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A field experiment was conducted during the year 2018-2019 at the vital place of Bangladesh Jute Research Institute (BJRI), Dhaka. The aim of the study was to evaluate the influence of Mn fertilizer on the dry matter yield and potential nutrient uptake of the newly developed jute variety BJRI Tossa jute-7. The treatments used in the experiments were T1: Control, T2: RDF sole (N90P10 K30 S20kg/ha), T3: RDF + 1 kg Mn/ha, T4: RDF + 2 kg Mn/ha, T5: RDF + 3 kg Mn/ha and T6: RDF + 4 kg Mn/ha. Mn application had a favorable effect on the dry matter yield of jute. Among the fertilizers of Mn with RDF, interaction promoted the dry yield biomass compared to sole RDF and control. Maximum dry matter yield (leaves + shoots + roots) achieved (20000 kg/ha) with T5 (RDF + 3 kg Mn/ha) and minimum (6421 kg/ha) in T1 (control). Study indicated that it needs to apply Mn to obtain optimum yield production. The interaction effect of Mn, NPK and S fertilizer significantly influenced nutrient uptake by the jute plant. The uptake of nutrient found highest N (218.9 kg/ha) in T5, P (92.75 kg/ha) in T3, K (239.73 kg/ha) with T6, S (19.59 kg/ha) in T4, Fe (0.64 kg/ha) in T5, Zn (0.38 kg/ha) in T5 and Mn (0.63 kg/ha) recorded in T6. The amount of micro nutrient uptake may be arranged in the order of Mn >Zn> Fe. Considering the highest uptake of major element, it can be kept as in the order of K > N>P>S. Study indicated that a higher rate of nutrient uptake was promoted dry matter accumulation in jute. Research revealed that the rate of nutrient uptake maximized with the addition of Mn in the RDF treatment. Hence, it could be concluded that applying RDF + 3 kg Mn/ha may be a suitable dose for optimum yield production of jute. Yet, in order to draw a sound conclusion, repeating the experiment is needed. In the future, the study will be a guideline for further investigation.

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.

Live bird markets (LBMs) are critical for poultry trade in many developing countries that are regarded as hotspots for the prevalence and contamination of avian influenza viruses (AIV). Therefore, we conducted weekly longitudinal environmental surveillance in LBMs to determine annual cyclic patterns of AIV subtypes, environmental risk zones, and the role of climatic factors on the AIV presence and persistence in the environment of LBM in Bangladesh. From January 2018 to March 2020, we collected weekly fecal and offal swab samples from each LBM and tested using rRT-PCR for the M gene and subtyped for H5, H7, and H9. We used Generalized Estimating Equations (GEE) approaches to account for repeated observations over time to correlate the AIV prevalence and potential risk factors and the negative binomial and Poisson model to investigate the role of climatic factors on environmental contamination of AIV at the LBM. Over the study period, 37.8% of samples tested AIV positive, 18.8% for A/H5, and A/H9 was, for 15.4%. We found the circulation of H5, H9, and co-circulation of H5 and H9 in the environmental surfaces year-round. The Generalized Estimating Equations (GEE) model reveals a distinct seasonal pattern in transmitting AIV and H5. Specifically, certain summer months exhibited a substantial reduction of risk up to 70–90% and 93–94% for AIV and H5 contamination, respectively. The slaughtering zone showed a significantly higher risk of contamination with H5, with a three-fold increase in risk compared to bird-holding zones. From the negative binomial model, we found that climatic factors like temperature and relative humidity were also significantly associated with weekly AIV circulation. An increase in temperature and relative humidity decreases the risk of AIV circulation. Our study underscores the significance of longitudinal environmental surveillance for identifying potential risk zones to detect H5 and H9 virus co-circulation and seasonal transmission, as well as the imperative for immediate interventions to reduce AIV at LBMs in Bangladesh. We recommend adopting a One Health approach to integrated AIV surveillance across animal, human, and environmental interfaces in order to prevent the epidemic and pandemic of AIV.

Trees in urban forests are able to better air quality by removing particulate matter (PM) from the atmosphere through the accumulation of particles on their leaf surfaces. When exposed to air pollutants, the physiology, morphology, and biochemistry of a plant may be affected, which will result in alterations to that plant’s function and growth. In this study, we assessed, for the first time, the tolerance or sensitivity of four evergreen trees (Ficus benghalensis, Ficus religiosa, Mangifera indica, and Polyalthia longifolia) towards air pollution by employing several indices. The trees, which are commonly grown along the roadside in Dhaka, Bangladesh, were evaluated by using the air pollution tolerance index (APTI), the anticipated performance index (API), and the metal accumulation index (MAI). The deposition of four heavy metals (Cd, Cr, Pb, and Ni) on the leaves of four aforementioned tree species was studied employing ICP-MS, and subsequently, a predictive foliar MAI was created. APTI values of the studied plants varied from 10.31 to 12.51 implying that they were either intermediately tolerant or sensitive. A significantly strong positive correlation was obtained between APTI and relative water content (RWC) (r = 0.864; p

Waterfowl are considered to be natural reservoirs of the avian influenza virus (AIV). However, the dynamics of transmission and evolutionary patterns of AIV and its subtypes within duck farms in Bangladesh remain poorly documented. Hence, a cross-sectional study was conducted in nine districts of Bangladesh between 2019 and 2021, to determine the prevalence of AIV and its subtypes H5 and H9, as well as to identify risk factors and the phylodynamics of H5N1 clades circulating in domestic duck farms. The oropharyngeal and cloacal swab samples were tested for the AIV Matrix gene (M-gene) followed by H5, H7, and H9 subtypes using rRT-PCR. The exploratory analysis was performed to estimate AIV and its subtype prevalence in different production systems, and multivariable logistic regression model was used to identify the risk factors that influence AIV infection in ducks. Bayesian phylogenetic analysis was conducted to generate a maximum clade credibility (MCC) tree and the maximum likelihood method to determine the phylogenetic relationships of the H5N1 viruses isolated from ducks. AIV was detected in 40% (95% CI: 33.0–48.1) of the duck farms. The prevalence of AIV was highest in nomadic ducks (39.8%; 95% CI: 32.9–47.1), followed by commercial ducks (24.6%; 95% CI: 14.5–37.3) and backyard ducks (14.4%; 95% CI: 10.5–19.2). The H5 prevalence was also highest in nomadic ducks (19.4%; 95% CI: 14.0–25.7). The multivariable logistic regression model revealed that ducks from nomadic farms (AOR: 2.4; 95% CI: 1.45–3.93), juvenile (AOR: 2.2; 95% CI: 1.37–3.61), and sick ducks (AOR: 11.59; 95% CI: 4.82–32.44) had a higher risk of AIV. Similarly, the likelihood of H5 detection was higher in sick ducks (AOR: 40.8; 95% CI: 16.3–115.3). Bayesian phylogenetic analysis revealed that H5N1 viruses in ducks belong to two distinct clades, 2.3.2.1a, and 2.3.4.4b. The clade 2.3.2.1a (reassorted) has been evolving silently since 2015 and forming at least nine subgroups based on >90% posterior probability. Notably, clade 2.3.4.4b was introduced in ducks in Bangladesh by the end of the year 2020, which was genetically similar to viruses detected in wild birds in Japan, China, and Africa, indicating migration-associated transmission of an emerging panzootic clade. We recommend continuing AIV surveillance in the duck production system and preventing the intermingling of domestic ducks with migratory waterfowl in wetlands.

The impacts of the avian influenza virus (AIV) on farmed poultry and wild birds affect human health, livelihoods, food security, and international trade. The movement patterns of turkey birds from farms to live bird markets (LBMs) and infection of AIV are poorly understood in Bangladesh. Thus, we conducted weekly longitudinal surveillance in LBMs to understand the trading patterns, temporal trends, and risk factors of AIV circulation in turkey birds. We sampled a total of 423 turkeys from two LBMs in Dhaka between May 2018 and September 2019. We tested the swab samples for the AIV matrix gene (M-gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We used exploratory analysis to investigate trading patterns, annual cyclic trends of AIV and its subtypes, and a generalized estimating equation (GEE) logistic model to determine the factors that influence the infection of H5 and H9 in turkeys. Furthermore, we conducted an observational study and informal interviews with traders and vendors to record turkey trading patterns, demand, and supply and turkey handling practices in LBM. We found that all trade routes of turkey birds to northern Dhaka are unidirectional and originate from the northwestern and southern regions of Bangladesh. The number of trades from the source district to Dhaka depends on the turkey density. The median distance that turkey was traded from its source district to Dhaka was 188 km (Q1 = 165, Q3 = 210, IQR = 45.5). We observed seasonal variation in the median and average distance of turkey. The qualitative findings revealed that turkey farming initially became reasonably profitable in 2018 and at the beginning of 2019. However, the fall in demand and production in the middle of 2019 may be related to unstable market pricing, high feed costs, a shortfall of adequate marketing facilities, poor consumer knowledge, and a lack of advertising. The overall prevalence of AIV, H5, and H9 subtypes in turkeys was 31% (95% CI: 26.6–35.4), 16.3% (95% CI: 12.8–19.8), and 10.2% (95% CI: 7.3–13.1) respectively. None of the samples were positive for H7. The circulation of AIV and H9 across the annual cycle showed no seasonality, whereas the circulation of H5 showed significant seasonality. The GEE revealed that detection of AIV increases in retail vendor business (OR: 1.71; 95% CI: 1.12–2.62) and the bird’s health status is sick (OR: 10.77; 95% CI: 4.31–26.94) or dead (OR: 11.33; 95% CI: 4.30–29.89). We also observed that winter season (OR: 5.83; 95% CI: 2.80–12.14) than summer season, dead birds (OR: 61.71; 95% CI: 25.78–147.75) and sick birds (OR 8.33; 95% CI: 3.36–20.64) compared to healthy birds has a higher risk of H5 infection in turkeys. This study revealed that the turkeys movements vary by time and season from the farm to the LBM. This surveillance indicated year-round circulation of AIV with H5 and H9 subtypes in turkey birds in LBMs. The seasonality and health condition of birds influence H5 infection in birds. The trading pattern of turkey may play a role in the transmission of AIV viruses in the birds. The selling of sick turkeys infected with H5 and H9 highlights the possibility of virus transmission to other species of birds sold at LBMs and to people.

ABSTRACT Whole-genome sequencing (WGS) of influenza A virus (IAV) is crucial for identifying diverse subtypes and newly evolved variants and for selecting vaccine strains. In developing countries, where facilities are often inadequate, WGS is challenging to perform using conventional next-generation sequencers. In this study, we established a culture-independent, high-throughput native barcode amplicon sequencing workflow that can sequence all influenza subtypes directly from a clinical specimen. All segments of IAV in 19 clinical specimens, irrespective of their subtypes, were amplified simultaneously using a two-step reverse transcriptase PCR (RT-PCR) system. First, the library was prepared using the ligation sequencing kit, barcoded individually using the native barcodes, and sequenced on the MinION MK 1C platform with real-time base-calling. Then, subsequent data analyses were performed with the appropriate tools. WGS of 19 IAV-positive clinical samples was carried out successfully with 100% coverage and 3,975-fold mean coverage for all segments. This easy-to-install and low-cost capacity-building protocol took only 24 h complete from extracting RNA to obtaining finished sequences. Overall, we developed a high-throughput portable sequencing workflow ideal for resource-limited clinical settings, aiding in real-time surveillance, outbreak investigation, and the detection of emerging viruses and genetic reassortment events. However, further evaluation is required to compare its accuracy with other high-throughput sequencing technologies to validate the widespread application of these findings, including WGS from environmental samples. IMPORTANCE The Nanopore MinION-based influenza sequencing approach we are proposing makes it possible to sequence the influenza A virus, irrespective of its diverse serotypes, directly from clinical and environmental swab samples, so that we are not limited to virus culture. This third-generation, portable, multiplexing, and real-time sequencing strategy is highly convenient for local sequencing, particularly in low- and middle-income countries like Bangladesh. Furthermore, the cost-efficient sequencing method could provide new opportunities to respond to the early phase of an influenza pandemic and enable the timely detection of the emerging subtypes in clinical samples. Here, we meticulously described the entire process that might help the researcher who will follow this methodology in the future. Our findings suggest that this proposed method is ideal for clinical and academic settings and will aid in real-time surveillance and in the detection of potential outbreak agents and newly evolved viruses.

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.

Avian influenza viruses (AIV) have been frequently detected in live bird markets (LBMs) around the world, primarily in urban areas, and have the ability to spillover to other species, including humans. Despite frequent detection of AIV in urban LBMs, the contamination of AIV on environmental surfaces in rural and peri-urban LBMs in Bangladesh is poorly documented. Therefore, we conducted this study to determine the prevalence of AIV subtypes within a subset of peri-urban and rural LBMs in Bangladesh and to further identify associated risk factors. Between 2017 and 2018, we collected faecal and offal samples from 200 stalls in 63 LBMs across four sub-districts. We tested the samples for the AIV matrix gene (M-gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We performed a descriptive analysis of market cleanliness and sanitation practices in order to further elucidate the relationship between LBM biosecurity and AIV subtypes by species, sample types, and landscape. Subsequently, we conducted a univariate analysis and a generalized linear mixed model (GLMM) to determine the risk factors associated with AIV contamination at individual stalls within LBMs. Our findings indicate that practices related to hygiene and the circulation of AIV significantly differed between rural and peri-urban live bird markets. 42.5% (95% CI: 35.56–49.67) of stalls were positive for AIV. A/H5, A/H9, and A HA/Untyped were detected in 10.5% (95% CI: 6.62–15.60), 9% (95% CI: 5.42–13.85), and 24.0% (95% CI: 18.26–30.53) of stalls respectively, with no detection of A/H7. Significantly higher levels of AIV were found in the Sonali chicken strain compared to the exotic broiler, and in offal samples compared to fecal samples. In the GLMM analysis, we identified several significant risk factors associated with AIV contamination in LBMs at the stall level. These include: landscape (AOR: 3.02; 95% CI: 1.18–7.72), the number of chicken breeds present (AOR: 2.4; 95% CI: 1.01–5.67), source of birds (AOR: 2.35; 95% CI: 1.0–5.53), separation of sick birds (AOR: 3.04; 95% CI: 1.34–6.92), disposal of waste/dead birds (AOR: 3.16; 95% CI: 1.41–7.05), cleaning agent (AOR: 5.99; 95% CI: 2.26–15.82), access of dogs (AOR: 2.52; 95% CI: 1.12–5.7), wild birds observed on site (AOR: 2.31; 95% CI: 1.01–5.3). The study further revealed a substantial prevalence of AIV with H5 and H9 subtypes in peri-urban and rural LBMs. The inadequate biosecurity measures at poultry stalls in Bangladesh increase the risk of AIV transmission from poultry to humans. To prevent the spread of AIV to humans and wild birds, we suggest implementing regular surveillance at live bird markets and enhancing biosecurity practices in peri-urban and rural areas in Bangladesh.

This study was undertaken to investigate the current pollution status of the Gomti River in Bangladesh by collecting water samples from twenty sites along the 120 km stretch of the Gomti River during the rainy (June 2019) and dry (January 2020) seasons. The collected samples were examined for their physicochemical parameters (pH, EC, TDS, TOC, and turbidity), heavy metals, namely chromium (Cr), copper (Cu), iron (Fe), nickel (Ni), lead (Pb), and zinc (Zn), and bacteriological parameters (aerobic heterotrophic bacteria and total coliforms). All the physicochemical parameters obtained were within the recommended guidelines set by the World Health Organization (WHO, 2011). Among the metals, Fe, Ni, and Zn were found to be higher than the recommended guideline values in the dry season. A significant (p

Objectives: The democratization of diagnostics is one of the key challenges towards containing the transmission of coronavirus disease 2019 (COVID-19) around the globe. The operational complexities of existing PCR-based methods, including sample transfer to advanced central laboratories with expensive equipment, limit their use in resource-limited settings. However, with the advent of isothermal technologies, the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possible at decentralized facilities. Methods: In this study, two recombinase-based isothermal techniques, reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription recombinase-aided amplification (RT-RAA), were evaluated for the detection of SARS-CoV-2 in clinical samples. A total of 76 real-time reverse transcription PCR (real-time RT-PCR) confirmed COVID-19 cases and 100 negative controls were evaluated to determine the diagnostic performance of the isothermal methods. Results: This investigation revealed equally promising diagnostic accuracy of the two methods, with a sensitivity of 76.32% (95% confidence interval 65.18–85.32%) when the target genes were RdRP and ORF1ab for RT-RPA and RT-RAA, respectively; the combination of N and RdRP in RT-RPA augmented the accuracy of the assay at a sensitivity of 85.53% (95% confidence interval 75.58–92.55%). Furthermore, high specificity was observed for each of the methods, ranging from 94.00% to 98.00% (95% confidence interval 87.40–9.76%). Conclusions: Considering the diagnostic accuracies, both RT-RPA and RT-RAA appear to be suitable assays for point-of-need deployment for the detection of the pathogen, understanding its epidemiology, case management, and curbing transmission.

Abstract 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

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.

IntroductionThe high burden of respiratory syncytial virus (RSV) infection in young children disproportionately occurs in low- and middle-income countries (LMICs). The PROUD (Preventing RespiratOry syncytial virUs in unDerdeveloped countries) Taskforce of 24 RSV worldwide experts assessed key needs for RSV prevention in LMICs, including vaccine and newer preventive measures.MethodsA global, survey-based study was undertaken in 2021. An online questionnaire was developed following three meetings of the Taskforce panellists wherein factors related to RSV infection, its prevention and management were identified using iterative questioning. Each factor was scored, by non-panellists interested in RSV, on a scale of zero (very-low-relevance) to 100 (very-high-relevance) within two scenarios: (1) Current and (2) Future expectations for RSV management.ResultsNinety questionnaires were completed: 70 by respondents (71.4% physicians; 27.1% researchers/scientists) from 16 LMICs and 20 from nine high-income (HI) countries (90.0% physicians; 5.0% researchers/scientists), as a reference group. Within LMICs, RSV awareness was perceived to be low, and management was not prioritised. Of the 100 factors scored, those related to improved diagnosis particularly access to affordable point-of-care diagnostics, disease burden data generation, clinical and general education, prompt access to new interventions, and engagement with policymakers/payers were identified of paramount importance. There was a strong need for clinical education and local data generation in the lowest economies, whereas upper-middle income countries were more closely aligned with HI countries in terms of current RSV service provision.ConclusionSeven key actions for improving RSV prevention and management in LMICs are proposed.

The avian influenza virus (AIV) impacts poultry production, food security, livelihoods, and the risk of transmission to humans. Poultry, like pigeons and quail farming, is a growing sector in Bangladesh. However, the role of pigeons and quails in AIV transmission is not fully understood. Hence, we conducted this study to investigate the prevalence and risk factors of AIV subtypes in pigeons and quails at live bird markets (LBMs) in Bangladesh. We collected oropharyngeal and cloacal swab samples from 626 birds in 8 districts of Bangladesh from 2017 to 2021. We tested the swab samples for the matrix gene (M gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We then used exploratory analysis to investigate the seasonal and temporal patterns of AIV and a mixed effect logistic model to identify the variable that influences the presence of AIV in pigeons and quails. The overall prevalence of AIV was 25.56%. We found that the prevalence of AIV in pigeons is 17.36%, and in quail is 38.75%. The prevalence of A/H5, A/H9, and A/H5/H9 in quail is 4.17, 17.92, and 1.67%, respectively. Furthermore, the prevalence of A/H5, A/H9, and A/H5/H9 in pigeons is 2.85, 2.59, and 0.26%. We also found that the prevalence of AIV was higher in the dry season than in the wet season in both pigeons and quail. In pigeons, the prevalence of A/untyped (40%) increased considerably in 2020. In quail, however, the prevalence of A/H9 (56%) significantly increased in 2020. The mixed-effect logistic regression model showed that the vendors having waterfowl (AOR: 2.13; 95% CI: 1.04–4.33), purchasing birds from the wholesale market (AOR: 2.96; 95% CI: 1.48–5.92) instead of farms, mixing sick birds with the healthy ones (AOR: 1.60; 95% CI: 1.04–2.45) and mingling unsold birds with new birds (AOR: 3.07; 95% CI: 2.01–4.70) were significantly more likely to be positive for AIV compared with vendors that did not have these characteristics. We also found that the odds of AIV were more than twice as high in quail (AOR: 2.57; 95% CI: 1.61–4.11) as in pigeons. Furthermore, the likelihood of AIV detection was 4.19 times higher in sick and dead birds (95% CI: 2.38–7.35) than in healthy birds. Our study revealed that proper hygienic practices at the vendors in LBM are not maintained. We recommend improving biosecurity practices at the vendor level in LBM to limit the risk of AIV infection in pigeons and quail 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.

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.

Canine coronavirus (CCoV) is widespread among the dog population and causes gastrointestinal disorders, and even fatal cases. As the zoonotic transmission of viruses from animals to humans has become a worldwide concern nowadays, it is necessary to screen free-roaming dogs for their common pathogens due to their frequent interaction with humans. We conducted a cross-sectional study to detect and characterize the known and novel Corona, Filo, Flavi, and Paramyxoviruses in free-roaming dogs in Bangladesh. Between 2009–10 and 2016–17, we collected swab samples from 69 dogs from four districts of Bangladesh, tested using RT-PCR and sequenced. None of the samples were positive for Filo, Flavi, and Paramyxoviruses. Only three samples (4.3%; 95% CI: 0.9–12.2) tested positive for Canine Coronavirus (CCoV). The CCoV strains identified were branched with strains of genotype CCoV-II with distinct distances. They are closely related to CCoVs from the UK, China, and other CoVs isolated from different species, which suggests genetic recombination and interspecies transmission of CCoVs. These findings indicate that CCoV is circulating in dogs of Bangladesh. Hence, we recommend future studies on epidemiology and genetic characterization with full-genome sequencing of emerging coronaviruses in companion animals 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.