Determining suitable machine learning classifier technique for prediction of malaria incidents attributed to climate of Odisha.

This study investigated the influence of climate factors on malaria incidence in the Sundargarh district, Odisha, India. The WEKA machine learning tool was used with two classifier techniques, Multi-Layer Perceptron (MLP) and J48, with three test options, 10-fold cross-validation, percentile split, and supplied test. A comparative analysis was carried out to ascertain the superior model among malaria prediction accuracy techniques in varying climate contexts. The results suggested that J48 had exhibited better skill than MLP with the 10-fold cross-validation method over the percentile split and supplied test options. J48 demonstrated less error (RMSE = 0.6), better kappa = 0.63, and higher accuracy = 0.71), suggesting it as most suitable model. Seasonal variation of temperature and humidity had a better association with malaria incidents than rainfall, and the performance was better during the monsoon and post-monsoon when the incidents are at the peak.

Development of a pair of real-time loop mediated isothermal amplification assays for detection of Yersinia pestis, the causative agent of plague.

Yersinia pestis, the causative agent of plague mainly infects rodents, while humans are the accidental host. The conventional diagnostic methods available for Y. pestis exhibit cross-reactivity with other enteropathogenic bacteria which makes its detection difficult. Rapid and reliable point-of-care detection of Y. pestis is essential for timely initiation of medical treatment. In the present study, a pair of loop mediated isothermal amplification (LAMP) assays has been developed for rapid detection of Y. pestis. Two sets of LAMP primers, each containing 6 primers were specifically designed targeting caf1 and 3a genes located on pFra plasmid and chromosome of Y. pestis, respectively. Isothermal amplification was accomplished at 65 °C for 40 min for caf1 target, and at 63 °C for 50 min for 3a choromosomal target. The analytical sensitivity of the assay for the caf1 and 3a targets was found to be 500 fg and 100 fg genomic DNA of Y. pestis, respectively. The caf1 and 3a LAMP assays detected as few as 100 copies of caf1 and 10 copies of 3a gene targets harboured in the respective recombinant plasmids. The amplified products were detected visually under visible and UV light using SYBR Green 1 dye. The assay pair was found to be highly specific as it did not cross-react with closely related and other bacterial species.

BA3338, a surface layer homology domain possessing protein augments immune response and protection efficacy of protective antigen against Bacillus anthracis in mouse model.

AIM: Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates challenges for global security and public health. In this study, development of a safe and ideal next-generation subunit anthrax vaccine has been evaluated in mouse model. METHOD AND RESULTS: Protective antigen (PA) and BA3338, a surface layer homology (SLH) domain possessing protein were cloned, expressed in heterologous system and purified by IMAC. Recombinant PA and BA3338 with alum were administered in mouse alone or in combination. The humoral and cell-mediated immune response was measured by ELISA and vaccinated animals were challenged with B. anthracis spores via intraperitoneal route. The circulating IgG antibody titre of anti-PA and anti-BA3338 was found significantly high in the first and second booster sera. A significant enhanced level of IL-4, IFN-γ and IL-12 was observed in antigens stimulated supernatant of splenocytes of PA + BA3338 vaccinated animals. A combination of PA and BA3338 provided 80% protection against 20 LD50 lethal dose of B. anthracis spores. CONCLUSION: Both antigens induced admirable humoral and cellular immune response as well as protective efficacy against B. anthracis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has been evaluated for the first time using BA3338 as a vaccine candidate alone or in combination with well-known anthrax vaccine candidate PA. The findings of this study demonstrated that BA3338 could be a co-vaccine candidate for development of dual subunit vaccine against anthrax.

Geostatistical predictive modeling for asthma and chronic obstructive pulmonary disease using socioeconomic and environmental determinants.

The spatial distribution of the prevalence of asthma and chronic obstructive pulmonary disease (COPD) remains under the influence of a wide array of environmental, climatic, and socioeconomic determinants. However, a large proportion of these influences remain unexplained. In completion, this study examined the spatial associations between asthma/COPD morbidity and their determinants using ordinary least squares (OLS) and geographically weighted regressions (GWR). Inpatient records collected from the secondary and tertiary care hospitals in Kandy from 2010 to 2014 were considered as the dependent variable. Potential risk factors (explanatory variables) were identified in four distinguished classes: 1) meteorological factors, (2) direct and indirect factors of air pollution, (3) socioeconomic factors, and (4) characteristics of the physical environment. All possible combinations of candidate explanatory variables were evaluated through an exploratory regression. A comparison between the regression models was also explored. The best OLS regression models revealed about 55% of asthma variation and 62% of COPD variation while GWR models yielded 78% and 74% of the variation of asthma and COPD occurrences respectively. Relative humidity, proximity to roads (0-200 m), road density, use of firewood as a source of fuel, and elevation play a vital role in predicting morbidity from asthma and COPD. Both local and global regression models are important in assessing spatial relationships of asthma and COPD. However, the local models exhibit a better prediction capability for assessing non-stationary relationships of asthma and COPD than global models. The geostatistical aspects used in this study may also provide insights for evaluating heterogeneous environmental risk factors in other epidemiological studies across different spatial settings.

In vitro antibacterial, antioxidant activity and total phenolic content of some essential oils.

In vitro antibacterial activity of 16 essential oils was investigated by disc diffusion method against two Gram positive bacteria Bacillus subtilis and Staphylococcus aureus and two Gram negative bacteria, Shigella flexneri and Escherichia coli. Oils of Cymbopogon citratus and Ocimum basilicum showed highest antibacterial activity. Gram positive bacteria were found to be more sensitive than Gram negative. Antioxidant activities were tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and ABTS radical cation decolourization assay while Folin-Ciocalteu method was used to determine the total phenolic content. In DPPH assay, highest antioxidant activity was observed in 0. basilicum oil followed by Azeratum conyzoides, A. marmelos and C. citratus, with percent inhibition and IC50 value ranging from 66.11-71.93% and 14.10-17.92 µl ml(-1) respectively. In ABTS assay, similar results were obtained but with higher percent inhibition which ranged from 67.48-76.23% and lower IC50 value (12.12-17.21 µ ml(-1)). Moreover, radical scavenging activity of essential oils was lower than that observed for the synthetic antioxidant BHA and BHT. The total phenolic content of the essential oils as GAE in mg 100 µl(-1) of EO was found to be highest in O. basilicum (0.406) oil followed byA. conyzoides (0.322), A. marmelos (0.238) and C. citratus (0.231). The results provide evidence that the oils of C. citratus and O. basilicum can be further commended for treatment of infections caused by these bacterial pathogens and are potential source of natural antioxidants having appreciable amount of total phenolic content.

Ecosystem evaluation (1989-2012) of Ramsar wetland Deepor Beel using satellite-derived indices.

The unprecedented urban growth especially in developing countries has laid immense pressure on wetlands, finally threatening their existence altogether. A long-term monitoring of wetland ecosystems is the basis of planning conservation measures for a sustainable development. Deepor Beel, a Ramsar wetland and major storm water basin of the River Brahmaputra in the northeastern region of India, needs particular attention due to its constant degradation over the past decades. A rule-based classification algorithm was developed using Landsat (2011)-derived indices, namely Normalised Difference Water Index (NDWI), Modified Normalised Difference Water Index (MNDWI), Normalised Difference Pond Index (NDPI), Normalised Difference Vegetation Index (NDVI) and field data as ancillary information. Field data, ALOS AVNIR and Google Earth images were used for accuracy assessment. A fuzzy accuracy assessment of the classified data sets showed an overall accuracy of 82 % for MAX criteria and 90 % for RIGHT criteria. The rules were used to classify major wetland cover types during low water season (January) in 1989, 2001 and 2012. The statistical analysis of the classified wetland showed heavy manifestation in aquatic vegetation and other features indicating severe eutrophication over the past 23 years. This degradation was closely related to major contributing anthropogenic factors, such as a railway line construction, growing croplands, waste disposal and illegal human settlements in the wetland catchment. In addition, the landscape development index (LDI) indicated a rapid increase in the impact of the surrounding land use on the wetland from 1989 to 2012. The techniques and results from this study may prove useful for top-down landscape analyses of this and other freshwater wetlands.

Early evaluation of voice quality in glottic cancer (T1, T2) following curative radiotherapy.

BACKGROUND: This is prospective study analyzing the subjective and objective quality of voice and voice related quality of life in patients of early glottic cancer (T1, T2 disease) before and after receiving curative radiotherapy. METHODS: Fifteen patients of early glottic carcinoma (T1, T2) underwent voice assessment using multidimensional voice protocol based on recommendation by European Laryngological Society which included Perceptual analysis of voice by speech therapist and otolaryngologist, acoustic analysis; aerodynamic efficiency analysis-Maximum phonation time; patient's self perception of voice analysis--Voice handicap index; and videolaryngostroboscopy. Assessment was done prior to commencement of radiation therapy and at 1 month and 3 months following radio- therapy. RESULTS: There was significant improvement in majority of the voice parameters post radiotherapy. Perceptual analysis showed significant improvement in GRBAS score following radiotherapy. Perturbation measures (jitter, shimmer, SNR, HNR) showed improvement post radiotherapy though remained inferior compared to controls. Mean fundamental frequency (Mean F0) and habitual frequency (habitual F0) decreased post radiotherapy. Intensity of voice increased following radiotherapy which was statistically significant (p < 0.05). There was significant improvement in the patient's perception of their quality of voice and voice related quality of life post radiotherapy. Maximum phonation time showed statistically significant improvement post-radiotherapy. Perceptual analysis of voice by professional observer correlated well with patients self perception of his own voice. CONCLUSION: Voice quality improves following radiotherapy but not all the patients regain normal voice. Improvement in voice quality improves quality of life of patients shown by improved voice handicap index.

Repellent activity of some essential oils against two stored product beetles Callosobruchus chinensis L. and C. maculatus F. (Coleoptera: Bruchidae) with reference to Chenopodium ambrosioides L. oil for the safety of pigeon pea seeds.

Essential oils from 35 aromatic and medicinal plant species of Gorakhpur Division (U. P., India) were evaluated for their repellent activity against pulse bruchids Callosobruchus chinensis L. and C. maculatus F. of stored pigeon pea seeds. The oil concentration was at 0.36 µl/ml. Out of 35 essential oils, Adhatoda vasica Ness and Chenopodium ambrosioides L. oils showed absolute (100 %) insect repellency. Chenopodium oil exhibited 100 % mortality for both the test insects at 10 µl concentration (LD50 = 2.8 µl for C. chinensis & 2.5 µl for C. maculatus) and more toxic than Adhatoda oil (LD50 = 6.8 µl for C. chinensis & 8.4 µl for C. maculatus). During in vivo evaluation, 0.29 and 0.58 µl/ml of Chenopodium oil significantly enhanced feeding deterrence in insects and reduced the seed damage as well as weight loss of fumigated pigeon pea seeds up to 6 months of storage as compared to control set. Thus, Chenopodium oil can be used as an effective option of commercial fumigants for the storage of pigeon pea seeds against pulse bruchids.

Ecological study of revegetated coal mine spoil of an Indian dry tropical ecosystem along an age gradient.

Mineral nitrogen (MN), belowground (root) biomass (BGB), soil nitrogen (N) mineralization (NM), microbial biomass N (MBN) and mine dump stability of a revegetated mine spoil were studied after 2, 6, 10 and 12 years of re-vegetation on coal mine spoil site. MN in revegetated mine spoil ranged from 7.4 to 11.6 kg ha(-1), NM from 38.4 to 252 kg ha(-1) year(-1), MBN from 86 to 426 kg ha(-1), and BGB from 380 to 3,750 kg ha(-1). Mining caused decline of physico-chemical characteristics of soil like MN by 46 %, N-mineralization by 92 %, MBN values by 91 %, respectively compared to forest ecosystems and reduction of total plant biomass (above ground and below ground). Revegetation of mine spoil caused increase in MN values by 12, 36 and 76 %, BGB values by 380, 1770 and 3750 times, NM values by 0.6, 3.58 and 9.5 times and MBN values by 0.43, 2.77, and 6.07 times in 2, 6 and 12 years, respectively. BGB was highly correlated with MN and MBN. Clay content was positively correlated to MN, NM, and the age of revegetation (P < 0.01). Numerical modelling indicated that revegetation increased the dump slope stability with a factor of safety from 1.2 to 1.4, 1.7, 1.9 and 2.1 after 2, 6, 10 and 12 years, respectively. Thus, long-term revegetation was found to enhance the dump stability and the soil fertility status in mine spoil, where plant biomass and microbial biomass provide major contributions in ecological redevelopment of the mine spoil.

Characterization of different road dusts in opencast coal mining areas of India.

Dust from haul and transport roads are the major source of air pollution in opencast coal mining areas. Dust generated during mining operations pollutes air which causes different health problems. Various available techniques are implemented in the field to minimize and control dust in mining areas. However, they are not very effective because dust deposited on road surfaces are not removed by these techniques. For effective control of dust in opencast mining areas, it has to be regularly collected from road surfaces and may be converted into solid form, and subsequently can be used as a domestic fuel considering its physicochemical properties. The present paper describes a comparative study of qualitative and quantitative aspects of road dust samples of four coalfields of India. The pH of the dust was found to be in the range of 5.1-7.7. Moisture, ash, volatile matter, fixed carbon, water-holding capacity, bulk density, and specific gravity of dust samples were found to be in the range of 0.5-3.0%, 45-76%, 12.6-20.0%, 10.2-45.3%, 21.17-31.71%, 1.15-1.70, and 1.73-2.30 g cm(-3), respectively. Observing the overall generation and characteristics of coal dust, it is suggested that coal dust from haul and transport roads of mining areas can be effectively collected and used as domestic fuel.

Earthquake precursory detection using diurnal GPS-TEC and kriging interpolation maps: 12 May 2008, Mw7.9 Wenchuan case study.

This study presents an alternative vertical total electron content (VTEC) anomaly detection technique based on diurnal VTEC values. In order to assess the consistency of the results, Mw7.9 Wenchuan earthquake occurred on May 12, 2008 was chosen as case study because several researches were performed on this earthquake event. In this detection technique, the daily mean of VTEC (AVTEC) and standard deviation of diurnal VTEC (SVTEC) were adopted in the analytical process instead of quartile-based technique. The spatial distribution of VTEC was illustrated by AVTEC and SVTEC maps which created from ordinary Kriging interpolation technique. The anomalous day derived from AVTEC and SVTEC was observed on May 9, 2008. The anomalous zone significantly appeared within the earthquake preparation zone in the southeast of the epicenter. The results were corresponding to the previous studies in terms of time and space. Thus, AVTEC, SVTEC and instantaneous ionospheric maps created from ordinary Kriging interpolation technique should be an alternative approach for detecting ionospheric anomaly prior to earthquake occurrence.•Simplified seismo-ionospheric anomaly detection technique•Ionospheric distribution is modelled by ordinary Kriging interpolation maps•The results are consistent with the previous studies in terms of time and space.

Performance of AnnAGNPS model in predicting runoff and sediment yields in Nan Province, Thailand.

Land use changes such as deforestation and urban development influences the river discharge, soil erosion and sediment yield. It is important to evaluate tools which can be used to assess such impacts on water and sediment yield. Therefore, this study evaluated the Annualized Agricultural Non-Point Source Pollutant (AnnAGNPS) model's performance in simulating runoff and sediment loads in Nan Province, Thailand using seven years of continuous monitoring data. The river discharge and sediment yield data from 2011-2013 were used for calibration, and data from 2014-2017 were used for validation. Several input parameters were computed using methods suggested by other researchers and previous studies. In this study, the runoff curve number, soil erodibility factor (K), and RUSLE-C value were used to accurately simulate runoff and sediment loads. The results indicate that the model satisfactorily simulated runoff and sediment loads (R2 = 0.65 and NSE = 0.53 for runoff volume, and R2 = 0.62 and NSE = 0.60 for sediment yields). Moreover, the model estimated the total sediment yield, which contributed 12,932 hundred tons of material to the Nan River in 2017. The maximum sediment yield was obtained below the catchment (Na Noi sub-district, Na Noi district), which corresponds to areas with high crop densities. Cropland generated the highest soil erosion of all investigated land use (87.52% of total soil erosion). Thus, the AnnAGNPS model has the potential to use for investigating management practices to reduce soil erosion and controlling floods and droughts in Nan Province of Thailand.

Development of a set of three real-time loop-mediated isothermal amplification (LAMP) assays for detection of Bacillus anthracis, the causative agent of anthrax.

Bacillus anthracis, the causative agent of anthrax is a Gram-positive, non-motile, spore forming bacterium. Its spores can persist in soil and water for years and can also be aerosolized. A rapid, sensitive and specific method to detect B. anthracis is important for clinical management and preventing spread of anthrax. Loop-mediated isothermal amplification (LAMP) assay is a rapid technique that amplifies target DNA in isothermal conditions with high sensitivity and specificity. In this study, a LAMP assay set targeting a chromosomal and two plasmid markers was developed. The individual assays of the LAMP set targeting pXO1 plasmid (lef), pXO2 plasmid (capB), and chromosome (BA5345) sequences could detect 10, 250, and 100 fg of genomic DNA and 10, 100, and 50 copies of the DNA targets harboured in recombinant plasmids, respectively. The lef and capB LAMP assays could detect ≥ 1 × 103 CFU per mL of bacteria in spiked human blood samples, while BA5345 LAMP assay could detect ≥ 1 × 104 CFU of bacteria per mL of spiked blood. The amplification was monitored in real-time by turbidimeter, and visual detection was also accomplished under normal and UV light after adding SYBR Green 1 dye on completion of the reaction. The assay set was found to be highly sensitive and did not cross-react with the closely related Bacillus spp. and other bacterial strains used in the study.

Development of a PCR Lateral Flow Assay for Rapid Detection of Bacillus anthracis, the Causative Agent of Anthrax.

Bacillus anthracis, the causative agent of anthrax is one of the most potent listed biological warfare agents. The conventional microbiological methods of its detection are labor intensive and time consuming, whereas molecular assays are fast, sensitive and specific. PCR is one of the most reliable diagnostic tools in molecular biology. The combination of PCR with lateral flow strips can reduce the diagnostic/detection time. It gives an alternative to gel electrophoresis and offers easy and clear interpretation of results. In the present study, a PCR Lateral flow (PCR-LF) assay targeting cya gene present on pXO1 plasmid of B. anthracis has been developed. The forward and reverse primers were tagged with 6-carboxyflourescein (6-FAM) and biotin, respectively, at 5' end. The dual labeled PCR products were detected using lateral flow (LF) strips developed in this study. The PCR-LF assay could detect ≥ 5 pg of genomic DNA and ≥ 500 copies of target DNA harboured in a recombinant plasmid. The assay was able to detect as few as 103 and 10 CFU/mL of B. anthracis Sterne cells spiked in human blood after 6 and 24 h of enrichment, respectively.

Development of a PCR-lateral flow assay for rapid detection of Yersinia pestis, the causative agent of plague.

Plague is a zoonotic disease caused by Yersinia pestis, a Gram-negative, rod shaped coccobacillus, which is primarily found in rodents and can be transmitted to humans through flea bite. The disease has three major clinical forms bubonic (by flea bite), pneumonic (by respiratory droplets) and septicemic plague. Y. pestis is classified as a category 'A' agent by NIAID, USA due to its high mortality and easy person to person dissemination. The conventional diagnostic methods available for Y. pestis show cross-reactivity with other enteropathogenic bacteria making its detection difficult. There is a need to develop sensitive and specific molecular assay for accurate detection of Y. pestis. PCR is well suited molecular biology tool for rapid diagnosis of plague but after completion of thermal cycling steps, it requires additional time to analyze amplified product using agarose gel electrophoresis. In the present study, PCR assay coupled with lateral flow strips has been developed for rapid detection of Y. pestis. Lateral flow strips give an alternative to gel electrophoresis and permit easy and rapid detection of PCR products. The PCR was performed with 5' 6-FAM and biotin tagged primers specific for Y. pestis, targeting yihN gene located on chromosome. The PCR product was analyzed using lateral flow strips which yielded result within 2-3 minutes. The analytical sensitivity of PCR-lateral flow (PCR-LF) assay was 1 pg genomic DNA of Y. pestis and 500 copies of target DNA sequence harboured in a recombinant plasmid. The assay could detect Y. pestis DNA extracted from spiked human blood samples containing ≥104 CFU per mL of bacteria. The assay was found to be specific and did not cross react with other closely related bacterial species. The developed assay was highly specific, sensitive and also did not require agarose gel electrophoresis for post amplification analysis.

Impact of post-mining subsidence on nitrogen transformation in southern tropical dry deciduous forest, India.

The goal of our research was to assess the impact of post-mining land subsidence, caused due to underground coal mining operations, on fine root biomass and root tips count; plant available nutrient status, microbial biomass N (MBN) and N-mineralization rates of a Southern tropical dry deciduous forest of Singareni Coalfields of India. The changes were quantified in all the three (rainy, winter and summer) seasons, in slope and depression microsites of the subsided land and an adjacent undamaged forest microsite. Physico-chemical characteristics were found to be altered after subsidence, showing a positive impact of subsidence on soil moisture, bulk density, water holding capacity, organic carbon content, total N and total P. The increase in all the parameters was found in depression microsites, while in slope microsites, the values were lower. Fine root biomass and root tips count increased in the subsided depression microsites, as demonstrated by increases of 62% and 45%, respectively. Soil nitrate-N and phosphate-P concentrations were also found to be higher in depression microsite, showing an increase of 35.68% and 24.74%, respectively. Depression microsite has also shown the higher MBN value with an increase over control. Net nitrification, net N-mineralization and MBN were increased in depression microsite by 29.77%, 25.72% and 34%, respectively. There was a positive relation of microbial N with organic C, fine root biomass and root tips.

Comparison of a dipstick enzyme-linked immunosorbent assay with commercial assays for detection of Japanese encephalitis virus-specific IgM antibodies.

BACKGROUND: Japanese encephalitis (JE) is a major public health concern in Asia including India. OBJECTIVES: To evaluate an in-house developed dipstick enzyme-linked immunosorbent assay (ELISA) test vis-à-vis two commercial kits for detection of JE virus-specific IgM antibodies. SETTING AND DESIGN: Comparative study carried out in Research and Development centre. MATERIALS AND METHODS: A total of 136 specimens comprising 84 serum and 52 CSF samples were tested by in-house dipstick ELISA, Pan-Bio IgM capture ELISA (Pan-Bio, Australia) and JEV CheX IgM capture ELISA (XCyton, India). RESULTS: The overall agreement among all three tests was found to be 92% with both serum and cerebrospinal fluid (CSF) samples. The sensitivity of the dipstick ELISA was found to be 91% with serum and 89% with CSF samples respectively. The specificity of the dipstick ELISA with reference to both commercial assays was found to be 100% in serum and CSF samples in this study. CONCLUSIONS: The in-house dipstick ELISA with its comparable sensitivity and specificity can be used as a promising test in field conditions since it is simple, rapid and requires no specialized equipment.

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis, which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli. The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis. Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis.

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.