We prospectively studied persons attending the travel clinic of the Public Health Service Amsterdam during October 1, 2006–September 30, 2007. All persons >18 years of age were eligible if they planned to travel for 1–13 weeks to >1 countries to which dengue is endemic. Dengue endemicity was based on information from The Global Infectious Diseases and Epidemiology Online Network (14).

All participants were seen by a medical doctor or nurse who specialized in travel medicine. They received vaccinations, a prescription for antimalarial chemoprophylaxis if required, and oral and written information about how to avoid acquiring travel-related diseases, including mosquito-borne infections, according to Dutch National Guidelines on Traveler’s Health Advice (15).

Before departure (just before they received the required vaccinations) and 2–6 weeks after return, participants donated venous blood samples for serologic testing. Before departure,they completed a standard questionnaire to collect data on sociodemographic factors, including travel history, vaccination status, and the purpose of travel (tourism, work/education, or visits to friends and/or relatives).

Participants were given a digital thermometer and asked to take their temperature (axillary or rectally) when feeling feverish. They kept a structured travel and posttravel diary to record symptoms and signs of dengue illness (fever, myalgia, arthralgia, headache, retro-orbital pain, skin rash, signs and symptoms of gastrointestinal disease), use of insect repellent containing N,N-diethyl-meta-toluamide (DEET), and use of mosquito netting. Participants were asked to report both the presence and absence of symptoms and preventive measures on a daily basis; after travel, the diary was checked by a registered nurse in the participant’s presence.

To encompass the incubation period of DENV infection, we used data from the third day after arrival at the travel destination until 2 weeks after return from travel. With respect to symptomatic outcome, we used the term travel-related to refer to this period.

Destinations were grouped into regions by using the classification of the United Nations Development Agency (16). Travel duration was recorded as the total days spent in areas meeting the inclusion criteria.

The Medical Ethics Committee of the Academic Medical Centre Amsterdam approved the study protocol. Participants were included only if they provided informed and written consent.

Blood samples were immediately stored at 6°C, then centrifuged and frozen at –80°C within 24 h after collection. After all study participants had returned, all posttravel serum samples were thawed and tested for immunoglobulin (Ig) M antibodies to DENV antigen by using an IgM-capture ELISA and for IgG antibodies to DENV antigen serotypes 1, 2, 3, and 4 by using an indirect ELISA (Panbio Diagnostics, Brisbane, Queensland,Australia) according to the manufacturer’s instructions. For participants whose posttravel sample yielded positive test results for anti-DENV IgM or IgG, pretravel samples were also tested for anti-DENV IgM or IgG, respectively.

The ELISA for anti-DENV IgM has a sensitivity of 87%–100% and a specificity of 96% (17–19). The ELISA for anti-DENV IgG has a sensitivity of 99%–100% and a specificity of 96%–98% (17,20). These test characteristics concern the use of paired serum samples.

Serology suggestive for previous DENV infection was defined as an anti-DENV IgG >11 Panbio units in both the pretravel and the posttravel sample. The posttravel-to-pretravel anti-DENV IgG ratio was <4:1.

We defined seroconversion indicating recent DENV infection. Criteria were as follows: 1) posttravel anti-DENV IgM >11 Panbio units with pretravel anti-DENV IgM <11 Panbio units and anti-DENV IgG <11 Panbio units; and/or 2) posttravel anti-DENV IgG >11 Panbio units and pretravel anti-DENV IgG <11 Panbio units, with a posttravel-to-pretravel ratio of >4:1; and/or 3) anti-DENV IgG >11 Panbio units in both the pretravel and posttravel sample, with a posttravel-to-pretravel ratio of >4:1 (21).

To calculate risk factors for previous DENV infection, we used SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA) to obtain prevalence rates, univariate and multivariate prevalence rate ratios, and 95% confidence intervals (CIs) by using logistic regression modeling. All variables with an overall p value <0.05 in univariate analysis were included in multivariate analysis. Statistical interactions between variables were checked for all variables. If significant (p<0.05), they were included in multivariate analysis.

We calculated attack rates of recent DENV infection per 100 travelers by dividing the number of seroconversions by the total number of participants. Incidence rates (IR) per 1,000 person-months were calculated by dividing the number of seroconversions by the total number of months in which participants were at risk for DENV infection. To calculate risk factors for recent DENV infection, incidence rate ratios (IRR) were obtained as conditional maximum-likelihood estimates of the rate ratio, with mid-P exact test, 95% CIs, and p values. We quantified the application of a protective measure against mosquito bites by dividing the number of days the measure was applied by the number of travel days; it was dichotomized by using the mean of this proportion in the total study population as the cutoff.

Travel during the rainy months was assessed by using the midtravel dates. The rainy months per destination were defined as months with an average total precipitation of >100 mm, based on World Weather Information Service data from the United Nations’ World Meteorological Organisation (22).

We used χ² tests to evaluate the predictive value of travel-related signs and symptoms for recent DENV infection. Fever was defined as a body temperature >38°C. Dengue-like illness was defined as any episode of fever accompanied by >1 of the following: myalgia, arthralgia, headache, retro-orbital pain, or skin rash. A p value <0.05 was considered significant.

The study began with 1,276 participants who intended to travel to dengue-endemic areas. Of these, 69 (5.4%) were excluded: 33 were lost to follow-up; 23 had their travel arrangements cancelled; 7 did not supply the posttravel blood donation; 3 changed travel plans such that inclusion criteria were not met; 2 did not complete the structured diary; and for 1, the posttravel sample was lost.

Most of the remaining 1,207 participants were persons of Dutch origin who were traveling for tourism and previously had traveled to dengue-endemic areas (Tables 1, 2). The median travel duration was 21 days, with an interquartile range (IQR) of 16–28. The median interval between return from travel and posttravel blood donation was 25 days (IQR 21–29).

Twenty-six percent of travelers had received yellow fever vaccination for a previous journey, and 23% received a yellow fever vaccination for the current trip, just after blood donation (not shown in Table 1). Only 4 (0.3%) participants had ever received Japanese encephalitis vaccination, all for a previous journey. No one had ever received vaccination against tick-borne encephalitis.

Serologic results suggestive of previous DENV infection were found for 78 (6.5%; 95% CI 5.2%–8.0%) participants (Table 1). Previous DENV infection was positively correlated with increasing age. The rate was significantly higher for travelers born in a dengue-endemic country, for those who traveled frequently to dengue-endemic countries (all 78 reported >1 previous trips to such a country), and for those whose purpose was to visit friends or relatives. In multivariate analysis, previous DENV infection was not related to yellow fever vaccination for previous travel because it was confounded by previous trips to a developing country. We found no interactions indicating effect modification.

Fourteen participants (1.2%; 95% CI 0.66%–1.9%) had recent DENV infection, of whom 5 seroconverted only for IgM; 7 seroconverted only for IgG, with a posttravel-to-pretravel ratio of >4:1; and 2 seroconverted for both IgM and IgG (Table 2). Thus, the attack rate for recent DENV infection was 1.2% (95% CI 0.66%–1.9%). The incidence rate was 14.6 per 1,000 person-months (95% CI 8.3–23.9) (Table 3).

All 14 persons with recent infection were born in non–dengue-endemic countries; 9 were male, and 11 had traveled previously to a dengue-endemic country (Table 3). The median age was 36 years (IQR 27–56 years). The primary purpose of travel was tourism (12 persons) or work/education (2 persons). The median travel duration was 24 days (IQR 19–30).

The incidence rate was significantly higher for travel during the rainy months (23.6 vs. 7.5/1,000 person-months; IRR 3.2, 95% CI 1.01–11.6; p = 0.048). Travel during the rainy months was not related to use of insect repellent containing DEET or use of a mosquito net (odds ratio 1.0; p>0.05).

The incidence rate appeared to be higher for male travelers (22.3 vs. 9.0; IRR 2.5, 95% CI 0.83–8.2; p = 0.11), persons who traveled for work or education (23.6 vs. 13.7; IRR 1.7, 95% CI 0.26–6.8; p = 0.47), and those who used insect repellent containing DEET <45% of travel days (18.6 vs. 9.5; IRR 2.0, 95% CI 0.63–7.2; p = 0.26), but differences were not significant. With regard to age, previous travel to a dengue-endemic country, yellow fever vaccination for the current trip, and use of a mosquito net, the IRRs equaled 1.0 (p>0.05).

Recent DENV infection was positively correlated with fever, retro-orbital pain, myalgia, arthralgia, and skin rash (Table 4). Dengue-like illness was reported by 5 (36%; 95% CI 14%–62%) of 14 participants. Evidence for recent DENV infection was 7.3× more prevalent among those with than without dengue-like illness. No traveler reported symptoms signs or suggestive of dengue hemorrhagic fever.