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BackgroundBangladesh 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.ResultsOur 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.ConclusionsTargeting 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.Electronic supplementary materialThe online version of this article (10.1186/s12863-017-0594-3) contains supplementary material, which is available to authorized users.

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.

To identify the circulating serotype(s) of dengue viruses in Bangladesh, a retrospective molecular identification was performed on stored serum samples of dengue surveillance during the period of 2013-2016. Real time RT-PCR was performed on serum samples collected from the patients with less than 5 days fever for detection of dengue virus nucleic acid. The samples, positive for dengue PCR were further analyzed for serotypes by real time RT-PCR. The overall prevalence of dengue virus infection was varied among 13-42% in study years with a single peak flanked by April to September. Among the four dengue serotypes DEN1 and DEN2 were in the circulation in three metropolitan cities with sequential emergence of DEN1 where DEN2 was persisted constantly during the study period. Persistence of all four serotypes in the neighboring country makes Bangladesh vulnerable for devastating secondary infection by introduction of new serotype(s) other than currently circulating viruses in the country. Thus continuous virological surveillance is crucial for early warning of emergence of new serotype in the circulation and public health preparedness.

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.