Zika Virus Seropositivity in 1–4-Year-Old Children, Indonesia, 2014

We assessed Zika virus seroprevalence among healthy 1–4-year-old children using a serum sample collection assembled in 2014 representing 30 urban sites across Indonesia. Of 662 samples, 9.1% were Zika virus seropositive, suggesting widespread recent Zika virus transmission and immunity. Larger studies are needed to better determine endemicity in Indonesia.

Z ika virus, first isolated in 1947 (1), is a flavivirus phylogenetically related to dengue virus (DENV) that is, like DENV, also transmitted by Aedes mosquitoes. Because of the epidemic that swept through the Americas in 2016, Zika virus infection is known to cause microcephaly, as well as other congenital defects and Guillain-Barré syndrome (2). Zika virus has long been known to be endemic in Southeast Asia (3,4), but laboratory confirmation of infection can be challenging. Acute infections are often asymptomatic. In those who are symptomatic, viral RNA typically persists in blood <7 days and in urine <10 days after symptom onset, limiting the usefulness of nucleic acid testing (5). Zika virus antibody cross-reacting with DENV can confuse results of tests conducted in regions where the viruses co-circulate (6). Virus-specific neutralization assays can more accurately detect and measure Zika virus antibody, but because of their complex requirements, these tests have seldom been used in epidemiologic studies (7).
Acute Zika virus cases have been reported in Indonesia (8), Singapore (9), Malaysia (10), Vietnam (11), and Thailand (12). However, little is known about Zika virus prevalence in the region. Limited retrospective testing of archived specimens collected from clinically ill patients in Thailand (12) and Cambodia (13) suggest that incidence in these countries is low. However, given the limited number of samples tested and lack of confirmatory testing in these studies, information on prevalence and distribution is challenging to assess. Likewise, little is known about the prevalence and geographic distribution of Zika virus in Indonesia, the biggest country in Southeast Asia. DENV and chikungunya virus, also transmitted by Aedes mosquitoes, are endemic throughout Indonesia, suggesting the ecologic conditions exist for Zika virus transmission as well. An estimated 80% of the population in Indonesia is infected with >1 DENV by the age of 10 years (14). In our study, we assessed Zika virus seroprevalence among healthy 1-4-year-old children to determine the prevalence and distribution of Zika virus in Indonesia.

The Study
We used serum samples collected during October-November 2014 for a previous population-based, cross-sectional cluster survey conducted to assess DENV seroprevalence; in the study, 3,312 samples were collected from 1-18-yearold children in 30 urban districts in 14 provinces of Indonesia (14). In our study, we assessed only the children 1-4 years (range 12-59 months) of age because these children were least likely to have cross-reactive DENV antibodies. Ethics clearance was obtained from the Health Research Ethics Committee of the Faculty of Medicine, Universitas Indonesia, and the US Centers for Disease Control and Prevention (CDC; Atlanta, Georgia, USA).
Plaque reduction neutralization tests (PRNTs) that could differentiate Zika virus neutralizing antibodies from those produced in response to DENV infection were adapted from protocols developed by the CDC (online Technical Appendix, https://wwwnc.cdc.gov/EID/article/24/9/18-0582-Techapp1.pdf). The challenge virus used in the PRNT was Zika virus JMB-185, acquired from a patient in 2014 (8). Convalescent serum from this same patient was used as a PRNT positive control. We subjected all specimens to 2 tiers of testing by PRNT 90 (i.e., a PRNT in which serum samples suppressing >90% of challenge virus were considered positive for neutralizing antibody). In the first tier, we tested serum samples diluted 1:10. Samples that suppressed >90% of Zika virus PFUs were considered potentially positive for Zika virus antibodies because DENVspecific antibodies, if present, could have cross-reacted and neutralized Zika virus. We then subjected the specimens considered potentially positive to a second PRNT 90 , in which we tested serum samples against Zika virus and all 4 DENV serotypes (online Technical Appendix). Specimens that tested positive for Zika virus neutralizing antibody and negative for DENV neutralizing antibody by PRNT 90 were classified as Zika virus seropositive, as were specimens that had Zika virus PRNT 90 titers >4-fold higher than all DENV PRNT 90 titers. We categorized specimens as flavivirus seropositive when Zika virus neutralizing antibodies were present but at titers <4-fold higher than any DENV neutralizing antibody titer (online Technical Appendix Table). We also tested a subset of samples for Japanese encephalitis virus antibody by PRNT 90 ; none of the samples tested had a titer >20, and none of the sample classifications were changed after testing.
Our assessment, involving use of the PRNT 90 , which is highly specific for Zika virus antibodies, indicates widespread, recent Zika virus infection in much of western and central Indonesia. Our criterion for confirmed Zika virus antibodies (i.e., PRNT 90 titer for Zika virus >4-fold higher than that for any DENV in the same specimen) is the international standard. In just 2% (12/662) of specimens, we could not determine whether the antibodies were Zika virus or DENV specific. When using the more conservative criterion of only classifying a sample as positive for Zika virus antibodies if no DENV-specific neutralizing antibodies are detected, the number of Zika virus antibody-positive samples decreases by only 6, leaving 54 samples still classified as Zika virus seropositive. Further evidence for the validity of the PRNT 90 was that DENV neutralizing antibody-positive samples were negative for the presence of Zika virus neutralizing antibodies across a range of titers (R.T. Sasmono, unpub. data).
Although our data provide some evidence regarding geographic distribution, no information is presented regarding a specific threshold titer associated with clinical illness or a correlation between geography and titer. Further studies involving larger sample sets would be necessary to address these topics. The single age group, relatively small number of specimens, and limited number of sites, particularly from eastern parts of the country, do not give a comprehensive picture of endemicity throughout Indonesia. The small numbers of specimens available from most localities did not enable accurate estimation of the proportional differences between localities. We could perform PRNT 90 with samples from the remaining cohort (the 5-18-year-olds), but we expect higher percentages of nonspecific flavivirus seropositivity in the samples from this older age group.