High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.

TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.

Coding-complete genomes of 18 SARS-CoV-2 Omicron JN.1, JN.1.4, and JN.1.11 sub-lineages in Bangladesh.

We report 18 coding-complete genome sequences of emerging SARS-CoV-2 Omicron sub-lineages JN.1, JN.1.4, and JN.1.11 from Bangladesh. Nasopharyngeal swab samples were obtained from individuals with COVID-19 symptoms between December 2023 and January 2024. Whole genome sequencing was performed following the ARTIC Network-based protocol using Oxford Nanopore Technology.

Investigation of the impact of nonsynonymous mutations on thyroid peroxidase dimer.

Congenital hypothyroidism is one of the most common preventable endocrine disorders associated with thyroid dysgenesis or dyshormonogenesis. Thyroid peroxidase (TPO) gene defect is mainly responsible for dyshormonogenesis; a defect in the thyroid hormone biosynthesis pathway. In Bangladesh, there is limited data regarding the genetic etiology of Congenital Hypothyroidism (CH). The present study investigates the impact of the detected mutations (p.Ala373Ser, and p.Thr725Pro) on the TPO dimer protein. We have performed sequential molecular docking of H2O2 and I- ligands with both monomers of TPO dimer to understand the iodination process in thyroid hormone biosynthesis. Understanding homodimer interactions at the atomic level is a critical challenge to elucidate their biological mechanisms of action. The docking results reveal that mutations in the dimer severely disrupt its catalytic interaction with essential ligands. Molecular dynamics simulation has been performed to validate the docking results, thus realizing the consequence of the mutation in the biological system's mimic. The dynamics results expose that mutations destabilize the TPO dimer protein. Finally, principal component analysis exhibits structural and energy profile discrepancies in wild-type and mutant dimers. The findings of this study highlight that the mutations in TPO protein can critically affect the dimer structure and loss of enzymatic activity is persistent. Other factors also might influence the hormone synthesis pathway, which is under investigation.

Molecular investigation of TSHR gene in Bangladeshi congenital hypothyroid patients.

The disorder of thyroid gland development or thyroid dysgenesis accounts for 80-85% of congenital hypothyroidism (CH) cases. Mutations in the TSHR gene are mostly associated with thyroid dysgenesis, and prevent or disrupt normal development of the gland. There is limited data available on the genetic spectrum of congenital hypothyroid children in Bangladesh. Thus, an understanding of the molecular aetiology of thyroid dysgenesis is a prerequisite. The aim of the study was to investigate the effect of mutations in the TSHR gene on the small molecule thyrogenic drug-binding site of the protein. We identified two nonsynonymous mutations (p.Ser508Leu, p.Glu727Asp) in the exon 10 of the TSHR gene in 21 patients with dysgenesis by sequencing-based analysis. Later, the TSHR368-764 protein was modeled by the I-TASSER server for wild-type and mutant structures. The model proteins were targeted by thyrogenic drugs, MS437 and MS438 to perceive the effect of mutations. The damaging effect in drug-protein complexes of mutants was explored by molecular docking and molecular dynamics simulations. The binding affinity of wild-type protein was much higher than the mutant cases for both of the drug ligands (MS437 and MS438). Molecular dynamics simulates the dynamic behavior of wild-type and mutant complexes. MS437-TSHR368-764MT2 and MS438-TSHR368-764MT1 showed stable conformations in biological environments. Finally, Principle Component Analysis revealed structural and energy profile discrepancies. TSHR368-764MT1 exhibited much more variations than TSHR368-764WT and TSHR368-764MT2, emphasizing a more damaging pattern in TSHR368-764MT1. This genetic study might be helpful to explore the mutational impact on drug binding sites of TSHR protein which is important for future drug design and selection for the treatment of congenital hypothyroid children with dysgenesis.

SARS-CoV-2 antigen detection by saliva; an alternative to nasopharyngeal specimen: A cross-sectional study.

Background and Aims: Saliva samples are less invasive and more convenient for patients than naso- and/or oropharynx swabs (NOS). However, there is no US Food and Drug Administration-approved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid antigen test kit, which can be useful in a prolonged pandemic to reduce transmission by allowing suspected individuals to self-sampling. We evaluated the performances of High sensitive AQ+ Rapid SARS-CoV-2 Antigen Test (AQ+ kit) using nasopharyngeal swabs (NPs) and saliva specimens from the same patients in laboratory conditions. Methods: The real-time reverse transcription-polymerase chain reaction (rRT-PCR) test result was used for screening the inrolled individuals and compared as the gold standard. NP and saliva samples were collected from 100 rRT-PCR positives and 100 negative individuals and tested with an AQ+ kit. Results: The AQ+ kit showed good performances in both NP and saliva samples with an overall accuracy of 98.5% and 94.0%, and sensitivity of 97.0% and 88.0%, respectively. In both cases, specificity was 100%. AQ+ kit performance with saliva was in the range of the World Health Organization recommended value. Conclusion: xOur findings indicate that the saliva specimen can be used as an alternative and less invasive to NPs for quick and reliable SARS-CoV-2 antigen detection.

Nasal swab as an alternative specimen for the detection of severe acute respiratory syndrome coronavirus 2.

Background and Aims: The coronavirus disease 2019 (COVID-19) has brought serious threats to public health worldwide. Nasopharyngeal, nasal swabs, and saliva specimens are used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, limited data are available on the performance of less invasive nasal swab for testing COVID-19. This study aimed to compare the diagnostic performance of nasal swabs with nasopharyngeal swabs using real-time reverse transcription polymerase chain reaction (RT-PCR) considering viral load, onset of symptoms, and disease severity. Methods: A total of 449 suspected COVIDCOVID-19 individuals were recruited. Both nasopharyngeal and nasal swabs were collected from the same individual. Viral RNA was extracted and tested by real-time RT-PCR. Metadata were collected using structured questionnaire and analyzed by SPSS and MedCalc software. Results: The overall sensitivity of the nasopharyngeal swab was 96.6%, and the nasal swab was 83.4%. The sensitivity of nasal swabs was more than 97.7% for low and moderate C t values. Moreover, the performance of nasal swab was very high (>87%) for hospitalized patients and at the later stage >7 days of onset of symptoms. Conclusion: Less invasive nasal swab sampling with adequate sensitivity can be used as an alternative to nasopharyngeal swabs for the detection of SARS-CoV-2 by real-time RT-PCR.

Pre COVID-19 molecular epidemiology of respiratory syncytial virus (RSV) among children in Bangladesh.

Epidemiological data of specific respiratory pathogens from the pre-COVID-19 period are essential to determine the effects of the COVID-19 pandemic on other respiratory infections. In this study, we revealed the pre-COVID-19 molecular epidemiology of respiratory syncytial virus (RSV) among children in Bangladesh. We tested 3170 samples collected from 2008 to 2012 for a panel of respiratory viruses; RSV, human metapneumovirus (hMPV), human parainfluenza viruses (hPIV) 1, 2, 3, and adenovirus. Five hundred fifty-five samples (17.5 %) were positive for RSV, including 2.5% having co-infections with other viruses. Genotypic characterization of RSV showed that RSV-A (82%) contributed more acute respiratory infections than RSV-B (18%). Clinical features were similar with RSV-A and RSV-B infections. However, children with RSV-B were more likely to have upper respiratory infections (URI) (10% vs. 29%, p = 0.03). Among RSV-A cases, hospitalization was higher for ON1 cases (25%, ON1 vs. 8%, NA1, p = 0.04), whereas the recovery without a disability was higher among the NA1 cases (56%, ON1 vs. 88%, NA1, p = 0.02). The time to the most recent common ancestor (TMRCA) for RSV in Bangladesh was 1949 for RSV-A and 1944 for RSV-B. This study revealed the genotypic diversity and evolutionary relatedness of RSV strains in Bangladesh and provided pre-COVID molecular epidemiology data to understand better the COVID-19 impact on upcoming RSV epidemiology in Bangladesh.

COVID-19 reinfections among naturally infected and vaccinated individuals.

The protection against emerging SARS-CoV-2 variants by pre-existing antibodies elicited due to the current vaccination or natural infection is a global concern. We aimed to investigate the rate of SARS-CoV-2 infection and its clinical features among infection-naïve, infected, vaccinated, and post-infection-vaccinated individuals. A cohort was designed among icddr,b staff registered for COVID-19 testing by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). Reinfection cases were confirmed by whole-genome sequencing. From 19 March 2020 to 31 March 2021, 1644 (mean age, 38.4 years and 57% male) participants were enrolled; where 1080 (65.7%) were tested negative and added to the negative cohort. The positive cohort included 750 positive patients (564 from baseline and 186 from negative cohort follow-up), of whom 27.6% were hospitalized and 2.5% died. Among hospitalized patients, 45.9% had severe to critical disease and 42.5% required oxygen support. Hypertension and diabetes mellitus were found significantly higher among the hospitalised patients compared to out-patients; risk ratio 1.3 and 1.6 respectively. The risk of infection among positive cohort was 80.2% lower than negative cohort (95% CI 72.6-85.7%; p < 0.001). Genome sequences showed that genetically distinct SARS-CoV-2 strains were responsible for reinfections. Naturally infected populations were less likely to be reinfected by SARS-CoV-2 than the infection-naïve and vaccinated individuals. Although, reinfected individuals did not suffer severe disease, a remarkable proportion of naturally infected or vaccinated individuals were (re)-infected by the emerging variants.

Factors influencing the performance of rapid SARS-CoV-2 antigen tests under field condition.

BACKGROUND: Globally, real-time reverse transcription-polymerase chain reaction (rRT-PCR) is the reference detection technique for SARS-CoV-2, which is expensive, time consuming, and requires trained laboratory personnel. Thus, a cost-effective, rapid antigen test is urgently needed. This study evaluated the performance of the rapid antigen tests (RATs) for SARS-CoV-2 compared with rRT-PCR, considering different influencing factors. METHODS: We enrolled a total of 214 symptomatic individuals with known COVID-19 status using rRT-PCR. We collected and tested paired nasopharyngeal (NP) and nasal swab (NS) specimens (collected from same individual) using rRT-PCR and RATs (InTec and SD Biosensor). We assessed the performance of RATs considering specimen types, viral load, the onset of symptoms, and presenting symptoms. RESULTS: We included 214 paired specimens (112 NP and 100 NS SARS-CoV-2 rRT-PCR positive) to the analysis. For NP specimens, the average sensitivity, specificity, and accuracy of the RATs were 87.5%, 98.6%, and 92.8%, respectively, when compared with rRT-PCR. While for NS, the overall kit performance was slightly lower than that of NP (sensitivity 79.0%, specificity 96.1%, and accuracy 88.3%). We observed a progressive decline in the performance of RATs with increased Ct values (decreased viral load). Moreover, the RAT sensitivity using NP specimens decreased over the time of the onset of symptoms. CONCLUSION: The RATs showed strong performance under field conditions and fulfilled the minimum performance limit for rapid antigen detection kits recommended by World Health Organization. The best performance of the RATs can be achieved within the first week of the onset of symptoms with high viral load.

IgG antibody response demonstrates inverse correlation with viral load in Bangladeshi women with acute hepatitis E virus genotype 1 infection.

OBJECTIVES: To determine IgG immune responses and hepatitis E virus (HEV) viral load, and to explore the associations with pregnancy. METHODS: A total of 121 HEV-infected women (57 pregnant, 64 non-pregnant) were analysed. Quantitative reverse transcription PCR (RT-qPCR) was done for 78 HEV IgM-positive patients to determine viral load, and Sanger sequencing was performed for 62 HEV-RNA-positive patients to confirm genotyping. ELISA was conducted to determine HEV antibody and avidity indices. RESULTS: The HEV genotype was identified as variant 1. Significant negative correlations were observed between log HEV copy number and log hepatitis E virus IgG antibody index in the late acute phase of jaundice for both pregnant women (r = -0.7971, p = 0.0002) and non-pregnant women (r = -0.9117, p = 0.0002). Pregnant women had significantly higher serum log viral copy numbers and lower IgG antibody indices than non-pregnant women in the late acute phase of HEV-induced jaundice (p = 0.0196 and p = 0.0303, respectively). Moreover, pregnant women with acute HEV hepatitis had higher cross-reactive IgG antibodies compared to the non-pregnant women (p = 0.0017). Five patients with HEV hepatitis died, of whom four were pregnant. CONCLUSIONS: Pregnancy might be associated with higher viral loads and a lower IgG response in the HEV-induced late acute phase of jaundice.

Mutation Spectrum in TPO Gene of Bangladeshi Patients with Thyroid Dyshormonogenesis and Analysis of the Effects of Different Mutations on the Structural Features and Functions of TPO Protein through In Silico Approach.

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.

Age-Specific Cut-off Values of Amino Acids and Acylcarnitines for Diagnosis of Inborn Errors of Metabolism Using Liquid Chromatography Tandem Mass Spectrometry.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is used for the diagnosis of more than 30 inborn errors of metabolisms (IEMs). Accurate and reliable diagnosis of IEMs by quantifying amino acids (AAs) and acylcarnitines (ACs) using LC-MS/MS systems depend on the establishment of age-specific cut-offs of the analytes. This study aimed to (1) determine the age-specific cut-off values of AAs and ACs in Bangladesh and (2) validate the LC-MS/MS method for diagnosis of the patients with IEMs. A total of 570 enrolled healthy participants were divided into 3 age groups, namely, (1) newborns (1-7 days), (2) 8 days-7 years, and (3) 8-17 years, to establish the age-specific cut-offs for AAs and ACs. Also, 273 suspected patients with IEMs were enrolled to evaluate the reliability of the established cut-off values. Quantitation of AAs and ACs was performed on an automated LC-MS/MS system using dried blood spot (DBS) cards. Then the specimens of the enrolled clinically suspected patients were analyzed by the established method. Nine patients came out as screening positive for different IEMs, including two borderline positive cases of medium-chain acyl-CoA dehydrogenase deficiency (MCAD). A second-tier test for confirmation of the screening positive cases was conducted by urinary metabolic profiling using gas chromatography- mass spectrometry (GC-MS). Out of 9 cases that came out as screening positive by LC-MS/MS, seven cases were confirmed by urinary GC-MS analysis including 3 cases with phenylketonuria, 1 with citrullinemia type II, 1 with methylmalonic acidemia, 1 with isovaleric acidemia and 1 with carnitine uptake defect. Two borderline positive cases with MCAD were found negative by urinary GC-MS analysis. In conclusion, along with establishment of a validated LC-MS/MS method for quantitation of AAs and ACs from the DBS cards, the study also demonstrates the presence of predominantly available IEMs in Bangladesh.

Impaired acylcarnitine profile in transfusion-dependent beta-thalassemia major patients in Bangladesh.

Patients with beta-thalassemia major (BTM) suffer from fatigue, poor physical fitness, muscle weakness, lethargy, and cardiac complications which are related to an energy crisis. Carnitine and acylcarnitine derivatives play important roles in fatty acid oxidation, and deregulation of carnitine and acylcarnitine metabolism may lead to an energy crisis. The present study aimed to investigate carnitine and acylcarnitine metabolites to gain an insight into the pathophysiology of BTM. Dried blood spots of 45 patients with BTM and 96 age-matched healthy controls were analyzed for free carnitine and 24 acylcarnitines by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although medium chain acylcarnitine levels were similar in the patients with BTM and healthy controls, free carnitine, short chain acylcarnitines, long chain acylcarnitines, and total acylcarnitine levels were significantly lower in patients with BTM than in the healthy controls (P < 0.05). Moreover, an impaired fatty acid oxidation rate was observed in the patients with BTM, as manifested by decreased fatty acid oxidation indicator ratios, namely C2/C0 and (C2 + C3)/C0. Furthermore, an increase in the C0/(C16 + C18) ratio indicated reduced carnitine palmitoyltransferase-1 (CPT-1) activity in the patients with BTM compared with that in the healthy controls. Thus, a low level of free carnitine and acylcarnitines together with impaired CPT-1 activity contribute to energy crisis-related complications in the patients with BTM.

High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

BACKGROUND: Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. RESULTS: Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. CONCLUSIONS: Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.