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 (STANDARD^TM Q Dengue NS1 Ag, SD Biosensor, Korea) were included in this study. In a recent study, the sensitivity of the STANDARD^TM 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 STANDARD^TM M10 Arbovirus Panel with the STANDARD M10 system (SD Biosensor, Suwon-si, Gyeonggi-do, Korea) according to the manufacturer's protocol. The STANDARD^TM 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.

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).

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).

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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

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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

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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).

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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

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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).

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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

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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).

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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

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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.

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.