VIRAL IMMUNOLOGY Volume 30, Number 1, 2017 ª Mary Ann Liebert, Inc. Pp. 54–57 DOI: 10.1089/vim.2016.0044
Seroprevalence of Cytomegalovirus Infection Among a Rural Population of Coˆte d’Ivoire Augustin Etile Anoh,1,2 Arse`ne Mossoun,2,3 Chantal Akoua-Koffi,1 Emmanuel Couacy-Hymann,3 Maude Pauly,4,5,# Siv-Aina Leendertz,4 Eliezer Kouakou N’goran,2 Grit Schubert,4 Sabrina Weiss,4,x Jo¨rg Hofmann,6 Fabian H. Leendertz,4 and Bernhard Ehlers5
Human cytomegalovirus (HCMV) is a betaherpesvirus that can be pathogenic to humans. In particular, immunocompromised patients can develop life-threatening symptoms. In the present study, HCMV seroprevalence was investigated in a rural population of Western Coˆte d’Ivoire. Plasma samples collected from 166 apparently healthy subjects living in 8 villages surrounding the Taı¨ Forest National Park were tested for anti-HCMV immunoglobulin G and M antibody with two commercial enzyme-linked immunosorbent assays. Prevalence of anti-HCMV IgG and IgM antibody was 100% and 5.4%, respectively. Anti-HCMV IgM positive was 10.2% (5/ 49) of the children and adolescents and 3.4% (4/117) of the adults. This observed decrease of IgM seropositivity and the seroprevalence difference between males and females (3.8% vs. 6.1%) was not statistically significant. In plasma of one IgM-positive participant, a low CMV load was detected indicating low-level replication. A second IgM-positive participant showed signs of local CMV replication. The other seven IgM-positive plasma samples likely reacted nonspecifically or due to polyclonal stimulation. Taken together, the results indicate that HCMV infection is hyperendemic in Coˆte d’Ivoire. Keywords: seroprevalence, IgM, IgG, cytomegalovirus, rural population, Coˆte d’Ivoire sociodemographic factors (age, sex, and ethnicity) and social environment. They differ between countries and regions (5,7,14). HCMV seroprevalence has been investigated in several countries in the world (3,5,20), but limited data are available from African countries, especially from rural areas. In Coˆte d’Ivoire, a few serological studies on HCMV have been conducted in urban areas (7,14,31), but not in rural populations. The present cross-sectional study provides data about HCMV seroprevalence in the rural Taı¨ region of Western Coˆte d’Ivoire.
uman cytomegalovirus (HCMV) is a betaherpesvirus with a double-stranded DNA genome that is transmitted by contact with blood, saliva, and urine and by breastfeeding (13,16,18). It is an opportunistic herpesvirus that infects a majority of the world’s population by early adulthood and causes asymptomatic latent infection in healthy individuals (23). However, it can cause severe disease in the absence of an effective immune response, especially in patients with AIDS, immune-compromised solid organ, and bone marrow allograft recipients (19,29,34). Symptoms in these subjects typically consist of indisposition, fever, hepatitis, pneumonia, gastrointestinal disease, and retinitis (24,28). HCMV seroprevalence and congenital HCMV infection depend on socioeconomic factors and hygienic practices (3,20). Of importance are also
Materials and Methods Sample collection
During two field’s missions in 2012, 166 blood samples were collected from apparently healthy people living in 8
CHU de Bouake´/Centre de Recherche pour le De´veloppement, Universite´ Alassane Ouattara de Bouake, Bouake, Coˆte d’Ivoire. UFR Biosciences, Universite´ Fe´lix Houphoue¨t Boigny, Abidjan, Coˆte d’Ivoire. 3 LANADA/Laboratoire Central de Pathologie Animale, Bingerville, Coˆte d’Ivoire. 4 Project P3 ‘‘Epidemiology of Highly Pathogenic Microorganisms,’’ Robert Koch Institute, Berlin, Germany. 5 Division 12 ‘‘Measles, Mumps, Rubella and Viruses Affecting Immune-Compromised Patients,’’ Robert Koch Institute, Berlin, Germany. 6 Labor Berlin, Department of Virology, and Institut of Medical Virology, Charite´ University Medicine Berlin, Berlin, Germany. # Current Address: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg. x Current Address: European Public Health Microbiology (EUPHEM) Training Programme, European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden, and Public Health England (PHE), London, United Kingdom. 2
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villages along Taı¨ Park Forest (Western Coˆte d’Ivoire). Blood samples were collected in ethylenediaminetetraacetic acid containing tubes following a standardized procedure (26) and plasma obtained. One hundred fourteen (69%) of the participants were females, and 52 (31%) were males. The mean age of the male participants was 36.9 – 3 years (range 30.8–43.1 years) and that of the females was 31.8 – 1.5 years (range 28.8–34.9 years). Plasma samples were stored in nitrogen liquid at the field site and then stored at -80C at LANADA Bingerville before being transported to Germany on dry ice. Before collection of samples, the purpose of the study was explained to health district authorities, village leaders, and villagers. Written informed consent was signed or finger printed by each participant before enrolment. The study was approved by the ‘‘Comite´ National d’Ethique et de Recherche (CNER), Ministe`re de la Sante´ et de l’Hygie`ne Publique, Coˆte d’Ivoire (101-10/MSHP/CNER/P).’’ Serological analysis and quantitative HCMV PCR
Anti-HCMV IgG and IgM seroprevalence was evaluated in 2014 in plasma samples, diluted 1:100 (according to manufacturer’s instruction), using two commercial enzymelinked immunosorbent assays, the CMV IgG-ELA test PKS medac and the CMV-IgM ELA test PKS medac (MEDAC, Hamburg, Germany). The further analyses of CMV IgMpositive plasma samples were performed with two commercial immunoblot assays, recomLine CMV IgG/IgM (for IgG avidity determination) and recomLine Epstein–Barr virus (EBV) IgG/IgM (Mikrogen GmbH, Neuried, Germany). All kits were used according to manufacturer’s instructions. CMV viral loads in plasma were measured with quantitative PCR as previously described (11). Statistical analyses
Statistical analyses were performed in R software (version 3.1.0.; R Foundation for Statistical Computing, Vienna, Austria [https://www.r-project.org/]). Means and proportions of the sociodemographic characteristics were determined for CMV seropositive groups. Fisher’s exact test, odds ratio, and 95% confidence intervals were calculated to examine associations between anti-CMV IgM positivity and categorical variables, age and sex. p-Value 21 years) 117 70.5 4 3.4 Sex Male 52 31.3 2 3.8 0.7 0.06–3.38 0.61 Female 114 68.7 7 6.1 95% CI, 95% confidence interval; na, not applicable; OR, odds ratio.
for all nine IgM-positive plasma samples, and quantitative HCMV PCR was performed (Table 2). All avidity indices were above 50% suggesting absence of primary CMV infection. The PCR revealed eight aviremic subjects and only one with a low level viremia (sample ID: PAN521; viral load: 3,290 copies/mL). In a previous study, we detected HCMV DNA in two oropharyngeal swabs (sample IDs: TAI223 and PAN521) (1). Since study participant TAI223 was aviremic in the present study, we consider the detection of HCMV in the oropharynx as a local virus replication, but not as disseminated infection. Such locally restricted virus replication may cause low titer IgM values. Regarding participant PAN521, we consider this disseminated infection at low level and the positive oropharyngeal swab (1) as a putative HCMV infection in the upper respiratory tract. In samples with high avidity index and undetectable HCMV DNA, the CMV IgM could be caused by a nonspecific cross reaction or a polyclonal stimulation following an infection with an unknown pathogen. For lymphotropic viruses like HCMV and EBV, it is well known that a polyclonal stimulation may lead to IgM reactivity against both pathogens. Therefore, we performed EBV IgM immunoblot analysis for the nine HCMV IgM–positive plasma samples (Table 2). Four were positive for EBV IgM. This likely indicates a putative polyclonal stimulation of B cells caused by EBV itself or an infection with an unknown pathogen. The remaining five
Table 2. Human Cytomegalovirus Viral Load, Human Cytomegalovirus Avidity, and Anti-Epstein–Barr Virus IgM in Human Cytomegalovirus IgM–Positive Individuals from Rural Population of Taı¨ Region, Coˆte d’Ivoire HCMV viral load HCMV avidity, % EBV IgM PON112 PON153 TAI223 TAI229 TAI230 TAI240 SON360 SON362 PAN521
Neg Neg Neg Neg Neg Neg Neg Neg 3290 copies/mL
>50 >50 >50 >50 >50 >50 >50 >50 >50
pos neg neg neg pos neg neg pos pos
EBV, Epstein–Barr virus; HCMV, human cytomegalovirus.
ANOH ET AL.
samples were EBV IgM negative. Thus, a nonspecific cross reaction in the HCMV IgM immunoassay is rather likely. In case of PAN521 and TAI223, the disseminated low-level HCMV infection (PAN521) and the local virus replication (TAI223) may have caused B cell stimulation. Discussion
This first report on HCMV seroprevalence in a rural population of Coˆte d’Ivoire shows that HCMV is hyperendemic in the study region (100% anti-HCMV IgG). The study participants apparently have been previously exposed to HCMV, which indicates stable circulation of HCMV in Taı¨ National Park region. This is in accordance with previous reports on HCMV endemicity in urban populations in African countries (2,21,30,36). However, HCMV seroprevalences reported in Europe, North America, and Australia were mostly lower (40–60%) (8,9,12,17,32,33). This may be explained by differences in socioeconomic status and hygienic practices, known as determinants of HCMV transmission (4,6,23). For example, lack of access to clean drinking water likely facilitates contact with HCMV-positive secretions (35). The presence of anti-HCMV IgM antibodies suggests active infection (23). This can be primary or reinfection with HCMV or reactivation of latent HCMV (25,27). In the present study, 9 plasma samples (5.4%) showed IgM reactivity. This is within the range of values reported in comparable studies from Sudan (2.5%), Iran (2.6%), Kenya (8.1%), and Nigeria (11%) (8,10,15,22). However, for 7/9 IgM-positive plasma samples, CMV avidity testing, combined with CMV quantitative PCR and EBV IgM testing, revealed that most of the observed IgM reactions likely originated from polyclonal B cell stimulation or from nonspecific cross reaction and not from active HCMV infection. Of note, the samples of two participants showed signs of active (plasma PAN521) or local (oral swab TAI223) infection. As reported previously, the HCMV genotypes that circulate in the investigated region do not differ from those identified in other continents (1). Conclusion
Although the sample size of this seroepidemiological study is limited, our results indicate stable circulation of HCMV in Taı¨ region (Western Coˆte d’Ivoire). This should be considered in any healthcare planning in Coˆte d’Ivoire. More prospective studies are required to investigate if HCMV has an impact on the health condition of rural populations in West Africa. Acknowledgments
The authors acknowledge Tape Bozoua, Ange Gnoukpo, and Joel Simpore for technical assistance in the field, the health authorities of the districts, and leaders and communities of the villages for their cooperation. For their excellent assistance in the laboratory, the authors thank Cornelia Walter, Sonja Liebmann, and Ulla Thiesen. This work was supported by the ‘‘Deutsche Forschungsgemeinschaft’’ (DFG) grant LE1813/4-1. Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to: Dr. Bernhard Ehlers Division 12 ‘‘Measles, Mumps, Rubella and Viruses Affecting Immune-Compromised Patients’’ Robert Koch Institute Berlin 13353 Germany E-mail: [email protected]