Asymptomatic arbovirus and campylobacter infections in German travelers to Asia

The true risk for many travel diseases is unknown because most studies do not detect asymptomatic infections. In this study, we performed ELISA for dengue virus (DENV), chikungunya virus (CHIKV), Zika virus (ZIKV), hepatitis E virus (HEV), and Campylobacter jejuni on samples from 81 healthy Germans before and after they traveled to Asia. ELISA found five seroconversions for C. jejuni, two for DENV, one for ZIKV, and zero for HEV. For CHIKV, three subjects were positive before travel and negative afterwards. None had symptoms. These infections would have gone unnoticed by retrospective studies. Therefore, the risk for these infections may be higher than previously estimated.

We therefore longitudinally analyzed the sera of all individuals attending Hamburg-area travel health clinics before traveling to South or Southeast Asia.Blood samples were drawn before and after travelling, antibody titers for vectorborne diseases (VBDs) and food-and water-borne diseases (FWDs) endemic in that region were determined, and a questionnaire about clinical symptoms was filled out.
As examples of viruses causing VBDs, we chose dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) because they have recently caused several outbreaks, and they can be asymptomatic as well as highly symptomatic [6,7].Additionally, they can be detected easily using standardized commercial ELISA kits [8].
As examples of pathogens causing FWDs, we chose C. jejuni and hepatitis E virus (HEV) because they are endemic in Asia and can cause asymptomatic, mildly symptomatic, or highly symptomatic infections [9].They also can be diagnosed using standard ELISA kits [10,11].

Introduction
Over the last decades, worldwide travel has seen an exponential increase, with a peak of 1.3 billion tourist arrivals in 2017 [1].Correspondingly, travel-associated infections have multiplied as well [2].To assess the risk of such infections, most studies in this field retrospectively count spontaneously reported, symptomatic diseases in returning travelers [3,4].In contrast, little is known about the rate of asymptomatic infections in travelers [5].
None of the subjects reported previous infection with the investigated pathogens in the past.
During the time of the study, dengue outbreaks were reported in Nepal, Sri Lanka, and certain regions of India [12][13].There was a baseline prevalence of Chikungunya, and there were some outbreaks, but all of them occurred in countries to which the subjects did not travel.Sporadic cases of endemic and imported Zika virus infections were seen in many Asian countries, but there were no outbreaks like the ones in South America during that time.Likewise, hepatitis E virus and C. jejuni were stably endemic, with no sudden peaks during 2017 [14,15] (Table 1).
For each individual, a questionnaire was filled out, and antibody titers in serum were determined by ELISA.In brief, sera were screened for anti-HEV IgA, IgG, and IgM; DENV, ZIKV, and CHIKV IgG and IgM; and C. jejuni IgA and IgG according to the manufacturer's instructions.The DENV test that we used had a sensitivity of > 94% for IgG and 38% for DENV IgM [16].All of the test kits were obtained from Euroimmun AG, Lübeck, Germany).Statistical analysis was performed using GraphPad Prism (Graphpad Software Inc., version number 9.0).

Discussion
To determine the risk for apparent as well as inapparent travel infections in a real-life scenario, we chose a consecutive, longitudinal, single-center serological approach.
For VBDs, we found one ZIKV and at least two DENV infections.The fact that we only found IgG but no IgM seroconversions for DENV could be due to the low sensitivity of the IgM test we used, so the real numbers might even be higher.We still preferred this test over the more sensitive ones because of its ability rule out cross-reactivity with CHIKV by performing parallel tests from the same manufacturer [17].Regarding CHIKV, there was one individual who was IgM positive pre-travel and negative posttravel, which would theoretically suggest a symptomless infection at the beginning of the study from which the subject subsequently recovered.Moreover, three individuals were positive for anti-CHIKV IgG pre-travel and negative post-travel.Although anti-CHIKV IgG levels can decrease over time, it is unlikely that these three Germans had been infected in the past but all of them lost their seropositivity just while traveling to Asia [17].We rather suspect the varying specificity of that test kit to be the cause [8].Calculated incidences were 2% for DENV, 1.2% for ZIKV, and zero for CHIKV.This is slightly higher but still within the order of magnitude reported by others [6,7].
Regarding FWDs, we found five seroconversions for anti-C.jejuni IgG, but none for IgA.This is not surprising, since the rise in IgA is usually transient and can be missed in a subclinical infection.No seroconversions for HEV were seen.The calculated incidence of 6.5% for C. jejuni was higher than expected.The incidence of HEV is, naturally, lower [9].
Of note, none of the individuals reported any symptoms, and even those who seroconverted, when specifically asked, did not recall being ill.This would mean that none of these infections would have been detected in a retrospective study or in a surveillance system relying on spontaneously reported infections [2,55].It would also be a possible explanation for some of the "autochthonous" VBD occurrences in European countries, where the primary case could not be identified [18,19].
This small study has significant limitations.First, although we collected data on the time span traveled and the countries visited, some of the subjects provided only vague travel information such as "2017" or "India".We know from other studies that there are significant regional and seasonal differences in the incidence and prevalence of these pathogens, especially for dengue virus [14,20], and we were unable to determine whether these individuals were in the exact region of an outbreak at the time that it occurred.We also do not know the absolute number of people who travelled to a particular region because only individuals who spontaneously attended travel health clinics before departure were enrolled in the study.It is not clear if the study population was more or less at risk than those who did not seek advice.Finally, the low sensitivity of the DENV IgM test we used might have skewed the results [17], suggesting that the number of asymptomatic travel infections might actually have been higher than this study suggests.

Declarations
Conflict of interest The authors declare that no competing interests exist.
Compliance with ethical standards All procedures were in accordance with the standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent or substitute was obtained from all subjects for the purpose of publication.This study was approved by the ethics committee of the Hamburg Chamber of Physicians (#PV5262).
Informed consent was obtained from all individual participants included in the study.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this

Table 3
Seroprevalence for hepatitis E virus (HEV) and Campylobacter jejuni in absolute numbers and percentages.FWD, food and water-borne