Travel-related acquisition of diarrhoeagenic bacteria, enteral viruses and parasites in a prospective cohort of 98 Dutch travellers Travel Medicine and Infectious Disease

Background: Limited prospective data are available on the acquisition of viral, bacterial and parasitic diarrhoeagenic agents by healthy individuals during travel. Methods: To determine the frequency of travel associated acquisition of 19 pathogens in 98 interconti- nental travellers, qPCR was used to detect 8 viral pathogens, 6 bacterial enteric pathogens and 5 parasite species in faecal samples collected immediately before and after travel. Results: We found high pre-travel carriage rates of Blastocystis spp. and Dientamoeba fragilis of 32% and 19% respectively. Pre-travel prevalences of all other tested pathogens were below 3%. Blastocystis spp. (10%), Plesiomonas shigelloides (7%), D. fragilis (6%) and Shigella spp. (5%) were the most frequently acquired pathogens and acquisition of enteral viruses and hepatitis E virus in this relatively small group of travellers was rare or non-existent. Conclusions: Our ﬁ ndings suggest that the role of viruses as the cause of persisting traveller's diarrhoea is limited and bacterial pathogens are more likely as a cause of traveller's diarrhoea. The substantial proportion of travellers carrying Blastocystis spp. and D. fragilis before travel warrants cautious interpreta- tion of positive samples in returning travellers with gastrointestinal complaints.


Introduction
Although diarrhoea ranges in the top of the most frequently occurring travel associated conditions [1], limited data are available on the acquisition of viral, bacterial and parasitic diarrhoeagenic agents by previously healthy individuals during travel. In addition to the epidemiological relevance, such data may help to understand the clinical relevance of detected pathogens in the era of extremely sensitive diagnostic testing with real-time PCR (qPCR). This study was initiated to determine the frequency of travel associated acquisition of 19 (entero)pathogens by qPCR.

Study population
The COMBAT-study is a multicenter longitudinal cohort study primarily focussing on the acquisition of ESBL-and carbapenemase-producing Enterobacteriaceae [2,3]. A total of 2001 Dutch adults travelling intercontinentally for 1e12 weeks were included from November 2012 until November 2013. All subjects received a faeces collection swab (Fecal Swab; Copan, Brescia, Italy) with transport medium and a questionnaire before and after travel. The questionnaires comprised information about previous travel, health, travel destination and behaviour during travel. Stool samples were stored at À80 C for future analysis. For this study, 100 travellers were randomly selected from the complete study population of 2001 travellers.

Extraction and PCR
Automated nucleic acid extraction was performed using the MagNA Pure 96 instrument (Roche Applied Science, Roche Diagnostics B.V., the Netherlands) with the inclusion of internal controls for generic RNA and DNA. Reverse-transcriptase PCR was applied immediately after extraction to form cDNA. qPCR was performed using the LightCycler480 (Roche Applied Science, Roche Diagnostics B.V., the Netherlands) to detect 8 viral pathogens, 6 bacterial enteric pathogens and 5 parasite species in faecal samples collected immediately before and after travel (see Table 1 for targeted micro-organisms and Supplementary Table for primer and probe sequences). A positive (Phocine Herpesvirus, PhoHV) and negative extraction control (STAR-buffer) were included in every qPCR run.

Descriptive and statistical analysis
Acquisition was defined as a negative pre-travel and a positive post-travel test result for the examined micro-organism, irrespective of signs and symptoms of illness. Acquisition rates were calculated for travellers with a negative pre-travel test: the population at risk. Traveller's diarrhoea (TD) was defined as three or more unformed stools within a 24-h period during travel.

Results
For one of the 100 randomly selected travellers a sample was missing, and for another one, DNA extraction failed. Therefore, 98 travellers were analysed. Distributions of the population characteristics were similar to the distributions in the full study population ( Table 2). The median age was 48 years (range 19e75) and 53% was female. Median travel duration was 20 (7e72) days. Most frequently visited sub-regions were South-Eastern Asia (n ¼ 30), South America (n ¼ 13) and Eastern Africa (n ¼ 10). Thirty-five percent of travellers (34/97) reported TD.
Pre-travel carriage rates for Blastocystis spp. and Dientamoeba fragilis were 31/98 (32%) and 19/98 (19%), respectively of which seven participants carried both species (Table 3). Pre-travel prevalences of the other tested species were much lower and ranged from 0 to 3%. A total of six travellers carried one or more pathogenic bacteria before travel. None of these six travellers had diarrhoeal complaints before travel.
Entamoeba histolytica, Cryptosporidium spp., Rotavirus (A, C), Adenovirus (40, 41, 52), Astrovirus and Sapovirus were neither detected in any of the pre-travel samples nor in the post-travel samples. Hepatitis E virus (HEV) was detected in 2 pre-travel samples only. Norovirus and Parechovirus were both acquired by two travellers and Enterovirus by one traveller, respectively. Of note, the traveller who acquired an Enterovirus travelled to China only and did not visit a polio-endemic area.

Discussion
This study shows that Dientamoeba fragilis and Blastocystis spp. were highly prevalent before travel. Blastocystis spp., Plesiomonas shigelloides, Dientamoeba fragilis and Shigella spp. were the most frequently acquired pathogens and acquisition of enteral viruses and HEV in this relatively small group of travellers was rare or nonexistent.
To our knowledge, travel associated acquisition of a wide spectrum of nineteen viral, bacterial and parasite species has not been reported before. The PCR's used for this study are validated and used for routine diagnostics and are considered very sensitive. However, as with all molecular tests, the detection of DNA does not necessarily mean that detected pathogens are viable. Also, the possibility exists that pathogens acquired during travel were already cleared by time the traveller returned and did the post travel sampling, and some brief intermittent acquisitions may therefore have been missed in this analysis. However, post-travel samples were provided within a mean of 3 days (range 0e19 days; median 2 days) and the mean duration between sample collection and processing in the laboratory was 2 days (range 1e23 days) so we do not regard this possibility as a significant source of bias. A major limitation of this study is the sample size that is probably too small to detect rarely acquired enteropathogens. Therefore, no firm conclusions on the acquisition of HEV and other rare pathogens can be drawn from this study. Also, an association between the acquisition of studied microorganisms and TD was not found, but because a limited number of travellers were tested, the statistical power to study this association was low. Unfortunately we were not able to study Cyclospora cayetanensis, which is considered an important parasitic cause of traveller's diarrhoea in some regions [4].
L€ a€ averi and co-workers performed a prospective study on bacterial TD pathogens in 382 Finnish travellers [5] and found acquisition of Salmonella spp. and Shigella/EIEC to be slightly lower than in the present study; namely 2.4% and 0.8% respectively; and Campylobacter was acquired by slightly more (6.8%) travellers. However, they did not test for Clostridium, Yersinia, Plesiomonas, parasites and viruses.
A non-prospective case-control study in 114 returning travellers with diarrhoea and 56 travellers without diarrhoea [6] found 19/ 170 (11.2%) to be positive by microscopy for Blastocystis spp. after travel and only one positive for Dientamoeba fragilis. Probably, these lower post-travel prevalences are the result of lower diagnostic sensitivity of microscopy compared to PCR. Interestingly, they

Giardia lamblia Parechovirus
Yersinia enterocolitica Rotavirus (A, C) Sapovirus found 10 cases of Giardia lamblia and 3 cases of Cryptosporidium spp., whereas in the present study only one acquisition of Giardia lamblia and no Cryptosporidium spp. was found. In their study, PCR for Norovirus was positive in 10.5% of the cases and 3.6% of the controls, whereas we found only 2.1% acquisition in the total studied population. The observed differences might be explained by a selection towards a more symptomatic population in the study by Paschke [6], because all of their participants presented as patients to the Department of Infectious Diseases of which two-third with diarrhoea [6].
Travel associated acquisition of HEV is a potential threat to blood safety [7], but no acquisitions were found in the present study. Accordingly, in a Dutch study, no seroconversions to anti-HEV were found in 1206 travellers to (sub)tropical countries [8]. An Australian study estimated the risk of acquiring HEV to be 0.01 to 18 per 10,000 travellers [7] indicating that our limited sample size likely is too small to reliably determine travel-associated acquisition of HEV. Furthermore, the incubation period of symptomatic HEV infections is approximately 40 days (range 2e10 weeks) [9], hence infections may be missed by analysis of samples that were collected shortly after return.
The low acquisition rates of enteric viruses during travel could indicate that the role of viruses as the cause of persisting TD is limited or that viral RNA or DNA is rapidly cleared before returning. For Norovirus however, virus is shed for a median of 28 days after inoculation in previously healthy persons [10] and for Rotavirus, median duration of shedding is approximately 3 weeks [11]. Since bacterial pathogens were acquired more often, they are likely to be more important as a cause of traveller's diarrhoea. For Blastocystis and Dientamoeba, pathogenicity of these parasites is under debate. To that end, a positive test in a symptomatic returning traveller must be interpreted with caution, keeping in mind that a substantial proportion probably would already have been carrier before travel.
Since Blastocystis spp were acquired relatively often, it would be interesting to study if acquisition could be assigned to specific travel destinations.  the 'population at risk'. b Denominator is the number travellers with and without TD.