Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices.

BACKGROUND: Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence. METHODS: An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls. RESULTS: A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil. CONCLUSIONS: The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

Development of a Novel Internally Controlled HrpB1 Gene-Based Real-Time qPCR Assay for Detection of Burkholderia pseudomallei.

BACKGROUND: Melioidosis, caused by category B bioterrorism agent Burkholderia pseudomallei, is a seasonal disease of tropical and subtropical regions with a high mortality rate. An early and culture-independent detection of B. pseudomallei is required for the appropriate disease management and prevention. The present study is designed to identify novel and unique sequences of B. pseudomallei and development of quantitative polymerase chain reaction (qPCR) assay. METHODS: A novel B. pseudomallei-specific target sequence was identified by in silico analysis for the qPCR assay development. The specificity of the developed assay was assessed using purified DNA of 65 different bacterial cultures, and the sensitivity was estimated using a cloned target gene. Further, a type III secretion protein HrpB1 (HrpB1) gene-based duplex qPCR assay incorporating suitable extraction and amplification control was developed, and its viability was assessed in different clinical and environmental matrices for the detection of B. pseudomallei. RESULTS: In this study, an 80-nucleotide-long B. pseudomallei-specific region within the gene HrpB1 was identified by computational analysis. The developed HrpB1-based qPCR assay was highly specific for B. pseudomallei detection when evaluated with 65 different bacterial cultures. The sensitivity of the qPCR assay with the HrpB1-recombinant plasmid was found to be five copies per qPCR reaction. The assay's detection limit was found to be 5 × 102 CFU/mL for human blood and urine, 5 × 101 CFU/mL in river water, and 2 × 103 CFU/gm in paddy field soil. CONCLUSION: The results of the study showed the applicability of a novel HrpB1-based qPCR assay for sensitive and specific detection of B. pseudomallei in diverse clinical and environmental samples.

Hybrid time series models with exogenous variable for improved yield forecasting of major Rabi crops in India.

Accurate and in-time prediction of crop yield plays a crucial role in the planning, management, and decision-making processes within the agricultural sector. In this investigation, utilizing area under irrigation (%) as an exogenous variable, we have made an exertion to assess the suitability of different hybrid models such as ARIMAX (Autoregressive Integrated Moving Average with eXogenous Regressor)-TDNN (Time-Delay Neural Network), ARIMAX-NLSVR (Non-Linear Support Vector Regression), ARIMAX-WNN (Wavelet Neural Network), ARIMAX-CNN (Convolutional Neural Network), ARIMAX-RNN (Recurrent Neural Network) and ARIMAX-LSTM (Long Short Term Memory) as compared to their individual counterparts for yield forecasting of major Rabi crops in India. The accuracy of the ARIMA model has also been considered as a benchmark. Empirical outcomes reveal that the ARIMAX-LSTM hybrid modeling combination outperforms all other time series models in terms of root mean square error (RMSE) and mean absolute percentage error (MAPE) values. For these models, an average improvement of RMSE and MAPE values has been observed to be 10.41% and 12.28%, respectively over all other competing models and 15.83% and 18.42%, respectively over the benchmark ARIMA model. The incorporation of the area under irrigation (%) as an exogenous variable in the ARIMAX framework and the inbuilt capability of the LSTM model to process complex non-linear patterns have been observed to significantly enhance the accuracy of forecasting. The performance supremacy of other hybrid models over their individual counterparts has also been evident. The results also suggest avoiding any performance generalization of individual models for their hybrid structures.

Development of a novel lectin-based gold nanoparticle point-of-care immunoassay for rapid diagnosis of patients with severe Dengue infection.

Rapid diagnosis of patients with severe Dengue infection can be useful for the efficient clinical management of cases caused by the Dengue virus. Lateral Flow Immunoassay (LFIA) have been broadly used for rapid Dengue diagnosis, because of their quick readouts with the human eye, simplicity of use, and affordability. Despite the availability of several commercial Dengue point-of-care assays, none has shown to be successful in discriminating between severe and nonsevere forms of Dengue infection. In the current study, for the first time, a novel lectin-based point-of-care assay for the early detection of patients with severe Dengue infection with gold-adorned sheets as detection labels is being reported. In this assay, Dengue severity was diagnosed by detecting the glycosylation profile of vitronectin, a known Dengue severity marker. Two lectins were employed namely DSA (Datura stramonium) and MAA (Maackia amurensis) that can recognize specific glycans like galactose Gal-(1-4) GlcNAc and sialic acid in an (α2-3) linkage, which displayed high sensitivity and high specificity, i.e. 90% and 85% for DSA and 90.91% and 95% for MAA. The new assay has a detection limit of 5 µg µl-1 and enables the quick (30 min) and sensitive detection of severe Dengue cases. The reported point-of-care immunoassay exhibits considerable promise for early identification of patients with Dengue severity.

Intensity Modulated Radiotherapy in Head and Neck Cancer: Initial Experience of the First Treated Cases from North-East India.

Head and neck cancer incidence is relatively higher in north-eastern states than in other parts of India. Intensity-modulated radiotherapy (IMRT) was first introduced in our institute in 2012 to cater the entire north-east. This study attempts to assess the treatment outcomes and prognostic factors of head and neck cancer patients who had been treated with definitive radiotherapy in our institute from 2012 to 2016 using IMRT. This is a single institutional retrospective study. Thirty-six patients of head and neck cancer other than nasopharynx primary treated with definitive radiotherapy using IMRT between 2012 to 2016 were evaluated. The survival was analyzed using the Kaplan-Meir method, and various clinicopathologic factors were compared. The median age of the study patients was 58 years (range 24-79 years). The majority of the patients (69.4%) were below the age of 50 years. Thirty-two patients (88.9%) were male, and only four (11.1%) were females. After a median follow-up time of 40 months (7-84 months), the 5-year overall survival (OS) was 42.1%. The stage at presentation and radiotherapy treatment time were found to be significant prognostic factors of the outcome. The treatment-related toxicities were within acceptable limits. This retrospective study has reported the outcome and treatment-related toxicities of initially treated HNC patients with IMRT from northeast India.

Comprehensive investigation of fever cases enrolled during 2019 dengue outbreaks from three hyperendemic regions of North 24 Parganas district of West Bengal, India.

For the past several decades, dengue fever has been emerging in epidemic proportions in several regions of the world. During August-September 2019, an increasing number of fever cases were being reported from some areas of North 24 Parganas district of West Bengal, India. Accordingly, outbreak investigation of fever cases from these affected areas of Bongoan, Barasat, and Habra was carried out. To characterize clinical and biochemical features of fever cases as well as to investigate the utility of CRP as a Dengue severity marker in resource-limited settings. We systematically enrolled 108 patients from the affected region of North 24 Parganas. Standard diagnostic assays along with routine serological and biochemical parameters were performed. Of the 108 patients, 77 (71%) were confirmed with Dengue infection followed by 22 (20%) DENV seronegative and 9 (8%) coinfected DENV cases. Among the 77 confirmed Dengue patients, 53 (69%) had primary infection while 24 (31%) had secondary infection. Among the DENV clinical symptoms, fever (r = 0.50; p = 0.004), headache (r = 0.40; p = 0.03) and abdominal pain (r = -0.40; p = 0.02) were found to bear significant correlation with DENV viral load. The predominant circulating serotype was found to be DENV2. CRP Dengue severity cut-off level of 10.15 mg/L (AUC: 0.85; 86% sensitivity, 77% specificity) was obtained. CRP had correlation with viral load (r = 0.4, p = 0.05) within febrile phase of infection. The performance of biomarkers can be influenced by local epidemiology, geography, and several patient factors, therefore, CRP Dengue severity cut-off value may be region-specific. This study for the first time attempts to estimate CRP Dengue severity cut-off value based on routine immunoturbidometric evaluation from Dengue Hyperendemic zones of North 24 Parganas, West Bengal, Eastern India.

US QE and the Indian Bond Market.

This paper examines the long- and short-run spillover effects of US quantitative easing (QE) on the benchmark 10-year Indian government bond (IGB) yield by Autoregressive Distributed Lag (ARDL) bounds testing co-integration approach using monthly data from September 2008 to June 2019. The results show that a 10%-point rise in US QE led to a 4 bp rise in yields. The counterfactual analysis shows that volatility of the yields would have been less without the QE. During the episodes of QE, the Reserve Bank of India (RBI) had to alter its policy rate and engage in open-market operations (OMOs) to simultaneously maintain liquidity in the system and reduce the volatility of interest rates. Spillover on the debt yield leads to mispricing of assets and partial loss of the monetary-policy autonomy of the RBI.

Clinical Outcomes and Prognostic Factors of Locally Advanced Nasopharyngeal Cancer Treated with Intensity Modulated Radiotherapy: First Experience Report from Northeast India.

The aim is to retrospectively evaluate the clinical outcomes and prognostic factors in patients of locally advanced nasopharyngeal cancer (NPC) treated with intensity-modulated radiotherapy (IMRT). The northeastern states report relatively more NPC cases in comparison to other states of India. This study is an attempt to assess the treatment outcomes and prognostic factors of locally advanced NPC who had been treated with definitive radiotherapy with or without chemotherapy in our institute from 2012 to 2016 using IMRT. This is a single institutional retrospective study. Thirty-one consecutive patients of locally advanced NPC treated with definitive chemoradiation using the IMRT technique between 2012 to 2016 were evaluated. The survival was analyzed using Kaplan-Meir method and their relations with various clinicopathologic parameters were compared. After a median follow-up time of 36 months, the 5-year overall survival (OS) and disease-free survival (DFS) was 47.3% and 26.1% respectively. The younger patients of < 50 years had improved OS (p = 0.05). Patients of stage IVA had inferior 5-year OS (p = 0.1) and 5-years DFS (0.02) than those of stage III. The patients who received neoadjuvant chemotherapy showed improved DFS at 5 years (p = 0.09). The treatment-related toxicities were within acceptable limit. This retrospective analysis has reported outcomes of locally advanced NPC patients treated with IMRT with concurrent chemotherapy when IMRT was first introduced in our institute. This is the first of its kind from the Northeastern region of India.

COVID surveillance robot: Monitoring social distancing constraints in indoor scenarios.

Observing social/physical distancing norms between humans has become an indispensable precaution to slow down the transmission of COVID-19. We present a novel method to automatically detect pairs of humans in a crowded scenario who are not maintaining social distancing, i.e. about 2 meters of space between them using an autonomous mobile robot and existing CCTV (Closed-Circuit TeleVision) cameras. The robot is equipped with commodity sensors, namely an RGB-D (Red Green Blue-Depth) camera and a 2-D lidar to detect social distancing breaches within their sensing range and navigate towards the location of the breach. Moreover, it discreetly alerts the relevant people to move apart by using a mounted display. In addition, we also equip the robot with a thermal camera that transmits thermal images to security/healthcare personnel who monitors COVID symptoms such as a fever. In indoor scenarios, we integrate the mobile robot setup with a static wall-mounted CCTV camera to further improve the number of social distancing breaches detected, accurately pursuing walking groups of people etc. We highlight the performance benefits of our robot + CCTV approach in different static and dynamic indoor scenarios.

Effect of corticosteroid ointment on radiation induced dermatitis in head and neck cancer patients: A prospective study.

BACKGROUND: Almost all the patients receiving curative radiotherapy for head and neck cancer develop radiation dermatitis, which many a times leads to treatment interruption and reduce patient compliance. In this study, we evaluated the effect of potent topical steroid (Betamethasone Valerate 0.1%) cream on acute radiation dermatitis in head and neck cancer patients receiving curative radiotherapy. METHODS: A total 106 patients of head and neck cancers were randomly divided into arm A (52 patients) and arm B (54 patients). The patient in study arm A were treated with topical betamethasone 0.1% twice daily during radiotherapy/chemo-radiotherapy and arm B was kept as control. The radiation reaction in both the groups was monitored weekly according to Radiation Therapy Oncology Group (RTOG) acute radiation dermatitis grading. RESULTS: Out of 106 patients, 85 (80.2%) patients completed treatment. Patient in control arm had earlier onset of grade 1 reaction (5.7% in arm A vs 16.7 % in arm B at 2nd week, P value 0.157 and 28.8% in arm A vs 50% in arm B at 3rd week, P value 0.028) and progression of radiation dermatitis. In 7th week patient in arm A had higher grade 1 reaction (17.3% in arm A vs 0% in arm B), while arm B had higher grade 2 reaction (66.7% arm B vs 55.8% in arm A). There was no difference in incidence of grade 3 and 4 reaction. No difference was observed in time taken for reaction to heal. CONCLUSION: Topical Betamethasone can delay the onset and progression of radiation dermatitis in head and neck cancer, without significant delay in wound healing.

Blockade of HMGB1 Attenuates Diabetic Nephropathy in Mice.

Activation of TLR2 or TLR4 by endogenous ligands such as high mobility group box 1 (HMGB1) may mediate inflammation causing diabetic kidney injury. We determined whether blockade of HMGB1 signaling by: (1) supra-physiological production of endogenous secretory Receptor for Advanced Glycation End-products (esRAGE), a receptor for HMGB1; (2) administration of HMGB1 A Box, a specific competitive antagonist, would inhibit development of streptozotocin induced diabetic nephropathy (DN). Wild-type diabetic mice developed albuminuria, glomerular injuries, interstitial fibrosis and renal inflammation. Using an adeno-associated virus vector, systemic over-expression of esRAGE afforded significant protection from all parameters. No protection was achieved by a control vector which expressed human serum albumin. Administration of A Box was similarly protective against development of DN. To determine the mechanism(s) of protection, we found that whilst deficiency of TLR2, TLR4 or RAGE afforded partial protection from development of DN, over-expression of esRAGE provided additional protection in TLR2-/-, modest protection against podocyte damage only in TLR4-/- and no protection in RAGE-/- diabetic mice, suggesting the protection provided by esRAGE was primarily through interruption of RAGE and TLR4 pathways. We conclude that strategies to block the interaction between HMGB1 and its receptors may be effective in preventing the development of DN.

Serum amyloid A receptor blockade and incorporation into high-density lipoprotein modulates its pro-inflammatory and pro-thrombotic activities on vascular endothelial cells.

The acute phase protein serum amyloid A (SAA), a marker of inflammation, induces expression of pro-inflammatory and pro-thrombotic mediators including ICAM-1, VCAM-1, IL-6, IL-8, MCP-1 and tissue factor (TF) in both monocytes/macrophages and endothelial cells, and induces endothelial dysfunction-a precursor to atherosclerosis. In this study, we determined the effect of pharmacological inhibition of known SAA receptors on pro-inflammatory and pro-thrombotic activities of SAA in human carotid artery endothelial cells (HCtAEC). HCtAEC were pre-treated with inhibitors of formyl peptide receptor-like-1 (FPRL-1), WRW4; receptor for advanced glycation-endproducts (RAGE), (endogenous secretory RAGE; esRAGE) and toll-like receptors-2/4 (TLR2/4) (OxPapC), before stimulation by added SAA. Inhibitor activity was also compared to high-density lipoprotein (HDL), a known inhibitor of SAA-induced effects on endothelial cells. SAA significantly increased gene expression of TF, NFκB and TNF and protein levels of TF and VEGF in HCtAEC. These effects were inhibited to variable extents by WRW4, esRAGE and OxPapC either alone or in combination, suggesting involvement of endothelial cell SAA receptors in pro-atherogenic gene expression. In contrast, HDL consistently showed the greatest inhibitory action, and often abrogated SAA-mediated responses. Increasing HDL levels relative to circulating free SAA may prevent SAA-mediated endothelial dysfunction and ameliorate atherogenesis.

Safety, feasibility, and effect of remote ischemic conditioning in patients undergoing lung transplantation.

BACKGROUND: Primary graft dysfunction (PGD) remains a significant problem after lung transplantation. Data from animal and clinical studies suggest that remote ischemic conditioning (RIC) may reduce ischemia-reperfusion injury in solid organ transplantation. METHODS: A pilot randomized controlled trial of 60 patients undergoing bilateral sequential lung transplantation assessed the utility of RIC in attenuating PGD. Treated recipients underwent 3 cycles of lower limb ischemic conditioning before allograft reperfusion. The primary outcome measure was a comparison of the partial pressure of arterial oxygen/fraction of inspired oxygen ratio (P/F ratio) between treatment groups. RESULTS: No adverse effects of tourniquet application were observed. The mean lowest P/F ratio during the first 24 hours after transplantation was 271.3 mm Hg in the treatment arm vs 256.1 mm Hg in the control arm (p = 0.46). PGD grade and severity and the rate of acute rejection also showed a tendency to favor the treatment arm. Sub-group analysis demonstrated a significant benefit of treatment in patients with a primary diagnosis of restrictive lung disease, a group at high risk for the development of PGD. RIC was not accompanied by systemic release of high-molecular-weight group box 1. Levels of cytokines, high-molecular-weight group box 1, and endogenous secretory receptor for advanced glycation end products peaked within 2 hours after reperfusion and likely reflected donor organ quality rather than an effect of RIC. CONCLUSIONS: RIC did not significantly improve P/F ratios or PGD in this randomized controlled trial. However, encouraging results in this small study warrant a large multicenter trial of RIC in lung transplantation.

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences.

The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences.

The sequence selectivity of DNA-targeted 9-aminoacridine cisplatin analogues in a telomere-containing DNA sequence.

In this study, the detailed DNA sequence specificity of four acridine Pt complexes was examined and compared with that of cisplatin. The DNA sequence specificity was determined in a telomere-containing DNA sequence using a polymerase stop assay, with a fluorescent primer and an automated capillary DNA sequencer. The Pt compounds included an acridine intercalating moiety that was modified to give a 9-aminoacridine derivative, a 7-methoxy-9-aminoacridine derivative, a 7-fluoro-9-aminoacridine derivative and a 9-ethanolamine-acridine derivative. Compared with cisplatin, the DNA sequence specificity was most altered for the 7-methoxy-9-aminoacridine compound, followed by the 9-aminoacridine derivative, the 7-fluoro-9-aminoacridine compound and the 9-ethanolamine-acridine derivative. The DNA sequence selectivity for the four acridine Pt complexes was shifted away from runs of consecutive guanines towards single guanine bases, especially 5'-GA dinucleotides and sequences that contained 5'-CG. The sequence specificity was examined in telomeric and non-telomeric DNA sequences. Although it was found that telomeric DNA sequences were extensively damaged by the four acridine Pt complexes, there was no extra preference for telomeric sequences.