Scalable noninvasive amplicon-based precision sequencing (SNAPseq) for genetic diagnosis and screening of ß-thalassemia and sickle cell disease using a next-generation sequencing platform.

ß-hemoglobinopathies such as ß-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire ß-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline.

Prevalence of Dental Caries With Salivary Assessment in Six to Twelve Years Old School-Going Children in Shahpura Tehsil, Jaipur.

Introduction Dental caries is a chronic, infectious, and irreversible disease of the calcified tissues of teeth, which demineralises the inorganic portion and destroys the organic substance of the tooth, which often leads to cavitation. Epidemiological studies measuring the prevalence and severity of dental caries have used modified versions of Klein and colleagues' decayed, missing, and filled (DMF) or Gruebbel's decayed, extraction indicated, and filled (def) indexes; however, these indexes only capture cavitated lesions. Saliva plays a vital role in caries prevention; significant reduction or deterioration of salivary function can aggravate the development of dental caries. Saliva affects the incidence of dental caries in four ways: as a mechanical cleansing agent that results in less accumulation of plaque, by reducing enamel solubility using calcium, phosphate, and fluoride, by buffering and neutralising the acids produced by cariogenic organisms, or by introducing directly through diet and by antibacterial activity. The study aims to assess the prevalence of dental caries and salivary parameters such as salivary pH, flow, and buffering capacity in six to 12 years old children of a rural tehsil of Jaipur. Materials and methods The study was done on a population consisting of 400 school-going children in the age group of six to 12 years. Oral examination was undertaken by a single examiner, who is the study's principal investigator, to avoid inter-examiner variability. Testing of resting saliva was done for evaluation of visual inspection of the level of hydration, saliva consistency, pH measurement, saliva quantity, and buffering capacity. Statistical analysis The data were analysed using the chi-square test, t-test, and statistical software SPSS version 17.00 (SPSS Inc., Chicago, IL). The chi-square test was used to compare and analyse qualitative data, whereas the unpaired t-test was used to analyse and compare quantitative data. Quantitative data were summarised as mean and standard deviation. A p-value of 0.001 or less was considered for standard significance. Results There was no significant difference in resting salivary flow rate between children with decayed, missing, and filled teeth (DMFT) scores less than 5 and DMFT scores of 5. The mean buffering capacity of stimulated saliva was found to be significantly more among children with DMFT scores less than 5 than children with DMFT scores of 5 or more. The mean pH of resting saliva was found to be significantly higher among children with DMFT scores less than 5 than children with DMFT scores of 5 or more. Conclusion The prevalence of caries based on age was maximum in mixed dentition and minimum in primary dentition. In contrast, the difference in severity based on age was maximum in permanent dentition. The prevalence of caries was higher in children whose parents were aware of dental health; the difference was more significant in children with primary and mixed dentition. This study showed that salivary parameters such as salivary flow rate, salivary pH, and salivary buffering capacity among school-going children correlated with the prevalence of caries.

A Simple, Cost-Effective, and Extraction-Free Molecular Diagnostic Test for Sickle Cell Disease Using a Noninvasive Buccal Swab Specimen for a Limited-Resource Setting.

Sickle cell disease (SCD) is the most prevalent life-threatening blood monogenic disorder. Currently, there is no cure available, apart from bone marrow transplantation. Early and efficient diagnosis of SCD is key to disease management, which would make considerable strides in alleviating morbidity and reducing mortality. However, the cost and complexity of diagnostic procedures, such as the Sanger sequencing method, impede the early detection of SCD in a resource-limited setting. To address this, the current study demonstrates a simple and efficient proof-of-concept assay for the detection of patients and carriers using extraction-free non-invasive buccal swab samples by isothermal DNA Amplification coupled Restrictase-mediated cleavage (iDAR). This study is a first of its kind reporting the use of buccal swab specimens for iDA in molecular diagnosis of a genetic disease, all the while being cost effective and time saving, with the total assay time of around 150 min at a cost of USD 5. Further, iDAR demonstrates 91.5% sensitivity and 100% specificity for detecting all three alleles: SS, AS, and AA, having a 100% concordance with Sanger sequencing. The applicability of the iDAR assay is further demonstrated with its adaptation to a one-pot reaction format, which simplifies the assay system. Overall, iDAR is a simple, cost-effective, precise, and non-invasive assay for SCD screening, with the potential for use in a limited resource setting.

Effectiveness of Plyometrics Along With Pilates Exercises in Increasing Vertical Jump Performance Among Basketball Players.

Introduction Basketball is an athletic court game sport played by five players from two sports teams each. Therefore, the objective of this study is aimed at gauging the potential impact of the combination of plyometrics and Pilates training along with the efficacy of these exercise regimes individually on raising vertical jump performance in basketball players. Methodology For this study, 45 subjects were enrolled and assigned into groups A, B, and C. Group A was given a plyometric program, group B was given a Pilates program, and group C was given a combination of plyometrics and Pilates program. All were tested for vertical jump and muscle endurance prior to starting the plyometric and Pilates training and plyometrics with Pilates program. All the subjects passed through six weeks of the training program and were retested. The program was given three days a week for six weeks. Result The data collected were statistically analyzed by applying the Wilcoxon signed-rank test and the one-way analysis of variance test. The analysis of variance in vertical jump ability among three groups was assessed. The average value of vertical jump ability before training was 260.60 for group A, 243.47 for group B, and 263 for group C. Training group C had a higher mean vertical jump value. All groups showed improvement in vertical jump, but group C (plyometrics with Pilates) showed greater improvement in the vertical jump height and trunk flexor and trunk extensor endurance test. Conclusion The finding suggested that group C (plyometrics with Pilates) is more effective than group A (plyometrics) and B (Pilates) in improving the vertical jump height.