Seroprevalence of Chikungunya in an Asymptomatic Adult Population in North and South Sulawesi, Indonesia

Chikungunya (CHIK) is an arthritogenic alphavirus from the Togaviridae family of arboviruses with an enveloped RNA and a positive-sense single-stranded 11.8-kb genome. Viral transmission occurs through the bite of infected Aedes mosquitoes, which are prevalent in both tropical and temperate regions. Viral replication occurs in many vertebrate hosts including monkeys and humans.¹ Chikungunya virus (CHIKV) manifests in humans with fever and polyarthralgia.² The rapid onset of CHIK fever is typically characterized by intense asthenia, myalgia, arthralgia, and headache, with or without rash. The abrupt onset of fever follows a mean incubation period of 3 days with body temperature often above 39°C. The onset of CHIK fever and the intensity of the acute infection have been shown to correlate with viremia. The illness usually lasts 1 week until viremia wanes.^3,4 IgM is detectable in the blood within a few days after symptom onset and persists for up to 3 months, whereas IgG is detectable from around day 4 postonset and might persist for years.⁵ As is true with most arboviruses, many infected people develop only mild or no symptoms.

Comprehensive CHIKV seroprevalence data are lacking in Indonesia despite the endemicity. Previous studies have reported the median IgG seroprevalence of anti-CHIKV antibodies to be 18.5%, with a wide range across studies between 0.0% and 73.1%.⁶ The objective was to determine CHIKV seroprevalence among asymptomatic individuals, including routine medical checkups and blood donors, in North and South Sulawesi, Indonesia. The asymptomatic and the high presymptomatic viral load of 10⁸ plaque-forming units per milliliter⁷ suggest that CHIKV can pose a threat to blood transfusion safety. As in many endemic countries, CHIKV screening is not routinely performed for blood donors in Indonesia. CHIKV infection among blood donors⁸ indicates that transfusion–transmission is probable although it has not been documented.⁹ Our study also aims to test archived blood donor specimens from North Sulawesi for CHIKV IgM and viremia.

Chikungunya virus seroprevalence was conducted on archived samples from North Sulawesi and South Sulawesi provinces (Figure 1). Inclusion criteria were participants aged 17 years old or older without acute infection and residing in the study sites for at least a year. Categorization for urban or rural is based on population density, percentage of agricultural households, and number of facilities such as hospitals and schools, according to the Sub-Directorate of Statistical Services and Promotion, Indonesia.¹⁰ Occupation was classified based on concepts of job into indoor workers including office workers, medical or laboratory technicians, and teachers, and outdoor workers including agricultural, fishery, and forestry workers.

Location of the study areas in Sulawesi, Indonesia.

Citation: The American Journal of Tropical Medicine and Hygiene 108, 2; 10.4269/ajtmh.22-0328

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Location of the study areas in Sulawesi, Indonesia.

Citation: The American Journal of Tropical Medicine and Hygiene 108, 2; 10.4269/ajtmh.22-0328

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Location of the study areas in Sulawesi, Indonesia.

Citation: The American Journal of Tropical Medicine and Hygiene 108, 2; 10.4269/ajtmh.22-0328

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• Download figure as PowerPoint slide

In North Sulawesi, samples collected from blood donors by Red Cross Indonesia, Manado, represented urban areas, while routine medical checkups at Noongan District Hospital, Langowan, represented rural areas. For South Sulawesi, the checkup samples were from the Health Laboratory Center (Balai Besar Laboratorium Kesehatan, BBLK), Makassar, to represent urban and the surrounding rural areas. All samples were collected from January 2019 to February 2020. Personal identification was removed from all samples except for demographic data such as sex, age, residency (rural or urban), and occupation (indoor or outdoor). Ethical approval was obtained from the Eijkman Institute for Molecular Biology Research Ethics Commission (Ethical Approval No. 156).

Samples were screened for anti-CHIKV IgG to determine exposure to CHIKV using an in-house IgG ELISA as previously described¹¹ with minor modifications. CHIKV IgM testing on a subset of samples that were age stratified and randomly selected from IgG-positive samples was conducted using a CHIKV-IgM kit (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany), with sensitivity and specificity of 99.2% and 98.2%, respectively, following the manufacturer’s instructions. To determine viremia in blood donor samples, CHIKV RNA was extracted from serum/plasma using the QIAamp viral RNA Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. CHIKV nucleic acid detection was done by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) following the protocol previously described.¹² Cycle threshold (Ct) value < 40 is considered positive. The data were analyzed using Statistical Package for Social Sciences version 20 (IBM, Armonk, NY). Categorical variables were expressed in ratios and frequencies, and χ² test was used to calculate significant differences between categorical variables. A P value < 0.05 was considered statistically significant.

Plasma/serum samples from 1,092 participants comprising 529 (48.4%) males and 563 (51.6%) females were analyzed in this study (Table 1). The mean age of participants was ±38.5 years with a range of 17 to 94 years. The age groups of > 60 years and 17–20 years were the least represented, with 93 (8.5%) individuals each representing the age distribution of the study population involving medical checkup and blood donor participants. Of the 600 participants from North Sulawesi, indoor and outdoor workers were 434 (72.3%) and 166 (27.6%), respectively. Information on participants’ occupations was not available for South Sulawesi. Overall, CHIKV IgG seroprevalence was 53.5% (584/1,092), with an average seropositivity of 53.2% and 53.9% in North Sulawesi and South Sulawesi, respectively. There was no significant difference in IgG seropositivity based on age group and indoor or outdoor occupation among participants in either North or South Sulawesi. The seropositivity in male participants was significantly higher in both study sites: 58% in males and 48.3% in females in North Sulawesi (P > 0.05) and 59.8% and 48.7% in South Sulawesi (P > 0.05). Anti-CHIKV IgG seroprevalence was seen in all age group participants, with approximately half of adults already seroconverted in their 20s in North Sulawesi and 30s in South Sulawesi. Seropositivity in all age groups might be linked to sustained local transmission although multiple smaller outbreaks could not be ruled out.

The overall seropositivity for anti-CHIKV IgM was 12.9% (39/303) (P > 0.05), with seroprevalence rates of 16.4% (28/171) and 8.3% (11/132) in North and South Sulawesi, respectively (Table 1). There was no significant difference in IgM seropositivity based on sex, region, and age groups; however, there was a statistically significant difference in seroprevalence regarding indoor or outdoor occupancy in North Sulawesi (P = 0.01). Additionally, the current study recorded two positive samples by qRT-PCR (Ct: 38.96 and 38.91) without positive IgM response.

Chikungunya virus has caused explosive outbreaks in Asia, especially in Southeast Asia, and, because of the ubiquity of Aedes aegypti, it is a global health concern. Several studies have been conducted in Indonesia, but these targeted symptomatic cases range from 1.4% to 3.2%.¹³ A serosurvey might be a better approach to determining endemicity, which could provide significant guidance for public health interventions. In our study, we evaluated the environmental factor (urban versus rural), demographic (age and sex), and occupation in eastern Indonesia, where the data are limited, and the burden of infectious diseases is high. In general, seroprevalence in Sulawesi was higher than that reported elsewhere in the region: 5.9% for Malaysia¹⁴; 1.9% for Singapore¹⁵; 34.5% for Myanmar¹⁶; 26.8% for Thailand¹⁷; and 13.4% for Vietnam.¹⁸ Comparing CHIK serosurveys across different countries and regions is undeniably challenging due to differences in the study population, demography, stratification of age groups, antibody detection method used, and study approach.

Our study shows that the CHIKV IgG seroprevalence in North and South Sulawesi was 53.2% and 53.9%, respectively. The IgG seroprevalence in our study was remarkably high compared with reported rates of 8.1% in North Sulawesi and an overall prevalence of 34.9–73.1%.^6,19 The current study also shows that IgM seropositivity in North and South Sulawesi (16.4% and 8.3%, respectively) during a non-outbreak period was higher than the overall prevalence of 7.3%.⁶ The difference may be attributed to demographic differences among patients recruited, different periods when the studies were carried out, as well as study design.

Chikungunya virus IgG seroprevalence in rural and urban areas were 49.3% and 57.0% for North Sulawesi and 57.8% and 51.3% for South Sulawesi, respectively; the finding of increased seroprevalence in urban areas is consistent with earlier reports,^20,21 and is likely due to the adaptation of Ae. aegypti to domestic environment. Overall, seroprevalence was higher in adults because they tend to be the most active groups in society. Similarly, associations between seropositivity and age have been reported,^14,15,22 which may be linked with a higher likelihood of exposure to mosquitoes over their lifetime.

The majority of anti-CHIKV IgM positivity was associated with indoor occupation (Table 1). Differences in exposure could reflect adaptation of the Aedes vector for resting and breeding inside water-holding containers situated indoors or outdoors.²³ IgG seropositivity tends to be higher in males than females (Table 1); similar trends were reported from Myanmar and the southern part of Thailand,^16,17 which may reflect similarities in cultural habits and behavior patterns that predispose males to more bites by Aedes species. Nevertheless, these results must be interpreted with caution because there was a preponderance of males in this study, which may have biased the outcomes. Conversely, IgM seropositivity was higher in females in North and South Sulawesi (Table 1), which differs from a recent regional report in Myanmar.¹⁶ There are a few notable limitations in this study: lack of participant occupation data from South Sulawesi and unequal participant distribution between North and South Sulawesi for a comprehensive analysis. In addition, a confirmatory neutralization assay was not conducted to exclude other alphaviruses. However, the in-house IgG ELISA did not cross-react with Ross River virus¹¹ and the IgM ELISA kit showed comparable performance to the reference assay.²⁴ Moreover, there is currently no evidence of co-circulation with other alphaviruses in Indonesia.^11,19 Finally, the binding affinity of CHIKV IgG to viral proteins of different lineages has not been established. However, it might not notably affect our seroprevalence results, because the ELISA used antigen from an Asian lineage, which is the predominant circulating strain in Indonesia.⁶

Asymptomatic CHIK poses a substantial threat to transfusion.^8,25 The current study recorded two positive low blood donor samples by qRT-PCR without positive IgM response, presumably from the initial phase of viremia and likely to be infectious. Whole-genome sequencing attempts were unsuccessful,²⁶ likely due to low virus concentration. Although transfusion-related CHIKV infection has not been documented, further studies using sensitive viral nucleic acid detection and IgM assays are required to assess transfusion safety threats during outbreaks and in highly endemic areas.

We found evidence of higher rates of CHIKV infection on Sulawesi than had been found earlier. There was evidence for disparate risk based on sex, domicile, and age. Especially concerning is the risk of asymptomatic blood donors.