Introduction

Aedes-borne diseases contribute significantly to the global burden of disease, and are especially prevalent in tropical and subtropical settings. Ae. aegypti, and to a lesser extent Ae. albopictus, are the major vectors of disease and three viruses of primary concern are dengue virus (DENV, from genus Orthoflavivirus), Zika virus (ZIKV, from genus Orthoflavivirus), chikungunya virus (CHIKV, from genus Alphavirus).

DENV, CHIKV, and ZIKV each represent significant public health challenges with distinct impacts and distributions. DENV, endemic in tropical and subtropical regions, is responsible for an estimated 400 million infections annually, with a subset of cases progressing to dengue hemorrhagic fever and dengue shock syndrome [1]. CHIKV was first detected in Tanzania in 1952 and in recent years has caused epidemics in Africa, Asia, Europe, and the Americas [2,3]. The disease is characterized by debilitating joint pain and arthritis that can last weeks to months, with significant economic implications on top of health impacts [4]. ZIKV was first detected in Uganda in 1947 and in the last decades there have been epidemics in Africa, Asia, the Americas, and the Pacific [5]. While most ZIKV infections are mild, the disease has been associated with congenital abnormalities (e.g., microcephaly) when transmitted from mother to fetus [6–8]. Collectively, these mosquito-borne viruses cause substantial morbidity, impact quality of life, and strain healthcare systems globally.

While these viruses cause considerable disease globally, infections are presumptively diagnosed in much of the world, based on symptoms and without laboratory confirmation of the cause of disease. DENV and CHIKV infections result in similar symptoms and may be confused in some settings in the absence of laboratory confirmation. ZIKV infections are often asymptomatic. All three may be drastically under-diagnosed in some settings and surveillance is lacking in much of the African continent so that the true burden of these diseases is unknown.

Seroprevalence surveys offer the possibility to evaluate both the long- and short-term epidemiology of these diseases. IgM antibodies typically emerge shortly after infection (normally within days for each virus) and can persist for months. IgG antibodies typically emerge weeks after infection and can persist for years (in the case of dengue, may be lifelong). Dengue virus has four distinct serotypes (DENV-1 through DENV-4), and infection with one serotype typically provides lifelong immunity to that serotype but not to the others. Seroprevalence estimates from IgM are therefore normally indicative of recent epidemiology of these diseases whereas seroprevalence estimates of IgG are indicative of long-term dynamics. In some high transmission settings, most of the population will be seropositive before reaching adulthood [9,10]. Cross-reactivity, particularly between flaviviruses such as dengue and Zika, can complicate seroprevalence surveys that attempt to distinguish exposure to specific viruses [11].

Several dengue fever outbreaks have been documented in Ethiopia since 2013, including reported outbreaks in Dire Dawa in 2013 and 2015, and in other parts of East Ethiopia since [12–18]. Limited surveillance and diagnostic capacity mean that many cases likely go undetected or unconfirmed, making it difficult to precisely define the timing and extent of outbreaks. Chikungunya outbreaks have also been reported more recently in the Eastern part of the nation [19,20], and seropositivity among symptomatic individuals has been reported from the Northwest [21] and more recently in the Northeast part of the nation in patients with suspected malaria infections [22]. Dire Dawa, a major city in the Eastern part of Ethiopia has been repeatedly impacted by dengue infections and more recently (in 2019) by a major chikungunya outbreak that infected over 40,000 individuals [19]. Entomological surveys have confirmed the presence of Ae. aegypti in this city as well [23]. Research on Zika infections in this setting is scarce [24]. Surveillance is lacking for all three viruses.

The primary objective of this research was to conduct a pilot study assessing the seropositivity to DENV, CHIKV, and ZIKV in parts of Dire Dawa, Ethiopia that have a history of dengue outbreaks. Since age patterns in seropositivity can reflect both recent and historical transmission intensity, our goal was also to examine potential associations between age and seropositivity. We conducted secondary analyses of cross-reactivity.

Data and methods

Results

Overall seroprevalence

A total of 339 individuals were recruited for the seroprevalence survey in Dire Dawa (Table 1). Seropositivity to dengue virus IgG was 76% (95% Cis: 0.71 – 0.80). Chikungunya and Zika IgG seropositivity were 44% (0.38 – 0.50) and 38% (0.31 – 0.44), respectively. Seropositivity to IgM antibodies, typically indicating recent infections, was much lower (Table 2). Dengue and chikungunya IgM was approximately the same (0.17 (0.13 – 0.21) versus 0.18 (0.13 – 0.22), respectively). Zika IgM was much lower, at approximately 0.03 (0.02 – 0.05).

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Table 1. Crude serological results from Dire Dawa (March 2024) by age group, virus, and antibody type (IgG or IgM). Counts represent the number of individuals testing positive for IgG or IgM antibodies using the DPP ZCD rapid test. A total of 339 individuals were tested across all age groups.

https://doi.org/10.1371/journal.pntd.0013357.t001

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Table 2. Seropositivity estimates and confidence intervals, from GEE models using an exchangeable correlation structure to account for potential clustering in households. The data are from a seroprevalence survey done in Dire Dawa, Ethiopia in March 2024.

https://doi.org/10.1371/journal.pntd.0013357.t002

In comparison, few individuals from the secondary seroprevalence survey in Addis Ababa (August 2024) tested positive (not enough for accurate seroprevalence estimates). A total of 180 individuals were recruited in this survey (Table A in S1 Text) and out of them 5 tested positive for DENV IgG, 2 for ZIKV IgG, and 1 for CHIKV IgG. Six tested positive for DENV IgM, 1 tested positive for ZIKV IgM, and 1 tested positive for CHIKV IgM. Again, each of the ZIKV IgG positives were also DENV IgG positive. A total of 11 individuals were found seropositive (combining IgG and IgM, and for all three viruses); these individuals ranged in age from 25 to 36 (mean = 30.2; median = 28), and each reported travel to other regions in Ethiopia (five specifically mentioned travel to Dire Dawa).

Age patterns in seropositivity

We first examined crude age group–specific seroprevalence estimates (Fig 1, Table 1), which showed a clear age-related increase in IgG seropositivity for all three arboviruses. These patterns were broadly consistent with modeled predictions using generalized estimating equations (GEE), which incorporated age as a continuous spline term and accounted for household clustering (Fig 2). By age 10, 52% of all sampled individuals were seropositive for IgG to DENV; this proportion rose to approximately 86% by age 40. For CHIKV, over half (51%) of individuals were seropositive by age 30, while ZIKV seropositivity reached 37% by age. The potential cross-over between IgM responses for dengue and chikungunya was likewise evident in these estimates (Fig 2), though the confidence bands around the estimates were sufficiently large to warrant caution in interpreting this result.

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Fig 1. Age-specific seroprevalence of IgG and IgM antibodies to dengue, Zika, and chikungunya viruses in Dire Dawa, Ethiopia (March 2024).

Bars represent the proportion of individuals in each age group who tested positive for each virus. Error bars indicate 95% confidence intervals calculated using the Wilson score method. IgG results reflect past exposure, while IgM results suggest more recent or acute exposure. A total of 339 individuals were included in the survey. Fig A in S1 Text presents age specific estimates with smaller age groups.

https://doi.org/10.1371/journal.pntd.0013357.g001

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Fig 2. Predicted probability of IgG seropositivity by age based on GEE models (Dire Dawa, March 2024).

Predicted probabilities of seropositivity for dengue, Zika, and chikungunya IgG are shown as smoothed curves generated from generalized estimating equations (GEEs), incorporating age as a natural spline (df = 3) and adjusting for household-level clustering. Shaded areas represent 95% confidence intervals. Estimates reflect modeled age-specific trends in seropositivity, rather than crude age group proportions.

https://doi.org/10.1371/journal.pntd.0013357.g002

Potential for cross reactivity

All Zika IgG positive individuals were also dengue IgG positive, suggesting a potential for cross-reactivity. Indeed, both are flaviviruses (i.e., genus Orthoflavivirus), and so cross-reactivity would not be surprising. A total of 253 of the tested individuals were dengue IgG seropositive and 126 were Zika IgG positive.

We used the raw quantitative values generated by the test reader as the outcome variables in our cross-reactivity analyses, rather than relying solely on the binary classifications of seropositivity. These values reflect the magnitude of the antibody signal detected by the commercial test kit, though they are not derived from laboratory-based titration methods such as PRNT (plaque-reduction neutralization test) or ELISA (enzyme-linked immunosorbent assay).

An analysis of the quantitative IgG responses for Zika and dengue showed that individuals who were categorized as Zika IgG seronegative but dengue IgG seropositive had very low Zika IgG values. Those who were seropositive for both Zika IgG and dengue IgG had much higher Zika IgG responses than individuals who were dengue IgG seropositive but Zika IgG seronegative (Fig B in S1 Text). Individuals who were dual-seropositive for dengue IgG and Zika IgG had significantly higher quantitative dengue IgG values compared to individuals who were dengue IgG seropositive but Zika IgG seronegative (Fig B in S1 Text). In general, cross-reactivity appeared stronger in the direction of ZIKV influencing DENV IgG levels than the reverse. We therefore hypothesize that Zika IgG seropositivity in this sample is not solely the result of cross-reactivity with dengue virus, but may reflect true prior infection with ZIKV. Furthermore, not all Zika IgM seropositive individuals were also dengue IgM seropositive, supporting this distinction. Given the lack of test validation in East African settings and the absence of confirmatory assays such as PRNT, these patterns should be interpreted cautiously. The observed ZIKV seropositivity may reflect true prior exposure or cross-reactive antibody responses, particularly among individuals with high DENV IgG levels.

We generated a Spearman’s correlation matrix from all possible immunological outcomes (Fig 3). The strongest associations were between Zika IgG and dengue IgG, chikungunya IgG and chikungunya IgM, followed by Zika IgG and dengue IgM, and dengue IgG and IgM.

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Fig 3. Spearman correlation matrix of quantitative IgG and IgM levels for each arbovirus.

https://doi.org/10.1371/journal.pntd.0013357.g003

Our linear mixed models analyses (Tables B and C in S1 Text) showed that Zika IgG was strongly associated with dengue IgG values (Table B in S1 Text). Chikungunya IgG was also associated but the association was smaller than with Zika IgG. Dengue IgG was strongly associated with Zika IgG. Chikungunya IgG was negatively associated with Zika IgG. Finally, Zika IgG was positively associated with chikungunya IgG values while dengue IgG was negatively associated with chikungunya IgG.

Zika IgM was positively associated with dengue IgM, as was chikungunya IgM (Table C in S1 Text). Likewise, dengue and chikungunya IgM values were positively associated with Zika IgM. Finally, dengue and Zika IgM were both positively associated with chikungunya IgM. IgG values were statistically associated with each IgM outcome, but their effect sizes were small.

Discussion

Using a cross-sectional seroprevalence survey from Dire Dawa, Ethiopia, we found and here report a high seroprevalence to three arboviruses of concern: DENV, CHIKV, and ZIKV. These results existed for both IgG and IgM suggesting both a history of widespread exposure to these viruses, and recent exposure. The age patterns for DENV and CHIKV IgG seroprevalence in particular suggest that most of the population in this setting has been exposed to these viruses by age 30 (over 50% seroprevalence to CHIKV IgG and over 80% to DENV IgG). We also found evidence of household clustering with regard to DENV and CHIKV exposure, but not ZIKV exposure.

While approximately 25% of all children ages 0 – 4 were seropositive for IgG to DENV, none were seropositive for IgG to CHIKV. In 2019 there was a major CHIKV outbreak in this setting, with over 40,000 individuals diagnosed with the disease [19]. It is therefore possible that much of the seroprevalence to CHIKV IgG that we found was the result of this previous epidemic, or to the 2019 epidemic and others. We did, however, find individuals in most ages (but not the youngest age group) who were seropositive for IgM to CHIKV, suggesting that there has potentially been some recent transmission of this disease or a related alphavirus, among individuals from the study location. IgM to Chikungunya has been shown to persist in some individuals over long periods of time (28 months post infection [29]) but we believe this is unlikely to explain the IgM seropositivity to CHIKV from our survey (now 5 years after the major 2019 epidemic).

While dengue and chikungunya epidemics have previously been reported in Ethiopia, exposure to Zika is poorly characterized because of limited surveillance. A large, nationally representative serosurvey conducted using multistage sampling and confirmatory plaque reduction neutralization tests (PRNT) detected low levels of flavivirus antibodies, including 0.4% ZIKV IgG positivity [24]. A smaller, community-level survey in Gambella (in West Ethiopia) estimated seroprevalence of 2.9, 15.6, and 27.3% seroprevalence for yellow fever, CHIKV, and ZIKV, respectively [30], though the absence of confirmatory testing raises concerns about potential cross-reactivity. Elsewhere in Ethiopia, most serological studies have focused on hospital-based or febrile populations, which may not accurately reflect broader community-level transmission.

In contrast, our survey employed a population-based household sampling strategy in a setting with known dengue and chikungunya outbreaks. We found high IgG seroprevalence, particularly for dengue, suggesting intense and sustained local transmission in Dire Dawa. These estimates may even underestimate true exposure given prior evaluations indicating low sensitivity of the rapid test we used [27]. Our secondary survey in Addis Ababa, where no outbreaks have been reported, served as a pragmatic control. The near-absence of seropositivity in this study population further supports the interpretation that the high values observed in Dire Dawa reflect localized transmission rather than test artifact.

We also observed substantial IgG reactivity to ZIKV in Dire Dawa. While this may indicate past ZIKV exposure, interpretation is complicated by well-documented cross-reactivity among flaviviruses, particularly between ZIKV and DENV. Without confirmatory tests we cannot definitively attribute these results to ZIKV infections. Yellow fever virus, another flavivirus, is endemic to parts of Ethiopia, and yellow fever vaccination can induce cross-reactive antibodies. However, there have been no yellow fever vaccination campaigns in Dire Dawa or Addis Ababa, making it unlikely to explain the observed ZIKV reactivity in our sample. Notably, individuals who were ZIKV IgG–positive had higher DENV IgG titers compared to those who were ZIKV IgG–negative. This could reflect true sequential infections or cross-reactive antibody responses; it may also support the hypothesis that prior ZIKV exposure influences DENV IgG responses. However, an equally plausible explanation is that high DENV IgG itself increases the probability of a false-positive ZIKV result. We therefore interpret these ZIKV IgG findings cautiously and view them as hypothesis-generating, underscoring the need for confirmatory investigation of arbovirus exposure and co-exposure in this setting.

One limitation of our work is the use of a single screening kit. It might have been preferable to have another test (e.g., ELISA) for comparison on at least a subsample. A few other studies have used the same test kit in research settings. One study suggested general low sensitivity of this test in comparison to ELISA. However, specificity was high (sensitivity/specificity: DENV 43.92/100%; ZIKV 25.86/98.81%; CHIKV 37.78/99.35% [27]). These evaluations, however, were not conducted in East African populations and no published validation studies exist for this test in East African settings. If the same pattern holds true in this setting it would indicate that the estimated seroprevalence is an underestimate [27]. Another study focusing on IgM alone found sensitivity/specificity of the ZCD test to ZIKV IgM was 79.0/97.1%; DENV was 90.0/89.2%; and for CHIKV it was 90.6/97.2% [26]. We take the results of these previous studies to suggest that for ZIKV and CHIKV in particular, our results may be underestimates. However, the absence of gold-standard confirmatory tests in our setting limits our ability to assess test performance directly. This limitation is particularly important when interpreting ZIKV results, which may be influenced by cross-reactivity with other flaviviruses (including DENV).

Cross reactivity may have influenced some of our results. DENV and ZIKV are both flaviviruses, and cross-reactivity between the two is well-known but not completely understood [11,31]. If other alphaviruses are circulating in this setting, they could potentially lead to false-positive CHIKV seropositivity. Although we observed patterns consistent with differential responses (e.g., higher DENV responses among ZIKV positives), these may still reflect cross-reactive antibody responses rather than true prior ZIKV infection.

These complexities have direct implications for surveillance and clinical interpretation. In the absence of molecular confirmation or PRNT-based serotyping, distinguishing true infections from cross-reactive responses is difficult. Misclassification could compromise seroprevalence estimates and introduce uncertainty into outbreak detection, risk mapping, and vaccine eligibility assessments. This highlights the importance of continued assay validation, ideally in local populations, and careful interpretation of serological survey data in flavivirus-endemic settings.

Cross-reactivity can have clinical implications, as is already well-known within DENV infections across serotypes and has been hypothesized between DENV and ZIKV infections. While DENV infections are known to provide long-term immunity, the immunity is serotype specific and infection by another serotype can lead to more severe disease outcomes (including death) through antibody dependent enhancement (ADE) [32–34]. ADE may also occur across flaviviruses – with the potential for previous ZIKV infection leading to severe dengue disease even in the primary infection by DENV [35]. Current dengue vaccines are only provided to populations that already have previous exposure to DENV (i.e., the vaccines should only be provided to seropositive individuals) as vaccinating individuals who are seronegative can increase their risk of severe disease when they are infected by DENV [36].

Cross-reactivity is further complicated by the fact that these three viruses are all vectored by the same mosquito (e.g., Ae. aegypti), meaning that individuals who have been infected with dengue in this setting are likely to also have been at risk of chikungunya (which has been confirmed in this setting) and Zika virus infection, if those viruses were present or introduced. Although Ae. aegypti has been documented in this region, data on vector abundance, seasonality, and human-vector contact patterns remain scarce. This pilot study was designed in part to address that gap and to generate preliminary evidence that can motivate more detailed entomological and behavioral investigations in future work. That is, individuals in this setting who acquire dengue infections are likely to also be at risk of CHIKV or ZIKV infections, if those viruses are present, given the shared mosquito vector that is present in this setting.

This seroprevalence survey was designed to evaluate age-specific seroprevalence in kebeles in Dire Dawa known to have previously had dengue outbreaks. While we used a clustered random sampling approach (randomly selecting households within the kebeles), our results may not be representative of the entire city of Dire Dawa. We also do not have precise information on the total population size or geographic areas represented by the selected kebeles, which limits our ability to generalize these findings to the broader urban population. It would be valuable to conduct further surveys that would be representative of the city, as well as in other parts of Ethiopia.

The observed age-specific IgG seroprevalence patterns offer important epidemiological insight. The rapid rise in DENV IgG positivity by age 10 (Figs 1 and 2) likely reflects active or recent transmission, as seropositivity among young children must result from infections within their relatively short lifespans. In contrast, slower age-related increases in CHIKV or ZIKV IgG suggest a lower force of infection or more recent introduction of these viruses. The smooth, progressive increase in DENV IgG with age also supports the interpretation that exposure is accumulating gradually, consistent with endemic transmission rather than isolated outbreaks. While maternally derived antibodies may explain some reactivity in very young children, the broader pattern suggests widespread exposure during early childhood. Although the ZIKV findings are also intriguing, we remain cautious in interpreting those results because of the potential for cross-reactive antibody responses with DENV.

We also found evidence of household level clustering (from the ICCs) for DENV and CHIKV IgG responses and for CHIKV IgM responses. In contrast, ZIKV responses showed no measurable household clustering. These patterns could reflect differing transmission dynamics or the timing of exposure across viruses. Household clustering in IgG or IgM responses may point to shared environmental exposures such as mosquito breeding sites near houses, or to household-level behaviors and socioeconomic factors that influence risk of infection. Future interventions or surveillance efforts might benefit from recognizing and targeting such within-household transmission patterns.

In summary, we found evidence of widespread exposure to DENV, CHIKV, and ZIKV in Dire Dawa, Ethiopia. This aligns with reported outbreaks of dengue fever in this setting, and of the massive chikungunya fever outbreak in 2019. We also found evidence of exposure to ZIKV, which has rarely been reported from this nation. Not only did we find evidence of previous exposure to ZIKV, we also found evidence of recent exposure (i.e., IgM seropositivity) to all three arboviruses. While we cannot rule out the potential for cross-reactivity influencing our results, they are suggestive of a large risk of infection with Ae. aegypti-transmitted viruses in a setting where this important vector of disease is known to be present [23]. Repeated infections can in some cases lead to more severe disease outcomes, for example through ADE. More research into arboviral diseases and their mosquito vectors in this setting is warranted both to confirm our results and to develop a better understanding of the distribution of different arboviral infections, any potential age and gender associated risk factors, and all so that public health interventions can be formulated to address these diseases. As the world has experienced pandemic levels of Aedes-borne disease several times since 2019, it is increasingly important to have increased surveillance for these diseases and for public health agencies to plan accordingly.

Supporting information

Acknowledgments

We are thankful for the community members and community health workers in Dire Dawa and Addis Ababa who participated in this research.