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The first case of COVID-19 was detected in Bangladesh on 8 March 2020. Since then, the Government of Bangladesh (GoB) has implemented various measures to limit the transmission of COVID-19, including widespread testing facilities across the nation through a laboratory network for COVID-19 molecular testing. This study aimed to analyze the dynamics of SARS-CoV-2 in Bangladesh by conducting COVID-19 testing and genomic surveillance of the virus variants throughout the pandemic. Nasopharyngeal swabs were collected from authorized GoB collection centers between April 2020 and June 2023. The viral RNA was extracted and subjected to real-time PCR analysis in icddr,b’s Virology laboratory. A subset of positive samples underwent whole-genome sequencing to track the evolutionary footprint of SARS-CoV-2 variants. We tested 149,270 suspected COVID-19 cases from Dhaka (n = 81,782) and other districts (n = 67,488). Of these, 63% were male. The highest positivity rate, 27%, was found in the >60 years age group, followed by 26%, 51–60 years, 25% in 41–50 years, and the lowest, 9% in under five children. Notably, the sequencing of 2742 SARS-CoV-2 genomes displayed a pattern of globally circulating variants, Alpha, Beta, Delta, and Omicron, successively replacing each other over time and causing peaks of COVID-19 infection. Regarding the risk of SARS-CoV-2 infection, it was observed that the positivity rate increased with age compared to the under-5 age group in 2020 and 2021. However, these trends did not remain consistent in 2022, where older age groups, particularly those over 60, had a lower positivity rate compared to other age groups due to vaccination. The study findings generated data on the real-time circulation of different SARS-CoV-2 variants and the upsurge of COVID-19 cases in Bangladesh, which impacted identifying hotspots and restricting the virus from further transmission. Even though there is currently a low circulation of SARS-CoV-2 in Bangladesh, similar approaches of genomic surveillance remain essential for monitoring the emergence of new SARS-CoV-2 variants or other potential pathogens that could lead to future pandemics.

Abstract Background The coronavirus disease 2019 (COVID‐19) pandemic has had a severe impact on population health. The genetic determinants of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in southern Bangladesh are not well understood. Methods This study aimed to determine the genomic variation in SARS‐CoV‐2 genomes that have evolved over 2 years of the pandemic in southern Bangladesh and their association with disease outcomes and virulence of this virus. We investigated demographic variables, disease outcomes of COVID‐19 patients and genomic features of SARS‐CoV‐2. Results We observed that the disease severity was significantly higher in adults (85.3%) than in children (14.7%), because the expression of angiotensin‐converting enzyme‐2 (ACE‐2) diminishes with ageing that causes differences in innate and adaptive immunity. The clade GK (n = 66) was remarkable between June 2021 and January 2022. Because of the mutation burden, another clade, GRA started a newly separated clustering in December 2021. The burden was significantly higher in GRA (1.5‐fold) highlighted in mild symptoms of COVID‐19 patients than in other clades (GH, GK, and GR). Mutations were accumulated mainly in S (22.15 mutations per segment) and ORF1ab segments. Missense (67.5%) and synonymous (18.31%) mutations were highly noticed in adult patients with mild cases rather than severe cases, especially in ORF1ab segments. Moreover, we observed many unique mutations in S protein in mild cases compared to severe, and homology modeling revealed that those might cause more folding in the protein's alpha helix and beta sheets. Conclusion Our study identifies some risk factors such as age comorbidities (diabetes, hypertension, and renal disease) that are associated with severe COVID‐19, providing valuable insight regarding prioritizing vaccination for high‐risk individuals and allocating health care and resources. The findings of this work outlined the knowledge and mutational basis of SARS‐CoV‐2 for the next treatment steps. Further studies are needed to confirm the effects of structural and functional proteins of SARS‐CoV‐2 in detail for monitoring the emergence of new variants in future.

Abstract Background Wastewater-based epidemiological surveillance has been considered a powerful tool for early detection and monitoring of the dynamics of SARS-CoV-2 and its lineages circulating in a community. This study is aimed to investigate the complexity of SARS-CoV-2 infection dynamics in Dhaka city by examining its genetic variants in wastewater. Also, the study seeks to determine a connection between the SARS-CoV-2 variations detected in clinical testing and those found in wastewater samples. Results Out of 504 samples tested in RT-qPCR, 185 (36.7%) tested positive for SARS-CoV-2 viral RNA. The median log10 concentration of SARS-CoV-2 N gene copies/Liter of wastewater (gc/L) was 5.2, and the median log10 concentration of ORF1ab was 4.9. To further reveal the genetic diversity of SARS-CoV-2, ten samples with ORF1ab real-time RT-PCR cycle threshold (Ct) values ranging from 28.78 to 32.13 were subjected to whole genome sequencing using nanopore technology. According to clade classification, sequences from wastewater samples were grouped into 4 clades: 20A, 20B, 21A, 21J, and the Pango lineage, B.1, B.1.1, B.1.1.25, and B.1.617.2, with coverage ranging from 94.2 to 99.8%. Of them, 70% belonged to clade 20B, followed by 10% to clade 20A, 21A, and 21J. Lineage B.1.1.25 was predominant in Bangladesh and phylogenetically related to the sequences from India, the USA, Canada, the UK, and Italy. The Delta variant (B.1.617.2) was first identified in clinical samples at the beginning of May 2021. In contrast, we found that it was circulating in the community and was detected in wastewater in September 2020. Conclusion Environmental surveillance is useful for monitoring temporal and spatial trends of existing and emerging infectious diseases and supports evidence-based public health measures. The findings of this study supported the use of wastewater-based epidemiology and provided the baseline data for the dynamics of SARS-CoV-2 variants in the wastewater environment in Dhaka, Bangladesh.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) continues to spread and evolve, giving rise to new surges in cases and deaths. Although nasopharyngeal swab (NPS) samples are the gold standard for diagnosing SARS-CoV2, NPS sampling is invasive and requires skilled staff, specialized transport medium, and nucleic acid extraction. The SalivaDirect method uses saliva samples and a simplified, flexible approach to overcome these drawbacks. In our study conducted at the COVID-19 Screening Unit of the Dhaka Hospital of icddr,b (International Centre for Diarrheal Disease Research, Bangladesh), the diagnostic performance of SalivaDirect and NPS was compared using paired samples. SARS-CoV2 was detected by performing RT-qPCR using RNA extracted from saliva samples with the SalivaDirect method and from the NPS with extracted RNA. A total of 200 participants were enrolled from February to March 2021, among whom 78 (39.0%) tested positive for SAR-CoV2 from at least one sample. Among the 78 participants testing positive, 65 (83.3%) tested positive in both specimens, eight (10.3%) tested positive only in NPS, and five (6.4%) tested positive only in saliva. SalivaDirect had a sensitivity of 89.0% and specificity of 96.1%, with NPS as the reference test. The sensitivity of NPS and SalivaDirect was 93.6% and 89.7% respectively, with a composite reference standard (where patients are defined as positive if tested positive in either method) as the reference. Our findings demonstrated that the SalivaDirect method can be used as an alternative to NPS in our clinical setting and also supports the use of SalivaDirect in other settings. IMPORTANCE Affordable and accessible tests for COVID-19 allow for timely disease treatment and pandemic management. SalivaDirect is a faster and easier method to implement than NPS sampling. Patients can self-collect saliva samples at home or in other non-clinical settings without the help of a healthcare professional. Sample processing in SalivaDirect is less complex and more adaptable than in conventional nucleic acid extraction methods. We found that SalivaDirect has good diagnostic performance and is ideal for large-scale testing in settings where supplies may be limited or trained healthcare professionals are unavailable.

Abstract 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 Ct 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.

ABSTRACT Accurate and early diagnoses are prerequisites for prompt treatment. For coronavirus disease 2019 (COVID-19), it is even more crucial. Currently, choice of methods include rapid diagnostic tests and reverse transcription polymerase chain reaction (RT-PCR) using samples mostly of respiratory origin and sometimes saliva. We evaluated two rapid diagnostic tests with three specimen types using viral transport medium (VTM) containing naso-oropharyngeal (NOP) swabs, direct nasal and direct nasopharyngeal (NP) samples from 428 prospective patients. We also performed RT-PCR for 428 NOP VTM and 316 saliva samples to compare results. The sensitivity of the SD Biosensor Standard Q COVID-19 antigen (Ag) test kit drastically raised from an average of 65.55% (NOP VTM) to 85.25% (direct nasal samples), while RT-PCR was the gold standard. For the CareStart kit, the sensitivity was almost similar for direct NP swabs; the average was 84.57%. The specificities were ≥95% for both SD Biosensor Standard Q and CareStart COVID-19 Ag tests in all platforms. The kits were also able to detect patients with different variants as well. Alternatively, RT-PCR results from saliva and NOP VTM samples showed high sensitivities of 96.45% and 95.48% with respect to each other as standard. The overall results demonstrated high performance of the rapid tests, indicating the suitability for regular surveillance at clinical facilities when using direct nasal or direct NP samples rather than NOP VTM. Additionally, the analysis also signifies not showed that RT-PCR of saliva can be used as an choice of method to RT-PCR of NOP VTM, providing an easier, non-invasive sample collection method. IMPORTANCE There are several methods for the diagnosis of coronavirus disease 2019 (COVID-19), and the choice of methods depends mostly on the resources and level of sensitivity required by the user and health care providers. Still, reverse transcription polymerase chain reaction (RT-PCR) has been chosen as the best method using direct naso-oropharyngeal swabs. There are also other methods of fast detection, such as rapid diagnostic tests (RDTs), which offer result within 15 to 20 min and have become quite popular for self-testing and in the clinical setting. The major drawback of the currently used RT-PCR method is compliance, as it may cause irritation, and patients often refuse to test in such a way. RDTs, although inexpensive, suffer from low sensitivity due to technical issues. In this article, we propose saliva as a noninvasive source for RT-PCR samples and evaluate various specimen types at different times after infection for the best possible output from COVID-19 rapid tests.

Background: The emergence of novel variants has been a great deal of international concern since the recently published data suggest that previous infections with SARS-CoV-2 may not protect an individual from new variants. We report a patient had two distinct episodes of COVID-19 with different variants of SARS-CoV-2.Methods: The nasopharyngeal samples collected from the two episodes were subjected to whole-genome sequencing and comparative genome analysis.Results: The first infection presented with mild symptoms, while the second infection presented with severe outcomes which occurred 74 days after the patient recovered from the first episode. He had elevated C-reactive protein, ferritin, and bilateral consolidation as a sign of acute infection. Genome analysis revealed that the strains from the first and second episodes belonged to two distinct Nexstrain clades 20B and 20I and Pangolin lineages B.1.1.25 and B.1.1.7, respectively. A total of 36 mutations were observed in the episode-2 strain when compared with the reference strain Wuhan-Hu-1. Among them, eight mutations were identified in the receptor-binding domain (RBD).Conclusion: Our findings concern whether the immunity acquired by natural infection or mass vaccination could confer adequate protection against the constantly evolving SARS-CoV-2. Therefore, continuous monitoring of genetic variations of SARS-CoV-2 strains is crucial for interventions such as vaccine and drug designs, treatment using monoclonal antibodies, and patient management.

ABSTRACT Coronavirus disease 19 (COVID-19)—caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—has spread rapidly around the world. The global shortage of equipment and health care professionals, diagnostic cost, and difficulty in collecting nasopharyngeal swabs (NPSs) necessitate the use of an alternative specimen type for SARS-CoV-2 diagnosis. In this study, we investigated the use of saliva as an alternative specimen type for SARS-CoV-2 detection. Participants presenting COVID-19 symptoms and their contacts were enrolled at the COVID-19 Screening Unit of Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), from July to November 2020. Paired NPS and saliva specimens were collected from each participant. Reverse transcription-quantitative PCR (RT-qPCR) was performed to detect SARS-CoV-2. Of the 596 suspected COVID-19-positive participants, 231 (38.7%) were detected as COVID-19 positive by RT-qPCR from at least 1 specimen type. Among the positive cases, 184 (79.6%) patients were identified to be positive for SARS-CoV-2 based on NPS and saliva samples, whereas 45 (19.65%) patients were positive for SARS-CoV-2 based on NPS samples but negative for SARS-CoV-2 based on the saliva samples. Two (0.5%) patients were positive for SARS-CoV-2 based on saliva samples but negative for SARS-CoV-2 based on NPS samples. The sensitivity and specificity of the saliva samples were 80.3% and 99.4%, respectively. SARS-CoV-2 detection was higher in saliva (85.1%) among the patients who visited the clinic after 1 to 5 days of symptom onset. A lower median cycle threshold (CT) value indicated a higher SARS-CoV-2 viral load in NPS than that in saliva for target genes among the positive specimens. The study findings suggest that saliva can be used accurately for diagnosis of SARS-CoV-2 early after symptom onset in clinical and community settings. IMPORTANCE As the COVID-19 pandemic erupted, the WHO recommended the use of nasopharyngeal or throat swabs for the detection of SARS-CoV-2 etiology of COVID-19. The collection of NPS causes discomfort because of its invasive collection procedure. There are considerable risks to health care workers during the collection of these specimens. Therefore, an alternative, noninvasive, reliable, and self-collected specimen was explored in this study. This study investigated the feasibility and suitability of saliva versus NPS for the detection of SARS-CoV-2. Here, we showed that the sensitivity of saliva specimens was 80.35%, which meets the WHO criteria. Saliva is an easy-to-get, convenient, and low-cost specimen that yields better results if it is collected within the first 5 days of symptom onset. Our study findings suggest that saliva can be used in low-resource countries, community settings, and vulnerable groups, such as children and elderly people.

The rapid and early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is key to control the current Coronavirus disease 2019 (COVID-19) pandemic. The present study was conducted to clinically evaluate a rapid diagnostic test (RDT) kit, Standard Q COVID-19 Ag Test (SD Biosensor®, Republic of Korea), with reference to the standard real-time RT-PCR for detection of COVID-19 cases in Bangladesh. Nasopharyngeal swabs were taken from 900 COVID-19 suspected patients. Among them, 34.11% (n = 307) were diagnosed as COVID-19 cases by RT-PCR assay, of which 85% (n = 261) were also detectable using the RDT. The overall sensitivity and specificity of the RDT compared to RT-PCR were 85.02% and 100%, respectively, regardless of age, sex, and type of SARS-CoV-2 variants. Most of the RT-PCR positive cases (94%) were found within the first five days of disease onset, and the sensitivity of RDT was 85.91% for the same samples. The positive predictive value (PPV) of the RDT was 100%, and the negative predictive value (NPV) was 92.8%. The Cohen's kappa value of 0.882 indicated excellent agreement between the RDT and RT-PCR assays. The findings of this study showed the potential use of SARS-CoV-2 antigen-based RDT to expedite the diagnostic process and onward COVID-19 management in Bangladesh.

Accurate diagnosis at the right moment is the prerequisite for treatment of any disease. Failure to correctly diagnose a disease can result in highly detrimental effects, unmistakably a crucial factor during the COVID-19 pandemic. RT-PCR is the gold standard for COVID-19 detection while there are other test procedures available, such as LAMP, X-Ray, and ELISA. However, these tests are expensive, require sophisticated equipment and a highly trained workforce, and multiple hours or even days are often required to obtain the test results. A rapid and cheap detection system can thus render a solution to the screening system on a larger scale and be added as an aid to the current detection processes. Recently, some rapid antigen-based COVID-19 tests devices have been developed and commercialized. In this study, we evaluated the clinical performance of a new rapid detection device (OnSite® COVID-19 Ag Rapid Test by CTK Biotech Inc., Poway, CA, USA) on COVID-19 symptomatic patients (n = 380). The overall sensitivity and specificity were 91.0% (95% CI: 84.8–95.3%) and 99.2% (95% CI: 97.1–99.9), against gold standard RT-PCR. The kit was capable of detecting patients even after 06 days of onset of symptoms and the sensitivity can be maximized to 98% in samples with an average RT-PCR Ct ≤ 26.48, demonstrating a high potential of the kit for clinical diagnosis of symptomatic patients in healthcare facilities.

To date, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 80 million people globally. We report a case series of five clinically and laboratory confirmed COVID-19 patients from Bangladesh who suffered a second episode of COVID-19 illness after 70 symptom-free days. The International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), is a leading public health research institution in South Asia. icddr, b staff were actively tested, treated and followed-up for COVID-19 by an experienced team of clinicians, epidemiologists, and virologists. From 21 March to 30 September 2020, 1370 icddr,b employees working at either the Dhaka (urban) or Matlab (rural) clinical sites were tested for COVID-19. In total, 522 (38%) were positive; 38% from urban Dhaka (483/1261) and 36% from the rural clinical site Matlab (39/109). Five patients (60% male with a mean age of 41 years) had real-time reverse transcription-polymerase chain reaction (rRT-PCR) diagnosed recurrence (reinfection) of SARS-CoV-2. All had mild symptoms except for one who was hospitalized. Though all cases reported fair risk perceptions towards COVID-19, all had potential exposure sources for reinfection. After a second course of treatment and home isolation, all patients fully recovered. Our findings suggest the need for COVID-19 vaccination and continuing other preventive measures to further mitigate the pandemic. An optimal post-recovery follow-up strategy to allow the safe return of COVID-19 patients to the workforce may be considered.