Seroepidemiology of Dengue Viruses in Basrah, Southern Iraq
ShantA Sinbat1, Alaa K Mousa2, Hassan J Hasony1
1Department of Microbiology, 2 Dept of Medicine, Basrah medical college,University of Basrah, Iraq
Abstract
Background: Dengue virus infection has been an important and serious public health concern in southeast asia, where local outbreaks of dengue fever occurred almost every year.
To our knowledge, no nationwide investigation has been carried out to determine the actual extent of infection in the general population.
Methods: A total of 282 random serum samples were collected from the general population in Basrah in 2019-2020. The antibody-captured enzyme-linked immunosorbent assays were used to detect DENV-specific IgM and IgG. Demographics data were used for risk analysis.
Results: The weighted overall seroprevalence was 30.9% for anti-DENV IgM, and 64.9% for anti-DENV IgG, respectively. A significant rise of DENV IgG seropositive rate had been noted since early adulthood stage, from 49.4% at the age group of 10–30 years to 68.8% at the age group of 31–50 years. For people aged over 50 years, the seropositive rate reached 81.4%. Age, and regions of residency were associated with the IgG seropositivity. There was no statistically significant difference in seroprevalence of anti-Dengue IgM, indicating recent infection, among predictors including gender, and residency.
Conclusions: Our results indicated that the majority of population inBasrah exposed to dengue virus and the prevalence of IgG antibody against dengue virus rises with age. and regions of residency are associated with the exposure of population to infection by dengue viruses. Further studies are needed to realize the current situation of seroprevalence of dengue fever in southern Iraq.
Keywords: Seroepidemiology, Dengue fever, southern Iraq INTRODUCTION
Dengue, one of the most threatening mosquito-borne diseases of humans, is caused by any of the four-serotypes (DEN-1 to 4) of dengue virus, a positive-strand RNA virus. Two clinical forms of dengue infection have been recognized: dengue fever, a relatively mild, self limiting febrile illness and dengue hemorrhagic fever/dengue shock-syndrome (DHF/ DSS), a severe infection with vascular and haemostatic abnormalities that can lead to death1 Dengue virus infection constitutes amajor public health concern and is estimated to cause about300million cases in a year across the globe. About 3.97 billionpeople, who are living in the epidemic areas in the tropicaland subtropical regions, are at risk of acquiring the viralinfection 2,3 Environmental changes such as rising temperatures, increased rainfall, accelerated urbanization and industrialization, populationgrowth, and poor waste and water management, which leadsto the proliferation of mosquito species (Aedesaegypti and A.albopictus) that transmit DENV, are all possible reasons for the upsurge of dengue cases 3,4, . Dengue transmission has increased worldwide, particularly in Asia and Latin America since the 1970s5, but limited information on the disease is available from the Middle East. Saudi Arabia6 and Yemen7,8 have reported a few epidemics of dengue. Climate conditions in the Middle East not all are favourable for the disease vector, but all other risk factors for dengue are potentially increasing9. The existence of a large immigrant work force from dengue- endemic countries, increased travel from and to dengue-endemic countries and increased
urbanization are expected to increase the likelihood of the emergence of dengue in the Middle East.
Seroprevalence studies help identify populations previouslyinfected by dengue. This is important because thelifelong immunity developed after infection with one ofthe four DENV serotypes is type-specific; secondaryinfection by heterologous serotype is frequently, not associated with severe dengue manifestations2,10. Seroprevalence studies reveal subclinical DENVinfections, as the majority of the infections are subclinicaland case reporting underestimates the true rate 2.Patients with subclinical infections can contribute to theoverall DENV transmission cycle 11. Additionally, estimatingboth clinical and subclinical infections providesreliable information to support modeling for future vaccinedemand and delivery strategies 2.
The aim of this study is to determine theseroprevalence of DENV infections in symptomatic and healthy adultpopulation in districts of Southern Iraq, andto identify factors associated with seropositivity.
Materials and methods:
The study site, Basrah is located in southern Iraq, has an estimated population of ∼3.796.000.
This study was conducted in four sites namely;A=north (served by Al- Mawanaateachinghospital, Al-Faihaateachinghospital),B=south(Al-sader teaching hospital),C=west(Al-basrah teaching hospital) D=East, (Paediatric and Oncology center) these sites were selected to represent almost all population in Basrah, there is a likelihood of transmission to humans, from lakes and river in these areas may act as breeding sites for the vectors and a habitat for arboviruses reservoirs.
Serum samples of 282patients that live in Basrsh region were collected in the years 2019 and 2020. The samples were collected from patients during their visit to hospitals in Basrah city.
The mean age of the study population was 45years (range = 10–80). Male to female ratio was 1:1.6 (39.3% males and 60.6% females) of the total study population.
The study utilizes thosesamples from a hospital-based cross-sectional descriptive survey and retrospective data from an ongoing study that started in December 2019. To augment the collection samples of febrile illness patients from identified hospitals were collected with the assistance of clinical officers in the four respective study districts.
A total volume of approximately 5 mL of blood was collected from the participants. The blood samples were incubated in room temperature for 1 hour, followed by 15 minutes centrifugation at 3,000 rpm and collection of the serum fraction. The serum samples were stored at −70°C until use.
Known as IgM Antibody Capture Enzyme-Linked Immunosorbent assay.All the Serum samples brought to the laboratory were screened for exposure to DENV. This was done using commercial kits namely; MAC-ELISA Kit of Arboviruses (DENV IgM Capture (DxSelectTM, FOCUS Diagnostic, USA).
An enzyme-linked immunosorbent assay (ELISA) specific for detection of Anti human IgG Immunoglobulin to DENV was used and the procedure followed according to the manufacturer's instructions( DxSelectTM, FOCUS Diagnostic, USA).
Chi-squared test was used to compare the seroprevalence between genders and between age groups.
Results:
Antibodies against dengue virus(Flaviviridae,) as specified below,were detected in87 of 282 (30.9%) samples positive for IgM antibody and 183 of 282 (64.9%) samples were positive for IgG antibody. There were a gradual increase in IgM and IgG seropositivity as the age increased where the highest IgG seropositivity was detected among age group of greater than 50 years of age(81.4%) and the least among early age group (49.4%). The difference was statistically significant (P<0.05).However, there were no differences related to gender(Table- 1). Recent infections was more among rural community (35.8%) compared to urban group (20.6%) while old infections (IgG) was dominant among urban community (80.4%) compared to (57.4%) in rural group.The differences in term of the rates of exposure between urban and rural community was statistically significant (P<0.05). Areas D and A represent the focuses of highly exposed peoples in these areas which was 73.3% and 68.6%
respectively, compared to areas B and C (Table-1 and Table-2).
Table-1: The demographic characteristics of study population
No. IgG+ve (%) No. IgM+ve (%)
No. tested (%) Character
Age group
46(49.4) 11(11.8)
93 10 -30
93(68.8) 50(37.0)
31- 50 135
44(81.4) 26(48.1 )
54 51-80
Gender
73(67.5) 30(27.7)
108 Male
110(63.2) 57(32.7)
174 Female
Residence
109(57.4%) 68(35.8%)
190 Rural
74(80.4%) 19(20.6%)
Urban 92 Study sites
103(68.6%) 54(36.0)
A 150
36(54.5%) 18(27.2%)
66 B
33(64.7%) 9(17.6%)
51 C
11(73.3%) 6 (40%)
15 D
183 (64.9%) 87(30.9%)
282 Total
Table-2: Distribution of dengue virus IgM and IgG antibody according to ELISA test of different regions of Basra province.
IgG+ve N(%) IgM+ve
N(%) Examined
Area of samples collection Area
103 (68.6%) 54 (36.0)
150 North(Served by Al-
Mawanae and Al-
FaihaTeachinh hospital) A
Table -3: Symptomatic status of study population in relation to DENV seropositivity DENV–IgG N(%)+ve DENV–IgM
N(%)+ve No. tested
Symptoms
81/174(46.5) 49/174(28)
174(61.7%) Symptomatic
102/108 (94.4) 38/108 (35)
108(38.3%) Asymptomatic
183/282(64.8) 87/282(30.8)
282 Total
Dengue virus seropositivity was higher among asymotomatic individual collectively for IgM (35%) and IgG (94.4%) compared to patient with febrile illness (symptomatic) which was 28% and 46.5% for IgM and IgG respectively (Table-3).
Table-4: - Clinical presentation of DENV symptomatic patients
Symptoms No. Percentage DENV –IgM DENV –IgG
Fever/ Headache 139 80 % 39(28%) 64(46%)
Intense abdominal pain &
tenderness
104 60 % 29(28%) 48(46%)
Persistent vomiting & GIT 87 50 % 24(28%) 40(46%) Bleeding of mucosal membranes 2 1.2 % 0 1(50%)
Joint pain 104 60 % 29(28%) 48(46%)
Postural hypotension (lipothymia) 17 10 % 5(29%) 8(47%) Thrombocytopenia & atypical
lymphocyte
17 10 % 5(29%) 8(47%)
Progressive increase in hematocrit 9 5 % 0 0
Total 174/282 (61.7%)
36(54.5%) 18(27.2%)
66 South(Served by AlSader Teaching Hospital)
B
33(64.7%) 9(17.6%)
51 West(Basrah General Hospital)
C
11(73.3%) 6 (40%)
15 East (Pediatric & Oncology hospital )
D
183(64.9%) 87(30.9%)
282 Total
Table -4 show the clinical presentation of symptomatic patient . There were no significant differences between patients at various clinical presentation almost the same rates of seropositivity to IgM (28%) and IgG (46%) antibodies for dengue virus.Fever and headache was presented in 70% and 80% respectively. However, 50% of patients complain of joint pain and vertigo, severe malaise, chills, cough and abdominal pain presented in 30%, 28%, 18%, 16% and 14% respectively (Table-4). The major complain of seropositive to DFV was fever (70%), headache (80%), joint pain (50%) and vertigo (30%).
Table- 5: Seasonal distribution of DENV infections in relation to seropositivity DENV –IgG DENV –IgM
No. tested (%)
Season
29(64.4%) 7(15%)
Winter 45
33(55.9%) 11(18.6%)
Spring 59
63(64.3%) 37(37.7%)
Summer 98
58(72.5%) 32(40%)
Autumn 80
183(64.9%) 87(30.9%)
282 Total
The majority of cases of DENV was detected during hot season (Summer and autumn which was 64.3% and 72.5% respectively ) although the rates of exposure to infections occur all the year around in our areas( Table-5).However, recent infections presented with high rate of IgM seropositivity during summer (37.7%) and autumn (40%) compared to winter (15%) and spring (18.6%).The difference between rates at hot seasons and cold seasons were statistically significant (p<0.05).
Table-6:DENV antibody levels: the proportion of susceptible No. +ve (%) Type of responses to DENV
87/282(30.9%) Recent : IgM
119/282(42.2%) High titer of IgG
64/282(22.7%) Low titer of IgG
270/282(95.7%) Total
The majority of individuals in the community has been exposed at a time to DEV (95.7%) although the presence of antibody could be not protective (Table-6). Suppose that individuals possesIgM and/or IgM and high titer of anti-DENV IgG which was 30.9% for IgM and 42.2% for high IgG titer could be protected leaving the rest of > 50% susceptible to DENV infection.
Table-7: The main hematological investigation in DENV infections
Low High
Normal No. Total
+ve Investigation (normal values)
35/270(13%) 65/270(24%)
170/270(63%) Hb% (Male 13-17,
Female 12-15)
35/270(13%) 65/270(24%)
170/270(63%) 270/282
(95.7%) PCV (Male 40-52,
Female 37-47 )
40/270(15%) 75/270(28%)
155/270(57%) Platelets ( 150-450 )
25/270(9%) 105/270(39%)
140/270(52%) Total WBCs (4500-
11000 )
The main hematologic investigations for the responders of participants(95.7%) is presented in table-7. The majority of DENV infected are presented with almost normal hematological findings except for platelet count and total WBCs which showed marginal increase among 28% and 39% respectively.On the other hand, small proportion of patients presented with low hematologic parameters.
DISCUSSION
Despite having relatively low incidence of reported dengue cases in Basrah district, an overwhelming64.9% of participants had serological evidence of previousDENV exposure, out of which 30.8% were recentexposures. This discrepancy could be attributed to thedengue notification system which is basedon passive reporting and may not reflect the actual disease burden due to underreporting, misreporting 5,12,and the failure to capture subclinical infections 2. They were not diagnosed clinically bya healthcare professional, as they were either asymptomaticor had minimal symptoms not necessitating medicalattention or hospitalization. The nonspecific nature ofsymptoms misattributing it to other febrile illnesses maypartly account for this 12. Our findings suggest a possibilitythat a high number of subclinical dengue cases arepresent in this community. This corroborates to that most dengue infections are clinicallyunapparent and are consistent with high rates reportedin countries such as Singapore 14, South India 12, and Saudi Arabia 15 in contrast to that reported in Iraq by Raddle et al( 2008)16 .
The prevalence of IgG antibodies increased withincreasing age, with >81% of the population having beenexposed to dengue after the age of 55 years. Severalstudies have shown an association between age and IgGdengue seropositivity12,15,17,18
, consistentwith the long-term persistence of anti-DENV IgG once aperson is infected . The increased seroprevalence withage suggests that the longer a person resides in anendemic area, the higher the chance of being infected byDENV 15,17..
Dengue has now become hyperendemic in many countries including Middle eastern area with all four DENV serotypesco-circulating, with fluctuations of the dominant serotypesover time and location 6,7. In recent years, therehas been a shift observed in the age distribution ofdengue cases toward older age groups, involving adultsin the productive age group 5,17. Another notable shiftis the rise in dengue incidence in rural areas, which isgradually approaching the high levels observed in urbancommunities 17.With the changing epidemiology of DENV infections world-wide, it has become increasingly important toobtain up-to-date seroprevalence data to further understandthe distribution and burden of DENV infections.An improved understanding of dengue epidemiology, burden and its dynamic characteristicsare important for public health planning. Seroprevalence studies in healthy volunteers provideinformation on infection history in the population, from which inferences about diseaseburden may be drawn. Since age reflects duration of exposure, age- stratified data provideinsights into transmission dynamics18,19 . There is a lack of dengue
seroepidemiologicaldata from Iraq and no previous study has used a population representative sample ofurban population.
Clinical manifestations of dengue virus infection rangefrom asymptomatic, mild flu-like symptoms, to severelife-threatening dengue complications such as dengueshock syndrome (DSS) and dengue hemorrhagic fever(DHF) 21. DHF/DSS cases are associated with a secondary-type dengue antibody response, which makesthe second dengue infection worse than the first due to
“antibody-dependent enhancement of infection” 21.Those asymptomatic infection cases, which induce anantibody response but lacks clinical symptoms requiringa medical consultation, pose challenges to disease prevention
programs.The incidence of dengue has grown dramatically worldwide
in recent years.22,23. The majority of outbreaks were reported in northern of Basrah .Notably, due to the special geographic and habitat nature of the area.
In recent years, arboviruses that cause dengue, chikungunya,and Zika illnesses have rapidly expanded across the globe, of which the immunopathogenesisis enhanced in the setting of high seroprevalenceof dengue antibodies as the case in the present study due to structuralsimilarities between Zika and dengue virus 24,25. It isimportant to study dengue virus seroprevalence to projectfuture epidemic patterns in Basrah. There havebeen scanty studies of seroprevalence of dengue virus from Iraq, and few results were published inthe literature16. Considering the massive population morbidityand the high rates of asymptomatic cases, existingstudies were not enough for determining the current immunestatus of dengue in the community 26,27,. Thisstudy was the first study to investigate the seroprevalence,measured by the presence of IgG and IgMantibodies of dengue virus in general populations fromdifferent geographical areas in Basrah to figure out thewhole picture of dengue disease prevalence in southern Iraq.
In countries with the presence of dengue vectors, with dengue virus circulation, WHO suggested the preparednessand response plans should focus on strategies to reduce risk of transmission. Rapid investigation of allclinically suspected casesshould be carried out as well as laboratory testing for confirmingthe presence of dengue virus28
ThepresentstudyandtheotherstudiesconductedinBrazil20,Singapore14,andSaudiArabia15foundth atdenguemostlyaffectedadultsintheagegroupof21–
70yearsold.Thismaybebecauseadultgroups(>20yearsold)engageinmoreoutdooractivities,givin gthemmorechancesofbeingexposedtoinfectedmosquitoesthantheyoungeragegroup(<20yearsol d)29.Inaddition,mildormainlyasymptomaticDVinfectionwas
normallyfoundinchildren30.Previousstudiesfromsouth Asian countries21foundthatmalesaremorepronetoDFthanfemales,suggestingthatthemorecommonout- doorworkhabitsofmalesgavethemmorechancesthanfemalestobebittenbymosquitoes29,31.Howev er,anequaldengueinfectionratewasobservedinmaleandfemalepopulationsin
Basrahinthisstudy,revealingnodifferencesinvectorexposurefrequencyorhealthcare- seekingbehaviorbetweenmalesandfemales.Morestudiesareneededtoverifythis point.
Dengueinfectionhasa wide clinical
spectrumthatvarieswithdifferentregionsandagegroups5,12.Earlyrecognitionandunderstandingof theclinicalproblemsinthefebrilephaseinaparticularregionleadstoearlydiagnosis,notificationand controlofa
dengueoutbreak.Inpreviousstudies,investigatorsobservedthatthemostfrequentsymptomswerefe
ver,vomiting,thrombocytopeniaand leucopenia
inSaudiArabia15,whilefever,myalgia,arthralgiaandheadachewerethemostfrequentinMalaysia22; however,inthepresentstudy,therewerenosignificantdifferencesinmostclinicalsymptomsassociat edwithDF.Thestudyhasshownthattheclinicalmanifestationsindenguepatientsvariedwithdiffere ntDVserotypes,probablyduetodifferencesintheirpathogeneses,replicationabilityandinfectionac tivity12..Inaddition,somechronicdiseases,suchashypertension,diabetes,renaldiseases,hepaticdis eases,cardiovasculardiseases,thyroiddiseaseandarthritis,wereestablishedtoberiskfactorsforDH FinpreviousstudybyFigueiredoetal32.
Conclusions: Our results indicated that the majority of population in Basrah exposed to dengue virus and the prevalence of IgG antibody against dengue virus rises with age and regions of residency are associated with the exposure of population to infection by dengue viruses. Further studies are needed to realize the current situation of seroprevalence of dengue fever in southern Iraq
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