SEROPREVALENCE OF DENGUE FEVER VIRUS AMONG SUSPECTED PATIENTS IN TAIZ GOVERNORATE-YEMEN

Background: Dengue Fever virus (DENV) considers one of the most important mosquito-borne viral diseases in the world and it is endemic in more than 100 countries. 
Objective: This study aimed to determine the seroprevalence of DENV infection among suspected patients and to investigate some associated risk factors with dengue fever infection in Taiz governorate, Yemen. 
Methods: This study was cross-sectional, descriptive, and experimental, combining the use of a structured questionnaire and analysis of serum samples obtained from 300 suspected patients attending at many hospital and clinic centers in Taiz during the period from July to November 2016. The serum samples were tested for anti-dengue immunoglobulin (IgM) and (IgG) by Enzyme-linked Immunosorbent Assay (ELIZA). 
Results: Out of 300 suspected febrile cases, it was found that 49(16.3%), 68(22.7%), and 17(5.7%) cases were showed positive for the IgM, IgG, and both IgM and IgG antibodies, respectively, while 166 (55.3%) cases were negative. The incidence rate was more in males than in females. The most affected age group with dengue fever infection were (21–30) years. Dengue Fever was more frequent among patients coming from the urban area, having secondary school, and low-income status people. Also, there was statistical significant between DENV infections with a place of residencyand gender (P<0.05) and not-statistical significance between DENV infections and other factors (P>0.05). 
Conclusion: Taiz governorate become one of the endemic governorates in Yemen particularly the Taiz city which should be brought to the attention of public health authorities   
Peer Review History: 
Received: 5 September 2020; Revised: 15 October; Accepted: 26 October, Available online: 15 November 2020 
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Received file 
 
Average Peer review marks at initial stage: 6.0/10 
Average Peer review marks at publication stage: 7.0/10 
Reviewer(s) detail: 
Dr. Rola Jadallah, Arab American University, Palestine, rola@aauj.edu 
Dr. Dulger Gorkem, Duzce University, Turkey, gorkemdulger@yandex.com     
Comments of reviewer(s): 
 
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INTRODUCTION
Dengue Fever virus (DENV) is one of the significant commonest mosquito-borne viral diseases in the world and it is endemic in more than 100 countries that estimated about 100 million infected cases and about 25,000 deaths per year worldwide 1 . DENV is an enveloped, positive-sense, single-stranded RNAgenome that belongs to the family Flaviviridae, genus Flavivirus 2 . One of the four serotypes of dengue viruses (DENV1-4) able to cause dengue fever. These types of viruses are transmitted by an infected females of Aedes egypti (major vector) and Aedes albopictus during biting the human host for obtained the meal of blood 3 . The virus transmission from person-to-person by the direct method has not been documented. Although few reports have been illustrated about the route of DENV transmission through exposure to DENV-infected organs, blood, or other tissues from solid organ transplants, blood transfusions, or bone marrow transplants 4, 5 . Dengue virus causes primary and secondary infections. Primary infection is an acute feverish illness known as Dengue Fever (DF) which mostly eliminates around seven days by a composite ISSN: 2456- 8058 22 CODEN (USA): UJPRA3 immune response, while the secondary infection is additional rigorous and causes Dengue Hemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS) 6, 7 .The majority of deaths that result from dengue infection result from Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) 8 . In Yemen, the early first recorded of dengue-like epidemics in Aden was reported by Hirsch between 1870 and 1873 years 9 . Also, the prevalence of dengue fever was98% of the studied population recorded in Al-Hudidah governorate in 1954 10 . After that dengue fever became an epidemic in the coastal planes of the Tehama (Hudidah), then outbreaks increased since 2005 and the disease has spread to new governorates 11 .
All three dengue viral serortyp1-3 were reported in Yemen but type 2 of the Dengue virus was the most predominant serotype in Yemen 12 . Interestingly DENV serotype 4 was identified and confirmed the first time in Al-Hudidah-Yemen by Alahdal et al., 13 . In the Taiz governorate, the infection rate varied from year to year. Recently, WHO 14 documented an extreme prevalence in cases of dengue fever in Taiz governorate were reached to1328 suspected cases compared with last years. But the seroprevelance of DENV is not well documented so far. Therefore, this work aimed to evaluate the seroprevalence of dengue fever and to reveal some possible risk factors associated with DENV infection in Taiz governorate, Yemen.

Study area
In the place of southwestern of Yemen, Taiz governorate locatesin East of GMT. It is the thirdlargest governorate in Yemen and its population is 2,393,425 according to the census of the 2004 year 15 . Administratively, Taiz is among the largest governorates as it includes 23 districts in its frame. Taizclimate is featuring by diversity and the average temperature is up (21°C). The rain falls on all parts of the governorate in the summer while in some districts in the winter 16 .

Study design
This is a cross-sectional, descriptive was conducted from July to November 2016, performed on suspected patients suffering undifferentiated fever (age range, 1 to 65 years) attending many hospitals and health centers at nine main locations in the Taiz governorate, which most patients coming seeking medical care.

Ethical approval
This study was approved by the Ethical Review Committee from the Department of Biology, Faculty of Science, Sana'a University.

Data collection
A structured questionnaire was intended to collect required data from suspected patients according to socio-demographics and risk-related data. The questionnaire was filled for each participant via face-to-face interview by a researcher to avoid any misunderstanding and confirm the accurate collection of data properly. Sample collection About 5 mL of blood specimen was collected from 300 suspected patients suffering undifferentiated fever by venipuncture and immediately transferred into a sterile anticoagulant-free sterile bottle for clotting. The clotted blood specimen was centrifuged (3000 rpm, 5 min), and the serum (the supernatant) was put in separate Eppendrof tubes with a specific study number (SNO) transferred inside a cooling box and stored at -20°C until required for use.

Serological Assay
Serum samples were examined for DENV-specific IgG and IgM antibodies by using IgG and IgM DRG Immunodiagnostic Kits(GmbH Germany) that were performed by Enzyme-Linked Immunosorbent Assay (ELISA)(Absorbance Microplate Reader/ELIZA-IRE96,SFRI, French) 17 .

Statistical analysis
The obtained results were analyzed by using the version 18.0 SPSS (Statistical Package for Social Science). A significant difference between the proportions and the groups or variables was determined by Chi-square test (≥3.9 considered significant) and Pvalue (< 0.05 considered significant).

RESULTS
In a total of 300 participants enrolled in this study, 202 (67.3%) were males and 98 (32.7%) were females. The highest participant groups in the present study were patients aged between (21-30) years with an average of 105 (35%). Most suspected patients 195 (65%) were living in urban areas. The suspected patients with secondary education levels were more than the third 113 (37.7%) of the total population in this study. On the other hand, more than half of the respondents 160 (53.3%) were of low-income status (  Figure 2). In the present results, the DENV IgM, IgG, and both IgM and IgG seropositivity were mostly observed in male patients, 39 (19.3%), 53 (26.2%), and 14 (6.9%), respectively, while the female patients were lower from that 10 (10.2%), 15 (15.3%), and 3 (3.1%), respectively. Also, the statistical analysis revealed a significant association between the gender and seroprevalence of DENV IgM and DENV IgG (P< 0.05) ( Table 2). In the current work, the higher prevalence rate of DENV IgM, IgG, and both (IgM and IgG) seropositivity were observed among patients aged from 21-30 years with 22 (21%), 29 (27.6%), and 8 (7.6%), respectively. While the lowest from those was among (> 50) years, 2 (10%), 3 (15%) and 0 (0 %), respectively. There was an association between age groups and seroprevalence of DENV was not significant (P>0.05) ( Table 2). The result regarding the resident area, it was found that 19.5% of positive anti-IgM, 26.2% of positive anti-IgG, and 8.2% of positive anti-IgM and anti-IgG antibodies were reported among patients coming from the urban area. On other hand, the seropositivity of DENV antibodies among cases living in the rural area was 10.5% for IgM, 16 Next, pools 6 (13.6%), 9 (20.5%) and 4 (9.1%) respectively. The lower abundant site was tires which recorded 7 (12.5%), 10 (17.9%) and 3 (5.4%), respectively of the breeding sites. There was no significant association between the seroprevalence of DENV and all breeding sites of mosquitoes (P> 0.05) ( Table 3). In the present finding, the most of infected cases that observed positive for Ig M, IgG, and both (IgM and IgG) antibodies were recorded in Taiz city with 31 (19.4%), 43 (26.9%) and 10 (6.9%), followed by Al-Barh 8 (17.7%), 11 (24.4%) and 3 (6.7%), then Al-Rahedah 4 (16.7%), 5 (20.8%) and 2 (8.3%), after that, Al-Makha 2 (12.5%), 3 (18.8%) and 1 (6.2%). Next, Al-Demna 2 (10%), 4 (20%) and 1 (5%), respectively, the lowest of that were Hajdah 1 (10%) for IgM, 2 (20%) for IgG and Mawyah 1 (10%) for IgM. While two locations were free of infection namely Al-Turbah and Al-Nashamah which no recorded any cases for IgM or IgG seropositivity (Table 4).   23 in Sudan. The possible reason for the high number of dengue infected cases among males due to the habit of males in the summer season, they did not cover their body whether at home or outside, spend more time outdoor and they have traveling history to that area where the dengue incidence is high. These habits make them more exposed to the bite of Aedes aegypti.
In the present study, it was revealed that the most susceptible age group for DENV infection were ( 26 . This result suggesting that the individuals in these age groups were more actively outdoor during the day which increased their chances of exposure to the infective DENV vector bite. According to the present study, the relationship between the dengue fever seropositivity and education levels which found higher in people with secondary education levels compared with lower among illiterates participants. This result is similar to a study by Abdullah et al., 19 conducted in Yemen. This finding may reflect their outdoor activity during the day, for playing, schooling, or picnic that increased their chances of exposure to the infective DENV vector bite. The prevalence of dengue fever infection was noted significantly correlated with place of residence (rural or urban) areas. The prevalence rate of dengue fever infection in the urban area was more than the rural areas. This finding is in agreement with earlier studies in Yemen by Abdullah et al., 19 and Bin Gluth et al., 21 . In the present study, the income statues were found non-significantly associated with dengue infection. But Dengue IgM, IgG and both seropositivity, were found highest with low-income statues cases (4-7 $ daily), it was rated (18.8%), (25.6%) and (6.3%), respectively. Whereas, the less infected people were with high-income statues (>20 $ daily), which rated (9.1%), (9.1%) and (0%) for IgM, IgG, and both, respectively. Similar results were reported byAl-Hemiree 27 and Abdullah et al., 19 in Yemen and by Muhammad et al., 26 in Pakistan. Furthermore, in this work the dengue mosquitoes breeding sites were found positive factors related to dengue IgM, IgG, and both seropositivity. The bogs were the most breeding associated with most of the  19 who noted that the highest breeding site factor was the bogs which rated (92.2%), while the lowest factor was the tires with an average (26.3%). Interestingly, the distribution of dengue fever results according to districts revealed that the of the most hotspot of dengue fever virus infection in Taiz governorate was concentrated in the city of Taiz, followed by Al-Barh, Al-Rahedah, Al-Makha, Al-Demna, Hajdah, and Mawyah. Whereas two districts namely Al-Turbah and Al-Nashamah were found free from DENV infection that may be due to climate change and the lack of suitable conditions for the mosquitoes breeding cycle in these areas. The low infected cases were found Al-Makha during this study that the infected case was low maybe contributing to the war most people replacement to another area within the governorate most infected case in Al-Barh which were coming from Alhodidah and Al-Makha.

CONCLUSION
In conclusion, the high prevalence of DENV antibodies in Taiz is becoming one of the most endemic governorates in Yemen which should be brought to the attention of public health authorities. Warm climate, rainfall also the war since 2015 until now contributed to destroying Yemen's healthcare, presence of breeding sites, lack of water supply systems and mosquito control measure, low-income status, and insufficient sanitation systems are the reasons that attributed to the increase of suspected cases of DENV among study area. Therefore, continuous surveillance for outbreaks of DENV infection required to identify early and in order to prevent and control the spread of infections among the community.