Jyoti Shukla

Development and evaluation of antigen capture ELISA for early clinical diagnosis of chikungunya.

The resurgence of chikungunya (CHIK) in the form of unprecedented explosive epidemic after a gap of 3 decades in India and Indian Ocean islands is a point of major public health concern. The laboratory diagnosis is essentially based on virus isolation, IgM ELISA, and reverse transcriptase polymerase chain reaction (RT-PCR). Although PCR-based methods are used for early and accurate diagnosis, the high cost of the assay and requirement of thermal cycler limit its application only to referral laboratories. The antibody-based IgM ELISA is found to be cost-effective, but it takes 5 to 6 days for the patient to develop antibody and, thus, has less implication for early clinical diagnosis and patient management. Therefore, a simple rapid, sensitive, and specific antigen detection system is reported for early and reliable clinical diagnosis as well as effective surveillance of CHIK. A double antibody sandwich system was designed for antigen capture ELISA, employing rabbit and mouse anti-CHIK IgG antibodies as capture and detector antibodies, respectively. An optimal assay condition with 0 background was established having no reactivity with healthy human serum and Cerebro spinal fluid (CSF) samples. The comparative evaluation with SYBR Green I-based real-time RT-PCR revealed an accordance of 96% with a sensitivity and specificity of 95% and 97%, respectively. The specificity of this assay was confirmed through cross-reactivity studies with confirmed dengue and Japanese encephalitis (JE) patient serum and CSF samples. The antigen capture ELISA reported in this study was able to detect the presence of viral antigen as early as the second day of fever and, thus, can be very useful for early clinical diagnosis of CHIK with acute phase patient serum and CSF samples. This can also be used for rapid screening of large numbers of clinical samples in endemic areas during epidemics.

Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India

BACKGROUND In late 2009/early 2010, approximately 2000 people were affected by a mysterious viral outbreak in a southern district of Tamil Nadu; this particularly affected those living in coastal areas. Blood samples from affected patients were sent for clinical analysis to determine the actual cause of the illness, but reports were inconclusive. METHODS The present study describes the clinical observations and laboratory investigations involving molecular methods performed on 170 of the 2000 clinically suspected cases. These were patients who were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assays were used to detect West Nile virus (WNV) infection. Further investigation of the genetic diversity of the WNV implicated in ocular complications was undertaken by sequence phylogeny. RESULTS Out of 170 samples, 25 (15%) were positive for chikungunya IgM antibody, 10 (6%) for chikungunya antigen, and 30 (18%) were positive for dengue IgM antibody. The remaining 105 seronegative samples were further processed for WNV detection by IgM capture ELISA and molecular methods. Out of the 105 samples, 35 (33%) were positive for WNV IgM antibody, 15 (14%) were positive for WNV by RT-PCR, and 27 (26%) were found to be positive for WNV by both real-time RT-PCR and RT-LAMP assays. Comparative evaluation with acute-phase patient serum samples revealed 100% concordance between the real-time RT-PCR and RT-LAMP assays. These assays had an overall higher sensitivity than the conventional RT-PCR as they picked up 12 additional samples with a low copy number of template. Further genotyping through sequence phylogeny revealed that all the WNV isolates were grouped in lineage I. CONCLUSIONS The association of West Nile virus with ocular infection in South India during an epidemic of mysterious fever in the first half of 2010 was clearly established through molecular approaches employing envelope gene-specific real-time RT-PCR and RT-LAMP assays followed by nucleotide sequencing.

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus.

The recent emergence of the swine-origin influenza A H1N1 virus (S-OIV) poses a serious global health threat. Rapid detection and differentiation of S-OIV from seasonal influenza is crucial for patient management and control of the epidemics. A one-step, single-tube accelerated and quantitative S-OIV-specific H1 reverse transcription loop-mediated isothermal amplification (RTLAMP) assay for clinical diagnosis of S-OIV by targeting the H1 gene is reported in this article. A comparative evaluation of the H1-specific RTLAMP assay vis-à-vis the World Health Organization-approved real-time polymerase chain reaction (RTPCR), involving 239 acute-phase throat swab samples, demonstrated exceptionally higher sensitivity by picking up all of the 116 H1N1-positive cases and 36 additional positive cases among the negatives that were sequence-confirmed as S-OIV H1N1. None of the real-time RTPCR-positive samples were missed by the RTLAMP system. The comparative analysis revealed that S-OIV RTLAMP was up to tenfold more sensitive than the World Health Organization real-time RTPCR; it had a detection limit of 0.1 tissue culture infectious dosage of 50/ml. One of the most attractive features of this isothermal gene amplification assay is that it seems to have an advantage in monitoring gene amplification by means of SYBR Green I dye-mediated naked-eye visualization within 30 minutes compared to 2 to 3 hours for a real-time reverse transcription polymerase chain reaction. This suggests that the RTLAMP assay is a valuable tool for rapid, real-time detection and quantification of S-OIV in acute-phase throat swab samples without requiring sophisticated equipment.

Comparative complete genome analysis of dengue virus type 3 circulating in India between 2003 and 2008

Dengue is endemic in most parts of the tropics including India. So far, complete genome information for Indian dengue isolates is not available. In the present study, we characterized the genome of three dengue type 3 viruses isolated from India. The genomes of all three viruses were found to be 10,707 bp long with an ORF encoding 3390 aa. Extensive molecular phylogenetic analysis based on comparison of the complete genome and envelope gene classified the recent Indian viruses into genotype III (lineage III), revealing a shift of lineage from lineage V. The sequence analysis revealed several non-conservative changes in major structural proteins. This study clearly indicates that the genotype III (lineage III) dengue type 3 viruses have been continuously circulating in major parts of India since 2003 and are responsible for the recent major outbreaks all over India. This is the first extensive study on complete genome analysis of dengue type 3 viruses in India.

Cloning and expression of domain III of the envelope gene of Japanese encephalitis virus: evaluation for early clinical diagnosis by IgM ELISA.

Japanese encephalitis virus (JEV) is the single largest cause of childhood viral encephalitis in the world with an estimated 50,000 cases and 10,000 deaths annually. The laboratory diagnosis is based essentially on IgM ELISA owing to low transient viremia making virus isolation difficult. In addition the requirement of cerebrospinal fluid (CSF) sample for confirmatory molecular diagnosis by reverse transcription-polymerase chain reaction (RT-PCR) makes IgM ELISA the test of choice for early clinical diagnosis. The development and evaluation of a highly sensitive and specific IgM ELISA using the recombinant domain III envelope protein (rJEV-DIII) for rapid, early and accurate diagnosis of JEV is reported in the present study. The gene coding for the envelope protein of JEV was cloned and expressed in pET 30a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rJEV-DIII was optimized that had no reactivity with healthy persons. The comparative evaluation accomplished with the JE-Dengue IgM Combo ELISA (PanBio, Brisbane, Australia) and JEV Chex (XCyton Diagnostic Ltd., Bangalore, India) ELISA kits, by subjecting 120 acute phase of clinical samples revealed more than 95% accordance. The rJEV-DIII ELISA and the PanBio ELISA were found to have a sensitivity and specificity of 98% and 96%, respectively. The compared positivity of the rJEV-DIII ELISA and SYBR green-I based real-time RT-PCR assay in CSF samples revealed higher positivity. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. The recombinant domain III envelope protein based JEV specific ELISA will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks.

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 102 to 1010 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.

Rapid and Real-time Detection of Human Viral Infections: Current Trends and Future Perspectives

The development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number of threats posed by emerging and re-emerging viruses in the recent past. The conventional identification methods require time-consuming culturing, and/or detection of antibodies, which are not very sensitive and specific. The recent advances in molecular biology techniques in the field of genomics and proteomics greatly facilitate the rapid identification with more accuracy. The real-time assays viz; SYBR green I based real time RT-PCR and RT-LAMP have been developed for rapid detection as well as typing of some of the emerging arboviruses of biomedical importance viz; Dengue, Japanese Encephalitis, Chikungunya, West Nile, SARS and Swine Flu etc. Both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity and specificity. One of the most important advantages of RT-LAMP is its field applicability, without requirement of any sophisticated equipments. The establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future.