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.

Prevalence of silver resistance determinants and extended-spectrum ß-lactamases in bacterial species causing wound infection: First report from Bangladesh.

Background: The use of silver is rapidly rising in wound care and silver-containing dressings are widely used along with other antibiotics, particularly ß-lactams. Consequently, concerns are being raised regarding the emergence of silver-resistance and cross-resistance to ß-lactams. Therefore, this study aimed to determine the phenotypic and genotypic profiles of silver-resistance and extended-spectrum ß-lactamases in isolates from chronic wounds. Methods: 317 wound swab specimens were collected from tertiary hospitals of Dhaka city and analysed for the microbial identification. The antibiotic resistance/susceptibility profiles were determined and phenotypes of silver resistant isolates were examined. The presence of silver-resistance (sil) genes (silE, silP, and silS) and extended-spectrum ß-lactamases (ESBL) (CTX-M-1, NDM-1, KPC, OXA-48, and VIM-1) were explored in isolated microorganisms. Results: A total of 501 strains were isolated with Staphylococcus aureus (24%) as the predominant organism. In 29% of the samples, polymicrobial infections were observed. A large proportion of Enterobacterales (59%) was resistant to carbapenems and a significantly high multiple antibiotic-resistance indexes (>0.2) were seen for 53% of organisms (P < 0.001). According to molecular analysis, the most prevalent types of ESBL and sil gene were CTX-M-1 (47%) and silE (42%), respectively. Furthermore, phenotypic silver-nitrate susceptibility testing showed significant minimum-inhibitory-concentration patterns between sil-negative and sil-positive isolates. We further observed co-occurrence of silver-resistance determinants and ESBLs (65%). Conclusions: Notably, this is the first-time detection of silver-resistance along with its co-detection with ESBLs in Bangladesh. This research highlights the need for selecting appropriate treatment strategies and developing new alternative therapies to minimize microbial infection in wounds.

Genotypic and phenotypic profiles of antibiotic-resistant bacteria isolated from hospitalised patients in Bangladesh.

OBJECTIVES: Characterisation of resistance phenotype and genotype is crucial to understanding the burden and transmission of antimicrobial resistance (AMR). This study aims to determine the spectrum of AMR and associated genes encoding aminoglycoside, macrolide and ß-lactam classes of antimicrobials in bacteria isolated from hospitalised patients in Bangladesh. METHODS: 430 bacterial isolates from patients with respiratory, intestinal, wound infections and typhoid fever, presenting to clinical care from 2015 to 2019, were examined. They included Escherichia coli (n = 85); Staphylococcus aureus (n = 84); Salmonella typhi (n = 82); Klebsiella pneumoniae (n = 42); Streptococcus pneumoniae (n = 36); coagulase-negative staphylococci (n = 28); Enterococcus faecalis (n = 27); Pseudomonas aeruginosa (n = 26); and Acinetobacter baumannii (n = 20). Reconfirmation of these clinical isolates and antimicrobial susceptibility tests was performed. PCR amplification using resistance gene-specific primers was done, and the amplified products were confirmed by Sanger sequencing. RESULTS: 53% of isolates were multidrug-resistant (MDR), including 97% of Escherichia coli. There was a year-wise gradual increase in MDR isolates from 2015 to 2018, and there was an almost twofold increase in the number of MDR strains isolated in 2019 (P = 0.00058). Among the 5 extended-spectrum ß-lactamases investigated, CTX-M-1 was the most prevalent (63%) followed by NDM-1 (22%); Escherichia coli was the major reservoir of these genes. The ermB (55%) and aac(6')-Ib (35%) genes were the most frequently detected macrolide and aminoglycoside resistance genes, respectively. CONCLUSION: MDR pathogens are highly prevalent in hospital settings of Bangladesh.

Frequency of Hepatitis B, C and HIV Infections among Transfusion-Dependent Beta Thalassemia Patients in Dhaka.

Transfusion transmitted infections have remained a major deterrent to public health, particularly among the patients with transfusion-dependent Beta thalassemia in developing countries. Although proper donor selection through adoption of WHO-advised infection panel has lowered the rate of infections, the multi-transfused patients are not free of risk. In this study, we screened 148 transfusion-dependent Beta thalassemia patients to determine the frequency of Hepatitis C Virus (HCV), Hepatitis B Virus (HBV) and Human Immunodeficiency Virus (HIV) using the ELISA method. Among them, infected cases with HCV, HBV and HIV were 13.51%, 3.37% and 0%, respectively. Moreover, 2% of the patients were found to be co-infected with both HBV and HCV. The percentage of infections in the patients with frequent transfusion interval (≤30 days) was significantly higher (p < 0.0005) than that in the patients with less frequent transfusion intervals (>30 days). Immunochromatography (ICT)-based rapid test kits are usually used to screen and confirm these infections in the blood of the patients. However, ICT-based tests are not sensitive enough to detect the infections. So, a combination of both Nucleic Acid testing (NAT) and serological testing are suggested to significantly reduce the risk of viral infections during blood transfusion.

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.

Nationwide carrier detection and molecular characterization of ß-thalassemia and hemoglobin E variants in Bangladeshi population.

BACKGROUND: ß-thalassemia is one of the most common inherited blood disorders in the world and a major deterrent to the public health of Bangladesh. The management of thalassemia patients requires lifelong frequent blood transfusion and the available treatment options are unsatisfactory. A national policy on thalassemia prevention is mandatory in Bangladesh. However, precise and up-to-date information on the frequency of ß-thalassemia carriers are missing due to lack of accurate diagnostic approaches, limited access to information and absence of national screening program. This study aims to determine the nationwide carrier frequency of hemoglobin E (HbE) and ß- thalassemia and mutation spectrum among the carriers using molecular, hematological and biochemical methods. METHODS: The study enrolled a total of 1877 individuals (60.1% male and 39.9% female) aged between 18 and 35 years. Total sample size and its division-wise breakdown were calculated in proportion to national and division-wise population. Venous blood was collected and subjected to CBC analysis and Hb-electrophoresis for each participant. Serum ferritin was measured to detect coexistence of iron deficiency anemia with thalassemia carrier. DNA-based High Resolution Melting (HRM) curve analysis was performed for confirmation of carrier status by mutation detection. RESULTS: Of 11.89% (95% CI, 10.43-13.35) carriers of ß-globin gene mutations, 8.68% (95% CI, 7.41-9.95) had HbE trait (ETT) and 2.24% (95% CI, 1.57-2.91) had beta-thalassemia trait (BTT). Among eight divisions, Rangpur had the highest carrier frequency of 27.1% (ETT-25%, BTT-2.1%), whereas Khulna had the lowest frequency of 4.2% (ETT-4.2% only). Moreover, α- thalassemia, HbD trait, HbE disease, hereditary persistence of HbF were detected in 0.11, 0.16, 0.43 and 0.16% participants, respectively. HRM could identify two individuals with reported pathogenic mutations in both alleles who were erroneously interpreted as carriers by hematological indices. Finally, a total of nine different mutations including a novel mutation (c.151A > G) were detected in the ß-globin gene. CONCLUSIONS: Since carrier frequency for both HbE and ß-thalassemia is alarmingly high in Bangladesh, a nationwide awareness and prevention program should be made mandatory to halt the current deteriorating situations. Mutation-based confirmation is highly recommended for the inconclusive cases with conventional carrier screening methods to avoid any faulty detection of thalassemia carriers.

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.

High resolution melting curve analysis enables rapid and reliable detection of G6PD variants in heterozygous females.

BACKGROUND: Like glucose-6-phosphate dehydrogenase (G6PD) deficient hemizygous males and homozygous females, heterozygous females could also manifest hemolytic crisis, neonatal hyperbilirubinemia or kernicterus upon exposure to oxidative stress induced by certain foods such as fava beans, drugs or infections. Although hemizygous males and homozygous females are easily detected by conventional G6PD enzyme assay method, the heterozygous state could be missed by the conventional methods as the mosaic population of both normal and deficient RBCs circulates in the blood. Thus the present study aimed to apply high resolution melting (HRM) curve analysis approach to see whether HRM could be used as a supplemental approach to increase the chance of detection of G6PD heterozygosity. RESULTS: Sixty-three clinically suspected females were evaluated for G6PD status using both enzyme assay and HRM analysis. Four out of sixty-three participants came out as G6PD deficient by the enzyme assay method, whereas HRM approach could identify nine participants with G6PD variants, one homozygous and eight heterozygous. Although only three out of eight heterozygous samples had G6PD enzyme deficiency, the HRM-based heterozygous G6PD variants detection for the rest of the samples with normal G6PD enzyme activities could have significance because their newborns might fall victim to serious consequences under certain oxidative stress. CONCLUSIONS: In addition to the G6PD enzyme assay, HRM curve analysis could be useful as a supplemental approach for detection of G6PD heterozygosity.

Bacterial and viral pathogen spectra of acute respiratory infections in under-5 children in hospital settings in Dhaka city.

The study aimed to examine for the first time the spectra of viral and bacterial pathogens along with the antibiotic susceptibility of the isolated bacteria in under-5 children with acute respiratory infections (ARIs) in hospital settings of Dhaka, Bangladesh. Nasal swabs were collected from 200 under-five children hospitalized with clinical signs of ARIs. Nasal swabs from 30 asymptomatic children were also collected. Screening of viral pathogens targeted ten respiratory viruses using RT-qPCR. Bacterial pathogens were identified by bacteriological culture methods and antimicrobial susceptibility of the isolates was determined following CLSI guidelines. About 82.5% (n = 165) of specimens were positive for pathogens. Of 165 infected cases, 3% (n = 6) had only single bacterial pathogens, whereas 43.5% (n = 87) cases had only single viral pathogens. The remaining 36% (n = 72) cases had coinfections. In symptomatic cases, human rhinovirus was detected as the predominant virus (31.5%), followed by RSV (31%), HMPV (13%), HBoV (11%), HPIV-3 (10.5%), and adenovirus (7%). Streptococcus pneumoniae was the most frequently isolated bacterial pathogen (9%), whereas Klebsiella pneumaniae, Streptococcus spp., Enterobacter agglomerans, and Haemophilus influenzae were 5.5%, 5%, 2%, and 1.5%, respectively. Of 15 multidrug-resistant bacteria, a Klebsiella pneumoniae isolate and an Enterobacter agglomerans isolate exhibited resistance against more than 10 different antibiotics. Both ARI incidence and predominant pathogen detection rates were higher during post-monsoon and winter, peaking in September. Pathogen detection rates and coinfection incidence in less than 1-year group were significantly higher (P = 0.0034 and 0.049, respectively) than in 1-5 years age group. Pathogen detection rate (43%) in asymptomatic cases was significantly lower compared to symptomatic group (P<0.0001). Human rhinovirus, HPIV-3, adenovirus, Streptococcus pneumonia, and Klebsiella pneumaniae had significant involvement in coinfections with P values of 0.0001, 0.009 and 0.0001, 0.0001 and 0.001 respectively. Further investigations are required to better understand the clinical roles of the isolated pathogens and their seasonality.

Examination of Huntington's disease with atypical clinical features in a Bangladeshi family tree.

Atypical manifestation of Huntington's disease (HD) could inform ongoing research into HD genetic modifiers not present in the primarily European populations studied to date. This work demonstrates that expanding HD genetic testing into under-resourced healthcare settings can benefit both local communities and ongoing research into HD etiology and new therapies.

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh.

High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute.

MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops.

In silico analysis reveals the presence of a large number of Ankyrin repeat containing proteins in Ectocarpus siliculosus.

Proteins with Ankyrin repeat motifs (ANK) are found to be associated with diverse biological processes and molecular functions in most of the studied organisms. Several studies have been done on the ANK-motif containing proteins of various model species, but similar studies on their counterparts in brown algae are not available. In this study, we have identified a total of 1,372 ankyrin repeats in 339 proteins of the model brown algae Ectocarpus siliculosus and the consensus sequence of the ANK repeats was determined. The proteins were classified into eight different subfamilies depending on their structural diversity. The data provided in this study may provide useful basis for future reverse genetics analysis of the members of this family.