peroxidation, depletion of glutathione (GSH), and expression of heat shock protein-70 indicated oxidative damage. We also evaluated the activity of antioxidant enzymes and compared the gene expression profile by quantitative real-time reverse transcriptase-polymerase chain reaction. Except for glutathione reductase (GR), there was a significant increase in activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase at 1 LD50 dose. At 2 LD50 dose, SOD showed decrease in activity, whereas GST, GPx, and catalase showed significant increase. In contrast, gene expression profile showed downregulation in GR, GPx, GST, and catalase at 1 LD50 dose. At 2 LD50 dose except GSH synthetase, all other genes were downregulated. The results clearly show oxidative stress as one of the mechanisms of T-2 toxin-mediated toxicity.

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.

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 a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection.

Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.

Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates.

The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented explosive epidemic after a gap of 32 years in India is a point of major public health concern. In 2007 again there was outbreak in Kerala, India, affecting more than 25,000 cases with many reported mortalities. To understand the molecular basis of this high virulence and its implication in large-scale epidemic, a detailed systematic serological, virological and molecular investigation was undertaken with the epidemic samples of Kerala-2007. The comparative analysis of full genome sequence of Chikungunya virus isolate of 2007 with 2006 revealed three unique substitutions in structural and non-structural genes of 2007 isolate [two in E1 region (V14A and A226V) and one in Nsp1 (M184T)]. Our finding further substantiates the association of A226V shift in E1 gene with evolutionary success possibly due to adaptation in the mosquito vector with progression of epidemic, as observed in Reunion Island. This A226V shift which was absent in all 2006 Indian isolates, is found to be present in the four 2007 isolates, analysed in this study. These unique molecular features of the 2007 isolates with the progression of the epidemic from 2005 to 2007 demonstrate their high evolutionary and epidemic potential and thereby suggesting possible implication in higher magnitude and virulence of this outbreak.

Clinical features and molecular diagnosis of Chikungunya fever from South India.

An epidemic of Chikungunya fever of unprecedented magnitude occurred in many parts of India in early 2006 after an interval of 33 years, and there has been a resurgence in some parts of South India since June 2007. The article highlights clinical manifestations of infection and various molecular tests that were used for diagnoses of Chikungunya virus infection. Of particular interest is the real-time loop-mediated isothermal amplification (RT LAMP) assay, which is rapid and cost-effective and can be adopted at ill-equipped laboratories. Clinical symptoms were characterized by a triad of fever, rash, and severe rheumatic manifestations. RT LAMP identified 20 additional Chikungunya virus-positive cases, compared with reverse-transcriptase polymerase chain reaction. Chikungunya virus was isolated from 20 randomly selected samples. Genotyping of the virus isolates revealed that the East Central South African genotype of Chikungunya virus was the etiologic agent of this epidemic. Molecular diagnosis is an important tool to identify such new vectorborne viral illnesses.

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

BACKGROUND: Dengue 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. RESULTS: An 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. CONCLUSION: These 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.

Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus.

The development of a one-step SYBR Green I-based real-time RT-PCR assay is reported for detection and quantification of Chikungunya virus (CHIKV) in acute-phase patient serum samples by targeting the E1 structural gene. A linear relationship was obtained between the virus concentration and cycle threshold (C(t)) value over a range of 10(7)-0.1PFU/ml. The reported assay was found to be 10-fold more sensitive compared to conventional RT-PCR with a detection limit of 0.1PFU/ml. The feasibility of this reported assay system for clinical diagnosis was validated with 51 suspected acute-phase serum samples of the recent CHIKV epidemic in southern India, 2006. The comparative evaluation with acute-phase patient serum samples revealed the higher sensitivity of real-time RT-PCR assay by picking up six additional samples with low copy number of template. None of the healthy serum samples analyzed in this study showed amplification. The quantification of the viral load in the acute-phase serum samples was also determined employing the standard curve, which varies from 0.1 to 10(7)PFU/ml. These findings demonstrated that the reported assay has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of Chikungunya virus in acute-phase patient serum samples.

Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus.

One-step SYBR Green I-based real-time RT-PCR assay for rapid detection as well as quantitation of Japanese encephalitis virus (JEV) in acute-phase patient CSF samples by targeting the NS3 gene was developed. The assay developed in this study was found to be more sensitive as compared to conventional RT-PCR. The specificity of the reported assay system was established through melting curve analysis as well as by cross-reactivity studies with related members of Flavivirus. The applicability of Real-time PCR assay for clinical diagnosis was validated with 32 suspected acute-phase CSF samples of Gorakhpur epidemic, India, 2005. The improved sensitivity of real-time RT-PCR was reflected by picking up 10 additional samples with low copy number of template in comparison to conventional RT-PCR. The quantitation of the viral load in acute-phase CSF samples was done using a standard curve obtained by plotting cycle threshold (C(t)) values versus copy numbers of the RNA template. This is the first report on the application of real-time RT-PCR for detection as well as quantitation of JEV from patient CSF samples. These findings demonstrate the potential clinical application of the reported assay as a sensitive diagnostic test for rapid and real-time detection and quantitation of JEV in acute-phase clinical samples.

Rapid and real-time detection of Chikungunya virus by reverse transcription loop-mediated isothermal amplification assay.

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 x 10(8) to 2 x 10(2) 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 x 10(8) to 2 x 10(1) 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 degrees 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.