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Journal of Clinical Microbiology, December 2005, p. 6126-6129, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6126-6129.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Noroviruses as a Cause of Traveler's Diarrhea among Students from the United States Visiting Mexico

GwangPyo Ko,1* Coralith Garcia,2,4 Zhi-Dong Jiang,2 Pablo C. Okhuysen,2,3 Jaime Belkind-Gerson,6 Roger I. Glass,7 and Herbert L. DuPont2,5

Seoul National University, School of Public Health, Institute of Health and Environment, Seoul, South Korea,1 University of Texas—Houston School of Public Health,2 Medical School,3 Baylor College of Medicine,4 St. Luke's Episcopal Hospital, Houston, Texas,5 Instituto Nacional de Salud Publica, Cuernavaca, Mexico,6 Centers for Disease Control and Prevention, Atlanta, Georgia7

Received 5 September 2005/ Accepted 9 September 2005


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ABSTRACT
 
Stool specimens from 124 international travelers with acute diarrhea were tested for the presence of enteropathogens. Noroviruses (NoVs) were the second most commonly identified enteric pathogen in diarrheal stool samples (21/124, 17%), exceeded only by enterotoxigenic Escherichia coli (50/106, 47%). This study indicates that NoV is an underappreciated cause of traveler's diarrhea.


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TEXT
 
Traveler's diarrhea (TD) is the most common medical complaint of international travelers (16, 24). The majority of TD cases are due to bacterial agents, which can be identified in up to 60% of cases (2, 14, 23, 25). A subset of TD cases appears to be caused by nonbacterial agents since antibiotic therapy fails to shorten illness duration (7, 8, 10). Also, in travelers with enteric disease, a viral type of clinical gastroenteritis syndrome is commonly seen (13). Noroviruses (NoVs) are recognized as the most common cause of acute nonbacterial gastroenteritis in persons over the age of 5 years and are responsible for more than 90% of nonbacterial gastroenteritis outbreaks (11). The aims of this study were to determine the frequency at which NoVs cause TD in U.S. students traveling to Mexico and to characterize the clinical symptoms associated with NoV infection.

Stool specimens from 124 students from the United States with acute diarrhea acquired during a short-term stay in Guadalajara, Mexico, during the summer of 2004 were collected and studied for the presence of enteric pathogens as previously described (9). Acute diarrhea was defined as described in previous studies (14, 23). The subjects were between 16 and 75 years of age (mean, 27 years; median, 22 years). Seventy (56%) were female, and 99 (80%) were white. Study subjects rated their signs or symptoms by severity scores according to the following scale: 1, mild irritation (no change in itinerary was required); 2, medium irritation (change in schedule was required); and 3, severe irritation (subject was disabled by the illness) (4). Informed consent was obtained from all participants, and the study was approved by University of Texas Committee for the Protection of Human Subjects.

Viral RNA was extracted from 10% fecal suspension in phosphate-buffered saline with the QIAamp viral RNA extraction kit (QIAGEN, Valencia, CA) by following the manufacturer's protocol and stored at –80°C. Two sets of primers for the capsid region of target viral genome were used as described previously (17). Briefly, the G1-SKF/G1-SKR and G2-SKF/G2-SKR primer sets were used for amplifying NoV genotypes I and II (GI and GII, respectively), generating a 330-bp and a 344-bp PCR product, respectively. The QIAGEN one-step reverse transcriptase PCR (RT-PCR) kit (QIAGEN, Valencia, CA) was then used for the RT-PCR. Viral RNA was reverse transcribed for 60 min at 42°C and then for 15 min at 95°C to activate the Taq polymerase. Thermocycling conditions for the reaction consisted of 40 cycles of 1 min each at 94°C, 1 min at 40°C, 1 min at 72°C, and a final extension of 72°C for 10 min. Amplified products were analyzed on ethidium bromide-stained 2% agarose gels. All RT-PCR products positive for NoV were purified by the QIAquick PCR purification kit (QIAGEN, Valencia, CA) and then sequenced using an ABI model 3730XL sequencer by a commercial company (SeqWright, Inc., Houston, TX).

The collected stool samples were processed in our field laboratory for enteric bacterial pathogens (enterotoxigenic E. coli [ETEC], enteroaggregative E. coli [EAEC], Salmonella spp., Shigella spp., Vibrio spp., Campylobacter jejuni, Yersinia enterocolitica, Aeromonas spp., and Plesiomonas shigelloides) and parasites (Giardia lamblia, Cryptosporidium species, and Entamoeba histolytica) by previously described methods (1, 14, 21).

Phylogenetic analyses were performed based on approximately 263-bp nucleic acid sequences of the RT-PCR-amplified NoV capsid region. Phylogenetic analyses were conducted using the MegAlign program from the LASERGENE software package (version 6.0; DNAstar, Madison, WI) and with MEGA version 3.0 software (http://www.megasoftware.net) (18). A multiple alignment was created using Clustal W software, and the neighbor-joining method was used for the construction of the phylogenetic tree.

Association between identified NoVs with clinical symptoms was measured by multivariate ordered probit regression analysis using STATA (College Station, TX) software.

At least one enteropathogen was found in 85 of 124 (69%) patients, and mixed infection (at least two enteric pathogens) was found in 28 (23%) (Table 1). A total of 21/124 (17%) patients were positive for NoV according to both RT-PCR and nucleic acid sequencing. Among the NoVs identified in 21 patients, 17 (81%) were positive for the GI group only, 3 (14%) were positive for GII only, and 1 (5%) was positive for both GI and GII. Among the other enteric pathogens identified in the tested specimens, the most frequently isolated organism was ETEC (45%, 54 of 119 subjects), followed by EAEC (15%, 18/119), Providencia spp. (8%, 9/119), Cryptosporidium parvum (5%, 5/119), Shigella spp. (4%, 4/119), Aeromonas sp. (1%, 1/119), and Plesiomonas shigelloides (1%, 1/119). Of the 54 ETEC isolates seen in this study, heat-labile enterotoxin (LT)-only and heat-stable enterotoxin (ST)-only toxins were found in 18 (33%) and 17 (31%), respectively. Both LT and ST toxins were found in 19 of 54 (35%) ETEC isolates studied. Salmonella spp., Campylobacter jejuni, Vibrio spp., and Giardia lamblia were not identified in the study.


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  		TABLE 1. Distribution of enteric pathogens in 124 cases of TD among U.S. students in Mexico during the summer of 2004

Mixed infections with NoV were common, and among 21 NoV-positive specimens, only 6 (29%) had NoV alone, whereas 15 (71%) were identified as coinfected with other enteric pathogens. The most frequent NoV-coinfecting organism was ETEC (11/21, 51%), with a variety of toxin types of LT only (4/21, 19%), ST only (3/21, 14%), ST/LT (4/21, 19%), EAEC (4/21, 19%), Providencia spp. (4/21, 19%), Aeromonas sp. (1/21, 5%), and Plesiomonas shigelloides (1/21, 5%). Multiple infections with three and four enteric pathogens were detected in 6 (5%) and 1 (1%) specimens, respectively.

The phylogenetic tree placed our 22 samples in two distinct genogroups, GI (n = 18) and GII (n = 4) (Fig. 1). Among the 18 GI NoV-positive specimens, strains 2, 3, and 12 had identical sequence fragments, and all four GII NoVs were identical.



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  		FIG. 1. Phylogenetic tree of NoV GI and GII isolates identified from TD patients in Mexico. The tree was constructed from partial sequences of the capsid region using Clustal W and the neighbor-joining method. The numbers in parentheses are the numbers of TD cases identified in this study.

We performed multivariate analysis (ordered probit regression analysis) to examine the association between clinical symptoms and identified enteric pathogens. In multivariate analysis, a clinical symptom was considered an ordinal-dependent variable (0, none; 1, mild; 2, moderate; 3, severe), and age, gender, and identified microbiological infections were considered independent variables (0, no infection; 1, infection). Both vomiting (P < 0.01) and nausea (P < 0.01) were positively associated with NoV infection. Vomiting was significantly associated with ST-producing ETEC (P = 0.04) as well, but the association was weaker than that seen with NoV infection. Abdominal pain was significantly associated with both infections by Shigella spp. (P < 0.01). Gas, fecal urgency, and tenesmus were not significantly associated with any pattern of microbial infection.

This study demonstrated that NoV was a major etiological enteric pathogen in U.S. students with traveler's diarrhea acquired in Mexico. Previous studies have suggested that bacterial enteric pathogens were responsible for up to 85% of traveler's diarrhea cases (6, 12, 14, 23). These previous studies either have demonstrated low rates of enteric viruses and parasites found in 5 to 10% of all cases or the agents were not sought (14, 15, 22, 23). Our data suggest that previous studies may have significantly underestimated the etiological role of NoV due to the limitation of detection methods. The role of NoV in traveler's illness could be even more important, because stool samples were collected only from travelers with acute diarrhea in this study and not from travelers with nausea and vomiting as the primary presenting symptoms.

High rates (>70%) of coinfections with other bacterial and parasite pathogens were found in the present study, as was the case in a recent study (3). In order to evaluate a causal relationship between NoV and clinical symptoms in a setting of a high rate of coinfection, we used a multivariate regression model, adjusting for infection by a variety of enteric pathogens. After adjusting for other enteric pathogens, we found that NoV was positively associated with both vomiting (P < 0.01) and nausea (P = 0.01). These results indicated that NoV was the etiological agent for this syndrome of gastroenteritis.

NoV GI was more prevalent than GII in this study. Our study coincided with the previous finding that only GI was found in 22 TD cases in Mexico (3). This finding is interesting because GII is reported as the predominant strain currently found in outbreaks worldwide (11, 19, 20).

In the past, a number of antibiotics and chemoprophylaxis trials were performed to control TD (7, 8, 10). In these studies, NoV infection was not tested by a sensitive detection method; however, antibiotic treatment was generally quite effective in the treatment and prevention of TD regardless of identified bacterial pathogens. Antibiotic treatment is currently recommended for treating TD and for targeted chemoprophylaxis (5). High rates of NoV infection would explain the failures of antibacterial drug therapy and chemoprophylaxis in these studies. We conclude that a significant proportion of TD cases without identified enteric pathogens in previous studies was likely caused by NoV.


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ACKNOWLEDGMENTS
 
We thank Robert Atmar, Mary Estes at Baylor College of Medicine, and Jan Vinje at the University of North Carolina at Chapel Hill for providing us with NoV-positive stool samples and helpful discussion for the study. We also thank the members of the field and laboratory teams in this study.

This work was supported in part by grant DK 56338 from Public Health Service, which funds the Texas Gulf Coast Digestive Diseases Center, grant NIH NCRR to the University of Texas General Clinical Research Center, and grants M01-RR-02558 and NIH NIAID R01 AI54948.

We do not have commercial or other associations that might pose a conflict of interest.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Environmental Health, School of Public Health, Seoul National University, 28 Yunkeun-Dong, Chongro-Ku, Seoul 110-799, South Korea. Phone: 82-2-3668-7881. Fax: 82-2-745-9104. E-mail: gko{at}snu.ac.kr. Back


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Journal of Clinical Microbiology, December 2005, p. 6126-6129, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6126-6129.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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