Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective.

The study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive control and synthetic version of human RP was done on Applied Biosystems (ABI) QuantStudioTM5 (conventional), ABI 7500 Fast Dx (designated), and CFX96 Touch Real Time Detection System, Bio-Rad (conventional). Finally, a performance evaluation of Fast PCR was performed in terms of sensitivity, specificity, and precision. For a 40-cycle PCR with optimized Fast PCR protocols on QuantStudioTM5, ABI 7500 Fast Dx, and CFX96 Touch (conventional), standard/regular versus Fast PCR run times (min) were 84 vs. 49, 96 vs. 48, and 103 vs. 61, thereby saving 35, 48, and 43 min, respectively. For each thermal cycler, Standard and Fast PCR generated identical shapes of fluorescence curves, Ct values, and (3) R2 (0.95 to 0.99) for 5 10-log dilution panels of each positive control. The fast PCR approach generated results with 100% sensitivity and specificity. Median test comparisons between standard PCR and Fast PCR Cts of COVID-19 samples did not produce significance (p>0.5), suggesting that Fast PCR and Standard PCR were comparable. Also, the median and mean of each target had closely-related values, further suggesting that the two approaches were comparable. That is, there is an equivalency between Conventional and Fast PCR instruments for detection of COVID-19.

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.

Development and performance evaluation of the first in-house multiplex rRT-PCR assay in Bangladesh for highly sensitive detection of SARS-CoV-2.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is posing a great threat to global health and economy. Due to the lack of broad diagnostic setup, consistent reagent supply lines, and access to laboratory instruments and equipment, it is undoubtedly an enormous burden for developing countries to face the crisis. OBJECTIVES: To develop a cost-effective, reliable and sensitive multiplex assay for SARS-CoV-2 screening which would expand the testing capacities of a developing and low-income country like Bangladesh. STUDY DESIGN: Initially a singleplex and then a multiplex real-time reverse-transcriptase PCR assays were developed targeting 2 nucleocapsid genes of SARS-CoV-2, and the human RNase P gene as an internal control using laboratory-made mastermixes. Three sets of primer- probes were designed for each of the target genes and one set was optimized for the final reaction set-up. Limit of detection, cross-reactivity and reproducibility were checked in order to assess the sensitivity and specificity of the assays, and validation was done using clinical specimens. RESULTS: Clinical evaluation of the new assays using 240 nasopharyngeal swabs showed 100 % sensitivity, specificity, and accuracy in detecting SARS-CoV-2 infection in human. Equal efficiency and concordant results were observed between the singleplex and multiplex approaches. Notably, the kit was able to detect SARS-CoV-2 RNA at very low concentration upto 5 copies/reaction. CONCLUSION: This is the first locally developed multiplex rRT-PCR kit in Bangladesh providing rapid and low-cost screening of COVID-19 which would be valuable for infection prevention and clinical management in the perspective of Bangladesh.

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.

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.

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.

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.