Molecular Laboratory Research Articles

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Molecular Characteristics of Regional Chromoblastomycosis in Guangdong, China: Epidemiological, Clinical, Antifungal Susceptibility, and Serum Cytokine Profiles of 45 Cases.

Chromoblastomycosis (CBM) is a chronic disease caused by several species of dematiaceous fungi. In this study, a regional collection of 45 CBM cases was conducted in Guangdong, China, a hyper-endemic area of CBM. Epidemiology findings indicated that the mean age of cases was 61.38 ± 11.20 years, long duration ranged from 3 months to 30 years, and the gender ratio of male to female was 4.6:1. Thirteen cases (29%) declared underlying diseases. Verrucous form was the most common clinical manifestation (n = 19, 42%). Forty-five corresponding clinical strains were isolated, and 28 of them (62%) were identified as F. monophora; the remaining 17 (38%) were identified as F. nubica through ITS rDNA sequence analysis. Antifungal susceptibility tests in vitro showed low MICs in azoles (PCZ 0.015–0.25 μg/ml, VCZ 0.015–0.5 μg/ml, and ITZ 0.03–0.5 μg/ml) and TRB (0.015–1 μg/ml). Itraconazole combined with terbinafine was the main therapeutic strategy used for 31 of 45 cases, and 68% (n = 21) of them improved or were cured. Cytokine profile assays indicated upregulation of IL-4, IL-7, IL-15, IL-11, and IL-17, while downregulation of IL-1RA, MIP-1β, IL-8, and IL-16 compared to healthy donors (p < 0.05). The abnormal cytokine profiles indicated impaired immune response to eliminate fungus in CBM cases, which probably contributed to the chronic duration of this disease. In conclusion, we investigated the molecular epidemiological, clinical, and laboratory characteristics of CBM in Guangdong, China, which may assist further clinical therapy, as well as fundamental pathogenesis studies of CBM.

Abstract P3-10-05: Feasibility of detecting long noncoding RNAs in clinical FFPE specimens using clinical grade RNA-sequencing pipeline

Abstract BACKGROUND: RNA-based predictive biomarkers are not assessed in the clinical diagnostic setting. Potential biomarkers include long noncoding RNAs (lncRNAs), a class of molecules emerging as critical regulators of diverse cellular functions, including therapy resistance. Advances in sequencing technologies have made whole transcriptome profiling (RNA-seq) possible in formalin fixed, paraffin embedded (FFPE) tissue. In this study, we assessed the feasibility of detecting lncRNAs in clinical FFPE specimens, using clinical grade RNA-seq technology offered at a CLIA-certified laboratory. Our second aim was to investigate expression of lncRNAs contributing to endocrine resistance in a cohort of metastatic breast cancer patients. METHODS: RNA-seq was performed on clinical FFPE specimens as a physician-ordered diagnostic test in a CLIA-certified laboratory. RNA-seq libraries were constructed using ribosomal RNA depletion method and &amp;gt; 100 M reads were sequenced for each library. We checked for contaminating sequences using fastqScreen against multiple genomes, including human and E. coli among others. To assess the quality of the sequencing libraries, we used the nf-core RNASeq pipeline. Trimmed reads were mapped to the GRCh38 reference and abundance of transcripts from the Gencode comprehensive annotation (v33) was estimated. Each library was assessed for quality control (QC). Mapped reads were merged into a matrix of counts per gene for each sample. Differential expression was assessed between metastatic breast cancers with and without endocrine resistance. RESULTS: We obtained twenty-five cases (23 patients) of ER-positive, HER2 negative metastatic breast carcinoma that had undergone whole transcriptome sequencing at a CLIA-certified molecular laboratory. Of these, 6 (24%) cases had failed QC: 5 patients’ specimens had low RNA content (all core biopsies); 1 patient had total RNA content comprised of less than 75% human reads (lumpectomy). The mean age for specimens that failed sequencing QC was significantly different from those that passed (637.2 days vs 193.7 days, t-test, p = 0.04). After QC, a total of 19 cases were analyzed for differentially expressed lncRNA profiles. Of these, 5 cases were clinically annotated as hormone sensitive; 14 cases had acquired secondary hormone resistance. Thirty-six lncRNAs were identified as differentially expressed (7 antisense; 25 intergenic; 3 intronic; 1 sense-overlapping). Of these, lncRNAs which were found to be over-expressed in endocrine resistant tumors compared to endocrine sensitive tumors are involved in various oncogenic pathways such as cell cycle regulation (Linc0051, MIR205HG, ST8SOA6-AS1), tumor progression (LINC00885, LINC00319), and chemoresistance (SIRLNT). LncRNAs which were downregulated in endocrine resistant tumors compared to endocrine sensitive tumors are involved in stem cell state (CCDC144NL.AS1) and endocrine resistance (MIR2052HG). CONCLUSION: It is feasible to detect lncRNA biomarkers in clinical FFPE tissue specimens, using a laboratory-developed RNA sequencing pipeline in a CLIA-certified genomic laboratory. FFPE-RNA-seq pipeline can potentially expand the molecular information that could be gleamed from clinical FFPE specimens to answer pertinent clinical questions, such as therapy resistance. Table 1.Summary data of metastatic ER+ breast cancer evaluated in this studyFailed RNA QC Success RNA QCNumber of samples (%)6 (24%)19 (76%)Mean specimen age (range)27 days (11-44)194 days (3-1331)Number of specimens which are biopsies (%)5 (83%)17 (89%)Number of specimens derived from bone source (%)2 (33%)1 (5%)Number of specimens derived from visceral organs (%)2 (33%)12 (63%)Number of specimens derived from soft tissue or lymphoid sources (%)2 (33%)6 (32%)Acquired endocrine resistance5 (83%)14 (74%)Endocrine sensitive1 (17%)5 (26%) Citation Format: Christina H. Wei, Denis O'Meally, Michele Kirschenbaum, Allen Mao, Chetan Kancharla, Kevin V. Morris, Daphne Stewart. Feasibility of detecting long noncoding RNAs in clinical FFPE specimens using clinical grade RNA-sequencing pipeline [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-05.

Prototype of a Transition Metal Visualization App for the Learning of Stereochemistry in a General Chemistry Course: Initial Findings and Reflections

As part of COVID-19 preparedness, a student-developed, Android-based app was used as a pre-laboratory learning aid for a molecular modeling laboratory in a first-year general chemistry course. A worksheet activity with trigger codes and questions related to spatial features of transition metal complexes was designed. Using the Transition Metal Visualization (TMVis) app, learners activated augmented reality models on their mobile phones. Actual models were then constructed in class. Mean stereochemistry post-laboratory grades increased with stronger worksheet grades, though differences between worksheet grades for app and non-app users were not significant. Feedback revealed that approximately 60% of respondents preferred the physical modeling kit, citing ease of visualization and hands-on interactivity as its key strengths. Improvements for future work were proposed in light of the results and limitations.

External quality assessment of COVID-19 real time reverse transcription PCR laboratories in India.

Sudden emergence and rapid spread of COVID-19 created an inevitable need for expansion of the COVID-19 laboratory testing network across the world. The strategy to test-track-treat was advocated for quick detection and containment of the disease. Being the second most populous country in the world, India was challenged to make COVID-19 testing available and accessible in all parts of the country. The molecular laboratory testing network was augmented expeditiously, and number of laboratories was increased from one in January 2020 to 2951 till mid-September, 2021. This rapid expansion warranted the need to have inbuilt systems of quality control/ quality assurance. In addition to the ongoing inter-laboratory quality control (ILQC), India implemented an External Quality Assurance Program (EQAP) with assistance from World Health Organization (WHO) and Royal College of Pathologists, Australasia. Out of the 953 open system rRTPCR laboratories in both public and private sector who participated in the first round of EQAP, 891(93.4%) laboratories obtained a passing score of > = 80%. The satisfactory performance of Indian COVID-19 testing laboratories has boosted the confidence of the public and policy makers in the quality of testing. ILQC and EQAP need to continue to ensure adherence of the testing laboratories to the desired quality standards.

Polyvinylchloride and polypropylene as adsorbents of the pesticide monocrotophos enhance oxidative stress in Eudrillus eugeniae (Kinberg)

The use of plastics has increased significantly with consequent rise in the generation of wastes. Microplastics (MPs) with particle size <5 mm are produced in natural terrestrial habitats by weathering of the discarded plastic debris and therefore are likely to impact soil biota. Earthworms are the dominant soil fauna which play vital role in soil formation and decomposition of organics. Since these animals are soil feeders, MP particles contaminating soil are likely to enter in to the gut of these animals affecting their physiology. MPs have been shown to be potent adsorbents of various other pollutants such as heavy metals and agrochemicals. This study reports the effects of two MPs, polyvinyl chloride (PVC) and polypropylene (PP) alone and in combination with the pesticide monocrotophos in soil on tissue protein, lipid peroxidation (LPX), activities of lactate dehydrogenase (LDH) and catalase (CAT) of an epigeic earthworm Eudrillus eugeniae over an exposure period of 48h. Results from molecular docking and laboratory experiment confirmed that both the MPs are potent adsorbents of the pesticide and enhanced oxidative stress on the animal with significant reduction in protein, increased LPX level and enzyme activities. PP indicated significantly higher pesticide adsorption relative to PVC.

The U.S. National Library of Medicine's Impact on Precision and Genomic Medicine.

Precision medicine offers the potential to improve health through deeper understandings of the lifestyle, biological, and environmental influences on health. Under Dr. Donald A.B. Lindberg's leadership, the U.S. National Library of Medicine (NLM) has developed the central reference resources for biomedical research and molecular laboratory medicine that enable precision medicine. The hosting and curation of biomedical knowledge repositories and data by NLM enable quality information reachable for providers and researchers throughout the world. NLM has been supporting the innovation of electronic health record systems to implement computability and secondary use for biomedical research, producing the scale of linked health and molecular datasets necessary for precision medicine discovery.

Anosmia with or without Ageusia as a Presenting Symptom in COVID-19 Positive Patients: A Single Center Study at Dhaka in Bangladesh

Background: The loss of smell and taste in COVID-19 patients is now acknowledged as one of the disease’s primary symptoms.&#x0D; Objective: The aim of this study was to investigate the incidence of loss of smell and taste in outpatients and hospitalized patients with SARS-CoV-2 infection that RT-PCR had confirmed.&#x0D; Methodology: This cross-sectional study was conducted in the in the Novus Molecular Lab, Dhaka between 5 June 2021 and 17 July 2021]. COVID-19 patients were included in this study who become RT-PCR positive in nasopharyngeal and oropharyngeal swabs in a single center. All of them were interviewed by a structured questionnaire over the phone.&#x0D; Results: Among 200 COVID-19-positive patients smell and taste dysfunction appeared among 75(37.5%) patients. Only smell dysfunction occurred in 57.0% of patients, and taste dysfunction appears in 34.0% patients, and both symptoms were present among 9.0% of patients with or without other symptoms of COVID-19. The smell and taste recovery time was distributed as 63.0% recovered within less than a week, 20.0% within 2 weeks, and 17.0% in 2 weeks.&#x0D; Conclusion: In conclusion significant number of COVID-19 patients are presented with loss of smell and taste.&#x0D; Bangladesh Journal of Infectious Diseases June 2021;8(1):32-35

Acute intermittent porphyria in adults: a clinical case

Background. Porphyria unites genetic pathologies related to abnormal haem (an intermediate product of haemoglobin metabolism) synthesis and its toxic products accumulation in human body. Symptoms can vary, from photosensitivity, skin rashes and chronic abdominal pain towards partial or complete paralysis and acute psychosis. This metabolic disorder is diagnosed with molecular genetic and laboratory biosample tests. Drug therapy aims at reducing toxic metabolites concentration in patient’s blood.Clinical Case Description. A 28-yo female patient had an acute atypical porphyria attack with a later onset of neurovisceral manifestations (acute abdominal pain, tachycardia) progressing post-drug-treatment into acute sensorimotor polyneuropathy with flaccid, predominantly proximal, hands-prevalent tetraparaesis. Biochemical urine tests at the National Research Center for Hematology (by 30.06.2020) revealed porphobilinogen 55.3 mg/L at norm &lt;3. Vital indications required an urgent haem arginate pathogenetic therapy (Normosang) in a 4-day course of 3 mg/kg/day drop infusion. The recommended course was well tolerated. Drug therapy and rehabilitation entailed a positive dynamics of restoring limb muscle strength towards an almost easy getting-up from chair and bed, and skin lightening. The patient was discharged on day 20 with diagnosis: “Acute intermittent porphyria. Axonal-demyelinating sensorimotor polyneuropathy. Severe flaccid asymmetric predominantly proximal hands-prevalent tetraparaesis. Subacute course, stabilisation phase. Condition after one course of haem arginate pathogenetic therapy (Normosang) at 3 mg/kg/day”. A resident haematologist surveillance was recommended, with a routine referral for inpatient examination and treatment at the Department of Orphan Diseases of the National Research Center for Hematology, Ministry of Health of Russia.Conclusion. Porphyria is relatively rarely diagnosed, about 12 cases per 100,000 people. The symptoms variety and nonspecificity conduce to a low detection rate, and untimely diagnoses can entail severe clinical manifestations, including lethal outcomes.

Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types.

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible.

Hidden Diversity-A New Speciose Gall Midge Genus (Diptera: Cecidomyiidae) Associated with Succulent Aizoaceae in South Africa.

Simple SummarySucculent Aizoaceae (often called “mesembs” or ice plants) form a dominant component of the Succulent Karoo in southern Africa, constituting one of the most species-rich families within the Greater Cape Floristic Region (GCFR). Despite the diversity and abundance of these plants, the diversity of insects specialized on them has never been surveyed methodically prior to this study. In a three-year study of the galling insects associated with succulent Aizoaceae in South Africa, we found that they support a rich community of gall midges (Diptera: Cecidomyiidae), virtually all of which are new to science. This is not surprising, given that knowledge of the Afrotropical fauna of gall midges is scarce, with most species described in the 1900s. Here, we describe the new genus Ruschiola with ten species from succulent Aizoaceae in Namaqualand, the Knersvlakte and the Cedarberg regions of South Africa based on morphological, molecular and life history data. The genetic data were particularly important in this study for differentiating taxa, given that Ruschiola species are very similar morphologically. Members of this genus develop in leaf galls or in plant tissues without visible gall formation, and are highly host specific.Aizoaceae (Caryophyllales) constitute one of the major floral components of the unique Greater Cape Floristic Region (GCFR), with more than 1700 species and 70% endemism. Within succulent Aizoaceae, the subfamily Ruschioideae is the most speciose and rapidly diversifying clade, offering potential niches for the diversification of specialized herbivorous insects. Nevertheless, insect diversity on these plants has not been studied to date, and knowledge of gall-inducing insects in the Afrotropics is generally scarce. Our recent observations indicate that succulent Aizoaceae in the GCFR support a rich and largely unstudied community of gall midges (Diptera: Cecidomyiidae). Here, we provide a first report of their diversity with a description of a new genus, Ruschiola Dorchin, and ten new species, based on morphological and molecular analyses of material collected during a three-year targeted survey across major GCFR vegetation types. A high degree of morphological uniformity in Ruschiola suggests recent diversification and necessitated the use of molecular data and laboratory rearing from host plants to verify species boundaries and host ranges.

Role of Quality Management System in Setting up and Sustaining a Molecular Diagnostic Laboratory during COVID-19 Pandemic

Introduction: Coronavirus Disease-2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2) catapulted the need to build, upgrade and expand the number of diagnostic laboratories having molecular capacity. Setting up and sustaining a molecular laboratory especially in the backdrop of a lockdown presented many challenges. The Department of Microbiology, in a tertiary level hospital in Mumbai was one of the first to start the molecular testing laboratory. All other tests performed in the department are accredited as per International Organisation for Standardisation (ISO) 15189:2012 since 2015. However, starting a molecular diagnostic facility for COVID-19 testing presented a unique set of challenges as the organism in question belonged to risk category 2 and had the potential for airborne transmission. Aim: To determine the challenges faced and activities undertaken especially with regards to the role of Quality Management System (QMS) in setting up and sustaining a molecular diagnostic facility during COVID-19 pandemic. Materials and Methods: A retrospective analysis was carried out of experiences and data generated from March 2020 to April 2021 at the Microbiology Department of a tertiary level medical college and hospital in Mumbai, Maharashtra, India. The article included the processes which required and data generated during setting up and sustaining a new molecular testing facility as per the QMS with special reference to the 12 Quality System Essentials (QSE). Quality Indicators (QI) were identified, objectives defined and monitored over the period of the study. It was a descriptive study and statistical analysis was not indicated. Results: All the objectives of the QI were met with. Only 4% staff needed corrective training. Specimen rejection rate pretest and post-test was 0.26% and 0.56%, respectively. Quality control failure was seen in 0.16% runs and Turnaround Time (TAT) deviated beyond 12 hours in 0.52% samples. The run contamination, equipment problems and laboratory associated infections were 0.08%, 0.56% and 0%, respectively. There were no External Quality Assessment (EQAS) failure and negative feedback. Laboratory contamination rate was 1.02%. Definite improvement was observed over time in all identified parameters. Conclusion: Implementation of QMS with specific reference to strengthening QSE is a necessary requirement for achieving quality standards.

Microsatellites based parental polymorphism survey for moisture stress in Rice (Oryza sativa L.) between the parental genotypes HUR-1309 and CR Dhan 801

The present investigation was conducted during Rabi 2018-19. The parental polymorphism research was done at the Molecular Drought Breeding Laboratory (Department of Genetics and Plant Breeding), Institute of Agricultural Sciences, BHU, and the Crop Improvement Division, ICAR-NRRI, Cuttack. The purpose of this research was to identify the parental polymorphism markers, analyze their chromosomal distribution, and determine the repetitive motifs. The polymorphic markers will be helpful to introgress the drought tolerant yield QTLs (qDTY) from the donor CR Dhan 801 into the background of HUR-1309, a popular aromatic short duration variety through marker-assisted backcross breeding program. Polymerase chain reaction (PCR), agarose gel electrophoresis, and genomic DNA separation were carried out according to standard procedures. A total of 510 microsatellites were used for evaluation of the parental polymorphism and 90 markers showed polymorphism among the parents and the rest 420 markers are monomorphic for the studied QTLs (qDTY1.1, qDTY2.1, and qDTY3.1). The parental polymorphism in the present study ranged between 11.43% to 30.61%. Considering the 12 chromosomes of rice, the average polymorphism was observed 17.65%. Out of 90 polymorphic markers, 59 were dinucleotide repeats, 24 were trinucleotide repeats and 4 were tetra nucleotide repeats. The dinucleotides were highly present on chromosomes 1 (12), 2 (9), and 3 (9) and repeated 12, 9, and 9 times respectively. The markers which are found to be polymorphic can be used in the marker-assisted backcross breeding (MABB) program for grain yield under moisture stress.

Design of de Laval nozzles for gas-phase molecular studies in uniform supersonic flow

A method to design de Laval nozzles to generate uniform supersonic flows for gas-phase molecular studies at very low temperature is presented. The nozzle design is optimized for the flows in argon, helium, or nitrogen, up to Mach 5 and down to a few kelvin. Experimental results have shown that flows exhibit a good uniformity in terms of speed, temperature, and density, with the length of the uniformity of the supersonic flows up to 50 cm which corresponds to a kinetic time of about 1 ms in nitrogen for nozzles with a throat of about 1 cm in diameter. The design of the de Laval nozzles is concentrated at the diverging section. The method is based on the calculation of an isentropic core as described in Owen's work [J. M. Owen, “An improved method of supersonic nozzle design for rarefied gas flows,” Ph.D. thesis (University of California, 1950)] of supersonic nozzle design for rarefied gas flows. The determination of the isentropic nozzle wall is carried out by the method of characteristics following Cronvich's algorithm [L. Cronvich, “A numerical–graphical methods of characteristics for axially symmetric isentropic flow,” J. Aeronaut. Sci. 15, 156–162 (1948)]. The laminar boundary layer is corrected by employing Michel's integral method [R. Michel, “Aérodynamique: Couches limites, frottement et transfert de chaleur” (ENSAE, 1963)]. This approach has already largely shown its potency and had been widely used for 30 years in the field of experimental molecular physics or laboratory astrophysics [sometimes known under the french acronym CRESU for Cinetique de Réaction en Écoulement Supersonique Uniforme (reaction kinetics in uniform supersonic flow)]. Based on this approach, an in-house computer program with graphical user interface to design de Laval nozzles for kinetic studies is published for the first time.

Investigating Causal Genetic Variation in the yellow Gene of Drosophila melanogaster as a Means of Teaching Foundational Molecular Genetic Concepts &amp; Techniques

How genetic variation influences phenotypic variation is of importance to many biological disciplines, including evolutionary biology, biomedicine, and agriculture. Nevertheless, students frequently struggle to make connections across levels of biological organization, which can make it challenging to facilitate understanding of how nucleotide variation gives rise to organismal variation. At the same time, biology students are now expected to gain early experience with cornerstone techniques from molecular biology, so that these skills can be reinforced and expanded upon. Here we describe a five-to-seven-week sequencing project that examines genetic and phenotypic variation in wild-type and yellow-bodied fruit flies and, in the process, exposes students to several foundational techniques in molecular biology. In addition, students analyze partial yellow gene sequences from PCR products using the freely available bioinformatics suite UGENE and in doing so are introduced to core bioinformatics skills. The entire project is framed around the axiom that if the yellow gene controls phenotypic differences in body color between wild-type and yellow-bodied flies, it should be possible to identify causal variation in yellow sequences from wild-type versus yellow-bodied flies. This project relies on guided inquiry and can be used in 1000- or 2000-level molecular biology courses and advanced high school laboratories.

CATALASE CRS-262T GENE POLYMORPHISM AND CHANGES IN VENTILATION LUNG CAPACITY IN CHILDREN-RESIDENTS OF RADIOACTIVELY CONTAMINATED TERRITORIES.

to determine the association of catalase С-262Т gene polymorphism with the presence of bronchial hyper-reactivity in children living in radioactively contaminated territories. There were examined school-age children-residents of radioactively contaminated territories (RCT), who did not have clinical signs of respiratory pathology. Catalase (CAT) С-262Т gene deletion polymorphism was studied in the molecular genetic laboratory of the State Institution «Reference Center for Molecular Diagnostic of Public Health Ministry of Ukraine». Determination of the polymorphic variant by the catalase С-262Т gene was performed by Polymerase Chain Reaction (PCR) using specific oligonucleotide primers, followed by Restriction Fragment Length Polymorphism (RFLP) analysis. The CAT С-262Т gene polymorphism in children living in RCT was compared with that in the reference group of practically healthy individuals. Ventilation lung capacity was performed by computer spirometry according to the analysis of the loop «the flow-volume». A pharmacological inhalation test with a bronchodilator that acts on β2-adrenergic receptors of the lungs was used to detect early changes in the ventilatory capacity of the lungs - bronchial hyperreactivity. Comparative analysis showed that in the presence of bronchial hyperreactivity in children living in RCT, the CT genotype was more common than in children without bronchial hyperreactivity, and the frequency of the CC genotype was correspondingly reduced. There was a trend towards a decrease in the frequency of the TT genotype. An analysis of the frequency distribution of allelic variants of the CAT С-262Т gene polymorphism in children living in the RCT revealed a tendency to increase in the frequency of the T-allele and according to the decrease in the frequency of C-allele in the presence of bronchial hyperreactivity.Сonclusions. Thus, among children living in RCT, CT-homozygotes of CAT С-262Т gene polymorphism had bronchial hyperreactivity probably more often than CC-heterozygotes. In the presence of bronchial hyperreactivity, there was a trend towards an increase in the frequency of the T-allele and, accordingly, a decrease in the frequency of the C-allele.

Rationale of using SARS-CoV-2 Rapid Antigen Test as a Point of care device in resource poor clinical set up: A comparative study in a peripheral health set up to meet up pandemic needs

COVID-19 testing with RT-PCR may result in late detection of disease. Rapid Antigen Detection Test is much faster, easy to perform and suitable for population wise testing strategies. Our study assesses the diagnostic accuracy of Rapid Antigen Test (RAT) compared to RT-PCR in peripheral health care set up. The study was aimed to detect the diagnostic accuracy of the COVID-19 antigen test such as to understand its position as a point of care device in a resource poor clinical set up during a rapidly spreading and high mortality ridden pandemic. In this study we compared SARS-CoV2 rapid antigen test and RT-PCR by considering that RT-PCR is the gold standard test in a rapidly arranged molecular diagnostic lab. We analyzed total 389 samples who have undergone both rapid antigen test and RT-PCR. The correlation between Ct values in RT-PCR test and RAT positivity was analyzed. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were found to be 75.47% (61.72-86.24%), 92.26% (88.87-94.88%), 33.92% (25.60-43.36%), 98.62% (97.8-99.14%) and 91.42% (88.19-94.19%), respectively. The k-coefficient of agreement between RAT and RTPCR was 0.615 ± 0.056 (0.504-0.724). For specimens with Ct value &lt; 20, 20-25, 26-30, 30-35 and &gt;35 were 100%, 90.3%, 80.3%, 60% for Ct value and 25% respectively. Our results suggest that RAT may be considered as an alternative to RT-PCR testing in peripheral health care set up. We reveal the importance of implementing rapid antigen test properly combined with clinical assessment of symptomatic patients so that it can be used in primary health care where RT-PCR testing facility is not available.

Subgenomic sequence analysis reveals emergence of new circulating recombinant forms of HIV-1 in Pakistan.

Background and Objective:Pakistan has witnessed a dramatic change in the increasing prevalence and emergence of HIV subtypes for more than two decades. Pakistani population is increasingly engaged in high-risk practices, and the prescribed drugs are potentially causing resistance. There are chances that these resistant strains are beginning to circulate from high-risk to the general population.Methods:The study was conducted at the section of Molecular Pathology Lab of Dow Diagnostic and Research Laboratory, Dow University of Health Sciences (DUHS) Karachi. In this study, we analyzed gene sequences of HIV for drug resistance and molecular epidemiology., along with amino acid sequence variability. Furthermore, we undertook phylogenetic analysis for possible geographic linkages of Pakistani HIV strains.Results:Our results demonstrate that A1 is the leading HIV subtype circulating in the country, whereas other emerging subtypes and recombinant forms, including subtype B, CRF02_AG, CRF10_CD CRF35_AD, and CRF11_cpx were also observed. Our sequences cluster with the Middle East, African, and a few European sequences according to geographical distribution. These sequences showed high-level resistance per drug resistance pattern, with 62.5% of patients exhibiting resistance to NNRTI drugs and 60% mutation at E138A and K103N, respectively, against NNRTI drugs. About 75% sequences showed resistance mutation at M184V against NRTI drugs. The antiretroviral drugs are now causing H-LR to the patients with no effect. Our results also revealed that certain regions of RT exhibited high sequence variability, especially at Amino Acids positions p.119, p.130, p.157, p.164.Conclusion:We hereby report major novel mutations and several minor mutations that may have a drastic change in the drug resistance pattern.

Assessing the in vitro Binding Affinity of Protein-RNA Interactions Using an RNA Pull-down Technique.

RNA is a vital component of the cell and is involved in a diverse range of cellular processes through a variety of functions. However, many of these functions cannot be performed without interactions with proteins. There are currently several techniques used to study protein-RNA interactions, such as electrophoretic mobility shift assay, fluorescence anisotropy, and filter binding. RNA-pulldown is a technique that uses biotinylated RNA probes to capture protein-RNA complexes of interest. First, the RNA probe and a recombinant protein are incubated to allow the in vitro interaction to occur. The fraction of bound protein is then captured by a biotin pull-down using streptavidin-agarose beads, followed by elution and immunoblotting for the recombinant protein with a His-tag-reactive probe. Overall, this method does not require specialized equipment outside what is typically found in a modern molecular laboratory and easily facilitates the maintenance of an RNase-free environment. This protocol was validated in: Nucleic Acids Res (2020), DOI: 10.1093/nar/gkaa029 Graphical abstract.

SARS-CoV-2 Positivity using Closed System RT-PCR at a Tertiary Care Medical Institute in Manipur, India

Introduction: Rapid cost effective, Point-of-Care (PoC) Truenat assay for the diagnosis of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) have been developed to shorten the Turn Around Time (TAT) of reporting with a wireless data transfer system. Aim: To explore the SARS-CoV-2 positivity using closed system Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Materials and Methods: An observational cross-sectional study was carried out in Molecular Laboratory of, Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal, Manipur, India, using Truenat RT-PCR (Molbio diagnostics) and data was entered from May 2020 to April 2021. Manufacturer’s literature was followed while performing the test. Screening of sample was done with Envelope (E) gene test and confirmed with RNA-dependent RNA polymerase gene (RdRP) gene test. Statistical analysis was done using Microsoft Excel sheet by calculating the percentage, proportions. Results: A total of 1,528 individuals were tested for SARSCov-2 and 73 tests were reported positive. The positivity rate by age was highest among 21-30 years. The positivity rate was higher among males than females. Among 1,105 asymptomatic individual, 27 (2.4%) were positive and among 423 symptomatic, 46 (10.9%) were positive. Conclusion: Using Truenat, positivity rate among symptomatic Coronavirus Disease 2019 (COVID-19) suspected persons was about four times more than positivity rate among exposed contact persons who are asymptomatic.