Dry- down probe free qPCR for detection of KFD in resource limited settings.

Kyasanur Forest Disease is a tick-borne flavivirus is endemic in the Southern India. The recent expansion and resurgence of sporadic outbreaks in southern parts of country is the most important concern. Although only formalin inactivated vaccine is available for treatment with limited efficacy the early detection and timely identification is a only way to prevent spread of cases. If the disease can be identified prior to infection in humans like in forest areas from ticks and vectors the disease spread supposed to be managed quickly. Here we have standardized a single tube ready to use dry-down probe free real time RT-PCR targeted against virus envelope gene for detection of KFDV infection. The assay was standardized in liquid format first, later it was converted into dry-down format with addition of stabilizers with a similar sensitivity and specificity (10RNA Copies/rxn). The sensitivity was comparable to the most widely used and accepted diagnostic platform i.e. TaqMan qRT-PCR. However as the reported assay here omit the need of probes makes it cost effective and dry-down reagents makes more stability to the developed assay in this study if compare to TaqMan qPCR. The assay was evaluated with KFD positive samples and healthy sample panel which revealed high concordance with TaqMan qRT-PCR. Stability was unaffected by temperature fluctuations during transportation even in cold chain free conditions, thus reduce the maintenance of strict cold storage. These findings demonstrated that the reported assay is convenient with 100% sensitivity and specificity to TaqMan qPCR. Thus this assay has the potential usefulness for diagnosis KFDV for routine surveillance in resource limited laboratory settings omitting the use costly and heat sensitive TaqMan qRT-PCR reagents without compromising the sensitivity and specificity of the diagnosis assay.

Computational prediction of B and T-cell epitopes of Kyasanur Forest Disease virus marker proteins towards the development of precise diagnosis and potent subunit vaccine.

Kyasanur Forest Disease (KFD), also known as 'monkey fever', caused by KFD Virus (KFDV), is a highly neglected tropical disease endemic to Western Ghat region of Karnataka, India. Recently, KFD, which is fatal for both monkeys and humans with a mortality rate of 2-10% has been found to spread from its epicenter to neighboring districts and states also. The current ELISA based KFD detection method is very non-specific due to cross-reactivity with other flaviviruses. Further, presently available formalin-inactivated vaccine has been found to be less effective leading to disease susceptibility and severity. To address these, the present study was aimed at predicting the potent specific B and T-cell epitopes of KFDV immunogenic marker proteins using diverse computational tools aiming at developing precise diagnostic method and an effective subunit vaccine. Here, we have chosen E, NS1 and NS5 proteins as markers of KFDV by taking into account of their differential and non-overlapping sequences with selected arboviruses. Based on the linear and nonlinear epitope prediction tools and important biophysical parameters, we identified three potential linear and ten nonlinear B-cell epitopes. We also predicted T-cell epitope peptides which binds to MHC class-I and class-II receptors for the effective T-cell activation. Thus, our molecular docking and molecular dynamics simulation analysis has identified six different TH-cell epitopes based on the distribution frequency of MHC-II haplotypes in the human population and one TC-cell epitope from NS5 protein that has maximum interaction with class-I MHC. Overall, we have successfully identified potential B and T-cell epitope marker peptides present in the envelope and two non-structural proteins.Communicated by Ramaswamy H. Sarma.

Development and application of a recombinant Envelope Domain III protein based indirect human IgM ELISA for Kyasanur forest disease virus.

Kyasanur forest virus disease (KFD) is a major public health concern in India. Its etiology KFD virus causes haemorrhagic fever with severe sequelae in humans. Due to continuous spatiotemporal expansion of KFD in last decade, the incidences of positive cases have been increasing in both humans and primates. Early diagnosis is of prime importance for disease management and epidemiological containment. In the present study, the highly immunogenic Envelope Domain III (EDIII) antigen was produced using prokaryotic expression system with an yield of 8 mg/L. The protein was purified using affinity chromatography and confirmed for its immuno-reactivity by western blot and UPLCMS/MS analysis. The recombinant EDIII was used as an antigen for the standardization of ELISA to detect anti KFD IgM antibodies in humans. The ROC curve was prepared to set the optimum cut-off OD for the assay. The comparative evaluation of the assay with a reference MAC ELISA revealed 86.96% concordance, 82.22% sensitivity and 91.48% specificity. Inter-rater agreement was performed with kappa index revealing significant agreement between the assays. This is the first study using safe recombinant protein antigen-based detection of anti KFDV antibodies in humans. This simple and scalable ELISA assay will be applicable for large scale screening of samples for combating the emerging threats of KFD in newer territories.

Heavy Metals and Pesticides in Chronic Kidney Disease - Results from a Matched Case-Control Study from a Rural Population in Shivamogga District in South India.

INTRODUCTION: There is a high prevalence of chronic kidney disease (CKD) in the rural agrarian population of South India and it often appears unrelated to major known causes such as diabetes or glomerulonephritis. METHODS: In a matched case-control study conducted in a rural population in Shivamogga district in South India, the association of heavy metals - lead (Pb), arsenic (As), cadmium (Cd) - and pesticides in CKD was studied. Blood and spot urine samples were tested quantitatively for heavy metals and qualitatively for pesticides. RESULTS: In all, 69 matched pairs (40 female, 58%) were recruited. The mean estimated glomerular filtration rate (mL/min/1.73 m2) was 60.1 (14.2) in cases and 83.4 (13.4) in controls. Elevated blood lead level >5 µg/dL was seen in 15 cases and 25 controls, respectively [P = 0.035, matched odds ratio (MOR) 0.5, 95% confidence interval (CI) 0.22-1.05]. Urinary Pb was elevated in 16 cases and 13 controls, respectively (P = 0.28, MOR 1.25, 95% CI 0.58-2.73). There was no significant association with As and Cd, while pesticide residues were undetectable in cases as well as controls. These results did not change even after excluding CKD cases with diabetes, stage 2 hypertension, and significant proteinuria. CONCLUSIONS: There was no statistical significant association between any of the studied heavy metals and CKD, although there was a significant burden of heavy metals in the studied subjects.

Kyasanur Forest disease outbreak and vaccination strategy,Shimoga District, India, 2013-2014.

We investigated a Kyasanur Forest disease outbreak in Karnataka, India during December 2013-April 2014. Surveillance and retrospective study indicated low vaccine coverage, low vaccine effectiveness, and spread of disease to areas beyond those selected for vaccination and to age groups not targeted for vaccination. To control disease, vaccination strategies need to be reviewed.

Kyasanur Forest disease, India, 2011-2012.

To determine the cause of the recent upsurge in Kyasanur Forest disease, we investigated the outbreak that occurred during December 2011-March 2012 in India. Male patients >14 years of age were most commonly affected. Although vaccination is the key strategy for preventing disease, vaccine for boosters was unavailable during 2011, which might be a reason for the increased cases.