Impact of relative humidity on SARS-CoV-2 RNA extraction using Nextractor automated extraction system.

This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity's impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing.

Innovative disposable in-tip cellulose paper (DICP) device for facile determination of pesticides in postmortem blood samples: A proof-of-concept study.

Accurately identifying and quantifying toxicants is crucial for medico-legal investigations in forensic toxicology; however, low analyte concentrations and the complex samples matrix make this work difficult. Therefore, a simplified sample preparation procedure is crucial to streamline the analysis to minimize sample handling errors, reduce cost and improve the overall efficiency of analysis of toxicants. To address these challenges, an innovative disposable in-tip cellulose paper (DICP) device has been developed for the extraction of three pesticides viz. Chlorpyrifos, Quinalphos and Carbofuran from postmortem blood samples. The DICP device leverages cellulose paper strips housed within a pipette tip to streamline the extraction process, significantly reducing solvent usage, time, and labor while maintaining high analytical accuracy. The extraction of pesticides from postmortem blood using the DICP device involves a streamlined process characterized by adsorption and desorption. The diluted blood samples were processed through the DICP device via repeated aspirating and dispensing calyces to adsorb the pesticides onto the cellulose paper. The adsorbed pesticides are then eluted using acetone, which is collected for GC-MS analysis. The method was meticulously optimized, achieving a limit of quantification in the range of 0.009-0.01 µg mL-1. The intra-day and inter-day precisions were consistently less than 5 % and 10 %, respectively, with accuracy ranging from 94-106 %. Relative recoveries for the analytes were observed to be between 60 % and 93.3 %, and matrix effects were determined to be less than 10 %. The method's sustainability was validated with a whiteness score of 98.8, an AGREE score of 0.64, a BAGI score of 70 and ComplexMoGAPI score of 77. Applicability was demonstrated through successful analysis of real postmortem blood samples and proficiency testing samples, highlighting its potential utility in forensic toxicology.

A retrospective analysis on seroprevalence of acute viral hepatitis observed among dengue patients attending a tertiary care centre in central India.

PURPOSE: The present study was conducted retrospectively to assess the frequency of acute viral hepatitis among the clinically suspected dengue cases presented at our tertiary care centre during 2021. METHODS: To determine the presence of acute viral hepatitis; Hepatitis A virus (HAV) and Hepatitis E virus (HEV) infections, 104 specimens were selected from the dengue-suspected clinical specimens received during 2021 on the basis of acute viral hepatitis symptoms. Following this, serological diagnosis was performed on those samples using anti-HAV IgM and anti-HEV IgM ELISA kits. RESULTS: Based on sero-positivity for IgM antibodies, 3 (5.3%) dengue virus (DENV) seropositive samples were positive for both HAV and HEV, while among DENV seronegative cases, 11 (22.91%) samples were positive for HEV and 1 (2.08%) sample was positive for HAV, pointing towards misdiagnosis due to overlapping symptoms. Additionally, co-infection of HAV & HEV in 1 sample was also observed in this study. CONCLUSIONS: This study revealed the presence of acute hepatitis infections among the dengue cases during monsoon and post-monsoon season. Overlapping of the clinical manifestations of these diseases can create misdiagnosis incidences raising risk for underreporting of the true cases of acute viral hepatitis infection. Dengue-suspected patients with selected symptoms during the monsoon and post-monsoon season should additionally be screened for acute hepatitis infections, as suggested in this study.

Maintenance of Certification-The Value to Patients and Physicians.

This Viewpoint discusses the ABIM's continuing efforts to innovate and streamline maintenance of certification, including the recently launched Longitudinal Knowledge Assessment (LKA), to better accommodate physicians' schedules and desires for flexibility.

Performance Evaluation of Different RT-PCR Kits for the Direct Detection of SARS-CoV-2 in Preheated Specimens.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created high demand for molecular kits and consumables for mass screening of suspected individuals. Direct real-time polymerase chain reaction (RT-PCR) assay without nucleic acid extraction has several advantages in saving testing time and cost and helps in the rapid reporting of SARS-CoV-2. The present study evaluated the analytical performance of four SARS-CoV-2 RT-PCR for direct RT-PCR testing using preheated specimens. Methods A total of 100 clinical specimens were selected and divided into three different groups: (1) group I: 20 SARS-CoV-2 positive specimens with high viral load, viz., low Ct values (< 30 Ct), (2) group II: 50 SARS-CoV-2 positive specimens with low viral load, viz., high Ct values (> 30 Ct), and (3) group III: 30 SARS-CoV-2 negative specimens. Specimens were heat-inactivated at 70°C for 10 minutes and cooled down at 4°C and were evaluated for standard and direct RT-PCR method by using ViralDtect-II Multiplex Real-Time PCR kit, TaqPath COVID-19 Combo kit, COVIDsure Pro Multiplex RT-PCR kit, and Hi-PCR Coronavirus (COVID-19) Multiplex Probe PCR kit. Results Results showed that except ViralDtect-II kit, the other three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit were able to amplify all the SARS-CoV-2 genes in the direct RT-PCR method using preheated specimens. In group I specimens, 100% sensitivity was observed in all three RT-PCR kits. In group II specimens, COVIDsure Pro kit was found to be superior among other kits. Conclusion Direct RT-PCR method during pandemic situation is valuable and cost effective for the detection of SARS-CoV-2. All three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit can be used for direct RT-PCR method and COVIDsure Pro kit performance was found to be superior among all.

A Study of Relationship Between Serum Lipids and Sensorineural Hearing Loss.

The modern life style poses new challenges on the normal physiological mechanisms of the human body. Drug abuse, tobacco smoking, and alcohol drinking, as well as lack of exercise may also increase the risk of developing certain diseases, especially older age.To study the relationship between degree of sensorineural hearing loss and serum lipid level (total cholesterol, triglyceride, low density lipoproteins, high density lipoproteins).A cross-sectional study was conducted in 150 patients aged between 15 to 60 years diagnosed with sensorineural hearing loss at Otorhinolaryngology outpatient department at Sir Sunder Lal hospital, IMS, BHU. All the 150 patients were amongst age group of 15 to 60 years who were registered from August 2019 to July 2021.The serum levels of total cholesterol, triglyceride and low-density lipoprotein directly correlate with the severity of sensorineural hearing loss significantly (p < 0.05) whereas the serum levels of high-density lipoprotein had no direct co-relation with the severity of sensorineural hearing loss. Hyperlipidemic state is a major risk factor for SNHL. Regular screening and monitoring of serum lipid might prevent morbid SNHL and improve patients' quality of life in long term.

A Non-Instrumental Green Analytical Method Based on Surfactant-Assisted Dispersive Liquid-Liquid Microextraction-Thin-Layer Chromatography-Smartphone-Based Digital Image Colorimetry(SA-DLLME-TLC-SDIC) for Determining Favipiravir in Biological Samples.

Favipiravir (FAV) has become a promising antiviral agent for the treatment of COVID-19. Herein, a green, fast, high-sample-throughput, non-instrumental, and affordable analytical method is proposed based on surfactant-assisted dispersive liquid-liquid microextraction (SA-DLLME) combined with thin-layer chromatography-digital image colourimetry (TLC-DIC) for determining favipiravir in biological and pharmaceutical samples. Triton X-100 and dichloromethane (DCM) were used as the disperser and extraction solvents, respectively. The extract obtained after DLLME procedure was spotted on a TLC plate and allowed to develop with a mobile phase of chloroform:methanol (8:2, v/v). The developed plate was photographed using a smartphone under UV irradiation at 254 nm. The quantification of FAV was performed by analysing the digital images' spots with open-source ImageJ software. Multivariate optimisation using Plackett-Burman design (PBD) and central composite design (CCD) was performed for the screening and optimisation of significant factors. Under the optimised conditions, the method was found to be linear, ranging from 5 to 100 µg/spot, with a correlation coefficient (R2) ranging from 0.991 to 0.994. The limit of detection (LOD) and limit of quantification (LOQ) were in the ranges of 1.2-1.5 µg/spot and 3.96-4.29 µg/spot, respectively. The developed approach was successfully applied for the determination of FAV in biological (i.e., human urine and plasma) and pharmaceutical samples. The results obtained using the proposed methodology were compared to those obtained using HPLC-UV analysis and found to be in close agreement with one another. Additionally, the green character of the developed method with previously reported protocols was evaluated using the ComplexGAPI, AGREE, and Eco-Scale greenness assessment tools. The proposed method is green in nature and does not require any sophisticated high-end analytical instruments, and it can therefore be routinely applied for the analysis of FAV in various resource-limited laboratories during the COVID-19 pandemic.

Cellulose paper sorptive extraction (CPSE): A simple and affordable microextraction method for analysis of basic drugs in blood as a proof of concept.

Aiming towards simplifying sample preparation procedure, the present work explores use of unmodified laboratory filter paper as sorbent for extraction of nine basic drugs (five antidepressants, four benzodiazepines, and ketamine) from human blood samples and their analysis by gas chromatography-mass spectrometry (GC-MS). The procedure termed as cellulose paper sorptive extraction (CPSE) is straightforward. It involves adsorption of target analytes from deproteinized diluted blood samples on the unmodified cellulose paper followed by elution into 2 mL of methanol. Multivariate optimization, consisting of Placket-Burman design (PBD) and central composite design (CCD), was used to screen and optimize significant factors for CPSE. The proposed method follows the principles of green analytical chemistry (GAC), as the unmodified filter paper used as the sorbent is inexpensive and biodegradable. The technique is easy to perform and requires only 2 mL of MeOH during the entire extraction procedure. Under the optimized conditions, the limit of detection and quantification for the target analytes were estimated to be in the range of 0.003-0.035 and 0.010-0.117 µg mL-1, respectively. In contrast, the relative standard deviations were consistently below 10 %. The calibration curves were linear in the range of 0.015-2 µg mL-1 with a coefficient of determination (R2) in the range of 0.995-0.999.Satisfactory recoveries ranging from 87 to 99 % was achieved. As proof of concept, the analysis of nine drugs in blood samples from the patients was performed to demonstrate the potential application of the proposed method.

Simple determination of dichlorvos in cases of fatal intoxication by gas Chromatography-Mass spectrometry.

Dichlorvos (DDVP) is an organophosphorous insecticide which is classified as "highly hazardous" Class 1B chemical by World Health Organization (WHO) and largely misused for the purpose of self-poisoning in developing countries. Forensic toxicology laboratories are routinely encountering cases of pesticide poisoning due to their fatal intoxication. Herein; a method is described based on vortex-assisted dispersive liquid-liquid microextraction (VA-DLLME) coupled with Gas Chromatography-Mass Spectrometry (GC-MS) for the determination of an organophosphorous insecticide; dichlorvos (DDVP) in human autopsy samples (blood, stomach content and liver). Under the optimum conditions, the method was found to be linear in the range of 0.5-10 µg mL-1 and 1.5-10 µg g-1 for blood and tissue samples, respectively. Limit of quantification was set at 0.55 µg mL-1 and 1.1 µg g-1 for blood and tissue samples, respectively. Intraday and inter-day precisions were less than 8 and 12 %, respectively. Good recoveries in the range of 86-95 % were obtained for the proposed procedure. The method has been satisfactorily applied for the determination of DDVP in autopsy samples from two different cases received in our laboratory. In comparison to previous methods; the proposed method is relatively short, high sample throughput, inexpensive and adheres to the principles of green analytical chemistry (GAC) for determination of DDVP in human autopsy samples. The method can be adopted in forensic toxicological laboratories for analysis of DDVP in autopsy samples. In addition, the green character of the proposed method was evaluated using ComplexGAPI procedure.

Rapid Determination of Non-Steroidal Anti-Inflammatory Drugs in Urine Samples after In-Matrix Derivatization and Fabric Phase Sorptive Extraction-Gas Chromatography-Mass Spectrometry Analysis.

Fabric phase sorptive extraction (FPSE) has become a popular sorptive-based microextraction technique for the rapid analysis of a wide variety of analytes in complex matrices. The present study describes a simple and green analytical protocol based on in-matrix methyl chloroformate (MCF) derivatization of non-steroidal anti-inflammatory (NSAID) drugs in urine samples followed by FPSE and gas chromatography-mass spectrometry (GC-MS) analysis. Use of MCF as derivatizing reagent saves substantial amounts of time, reagent and energy, and can be directly performed in aqueous samples without any sample pre-treatment. The derivatized analytes were extracted using sol−gel Carbowax 20M coated FPSE membrane and eluted in 0.5 mL of MeOH for GC-MS analysis. A chemometric design of experiment-based approach was utilized comprising a Placket−Burman design (PBD) and central composite design (CCD) for screening and optimization of significant variables of derivatization and FPSE protocol, respectively. Under optimized conditions, the proposed FPSE-GC-MS method exhibited good linearity in the range of 0.1−10 µg mL−1 with coefficients of determination (R2) in the range of 0.998−0.999. The intra-day and inter-day precisions for the proposed method were lower than <7% and <10%, respectively. The developed method has been successfully applied to the determination of NSAIDs in urine samples of patients under their medication. Finally, the green character of the proposed method was evaluated using ComplexGAPI tool. The proposed method will pave the way for simper analysis of polar drugs by FPSE-GC-MS.

A mechanistic framework for cardiometabolic and coronary artery diseases.

Coronary atherosclerosis results from the delicate interplay of genetic and exogenous risk factors, principally taking place in metabolic organs and the arterial wall. Here we show that 224 gene-regulatory coexpression networks (GRNs) identified by integrating genetic and clinical data from patients with (n = 600) and without (n = 250) coronary artery disease (CAD) with RNA-seq data from seven disease-relevant tissues in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study largely capture this delicate interplay, explaining >54% of CAD heritability. Within 89 cross-tissue GRNs associated with clinical severity of CAD, 374 endocrine factors facilitated inter-organ interactions, primarily along an axis from adipose tissue to the liver (n = 152). This axis was independently replicated in genetically diverse mouse strains and by injection of recombinant forms of adipose endocrine factors (EPDR1, FCN2, FSTL3 and LBP) that markedly altered blood lipid and glucose levels in mice. Altogether, the STARNET database and the associated GRN browser (http://starnet.mssm.edu) provide a multiorgan framework for exploration of the molecular interplay between cardiometabolic disorders and CAD.

Determining the optimum cut-off IgM/ IgG ratio for predicting secondary dengue infections: An observational hospital based study from Central India.

PURPOSE: The present study was conducted to determine an optimum cut-off IgM/IgG ratio for differentiating between primary and secondary Dengue. The calculated cut-off was used to estimate the relative prevalence of primary and secondary Dengue in central India during the 2021 outbreak. METHODS: To differentiate between primary and secondary Dengue, optimum cut-off IgM/IgG ratio was determined using ROC curve analysis and Area under the curve (AUC) was calculated. For this 84 IgM positive samples were selected during the study period and Dengue IgM and IgG testing was performed. RESULTS: Based on sero-positivity for IgG antibodies, we observed a prevalence of 77.4% for secondary Dengue during this outbreak. IgM/IgG ratio of 1.59 delivered a sensitivity of 100% and specificity of 90.8%. Comparison of clinical characteristics revealed significant differences in rigors (p â€‹= â€‹0.04) and haemorrhagic manifestations (p â€‹= â€‹0.01) between the two types of infection. CONCLUSIONS: This study revealed an overwhelming predominance of secondary Dengue during the reported outbreak, which hints at increasing endemicity in this region and raises the risk of severe clinical manifestations. An optimal IgM/IgG cut-off ratio, tailored according to the endemicity of this geographical region, is therefore suggested in this study.

Frequency of metastasis to the gastrointestinal tract determined by endoscopy in a community-based gastroenterology practice.

Metastasis to the gastrointestinal tract is rare. We performed a retrospective analysis to identify patients with metastatic disease to the gastrointestinal tract using two databases containing pathology results from all endoscopic procedures conducted by nearly 200 gastroenterologists in a community setting over a 14-year period. Forty-nine patients were diagnosed with metastasis to the gastrointestinal tract by endoscopy during the study period. Most were women (71%). The most common metastases to the gastrointestinal tract identified by endoscopy were breast cancers (n = 18), followed by melanomas (n = 12), ovarian cancers (n = 7), kidney cancers (n = 5), prostate cancers (n = 2), lung cancer (n = 1), and pancreatic cancer (n = 1). Three patients had unknown primary sites. Among women, the three leading known primary tumor sites were breast, ovary, and melanoma. Among men, the three leading primary tumor sites were melanoma, kidney, and prostate. The stomach was the most common portion of the gastrointestinal tract involved by metastases. Most affected women and were most frequently encountered in the stomach.

Serious health threats of novel adulterants of the street heroin: a report from India during the COVID-19 pandemic.

The COVID-19 pandemic and a consequent nationwide lockdown in India for several weeks had restricted the access to street heroin and treatment for substance abuse. Use of cutting agents to increase the volume or psychoactive effect has been widely practised under such circumstances. Our patient with opioid use disorder chased heroin with an unknown cutting agent to enhance psychoactive effect from the limited quantities of heroin. He suffered from an abrupt onset sedation, weakness, postural imbalance, slurred speech, cognitive dysfunctions and disinhibited behaviour. Symptoms rapidly reversed following abstinence and initiation of buprenorphine-naloxone. Gas chromatography-mass spectrometric analysis of the adulterant revealed high concentrations of benzodiazepines and barbiturates, alongside the usual cutting agents-caffeine and acetaminophen. Abrupt reduction in availability of 'street drugs' in conjunction with poor healthcare access can lead to the use of novel adulterants with potentially serious clinical and public health implications.

Bi2O3/ZnO nanocomposite: Synthesis, characterizations and its application in electrochemical detection of balofloxacin as an anti-biotic drug.

In the present work, a chemically modified electrode has been fabricated utilizing Bi2O3/ZnO nanocomposite. The nanocomposite was synthesized by simple sonochemical method and characterized for its structural and morphological properties by using XRD, FESEM, EDAX, HRTEM and XPS techniques. The results clearly indicated co-existence of Bi2O3 and ZnO in the nanocomposite with chemical interaction between them. Bi2O3/ZnO nanocomposite based glassy carbon electrode (GCE) was utilized for sensitive voltammetric detection of an anti-biotic drug (balofloxacin). The modification amplified the electroactive surface area of the sensor, thus providing more sites for oxidation of analyte. Cyclic and square wave voltammograms revealed that Bi2O3/ZnO modified electrode provides excellent electrocatalytic action towards balofloxacin oxidation. The current exhibited a wide linear response in concentration range of 150-1000 nM and detection limit of 40.5 nM was attained. The modified electrode offered advantages in terms of simplicity of preparation, fair stability (RSD 1.45%), appreciable reproducibility (RSD 2.03%) and selectivity. The proposed sensor was applied for determining balofloxacin in commercial pharmaceutical formulations and blood serum samples with the mean recoveries of 99.09% and 99.5%, respectively.