Abstract
Dengue is a serious epidemic for Bangladesh affecting thousands of lives. This study aimed to identify and determine the prevalence of the circulating variants of dengue virus (DENV) and their association with demographics and clinical manifestations among the dengue-infected patients. A total of 711 participants with NS1 antigen positivity were enrolled, followed by viral RNA extraction from the collected blood samples and a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay to determine the dengue virus serotypes. Of 711 dengue-infected patients, 503 (70.7%) were male. Among different age groups, most of the patients were 21–30 years old (n = 255, 35.9%). The DENV2 (n = 483, 67.9%) serotype was more prevalent than the DENV3 (n = 144, 20.3%) and DENV1 (n = 84, 11.8%). The duration of fever was highest in the DENV-1 patients (4.79 ± 1.84 days) in contrast to DENV-3 (4.48 ± 1.68 days) and DENV-2 (4.33 ± 1.45 days) (P = 0.039). Importantly, five highly populated areas were identified as dengue hotspots in Chittagong metropolitan city. Our results provide crucial insights into the patterns of dengue virus transmission and severity among southern Bangladeshi population, thereby aiding in the development of targeted public health interventions and management strategies to combat future outbreaks.
Introduction
Dengue is a viral infectious disease that can present with a variety of symptoms, ranging from mild fever to severe hemorrhagic fever. Humans are the primary host for this virus, which spreads through a human-mosquito-human cycle, predominantly in tropical and subtropical regions worldwide. This flu-like illness has spread to about more than 100 countries, with an annual estimation of 390 million dengue virus infections, of which only 96 million people manifest clinically [1]. The recent surge in dengue incidence has shown significant year-to-year fluctuations in the timing and enormity of seasonal peaks, with a constantly increasing case fatality rate every year [2]. The four structurally similar but antigenically distinct dengue virus (DENV) serotypes (DENV1-DENV4) have been found to be circulating in various types of population [3, 4]. Research indicates that the shifting of these serotypes during an outbreak can serve as an underlying cause of changes in clinical characteristics. Additionally, individuals infected with one serotype exhibit long-term immunity to that specific serotype, but not to the other serotypes, which can lead to multiple infections (secondary and tertiary), potentially resulting in dengue shock syndrome. This type of serotype-specific clinical manifestation and the related disease severity can be explained by the antibody-dependent enhancement theory [5, 6]. Multiple dengue serotypes have also been co-detected in recent outbreaks, underscoring the severity of the situation. Bangladesh, a densely populated South Asian country with over 165 million people has been reported as a dengue-endemic country since the first recorded outbreak in 2000. Due to multiple risk factors, Bangladesh has been experiencing successive major dengue outbreaks in recent years [7]. Between 2000 and 2022, 788 dengue related fatalities have been found in Bangladesh; with 492 (more than 62%) of these deaths occurring between 2019 and 2022 [8]. Although dengue is endemic in Bangladesh, it has been reported as a public health issue in Bangladesh due to its recurrent outbreaks by the World Health Organization in 2014. The fatality rate due to dengue infection is significantly associated with dengue serotypes. Since the largest outbreak in 2019, DENV-3 has been found to be the most prevalent circulating serotype. The recent re-emergence of the DENV-4 serotype, absent for over 20 years, could pose a significant public health threat to Bangladesh due to the potential for secondary infections [7]. Given the severity of the 2023 dengue outbreak in Bangladesh, as well as the lack of data on the prevalence of circulating DENV serotypes and genotypes, in this study, we aimed to identify and determine the prevalence of the circulating variants of DENV and their association with demographics and clinical manifestations among the dengue-infected patients in the Chittagong region. Our study highlights the genetic variations of circulating DENV, which could have implications for diagnostic and prophylactic interventions for dengue in Bangladesh.
Materials and methods
Study participants and sample collection
A total of 711 participants from the One Stop Emergency and Casualty Unit and Paediatrics Departments of Chattogram Medical College Hospital, Chittagong General Hospital, Bangladesh Institute of Tropical and Infectious Diseases, Asperia Health Care Ltd., and Fatikchari Health complex were enrolled in the study. Briefly, all the participants presenting with an acute, undifferentiated fever within 3 days of onset who tested positive for dengue NS1 antigen (STANDARDTM Q Dengue NS1 Ag, SD Biosensor, Korea) were included in this study. In a recent study, the sensitivity of the STANDARDTM Q Dengue NS1 Ag test kit was found to be 99.1% in rapid diagnostic tests for the detection of dengue infections [9]. The patients were prospectively reviewed for detailed demographics, signs and symptoms, comorbid conditions, secondary dengue infection, and so on. 3 ml of venous blood were collected from each patient in ethylenediamine tetraacetic acid (EDTA) containing a vacutainer providing a unique ID, and the collected blood samples with DENV transported to the lab at a proper temperature (2–8 °C) and stored at −80 °C until analysis.
Viral RNA extraction and real-time reverse transcription-polymerase chain reaction (RT-PCR)
Viral RNA was extracted from the collected blood samples by using the High Pure Viral RNA Kit, (Roche Diagnostics, Indianapolis, USA) following the manufacturer's instructions. The concentration and purity of the extracted viral RNA were measured using NanoDropTM 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Extracted viral RNA samples were used immediately for a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay on Light Cycler 96 (Roche Diagnostics, Switzerland) to determine the dengue virus serotypes by using the RealStar Dengue Type RT-PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany), as per the manufacturer's instructions. This RT-PCR kit was chosen based on availability as well as the findings of a recent published study on the evaluation of sensitivity (100% for DENV-3 and DENV-4, 86.7% for DENV-1 and DENV-2) and specificity (97.9%–100% for all four serotypes) [10]. Briefly, the reaction was carried out with a reaction volume of 30 μL containing 20 μL master mix and 10 μL extracted viral RNA. The cycling conditions were 55 °C for 20 min for the reverse transcription, followed by 2 min at 95 °C for the initial denaturation, and 45 cycles of amplification with cycling conditions of 95 °C for 15 s, 60 °C for 45 s, and 72 °C for 15 s. In the case of PCR result interpretation, a cycle threshold (Ct) < 38 was evaluated as positive. 20% (n = 142) of the positive test results for DENV were further validated by using the STANDARDTM M10 Arbovirus Panel with the STANDARD M10 system (SD Biosensor, Suwon-si, Gyeonggi-do, Korea) according to the manufacturer's protocol. The STANDARDTM M10 Arbovirus Panel is a molecular in vitro diagnostic test that detects and differentiates arboviruses such as dengue virus, zika virus, chikungunya virus, yellow fever virus, and west Nile virus RNA by nucleic acid amplification using RT-PCR. As a result, this number of DENV samples was chosen at random, focusing on positive samples with a Ct value greater than 35, which covered all three identified DENV serotypes by the RealStar Dengue Type RT-PCR Kit, in order to assess the kit's detection ability at extremely low Ct values close to negative for DENV and the sensitivity and specificity as well.
Statistical analysis
Windows-based software (GraphPad Prism 9) was used to produce a statistical analysis of the variables. The chi-square test was used to compare the categorical variables, while the numerical data were expressed as the mean with a standard deviation (±SD). 95% confidence intervals were calculated for these values. A P value less than 0.05 was considered statistically significant.
Ethical approval and consent to participate
The Institutional Review Board of the Chittagong Medical College, Chattogram, Bangladesh approved the study. All participants of the study were informed of the purpose and methods of the investigation. Each patient provided written consent before participating in the study. Participation in the study was voluntary. The investigators collected personal information and other pertinent medical data using a semi-structured questionnaire. Blood samples from each patient were submitted to the lab after they were assigned a unique identification code (ID, anonymous or numerical). The principal investigator documented the test findings by decoding the IDs and adding them to the main dataset.
Results
Demographics and dengue virus serotypes
A total of 711 laboratory-confirmed (NS1 antigen) dengue patients were enrolled in the study between June and September 2024, of whom 503 (70.7%) were male. Among different age groups, most of the dengue-infected patients were 21–30 years old (n = 255, 35.9%), followed by 11–20 years old (n = 180, 25.3%), 31–40 years old (n = 123, 17.3%), 0–10 years old (n = 51, 7.2%), 41–50 years old (n = 48, 6.8%), 51–60 years old (n = 36, 5.1%), 61–70 years old (n = 15, 2.1%), and 71–80 years old (n = 3, 0.4%). Of the 711 dengue-infected patients, RT-PCR results showed that 84 (11.8%) of them were infected with DENV-1, 483 (67.9%) were DENV-2, and 144 (20.3%) were DENV-3. The DENV-4 serotype was not identified among the patients. Notably, 15 (2.1%) of the patients had a history of previous infection with dengue virus, but the serotypes were not confirmed, and the majority (n = 705, 99.2%) of the dengue-infected patients did not take any medication to recover from dengue infection. Additionally, 126 (17.7%) of the patients had comorbid conditions, in whom diabetes (21.4%) and hypertension (21.4%) were mostly found, followed by eye problems (14.3%), asthma/bronchitis (7.1%), cardiovascular diseases (7.1%), renal diseases (4.8%), and others (9.5%). Moreover, 579 (81.4%) of the patients felt less active or irritable during their illness (Table 1).
Characteristics of laboratory-confirmed dengue patients (N = 711)
Characteristics | No. of patients | % of patients |
Sex | ||
Male | 503 | 70.7 |
Female | 208 | 29.3 |
Age group (years) | ||
0–10 | 51 | 7.2 |
11–20 | 180 | 25.3 |
21–30 | 255 | 35.9 |
31–40 | 123 | 17.3 |
41–50 | 48 | 6.8 |
51–60 | 36 | 5.1 |
61–70 | 15 | 2.1 |
71–80 | 3 | 0.4 |
>80 | 0 | 0 |
Dengue virus serotype | ||
DENV-1 | 84 | 11.8 |
DENV-2 | 483 | 67.9 |
DENV-3 | 144 | 20.3 |
DENV-4 | 0 | 0 |
History of previous dengue infection | ||
Yes | 15 | 2.1 |
No | 696 | 97.9 |
History of previous medication for dengue infection | ||
Yes | 6 | 0.8 |
No | 705 | 99.2 |
Comorbid conditions | ||
No | 585 | 82.3 |
Yes | 126 | 17.7 |
Allergy | 6 | 4.8 |
Asthma/Bronchitis | 9 | 7.1 |
Diabetes | 27 | 21.4 |
Cardiovascular diseases | 9 | 7.1 |
Hypertension | 27 | 21.4 |
Hypotension | 3 | 2.4 |
Jaundice | 3 | 2.4 |
Renal diseases | 6 | 4.8 |
Eye problem | 18 | 14.3 |
Tuberculosis | 3 | 2.4 |
Urinary tract infection | 3 | 2.4 |
Others | 12 | 9.5 |
Activity level during illness | ||
Normal | 132 | 18.6 |
Less Active/Irritable | 579 | 81.4 |
Significant correlation between age and infection with different serotypes was not found in the study (Fig. 1). Male patients had a greater proportion of infections with respective DENV serotypes compared with female patients. The gender distribution of DENV-1, DENV-2, and DENV-3 was 73.8% male and 26.2% female, 70.8% male and 29.2% female, and 68.8% male and 31.2% female, respectively (Fig. 2).
One-way ANOVA (mixed-effects analysis) for the comparison of ages among three dengue virus serotypes (DENV). P value less than 0.05 was considered as statistically significant
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00069
Distribution of gender infected with dengue virus serotypes DENV-1, DENV-2, and DENV-3
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00069
Comparative analysis between different age groups and infection with three dengue serotypes showed that the DENV-1 genotype was more prominent in the age groups of 0–10 years (10.7%), 41–50 years (10.7%), and 61–70 years (3.6%) compared with DENV-2 and DENV-3. The DENV-2 genotype was most prevalent in the age group of 21–30 years (37.3%) compared with other genotypes. In addition, the DENV-3 genotype was found mostly in the age group of 11–20 years (33.3%), followed by 31–40 years (22.9%), and 51–60 years (8.3%), compared with the DENV-2 and DENV-3 genotypes (Fig. 3).
Distribution of dengue virus serotypes DENV-1, DENV-2 and DENV-3 infected patients among different age groups
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00069
Dengue hotspots in Chittagong
Dengue risk zones in the Chittagong region were analyzed based on the detection of DENV serotypes among the study patients. 60% of dengue patients in Chittagong lived in five areas that are identified as hotspots, namely Bakalia, Chawkbazar, Kotwali, Double Mooring, and Baizid Bostami. Also, among the rural areas, the maximum number of patients were found in Sitakundu, Hathhazari, Patia, and Karnaphuli areas (Fig. 4).
Distribution of dengue hotspots in Chittagong region. A. Study area map of rural areas of Chattogram (outside City Corporation), B. Study map of urban areas of Chattogram (inside City Corporation)
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00069
Clinical manifestation
All the dengue cases (n = 711) infected with three different serotypes (DENV-1, DENV-2, and DENV-3) had fever (100%). Infected cases with DENV-3 were more likely to have headaches (75%), in contrast to DENV-2 and DENV-1, less likely to be observed. Instead, DENV-2 cases were more likely to present with muscle pain (54%), compared with DENV-1 (39.3%) and DENV-2 (47.9%). Anorexia was found mostly in cases with DENV-2 (73.9%), and cases with eye pain were mostly infected with DENV-3 (45.8%). Additionally, cases with DENV-1 infection had the highest proportion (28.6%) of abdominal pain, and 12.5% of the patients showed breathing difficulties who were infected with DENV-3. However, there were no significant differences in vomiting, watery stool, runny nose, rash, or bleeding among the three serotypes (Fig. 5).
Clinical manifestations by dengue virus serotypes DENV-1, DENV-2 and DENV-3 among the study patients
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00069
Table 2 shows the comparison of the duration of several clinical manifestations among the cases infected with three different dengue serotypes. Though DENV-2 (n = 483, 67.9%) was the most prevalent serotype among the cases, the duration of fever was highest in the patients infected with DENV-1 (mean ± SD, 4.79 ± 1.84 days) in contrast to DENV-3 (mean ± SD, 4.48 ± 1.68 days) and DENV-2 (mean ± SD, 4.33 ± 1.45 days) (P = 0.039). The duration of having a headache was higher in the cases with DENV-1 (mean ± SD, 4.60 ± 1.62 days) compared to the DENV-2 (mean ± SD, 3.93 ± 1.42 days) and DENV-3 (mean ± SD, 3.86 ± 1.60 days) (P = 0.0129). Cases with DENV-1 experienced eye pain for a longer period (mean ± SD, 4.89 ± 2.33 days) compared to DENV-2 (mean ± SD, 3.85 ± 1.39 days) and DENV-3 (mean ± SD, 3.64 ± 1.87 days) (P = 0.0032). In addition, the duration of having watery stool was highest in the patients infected with DENV-1 (mean ± SD, 3.22 ± 1.87 days) versus DENV-3 (mean ± SD, 3.18 ± 1.69 days) and DENV-2 (mean ± SD, 2.45 ± 1.01 days) (P = 0.0008). Moreover, cases with DENV-1 were longer compromised by runny noses (mean ± SD, 4.57 ± 1.18 days) compared to DENV-2 (mean ± SD, 3.73 ± 1.72 days) and DENV-3 infected patients (mean ± SD, 3.44 ± 1.84 days) (P = 0.0424). However, significant differences were not observed for the duration of having muscle pain, vomiting, or anorexia among the three dengue serotypes.
Comparison of duration of the clinical manifestations among the cases infected with three different dengue virus serotypes
Clinical manifestations | DENV-1 (N = 84) | DENV-2 (N = 483) | DENV-3 (N = 144) | P value* | |||||
N | Duration (days) | N | Duration (days) | N | Duration (days) | ||||
(mean ± SD) | (mean ± SD) | (mean ± SD) | DENV-1 vs. DENV-2 | DENV-1 vs. DENV-3 | DENV-2 vs. DENV-3 | ||||
Fever | 84 | 4.79 ± 1.84 | 483 | 4.33 ± 1.45 | 144 | 4.48 ± 1.68 | 0.035 | 0.322 | 0.566 |
Headache | 45 | 4.60 ± 1.62 | 309 | 3.93 ± 1.42 | 108 | 3.86 ± 1.60 | 0.014 | 0.015 | 0.905 |
Eye pain | 27 | 4.89 ± 2.33 | 186 | 3.85 ± 1.39 | 66 | 3.64 ± 1.87 | 0.007 | 0.003 | 0.620 |
Muscle pain | 33 | 4.73 ± 2.00 | 261 | 4.07 ± 1.60 | 69 | 4.26 ± 1.80 | 0.085 | 0.828 | 0.588 |
Vomiting | 45 | 3.27 ± 2.23 | 255 | 2.69 ± 1.53 | 81 | 2.74 ± 1.73 | 0.094 | 0.219 | 0.975 |
Watery stool | 27 | 3.22 ± 1.87 | 132 | 2.45 ± 1.01 | 51 | 3.18 ± 1.69 | 0.020 | 0.989 | 0.004 |
Runny nose | 27 | 4.57 ± 1.18 | 132 | 3.73 ± 1.72 | 48 | 3.44 ± 1.84 | 0.094 | 0.033 | 0.577 |
Anorexia | 33 | 4.81 ± 1.94 | 357 | 4.29 ± 1.44 | 87 | 4.34 ± 1.73 | 0.043 | 0.173 | 0.960 |
DENV = dengue virus serotype; N = no. of dengue virus infected patients; SD = standard deviation
*P value was calculated based on Tukey's multiple comparison test comparing each dengue virus serotype with the other two serotypes and P value less than 0.05 was considered statistically significant (marked bold).
Discussion
Bangladesh has been facing frequent dengue fever outbreaks since the dengue virus (DENV) emerged in 2000 [11]. In 2022, the nation faced the second-largest outbreak (62,382 cases and 281 deaths), followed by the most devastating dengue fever outbreak in the history of the nation in 2023, with the highest case fatality rate (321,179 cases and 1,705 deaths) [12].
Distribution of sex
Our study found a consistent disparity among the male and female patient numbers across all three serotypes, with male patients accounting for around 70% of the cases whereas females contributed nearly 30% (Table 1). This finding is consistent with previous studies reporting similar gender disparity patterns of dengue prevalence in Bangladesh and neighboring Asian countries [11, 13, 14]. It can be attributed to two main reasons: firstly, differences in behavioral patterns, outdoor activities, and work hours in the male population of South Asian countries contribute to increased exposure to dengue vectors, as men often spend more time outdoors at dawn and in the evening; secondly, disparity in seeking medical attention may also play a role, with men reportedly being more willing to seek medical care than women in both rural and urban areas [2].
Distribution of age groups
Throughout most of Southeast Asia, in the past, dengue was considered to be a disease that primarily affects children [15]. According to the current study, most dengue patients were adults (18–40 years old), consistent with several other recent studies carried out in South Asian countries, including Bangladesh, Singapore, Sri Lanka, India, and other nations [16–18]. Our study also found that the majority of dengue-infected patients were between the ages of 21 and 30, followed by other age groups (Table 1). Though adults are more prone to infections, there was no significant relationship between age and infection with different serotypes (Fig. 1).
Distribution of circulating DENV serotypes and comorbidities
Prior to 2018, DENV-2 was the predominant circulating serotype. In 2019, its prevalence was surpassed by DENV-3. However, in 2023, DENV-2 re-emerged as the primary circulating serotype (51%), with DENV-3 also remaining prevalent at 44%, which is consistent with our findings. According to our study, 67.9% of patients were infected with DENV-2, followed by DENV-3 (20.3%) and DENV-1 (11.8%) (Table 1). Additionally, distinct morbidities such as diabetes and hypertension were observed in the dengue classification group [16]. Among the patients who had comorbid conditions in our study sample, diabetes (21.4%) and hypertension (21.4%) had a higher prevalence, followed by the others (Table 1).
While DENV-2 was the most common serotype across all four age groups, comparative analysis of the age groups and serotypes revealed that the 21–30 years cohort was primarily infected with DENV-2, while the adjacent age groups were more susceptible to DENV-3. Children and older age groups were found to be predominantly infected with the DENV-1 serotypes (Fig. 3). Contrary to the current finding, earlier research on dengue outbreaks in Bangladesh has shown that DENV-3 was the most frequently circulating serotype from 2019 to 2024 across all age groups [19–21].
Distribution of dengue hotspots
This study represents the first comprehensive investigation into the identification and determination of DENV serotypes among patients with dengue infection, featuring a detailed map that delineates dengue hotspots within Chittagong, the second-largest city in Bangladesh, including its metropolitan areas (Fig. 4). We identified five metropolitan areas of Chittagong, including Bakalia, Chawkbazar, Kotwali, Double Mooring, and Baizid Bostami. These dengue hotspots are highly populated in comparison with other metropolitan areas (https://en.banglapedia.org/index.php/Chittagong_District). Additionally, among the rural areas, the maximum number of dengue patients were found in Sitakundu, Hathhazari, Patia, and Karnaphuli areas (Fig. 4).
Clinical features
In our study, fever was the most common clinical manifestation of DENV in all patients, regardless of the serotypes (100%); which has been observed among patients in previous dengue outbreaks as well [22]. While fever was the most prevalent clinical feature, other commonly associated symptoms, including headache, vomiting, muscle pain, anorexia, eye pain, watery stool, abdominal pain, and runny nose, were also observed in variable proportions based on different serotypes (Fig. 5). These are considered the general features of dengue fever [23] and demonstrate a strong correlation with related studies [24–26]. Although fever, pain, and rash are contemplated as triads of dengue fever, rash was observed with low frequency in recent outbreaks [16], including among all serotypes in our study sample. An increasing trend of anorexia was observed in most of the patients, with a high percentage in DENV-2 (73.9%), a very frequently reported gastrointestinal symptom of dengue fever along with vomiting in recent times [27]. Headache is the second most common symptom among dengue patients in all cases since its emergence [28]. Despite the highest number of patients having the DENV-2 serotype (N = 309), this study showed that headache as a symptom was more prevalent in the DENV-3 (75%) serotype compared to others. In addition, in many studies, respiratory distress is presented as being related to severe plasma leakage leading to serious complications [29]. However, this study observed a very low percentage of breathing difficulties in patients infected with three different serotypes (Fig. 5).
Although DENV2 was the most prevalent serotype in this study, the duration of clinical symptoms (fever, headache, watery stool, eye pain, anorexia) were significantly higher in patients carrying DENV1 (Table 2) which is consistent with a previous study conducted by Yung et al. [30].
This study investigates the demographic, epidemiological, and clinical characteristics of dengue patients infected with different circulating dengue virus serotypes during the 2023 outbreak in southern Bangladesh. However, the study recruited patients from hospitals or clinics, which may not reflect the full spectrum of dengue infection in the community, potentially underrepresenting milder or asymptomatic infections. This sampling bias may limit the generalizability of our findings to the broader population in southern Bangladesh. Future studies incorporating a more representative sample, including more outpatients and those with milder dengue, are crucial for a comprehensive understanding of serotype-specific disease characteristics.
Conclusion
Over the past 20 years, Bangladesh has experienced a noticeable change in the circulating DENV serotypes. Though recent major dengue outbreaks have been found to be associated with the prevalence of the serotype DENV-3, we found a higher prevalence of DENV-2 in the country's second-largest region. Variations in serotypes may have contributed to a rise in clinical presentation severity in recent times. Our results provide crucial insights into the patterns of dengue virus transmission and severity, thereby aiding in the development of targeted public health interventions and management strategies to combat future outbreaks.
Funding sources
This project was funded by Beximco Pharmaceuticals Ltd., Bangladesh (Dengue/Asperia/2023) and Asperia Healthcare Ltd (R&D/2023/001).
Author's contributions
Conceptualization: Adnan Mannan, Mohabbat Hossain, Abul Faisal Md. Nuruddin Chowdhury, H. M. Hamidullah Mehedi, Noor Mohammed, Mustafizur Rajman.
Data curation: Istiaq Uddin Ahmed, Kallyan Chakma, Mohabbat Hossain.
Methodology: Md. Abdur Rob, Mohabbat Hossain, Abul Faisal Md. Nuruddin Chowdhury, Mohammed Maruf ul Quader, Md. Zakir Hossain, Mustafizur Rahman, Adnan Mannan.
Formal analysis: Mohabbat Hossain.
Visualization: Mohabbat Hossain, Adnan Mannan.
Writing – original draft: Mohabbat Hossain, Susmita Barua, Silvia Naznin Etu, Uschash Sikder.
Writing – review & editing: Md. Abdur Rob, M. A. Sattar, Adnan Mannan.
Funding acquisition: Md. Abdur Rob, M. A. Sattar, Abul Faisal Md. Nuruddin Chowdhury, H. M. Hamidullah Mehedi, Noor Mohammed, Mohammed Maruf ul Quader, Adnan Mannan.
Investigation: Md. Abdur Rob, Mohabbat Hossain, Abul Faisal Md. Nuruddin Chowdhury, H. M. Hamidullah Mehedi, Noor Mohammed, Mohammed Maruf ul Quader, Md. Zakir Hossain, Susmita Barua, Silvia Naznin Etu, Uschash Sikder, Adnan Mannan.
Project administration: Md. Abdur Rob, Mohabbat Hossain, Abul Faisal Md. Nuruddin Chowdhury, H. M. Hamidullah Mehedi, Noor Mohammed, Mohammed Maruf ul Quader, Md. Zakir Hossain, Mustafizur Rahman, Adnan Mannan.
Supervision: Md. Abdur Rob, M. A. Sattar, Abul Faisal Md. Nuruddin Chowdhury, H. M. Hamidullah Mehedi, Noor Mohammed, Mohammed Maruf ul Quader, Md. Zakir Hossain, Adnan Mannan.
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgements
The authors would like to thank Chittagong Medical College, 250 bedded General Hospital, Asperia Health Research & Development Foundation (ARF) and Disease Biology and Molecular Epidemiology Research Group (dBme), Chattogram for their help at different stages of this research.
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