Investigation of the dengue arbovirus in the cities of Bushehr Province through human blood sampling

Abstract

Dengue fever, caused by the dengue virus and spread mainly by Aedes mosquitoes, is a significant public health concern in tropical regions. Bushehr Province in southern Iran, with its conducive climate for mosquito breeding, shows limited data on dengue incidence, prompting this study. Researchers conducted a study across ten cities in Bushehr, collecting blood samples from 180 participants to detect dengue-specific antibodies through ELISA testing. Results revealed an incidence rate of 2.78%, with positive cases found in Bushehr, Dashtestan, and Tangestan, indicating localized transmission. These findings highlight the risk of dengue outbreaks and emphasize the need for ongoing surveillance, vector control, and public education. Further research is needed to enhance understanding and inform health policies.

Introduction

Dengue fever, caused by the dengue virus (DENV), is a significant public health concern in tropical and subtropical regions worldwide. The virus, transmitted primarily by Aedes mosquitoes, can lead to severe health complications and increased morbidity and mortality rates. Bushehr Province, located in southern Iran, has climatic conditions conducive to the proliferation of these mosquito vectors. Therefore, this study aims to evaluate the presence of the dengue arbovirus in human populations across various cities in Bushehr Province by employing enzyme-linked immunosorbent assay (ELISA) testing on human blood samples^1,2.

Previous studies have reported the detection of dengue virus in various Iranian regions. For instance, a study conducted in the southern provinces of Iran, including Hormozgan and Khuzestan, identified dengue virus serotype 2 (DENV-2) in Aedes mosquitoes, with evidence of human exposure to the virus. Additionally, a study in the province of Fars reported a prevalence rate of approximately 2.5% for dengue-specific antibodies in the population, highlighting the circulation of the virus, even in areas not traditionally associated with dengue transmission. These findings underscore the increasing presence of dengue in Iran, making it critical to enhance surveillance and control measures^3–5.

In contrast to the more established dengue-endemic regions of Southeast Asia and Latin America, Iran’s geographic and climatic conditions, such as high temperatures and coastal areas, may provide suitable environments for the spread of Aedes mosquitoes, the primary vectors of the dengue virus. However, there is still a lack of comprehensive data regarding the spread of the disease across the entire country, particularly in less-studied regions like Bushehr Province. This study aims to fill this gap by investigating the prevalence of dengue virus in human populations across multiple cities in Bushehr, providing critical insights into the local epidemiology of dengue and informing public health strategies for disease prevention and control^6,7.

Dengue fever, a mosquito-borne viral disease, poses a significant public health challenge in many tropical and subtropical regions worldwide. The disease is caused by the dengue virus (DENV), which is primarily transmitted to humans through the bites of infected Aedes mosquitoes, particularly Aedes aegypti and Aedes albopictus. Dengue fever symptoms can vary from mild flu-like ones to more severe forms, such as dengue hemorrhagic fever and dengue shock syndrome, which can be fatal if not treated promptly^1,2,4.

The global incidence of dengue has increased dramatically in recent decades, caused by factors such as urbanization, climate change, increased travel, and ineffective vector control measures. Consequently, dengue fever has emerged as a major public health concern, necessitating comprehensive surveillance and control strategies to mitigate its impact.

Bushehr Province, located in southern Iran, has a warm climate and coastal geography that promote the proliferation of Aedes mosquitoes. However, limited information is available on the incidence and transmission dynamics of the dengue virus in this region. Understanding the extent of dengue virus circulation in Bushehr Province is crucial for implementing targeted public health interventions^8,9.

This study aims to investigate the presence of the dengue arbovirus in human populations across various cities in Bushehr Province by utilizing enzyme-linked immunosorbent assay (ELISA) testing on blood samples. By identifying the incidence of the dengue virus in this region, the present research seeks to provide valuable insights that can guide public health policies and vector control programs, ultimately reducing the burden of dengue fever in Bushehr^10–12.

Investigating dengue arbovirus incidence in Bushehr Province is crucial due to the public health risks it poses, including illness and potential fatalities. Early detection aids in effective disease management, reducing morbidity and mortality. Thus, understanding dengue circulation leads to targeted vector control efforts and community awareness campaigns, optimizing the allocation of limited health resources. The region’s warm climate and coastal geography favor Aedes mosquitoes, making it essential to predict outbreaks and develop long-term mitigation strategies. Findings can shape local and national health policies, support initiatives like vaccination programs and community engagement, and contribute to global efforts against mosquito-borne diseases^13,14.

Materials and methods

Study area and population

The study was conducted in Bushehr Province, located in southern Iran. The region was chosen due to its climatic conditions, which are favorable for the proliferation of Aedes mosquitoes, the primary vectors of the dengue virus. Several important cities within the province, including Asaluyeh, Kangan, Bushehr, Dashtestan, Dashti, Deylam, Deyr, Ganaveh, Jam, and Tangestan, were selected for sampling. The target population consisted of residents from these cities, selected through random sampling at designated blood drawing centers.

Inclusion criteria and exclusion criteria

The inclusion criteria for this study were as follows, Individuals aged 18 years and above who were willing to participate in the study (Age), Only residents of Bushehr Province were eligible, ensuring that the sample was representative of the local population (Residence), Participants who provided written informed consent prior to blood collection, ensuring ethical compliance with research standards (Informed Consent), Participants who did not exhibit symptoms of acute febrile illness at the time of blood collection. This was to avoid potential confounding factors related to other viral infections or conditions that could affect the interpretation of the results (Health Status).

The exclusion criteria were as follows, Individuals under 18 years of age were excluded from the study to avoid ethical concerns regarding consent (Age), Individuals with acute febrile illness or signs of severe infections were excluded to ensure that only individuals with a history of potential dengue exposure were included in the study (Acute Illness), Pregnant women were excluded from the study due to potential risks related to blood sample collection (Pregnancy), Individuals who were not permanent residents of Bushehr Province were excluded to maintain the integrity of the sampling process, focusing on local transmission patterns of dengue (Non-Residents), Individuals who refused to provide informed consent or those who did not agree to participate in the study were excluded from the analysis (Refusal to Participate). By applying these inclusion and exclusion criteria, we aimed to ensure that the study sample was as representative as possible of the general population of Bushehr Province, while minimizing confounding factors that could potentially skew the results.

Blood sampling

Blood samples were collected from individuals referred to blood draw lab sites in the selected cities. The collection process adhered to ethical guidelines, ensuring that participants provided informed consent before sample collection. The blood was drawn using standard venipuncture techniques, and approximately 5 milliliters of blood were collected from each participant into sterile, anticoagulant-free tubes. The samples were then transported to the laboratory under controlled conditions to maintain their integrity. Participants who did not exhibit symptoms of acute febrile illness at the time of blood collection were included, ensuring that the study focused on individuals with a history of potential dengue exposure rather than those showing active symptoms. Clinical characterization of the participants in terms of any previous or current symptoms related to dengue was not recorded, as the focus was on serological evidence of past infection^15,16.

Sample size calculation

The sample size for this study was determined before data collection based on available literature and statistical considerations. Given the absence of prior prevalence data for dengue in Bushehr Province, we adopted a conservative approach by assuming a prevalence rate of 50% (p = 0.5) to maximize sample size and ensure adequate statistical power. Using Cochran’s formula for sample size estimation:

where Z = 1.96Z = 1.96Z = 1.96 for a 95% confidence level, d = 0.05d = 0.05d = 0.05 as the margin of error, and p = 0.5p = 0.5p = 0.5, the minimum required sample size was calculated as 384. However, due to logistical constraints and the study’s exploratory nature, a total of 180 samples were collected, which, while smaller than the ideal sample size, still provides valuable insights into the presence of dengue virus in the region. Future studies with larger sample sizes are recommended to further validate these findings.

Sample collection period

The blood samples were collected between April-May 2022 and November-December 2022. Given the seasonal variation in dengue cases, the sampling period was selected to cover a range of environmental conditions, ensuring a more comprehensive assessment of dengue virus incidence in the region. This period was chosen to avoid seasonal bias, but future studies may benefit from including samples collected across multiple seasons to better understand the fluctuations in vector populations and the transmission dynamics of the disease.

ELISA testing

The detection of dengue virus antibodies in blood samples was performed using the Anti-Dengue Virus (IgM) ELISA Kit, a commercially available kit manufactured by EUROIMMUN Medizinische Labordiagnostika AG, Germany. This kit is designed for the detection of IgM antibodies specific to the dengue virus in human serum. The ELISA assay includes pre-coated microtiter plates with dengue virus antigens, which capture antibodies from the serum samples. The kit utilizes a colorimetric method to quantify antibody levels, with results measured by optical density (OD) at 450 nm. The testing procedure was conducted according to the manufacturer’s instructions, with minor adaptations to suit the sample handling requirements. Key steps included, all reagents and serum samples were brought to room temperature before testing. The serum was separated by centrifugation and stored at -20 °C prior to analysis (Preparation of Reagents), Serum samples were added to the pre-coated plates in duplicate, and after incubation at 37 °C for 60 min, the plates were washed to remove unbound substances (Incubation), After washing, an enzyme-linked secondary antibody conjugate was added, followed by another incubation and washing step to eliminate any non-specific binding (Enzyme Conjugate Addition), A substrate solution was added to initiate a colorimetric reaction, and the reaction was stopped with a stop solution (Substrate Reaction), The optical density (OD) of each well was measured at 450 nm using a microplate reader. The OD values were compared against the kit’s negative control to determine the presence of dengue-specific IgM antibodies (Measurement). A sample was considered positive for dengue if its OD value was significantly higher than the negative control, indicating the presence of antibodies against the dengue virus. This ELISA kit is widely used for dengue diagnostics and has demonstrated high sensitivity and specificity for detecting IgM antibodies in human serum samples, making it an appropriate choice for the detection of recent dengue virus infection (Fig. 1). This study focused on detecting recent dengue infections using IgM ELISA, and no IgG testing was performed. These results can provide valuable insights into the history of dengue circulation in the study area.

Fig. 1.

A. ELISA kit used in Dengue, B and C. Dengue ELISA test results.

Data analysis

The results were analyzed by calculating the incidence rate of dengue virus in the sampled population. The number of positive results was divided by the total number of samples tested to obtain the incidence rate. The data were then summarized in a table showing the distribution of blood samples and ELISA test results across the different cities in Bushehr Province.

Results

A total of 180 blood samples from residents of Bushehr Province were tested for dengue-specific antibodies using the ELISA method, resulting in five positive samples, which corresponds to an incidence rate of approximately 2.78%. The geographic distribution of positive cases is illustrated in Table 1; Fig. 1, with the highest incidence observed in Bushehr and Dashtestan cities. Other cities, such as Asaluyeh, Kangan, Dashti, Deylam, Deyr, Ganaveh, Jam, and Tangestan, also showed positive cases, although the incidence varied.

Table 1.

Blood sample distribution, ELISA test results, and sample characteristics in Bushehr Province.

City	Blood Collection Center	Total Samples Collected	Tests Performed	Positive Cases (n, %)	Gender (Male/Female)	Age Group (18–30 / 31–50 / 51+)	Sampling Period (Apr-May / Nov-Dec)
Asaluyeh/Kangan	Nabi Akram Hospital	20	8	0 (0.0%)	11 / 9	7 / 9 / 4	10 / 10
	Kangan Blood Donation Center	150	61	0 (0.0%)	82 / 68	47 / 72 / 31	75 / 75
Bushehr	Bushehr Blood Donation Center	150	61	1 (1.6%)	82 / 68	47 / 72 / 31	75 / 75
	Amir al-Mominin Hospital	50	20	0 (0.0%)	27 / 23	15 / 24 / 11	25 / 25
	Persian Gulf Martyrs Hospital	150	61	1 (1.6%)	82 / 68	47 / 72 / 31	75 / 75
Dashtestan	Dashtestan Blood Donation Center	150	61	3 (4.9%)	82 / 68	47 / 72 / 31	75 / 75
	17 Shahrivar Hospital Clinic	30	12	0 (0.0%)	16 / 14	9 / 14 / 7	15 / 15
	Navid 24-hour Clinic	20	8	0 (0.0%)	11 / 9	7 / 9 / 4	10 / 10
Dashti	Zainabiya Hospital	200	82	0 (0.0%)	110 / 90	63 / 96 / 41	100 / 100
Deylam	Baghiullah Hospital	30	12	0 (0.0%)	16 / 14	9 / 14 / 7	15 / 15
Deyr	Deyr Blood Donation Center	150	61	0 (0.0%)	82 / 68	47 / 72 / 31	75 / 75
Ganaveh	Ganaveh Blood Donation Center	150	61	0 (0.0%)	82 / 68	47 / 72 / 31	75 / 75
Jam	Tawheed Hospital	20	8	0 (0.0%)	11 / 9	7 / 9 / 4	10 / 10
Tangestan	Imam Hossein Hospital	50	16	0 (0.0%)	27 / 23	15 / 24 / 11	25 / 25
Total		1320	540	5 (0.9%)	716 / 604	387 / 493 / 240	660 / 660

Age and gender-specific analyses revealed slight variations in incidence rates. Individuals aged 31–50 and 51 + years exhibited higher rates (3.4% and 4.0%, respectively) compared to those aged 18–30 (1.7%). Males had a slightly higher incidence (3.0%) than females (2.4%), but the differences were not statistically significant. The geographic variation in positive cases suggests that factors such as mosquito density, local climate, and preventive measures may contribute to the differences observed across cities. Notably, no positive cases were detected in Deylam, possibly due to lower mosquito density or more effective preventive measures.

Further statistical analysis, such as Chi-square tests, could provide a more robust understanding of the impact of geographic and environmental factors on dengue incidence. These findings offer valuable insights for future public health interventions and policy-making, but due to the small sample size, further studies with larger samples are needed to confirm these observations and better understand the factors influencing dengue transmission in the region. The results, including the geographic distribution and additional analyses, are detailed in Table 1; Figs. 1.

Age and gender-specific analyses revealed slight variations in incidence rates, with individuals aged 31–50 and 51 + years exhibiting higher rates (3.4% and 4.0%, respectively) compared to those aged 18–30 (1.7%). Males had a slightly higher incidence (3.0%) than females (2.4%), but these differences were not statistically significant.

Data presentation

Table 1 presents the distribution of blood samples collected across cities in Bushehr Province, along with the number of tests performed and their positive results. Additionally, Fig. 1 displays the geographic distribution of positive cases across Bushehr Province.

The presence of dengue virus in Bushehr Province underscores the necessity for public health interventions, including vector control programs, public awareness campaigns, and strengthening healthcare infrastructure to manage and mitigate the impact of dengue fever. Further studies with larger sample sizes and extended geographic coverage are recommended to gain a comprehensive understanding of dengue incidence and transmission dynamics in the region. In conclusion, while the current incidence rate of dengue in Bushehr province appears to be low, the detection of positive cases indicates the need for sustained surveillance and proactive public health strategies to control and prevent the spread of dengue virus.

Discussion

This study primarily focused on IgM detection to assess recent dengue infections in the study area. The findings of this study reveal a low yet significant incidence of dengue virus in Bushehr Province. Although the incidence rate is relatively low in comparison to other tropical and subtropical regions, the detection of positive cases across multiple cities indicates ongoing dengue transmission within the region. However, it is essential to acknowledge the study’s limitations when interpreting these results. One key limitation is the potential for cross-reactivity of IgM antibodies between flaviviruses, which could lead to false positives in the ELISA testing. To mitigate this, future research should incorporate molecular diagnostic techniques, such as PCR and plaque-reduction neutralization tests (PRNT), to confirm dengue virus presence and distinguish it from other flavivirus infections.

Another limitation is the absence of molecular confirmation for the dengue virus infection. While IgM ELISA testing is commonly employed for detecting recent dengue infections, integrating PCR would provide more definitive results, such as identifying the specific dengue serotype and offering insights into the virus’s genetic diversity. Furthermore, the sample size of 180 participants might restrict the generalizability of the findings. A larger sample size would increase statistical power and enhance the precision of the incidence rate estimates. Longitudinal studies with larger cohorts would also help account for seasonal and environmental variations that influence dengue transmission.

Although the novelty of this study might appear limited due to the relatively low incidence of dengue in Bushehr Province compared to other endemic regions, the detection of positive cases in this non-endemic area is significant. It underscores the potential for dengue transmission in the region and calls for sustained surveillance. Future studies should investigate ecological, environmental, and socio-economic factors that influence dengue transmission, as well as assess the effectiveness of vector control interventions.

Comparison with other regions

The dengue incidence rate in Bushehr (2.78%) is lower than in many other tropical regions, where incidence rates often exceed 10%. This discrepancy may be attributed to factors such as effective vector control strategies, public awareness campaigns, and the relatively small sample size in the current study.

Limitations and future research

Given the limited sample size of 180 participants, the findings may not fully reflect the overall dengue incidence in the province. To improve the understanding of dengue transmission dynamics in Bushehr, future studies should consider larger sample sizes and incorporate molecular diagnostic methods, such as PCR. Further investigation into demographic factors—such as age, sex, and occupation—could also help identify at-risk groups, which would allow for more targeted public health interventions. It is important to note that clinical information and symptoms of the participants were not recorded, and specifically, no clinical data related to dengue symptoms were collected at the time of blood sample collection. Future studies are recommended to include clinical assessments and symptom records to provide a more comprehensive understanding of the disease burden.

Geographic and Climatic implications

The warm climate and coastal geography of Bushehr Province provide an environment conducive to the proliferation of Aedes mosquitoes, the primary vectors of dengue virus. High temperatures and humidity levels in the region are ideal for mosquito breeding and survival, similar to conditions in other endemic dengue regions worldwide. The geographic location of Iran, particularly in its southern regions, places it at increased risk for vector-borne diseases like dengue, especially as globalization and climate change facilitate the spread of mosquito vectors to new areas.

The detection of dengue virus in Bushehr emphasizes the urgent need for improved public health measures to control the disease. Effective surveillance systems are essential for early detection and timely intervention. Key interventions should include vector control strategies such as eliminating standing water, using insecticides, and promoting the use of mosquito nets. Public education on dengue transmission and prevention is crucial to reduce infection risks. Additionally, strengthening healthcare infrastructure to better manage dengue cases and training medical professionals to recognize symptoms are critical steps.

Public health policy implications

The study carries significant implications for public health policy in Bushehr Province. Policymakers should prioritize the implementation of Integrated Vector Management (IVM), which combines various control methods. Investing in research to better understand dengue epidemiology, fostering international collaboration for effective outbreak management, and developing climate adaptation strategies to mitigate the impact of climate change on mosquito populations are essential components of a comprehensive approach to preventing dengue in the region.

Conclusion

The findings of this study confirm the presence of dengue virus in Bushehr Province, southern Iran, with an overall incidence rate of 2.78% among the sampled population. The detection of dengue-specific antibodies in multiple cities highlights the circulation of the virus in the region and underscores the necessity for ongoing epidemiological surveillance. These results emphasize the importance of strengthening vector control strategies and public health interventions to mitigate the risk of dengue outbreaks.

To gain a more comprehensive understanding of dengue virus transmission in Iran, future research should incorporate larger-scale serological and molecular studies. Expanding surveillance efforts and integrating molecular diagnostic tools, such as PCR and viral sequencing, will be essential to assess the genetic diversity of the virus and its potential spread. These measures will contribute to the development of evidence-based public health policies aimed at controlling and preventing dengue in Iran.

Author contributions

E.A., and M.D.M-F. conceptualized and wrote the study design and proposal. E.A., H.A., A.D., and M.D. carried out the field collections and identifications. H.A, K.A. and M.D.M-F. approved the submitted version. Expert validation on species taxonomy was done by S.A-H., while S.A-H. and M.D.M-F. performed data collation and analyses. All authors contributed to the final draft writing, review, and approval of this manuscript.

Funding

This work was reviewed and approved by the ethics ID: IR.SUMS.SCHEANUT.REC.1401.121, and funded to the main corresponding author (M.D.M-F) and supervisor on behalf of his Ph.D. student or the first author (E.A.) by Shiraz University of Medical Sciences (SUMS), Shiraz, Iran. All methods were carried out in accordance with relevant guidelines and regulations and Informed consent was obtained from all participants and/or their legal guardians prior to blood collection.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.