Ankrah et al. BMC Res Notes (2019) 12:326 https://doi.org/10.1186/s13104-019-4371-4 BMC Research Notes RESEARCH NOTE Open Access Serological evidence of Zika virus infection in febrile patients at Greater Accra Regional Hospital, Accra Ghana Godson Aryee Ankrah1, Joseph Humphrey Kofi Bonney2* , Esinam Eudosia Agbosu2, Deborah Pratt2 and Theophilus Korku Adiku1,3 Abstract Objective: Increase in the evidence of global occurrence of Zika viral infection suggests that in Africa the circula- tion of the virus which causes 80% of asymptomatic infection could be undetected and/or overlooked. We sought to serologically detect Zika virus infection in febrile patients at Greater Accra Regional Hospital, Ghana. Results: Of the 160 patient serum samples analyzed, 33 were found to have antibodies against Zika virus infection. Among the sero-positives 30 (91%) of the cases were anti-Zika virus IgM with the 21–30-year age group recording the highest number of 8 (26%) and 2 (7%) cases being the least for the 61 years and above age group. All sero-positive febrile patients developed at least one symptom consistent with Zika virus infection: 33 (100%) fever, 25 (76%) muscle pain, 24 (73%) joint pain, and conjunctivitis 2 (6%). Digestive symptoms recorded include 16 (49%) nausea, 12 (36%) vomiting and diarrhea 18 (55%). In addition, 28 (85%) loss of appetite, 14 (75%) rapid respiration and chest pain 15 (42%) were reported by seropositive febrile patients. Our data indicates exposure to Zika virus which suggests the possible circulation of the virus among febrile patients in Ghana with a sero-prevalence rate of 20.6%. Keywords: Zika virus, Seroprevalence, Anti-Zika virus immunoglobulins M and G (IgM and IgG) antibodies, Enzyme linked immunosorbent assay (ELISA) Introduction [4] and in Asia, notably Indonesia, Malaysia and Thailand Febrile illnesses account for about 30% and 75% of [5]. Afterwards serological studies detected Zika virus healthcare visit by children [1] and adults [2] respec- infection in humans in Nigeria, Egypt, India, Pakistan, tively in Ghana. However, these febrile illnesses are North Vietnam and Philippines, spreading from Africa to mostly misdiagnosed for endemic diseases, malaria and Southeast Asia [6, 7]. Zika virus infection in Africa and typhoid fever. Zika virus infection shares overlapping Asia never got the deserved attention due to the sporadic signs and symptoms with most endemic diseases includ- nature of the infection coupled with mild short-term ing malaria and typhoid fever. The etiologic agent, Zika and low-grade fever (37.8–38.5  °C) and mostly self-lim- virus was first isolated in Uganda in 1947 from a rhe- iting symptoms including maculopapular and pruritic sus macaque monkey and Aedes africanus mosquito in rashes, conjunctivitis (non-purulent) and arthralgia [8]. 1948 [3]. First human cases of Zika virus infection were As a result, Zika virus infection appeared to have been reported in Africa and Asia. In Africa, Zika virus infec- neglected in tropical medicine and no efforts were made tion was documented in Uganda and Tanzania in 1952 to develop vaccine and drugs for treatment [7]. Although Zika virus infection has not been reported in Ghana, evidence of several outbreaks and exposure to the virus *Correspondence: Kbonney@noguchi.ug.edu.gh 2 have been reported in various African countries such as Virology Department, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, P.O. Box LG 581, Legon, Gabon [9] Cape Verde, Senegal [10, 11] and Ivory Coast Accra, Ghana which share a common border with Ghana. Thus, the Full list of author information is available at the end of the article evidence of exposure to the virus in our neighboring © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creati veco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ankrah et al. BMC Res Notes (2019) 12:326 Page 2 of 5 countries and the presence of active vector population of a specificity of 100% [12]. Manufacturer’s instructions or Aedes spp. of mosquitoes in Ghana have necessitated the recommendations were followed in all tests performed. need to document the exposure levels in febrile patients. Zika virus IgM and IgG negative control, Zika virus The mild or asymptomatic nature and the overlapping IgM and IgG positive control and the test serum samples clinical feature with other endemic disease conditions were first diluted with sample dilution buffer for Zika make it probable for typical Zika virus infection to be virus IgM and IgG. Thereafter, incubated at 37 °C for an missed out in diagnosis. Therefore, we set out to augment hour in micro-titre plates pre-coated with monoclonal and improve scientific data in the sub-region and create antibody bound to recombinant Zika virus antigen. After awareness to pre-empt possible outbreaks with the detec- subsequent washing, wells were treated with horserad- tion of antibodies against Zika virus circulating among ish peroxidase (HRP) Zika virus conjugate (peroxidase febrile patients at Greater Accra Regional Hospital, Accra labeled Zika virus antigen) except for the substrate blank Ghana. well and incubated for 30  min at 37  °C. Washing was repeated after which substrate solution 3,3′,5,5′-tetra- Main text methylbenzidine (TBM) hydrogen peroxide was added Methods and incubated in the dark at room temperature 20–25 °C Study design for 15  min. A stop solution was then added, and the This was a cross sectional study that involved 160 absorbance measured at 450 nm using ELISA microtiter archived serum samples from febrile patients at the plate reader (Human Diagnostic Worldwide, Germany) Greater Accra Regional Hospital between December within 30 min after terminating the reaction using a ref- 2016 and November 2017. These clinical specimens were erence wavelength of 620  nm. The ELISA tests results obtained as part of an ongoing project with the aim of were classified according to the interpretation provided using serological and molecular tools to detect Dengue, by the supplier, Abcam, Cambridge, UK. Chikungunya and other Arboviruses in febrile patients within selected health facilities in Ghana. A structured Statistical analysis case investigation form was used to collect information One Way-Anova was used to test the association within about demographic features, and clinical signs and symp- groups at significance level of P-value < 0.05 using SPSS toms of the febrile patients. version 20. Eligibility criteria Results Inclusion criteria for enrollment included a person with Characteristics of Zika virus sero‑positives fever (body temperature above 38 °C) and at least one of Of the 160 samples tested, 33 (20.6%) had positive sero- the following signs or symptoms: arthralgia; or arthri- logical evidence for Zika virus. Out of the 33 Zika virus tis; or conjunctivitis (non-purulent/hyperemic). Patients sero-positives, 30 (90.9%) were IgM positive whereas 3 positive for malaria by blood film diagnosis and patients (9.1%) were IgG positive. Of all the patients enrolled, 119 who refuse to submit samples after consenting were (74.4%) were females while 41 (25.6%) were males. Num- excluded from the study. Venous blood was collected into ber of anti-Zika virus IgM males was 11 (36.7%) and that heparinized tubes and centrifuged, and serum was har- of females was 19 (63.3%) (Table 1). Anti-Zika virus IgG vested, aliquoted into two separate cryogenic 1.8 ml vials antibodies were detected only in females, however Zika for each patient sample and stored at − 80 °C. virus infection was observed to affect males and females in a ratio of 1:2. Among the age groups identified, 8 Laboratory testing (26.7%) of the anti-Zika virus IgM were recorded in age The archived sera from febrile patients were tested by group 21–30 with 2 (6.7%) for the age group 61  years enzyme linked immunosorbent assay (ELISA) for anti- and above (Table  1). Anti-Zika virus IgG antibodies Zika virus IgM and IgG antibodies with a commer- were detected only in age groups 21–30 2 (66.7%) and cial ELISA kit, Zika virus IgM and IgG capture ELISA 41–50 1 (33.4%) (Table  1). There was significant differ- (Abcam, Cambridge, UK). The sensitivity of this ELISA ence between anti-Zika virus IgM among the age groups kit used (Abcam anti-Zika virus IgM and IgG) was (P = 0.004). reportedly assessed by evaluating the performances of 5 commonly used Zika virus immunoassays against an Monthly distribution of Zika virus antibodies ELISA (MAC ELISA) developed by the US Centers for Anti-Zika virus IgM detection peaked in March 7 (23.3%) Disease Control and Prevention and cross-Plaque reduc- and May 9 (30%) and lowed in September 1 (3.3%). How- tion neutralization test (PRNT) was reported as 57% and ever, anti-Zika virus IgG antibodies were detected only in Ankrah et al. BMC Res Notes (2019) 12:326 Page 3 of 5 Table 1 Demographic distribution of  seropositive febrile Table 2 Symptoms and  complications of  Zika virus patients seropositives (n = 33) Variables Zika virus antibodies, n = 33 Symptoms Zika positive cases Percentage IgM (30) P-value IgG (3) P-value Fever 33 100 Age groups Diarrhea 18 55 3–20 4 (13%) 0 Nausea 16 49 21–30 8 (26.7%) 2 (66.7%) Vomiting 12 36 31–40 6 (20%) 0 Loss of appetite 28 85 41–50 7 (23.3%) 0.004* 1 (33.3) 0.203 Muscle pain 25 76 51–60 3 (10%) 0 Joint pain 24 73 61 and above 2 (6.7%) 0 Conjunctivitis 2 6 Gender Chest pain 15 46 Male 11 (36.7%) 0 Rapid respiration 14 42 Female 19 (63.3%) 0.166 3 (100%) 0.500 Jaundice 1 3 Recent hearing loss 1 3 n number of Zika virus seropositive * Statistically significant difference between age groups (P = 0.004). P is significant at 0.05 virus infection dominated headlines in certain parts of the world, especially those with tropical climate. Data January and March 2 (66.7%) and 1 (33.3%) respectively presented by this study again suggests noticeable lev- (Fig. 1). els of susceptibility to Zika virus infections. This could mean many infected women subjects being at risk of giv- ing birth to babies with defects. Thus, an extended scope Symptoms presented by seropositive febrile patients to cover infected expectant mothers and diagnose birth Thirty-three (100%) of the seropositive febrile patients defects would have been interesting and will be consid- were presented with fever. The least 1 (0.8%) symptom ered in further studies. reported was conjunctivitis which was also not reported The sero-prevalence of Zika virus antibodies of 20.6% by any seropositive Zika virus female. All other symp- in this study was consistent with study findings from toms were reported in both males and females (Table 2). other African countries with a rate from as low as 0.1% in Gabon and Senegal and as high as 38% in Cameroon [13]. Discussion These differences could be due to inconsistency in the The results from the study indicate exposure levels to study participants’ inclusion criteria or diagnostic test Zika virus which suggest the possible circulation of the used. Zika virus infection was estimated to affect females virus among febrile patients in Ghana with a sero-preva- and males in a ratio of 2:1 which correlate well with sev- lence of 20.6%. Anti-Zika virus IgM prevalence observed eral studies [14]. Considering the findings of [14] and that is likely due to the recent (in the year 2016) wave of Zika of this study, there may be gender-related differences in Fig. 1 Monthly distribution of Zika virus antibodies Ankrah et al. BMC Res Notes (2019) 12:326 Page 4 of 5 Zika virus infection incidence, which might be due to immunoglobulin G/M; J-GRID: Japan Initiative for Global Research Network on exposure differences [15]. Activeness of females during Infectious Diseases; NMIMR: Noguchi Memorial Institute for Medical Research; TBM: 3,3′,5,5′-tetramethylbenzidine; US CDC: United States of America’s Cent- the early hours of the day exposes greater proportion ers for Disease Control and Prevention. of females to Zika virus-carrying Aedes spp. of mosqui- toes either at work or while travelling to and from work. Acknowledgements We are grateful to the clinicians and paramedical staff of the Public Health This may also be attributed to possible differences in who Division of Greater Accra Regional Hospital, Ridge for their technical assis- sought medical care following symptomatic Zika virus tance, the administrative support from the head and staff of the Department infection. High prevalence of IgM or IgG in age group of Microbiology, University of Ghana, Korle Bu and Department of Virology at Noguchi Memorial Institute for Medical Research. 21–30 compared to 61 and above support data from ear- lier studies by [14] and [16]. Possible explanation to this Authors’ contributions observation could be the activeness of age group 21–30 Conceived and designed the experiments: TKA, JHKB, GAA; Performed the experiments: GAA, JHKB, DP EEA; Analyzed the data: GAA, JHKB, DP, EEA; Con- during the early hours of the day which exposes them to tributed reagents/materials: JHKB; Wrote the paper: GAA, JHKB; Revised and the bites of Zika virus-carrying Aedes spp. of mosqui- edited manuscript: GAA, JHKB, TKA, DP, EEA. All authors read and approved the toes more frequently than age group 61 years and above. final manuscript. This could also suggest adults manifest with disease less, Funding as they become immune to Zika virus. The proportions This work was supported partly by funds from the Japan Initiative for Global of the IgG antibody titers recorded in age group 21–30 Research Network on Infectious Diseases (J-GRID) from the Japan Agency for Medical Research and Development (AMED) and by the Government of and 41–50 may suggest that, Zika virus infection is not Ghana through financial support from the University of Ghana Research Fund. endemic in Ghana, rather the virus had been introduced The funders did not contribute to the design of the study and collection, to a non-exposed population as endemicity is attained analysis, and interpretation of data and in writing the manuscript. This study was funded by Japan Agency for Medical Research and Development (AMED) when the adult infection decreases and only the new and by the Government of Ghana through financial support from the Univer- entrants into the population are affected more [16]. The sity of Ghana Research Fund (JP18fm0108010). significant difference between IgM among the age groups Availability of data and materials may be due to the pattern of erotic activities and expo- In this manuscript, the availability of data and material was not applicable. sure among the age groups. Active sexual activity and exposure begins early, increases simultaneously and Ethics approval and consent to participate The study was approved by both Institutional review Committees of the Col- declines as one ages. Monthly distribution of Zika virus lege of health Sciences, University of Ghana and Noguchi Memorial Institute infection with higher prevalence in the raining season as for Medical Research. A written consent was obtained from all eligible adult seen in this study is in harmony with reported pattern of patients or guardians of patients less than 18 years old before inclusion. All study participants were assigned with unique identification number through- Zika virus transmission [17]. The high frequency of IgM out the study. in the month of May and March could be due to the high amount of rainfall which provided temperatures suitable Consent for publication Not applicable. for virus survival, incubation, development and biting of Aedes aegypti and Aedes albopictus [18]. The compact Competing interests clusters of cases in March and May, and the high prev- The authors declare that they have no competing interests. alence of IgM against Zika virus are consistent with an Author details acute outbreak of Zika virus infection in the population 1 Department of Medical Microbiology, College of Health Sciences, University without previous immunity to Zika virus [19]. In this of Ghana, Korle Bu, Accra, Ghana. 2 Virology Department, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, study, commonly reported symptoms of Zika virus infec- P.O. Box LG 581, Legon, Accra, Ghana. 3 Department of Biomedical Sciences, tion documented were fever, muscle pain, joint pain and University of Health and Allied Sciences, Ho, Volta Region, Ghana. conjunctivitis. Strikingly, conjunctivitis often observed in Received: 30 April 2019 Accepted: 5 June 2019 Zika virus infection was not reported by any seropositive female. Digestive symptoms noted in Zika virus infected patients but rarely observed [20], however, found in this study were nausea, vomiting and diarrhea. References 1. Section on Clinical Pharmacology and, Therapeutics; Committee on, Drugs, Farrar HC, Sullivan JE. Fever and antipyretic use in children. Pediat- Limitation rics. 2011;127(3):580–7. The limitation of this study was the absence of plaque 2. 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