• 1

    Wolfe MS, 1990. Acute diarrhea associated with travel. Am J Med 88 :34S–37S.

  • 2

    Hill DR, 2000. Occurrence, and self-treatment of diarrhea in a large cohort of Americans traveling to developing countries. Am J Trop Med Hyg 62: 585–589.

    • Search Google Scholar
    • Export Citation
  • 3

    Yoder JS, Beach MJ, Centers for Disease Control, and Prevention, 2007. Cryptosporidiosis surveillance—United states, 2003–2005. MMWR 56: 1–10.

    • Search Google Scholar
    • Export Citation
  • 4

    Chappell CL, Okhuysen PC, 2002. Cryptosporidiosis. Curr Opin Infect Dis 15: 523–527.

  • 5

    Hashmey R, Genta RM, White AC Jr, 1997. Parasites and diarrhea. I: Protozoans and diarrhea. J Travel Med 4: 17–31.

  • 6

    Parisi MT, Tierno PT, 1995. Evaluation of new rapid commercial enzyme immunoassay for detection of Cryptosporidium oocysts in untreated stool specimens. J Clin Microbiol 33: 1963–1965.

    • Search Google Scholar
    • Export Citation
  • 7

    Marques FR, Cardoso LV, Cavasini CE, Almeida MC, Bassi NA, Almeida MT, Rossit AR, Machado RL, 2005. Performance of an immunoenzymatic assay for Cryptosporidium diagnosis of fecal samples. Braz J Infect Dis 9: 3–5.

    • Search Google Scholar
    • Export Citation
  • 8

    Morgan UM, Pallant L, Dwyer BW, Forbes DA, Rich G, Thompson RC, 1998. Comparison of PCR, and microscopy for detection of Cryptosporidium parvum in human fecal specimens: Clinical trial. J Clin Microbiol 36: 995–998.

    • Search Google Scholar
    • Export Citation
  • 9

    Balatbat AB, Jordan GW, Tang YJ, Silva J Jr, 1996. Detection of Cryptosporidium parvum DNA in human feces by nested PCR. J Clin Microbiol 34: 1769–1772.

    • Search Google Scholar
    • Export Citation
  • 10

    Kehl KS, Cicirello H, Havens PL, 1995. Comparison of four different methods for detection of Cryptosporidium species. J Clin Microbiol 33: 416–418.

    • Search Google Scholar
    • Export Citation
  • 11

    Bialek R, Binder N, Dietz K, Joachim A, Knobloch J, Zelck UE, 2002. Comparison of fluorescence, antigen, and PCR assays to detect Cryptosporidium parvum in fecal specimens. Diag Microbiol Infect Dis 43: 283–288.

    • Search Google Scholar
    • Export Citation
  • 12

    Zhu G, Marchewka MJ, Ennis JG, Keithly JS, 1998. Direct isolation of DNA from patient stools for polymerase chain reaction detection of Cryptosporidium parvum. J Infect Dis 177: 1443–1446.

    • Search Google Scholar
    • Export Citation
  • 13

    Miller WA, Gardner IA, Atwill ER, Leutenegger CM, Miller MA, Hedrick RP, Melli AC, Barnes NM, Conrad PA, 2006. Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus). J Microbiol Methods 65: 367–379.

    • Search Google Scholar
    • Export Citation
  • 14

    Sanchez-Vega JT, Tay-Zavala J, Aguilar-Chiu A, Ruiz-Sanchez D, Malagon F, Rodriguez-Covarrubias JA, Ordonez-Martinez J, Calderon-Romero L, 2006. cryptosporidiosis and other intestinal protozoan infections in children less than one year of age in Mexico City. Am J Trop Med Hyg 75: 1095–1098.

    • Search Google Scholar
    • Export Citation
  • 15

    Javier Enriquez F, Avila CR, Ignacio Santos J, Tanaka-Kido J, Vallejo O, Sterling CR, 1997. Cryptosporidium infections in Mexican children: Clinical, nutritional, enteropathogenic, and diagnostic evaluations. Am J Trop Med Hyg 56: 254–257.

    • Search Google Scholar
    • Export Citation
  • 16

    Solorzano-Santos F, Penagos-Paniagua M, Meneses-Esquivel R, Miranda-Novales MG, Leanos-Miranda B, Angulo-Gonzalez D, Fajardo-Gutierrez A, 2000. Cryptosporidium parvum infection in malnourished and non malnourished children without diarrhea in a Mexican rural population. Rev Invest Clin 52: 625–631.

    • Search Google Scholar
    • Export Citation
  • 17

    Bouckenooghe AR, Jiang ZD, De La Cabada FJ, Ericsson CD, DuPont HL, 2002. Enterotoxigenic Escherichia coli as cause of diarrhea among Mexican adults and US travelers in Mexico. J Travel Med 9: 137–140.

    • Search Google Scholar
    • Export Citation
  • 18

    Taylor DN, Bourgeois AL, Ericsson CD, Steffen R, Jiang ZD, Halpern J, Haake R, Dupont HL, 2006. A randomized, double-blind, multicenter study of rifaximin compared with placebo and with ciprofloxacin in the treatment of travelers’ diarrhea. Am J Trop Med Hyg 74: 1060–1066.

    • Search Google Scholar
    • Export Citation
  • 19

    Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, Kazmierczak JJ, Addiss DG, Fox KR, Rose JB, 1994. A massive out break in Milwaukee of Cryptospridium infection transmitted through the public water supply. N Engl J Med 331: 161–167.

    • Search Google Scholar
    • Export Citation
  • 20

    Weitzel T, Wichmann O, Muhlberger N, Reuter B, Hoof HD, Jelinek T, 2006. Epidemiological, and clinical features of travel-associated cryptosporidiosis. Clin Microbiol Inf 12: 921–924.

    • Search Google Scholar
    • Export Citation
  • 21

    Mohamed JA, DuPont HL, Jiang ZD, Belkind–Gerson J, Figueroa JF, Armitige LY, Tsai A, Nair P, Martinez–Sandoval FJ, Guo DC, Hayes P, Okhuysen PC, 2007. A novel single–nucleotide polymorphism in the lactoferrin gene is associated with susceptibility to diarrhea in North American travelers to Mexico. Clin Infect Dis 44: 945–952.

    • Search Google Scholar
    • Export Citation
  • 22

    DuPont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski W, 1995. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 332: 855–859.

    • Search Google Scholar
    • Export Citation
  • 23

    Chappell CL, Okhuysen PC, Sterling CR, DuPont HL, 1996. Cryptosporidium parvum: Intensity of infection, and oocyst excretion patterns in healthy volunteers. J Infect Dis 173: 232–236.

    • Search Google Scholar
    • Export Citation
  • 24

    Okhuysen PC, Chappell CL, Crabb JH, Sterling CR, DuPont HL, 1999. Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J Infect Dis 180: 1275–1281.

    • Search Google Scholar
    • Export Citation
  • 25

    Llorente MT, Clavel A, Goñi MP, Varea M, Seral C, Becerril R, Suarez L, Gómez–Lus R, 2007. Genetic characterization of Cryptosporidium species from humans in Spain. Parasitol Int 56: 201–205.

    • Search Google Scholar
    • Export Citation
  • 26

    Xiao L, Ryan UM, 2004. Cryptosporidiosis: an update in molecular epidemiology. Curr Opin Infect Dis 17: 483–490.

  • 27

    Alves M, Xiao L, Sulaiman I, 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41: 2744–2747.

    • Search Google Scholar
    • Export Citation
  • 28

    McLauchlin J, Amar C, Pedraza–Diaz S, Nichols GL, 2000. Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping Cryptosporidium spp. in 1,705 fecal samples from humans, and 105 fecal samples from livestock animals. J Clin Microbiol 38: 3984–3990.

    • Search Google Scholar
    • Export Citation
  • 29

    Fretz R, Svoboda P, Ryan UM, 2003. Genotyping of Cryptosporidium spp. isolated from human stool samples in Switzerland. Epidemiol Infect 131: 663–667.

    • Search Google Scholar
    • Export Citation
  • 30

    Tiangtip R, Jongwutiwes S, 2002. Molecular analysis of Cryptosporidium species isolated from HIV–infected patients in Thailand. Trop Med Int Health 7: 357–364.

    • Search Google Scholar
    • Export Citation
  • 31

    Yagita K, Izumiyama S, Tachibana H, Masuda G, Iseki M, Furuya K, Kameoka Y, Kuroki T, Itagaki T, Endo T, 2001. Molecular characterization of Cryptosporidium isolates obtained from human, and bovine infections in Japan. Parasitol Res 87: 950–955.

    • Search Google Scholar
    • Export Citation
  • 32

    Nichols RA, Campbell BM, Smith HV, 2003. Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters, and drinking waters by using a modified nested PCR–restriction fragment length polymorphism assay. Appl Environ Microbiol 69: 4183–4189.

    • Search Google Scholar
    • Export Citation
  • 33

    Learmonth JJ, Ionas G, Pita AB, Cowie RS, 2003. Identification and genetic characterization of Giardia, and Cryptosporidium strains in humans, and dairy cattle in the Waikato Region of New Zealand. Water Sci Technol 47: 21–26.

    • Search Google Scholar
    • Export Citation
  • 34

    Cama VA, Bern C, Sulaiman IM, Gilman RH, Ticona E, Vivar A, Kawai V, Vargas D, Zhou L, Xiao L, 2003. Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru. J Eukaryot Microbiol 50: 531–533.

    • Search Google Scholar
    • Export Citation
  • 35

    Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W, Cabrera L, Gilman RH, Lal AA, 2001. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis 183: 492–497.

    • Search Google Scholar
    • Export Citation
  • 36

    Bushen OY, Kohli A, Pinkerton RC, Dupnik K, Newman RD, Sears CL, Fayer R, Lima AA, Guerrant RL, 2007. Heavy cryptosporidial infections in children in northeast Brazil: Comparison of Cryptosporidium hominis, and Cryptosporidium parvum. Trans R Soc Trop Med Hyg 101: 378–384.

    • Search Google Scholar
    • Export Citation
  • 37

    Gatei W, Greensill J, Ashford RW, Cuevas LE, Parry CM, Cunliffe NA, Beeching NJ, Hart CA, 2003. Molecular analysis of the 18S rRNA gene of Cryptosporidium parasites from patients with or without human immunodeficiency virus infections living in Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. J Clin Microbiol 41: 1458–1462.

    • Search Google Scholar
    • Export Citation
  • 38

    Di Giovanni GD, Betancourt WQ, Hernandez J, Assadian NW, Flores Margez JP, Lopez EJ, 2006. Investigation of potential zooanthroponotic transmission of cryptosporidiosis, and giardiasis through agricultural use of reclaimed wastewater. Int J Environ Health Res 16: 405–418.

    • Search Google Scholar
    • Export Citation
  • 39

    Gasser RB, El–Osta YG, Chalmers RM, 2003. Electrophoretic analysis of genetic variability within Cryptosporidium parvum from imported, and autochthonous cases of human cryptosporidiosis in the United Kingdom. Appl Environ Microbiol 69: 2719–2730.

    • Search Google Scholar
    • Export Citation
  • 40

    Warren KS, Swan RA, Morgan–Ryan UM, 2003. Cryptosporidium muris infection in bilbies (Macrotis lagotis). Aust Vet J 81: 739–741.

  • 41

    Fayer R, Morgan U, Upton SJ, 2000. Epidemiology of Cryptosporidium: Transmission, detection, and identification. Int J Parasitol 30: 1305–1322.

    • Search Google Scholar
    • Export Citation
  • 42

    Kostrzynska M, Sankey M, Haack E, Power C, Aldom JE, Chagla AH, Unger S, Palmateer G, Lee H, Trevors JT, De Grandis SA, 1999. Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples. J Microbiol Methods 35: 65–71.

    • Search Google Scholar
    • Export Citation
  • 43

    Mayer CL, Palmer CJ, 1996. Evaluation of PCR, nested PCR and fluorescent antibodies for detection of Giardia, and Cryptosporidium species in wastewater. Appl Environ Microbiol 62: 2081–2085.

    • Search Google Scholar
    • Export Citation
  • 44

    Sturbaum GD, Reed C, Hoover PJ, Jost BH, Marshall MM, Sterling CR, 2001. Species–specific, nested PCR–restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts. Appl Environ Microbiol 67: 2665–2668.

    • Search Google Scholar
    • Export Citation
Past two years Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 207 114 36
PDF Downloads 28 21 4
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Epidemiology of Cryptosporidiosis in North American Travelers to Mexico

Parvathy NairThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Parvathy Nair in
Current site
Google Scholar
PubMed
Close
,
Jamal A. MohamedThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Jamal A. Mohamed in
Current site
Google Scholar
PubMed
Close
,
Herbert L. DuPontThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Herbert L. DuPont in
Current site
Google Scholar
PubMed
Close
,
Jose Flores FigueroaThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Jose Flores Figueroa in
Current site
Google Scholar
PubMed
Close
,
Lily G. CarlinThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Lily G. Carlin in
Current site
Google Scholar
PubMed
Close
,
Zhi-Dong JiangThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Zhi-Dong Jiang in
Current site
Google Scholar
PubMed
Close
,
Jaime Belkind-GersonThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Jaime Belkind-Gerson in
Current site
Google Scholar
PubMed
Close
,
Francisco G. Martinez-SandovalThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Francisco G. Martinez-Sandoval in
Current site
Google Scholar
PubMed
Close
, and
Pablo C. OkhuysenThe University of Texas Health Science Center at Houston Medical School and School of Public Health, Houston, Texas; Baylor College of Medicine, Houston, Texas; Instituto Nacional de Salud Publica, Cuernavaca, Mexico; Universidad Autonoma de Guadalajara, Jalisco, Mexico

Search for other papers by Pablo C. Okhuysen in
Current site
Google Scholar
PubMed
Close

We studied 1,179 North American travelers who visited Mexico from 2005 to 2007. Travelers’ diarrhea (TD) was reported by 521 (44%) participants. Among subjects with TD, 218 cases were examined for cryptosporidiosis by polymerase chain reaction (PCR) and enzyme-linked immunoassays (ELISA). There were 14 (6%) cases of cryptosporidiosis and 141 cases (64%) of bacterial diarrhea. Compared with bacterial diarrhea, a longer stay in Mexico was a risk factor for cryptosporidiosis. Additionally, Cryptosporidium cases passed greater number of watery stools (P < 0.05), suffered more episodes of diarrhea (P ≤ 0.05), and were more likely to experience tenesmus (P ≤ 0.05) compared with bacterial causes of TD. ELISA detected seven (3%) cases of Cryptosporidium, whereas PCR identified an additional seven cases (6%). Speciation by 18SrRNA sequencing showed that 13 cases were caused by C. parvum and only 1 case was caused by C. hominis. ELISA showed a sensitivity of 50% and specificity of 100% compared with PCR.

INTRODUCTION

Travelers’ diarrhea (TD) affects 20–50% of persons from developed nations who visit developing countries.1 Although bacterial pathogens as a group are the most common cause of acute diarrhea in travelers, parasites have been associated with persistent and chronic diarrhea. Common causal organisms of parasitic diarrhea in travelers are Cryptosporidium spp., Giardia spp., and Entamoeba spp.13 Travel acquired parasitic infections are often insidious in onset, and because of long incubation periods, are often diagnosed on return to the original country of departure.

In adults, seropositivity to Cryptosporidium is an indicator of past or recent infection and is also a marker of partial protection against the parasite.4,5 The rate of seropositivity in US adults is ~25% in contrast to 64% among the Latin American urban population.4,5

Over the past two decades, the methods for Cryptosporidium detection have advanced from simple acid-fast staining to enzyme-linked immunoassays (ELISA), and immunofluorescent assays (IFA) to molecular techniques based on polymerase chain reaction (PCR) amplification of specific gene loci.4 Fecal ELISA is a quick, easy, and convenient method to test large numbers of stools specimens that has found wide application in clinical settings.6,7 Several studies have documented a high sensitivity for DNA PCR-based methods in detecting Cryptosporidium in fecal samples.813 Although technically more demanding, the nested PCR based on the 18S rRNA gene followed by direct sequencing offers the added advantage of characterizing the infecting organism at the species level.13

Cryptosporidiosis is a common disease in Mexican children, as evidenced by a study done in infants with severe diarrhea, where 41% were found to be infected with Cryptosporidium.14 In many instances, the infection is asymptomatic, particularly in older children.15,16 In a previous study conducted by our group on 127 US travelers to Mexico and 183 Mexican adults with diarrhea, cryptosporidiosis as determined by ELISA was more commonly seen in US travelers than in Mexican residents (3% versus < 1%).17 Another study found a 7%Cryptosporidium isolation rate among 87 adult travelers to Mexico.18 Although epidemiologic features of Cryptosporidium in human outbreaks has been well described,3,19 there are few studies on the clinical characteristics, epidemiology, and genotypes of Cryptosporidium that affect international travelers.20

The aim of our study was to characterize the clinical features and risk factors associated with cryptosporidiosis among US travelers to Mexico. We compared the detection rate of Cryptosporidium with ELISA to the detection rate with PCR followed by direct sequencing.

MATERIALS AND METHODS

Human subjects.

Participants were travelers from North America who visited the Mexican cities of Cuernavaca and Guadalajara between June 2005 and January 2007 to learn the Spanish language. The eligibility for participation entailed a minimum age of 16 years after parental consent or being an adult with informed consent, good health, and a stay of at least 5 days in Mexico. Subjects with known pre-existing irritable bowel syndrome, lactose intolerance, or pregnancy or those taking antibiotic prophylaxis for TD were excluded from the study.

Recruitment and management.

Students were enrolled within 72 hours of arrival at Mexico. The cohort was observed for the incidence, severity, and etiology of diarrhea while in Mexico. Participants who had soft unformed or watery stools at a frequency of three or more times in a 24-hour period were asked to contact the study personnel and report to the clinic within 72 hours for stool sample collection. Fecal samples from travelers matched for age, sex, and duration of stay were studied as a control group. This study was approved by the University of Texas Committee for the Protection of Human Subjects.

Laboratory procedures.

For detection of enteric pathogens, stools were cultured for Shigella spp., Salmonella spp., Providencia spp., Plesiomonas spp., Serratia spp., and Campylobacter spp. Enterotoxigenic Escherichia coli was identified by colony PCR or colony hybridization, whereas enteroaggregative E. coli were detected by HEp2 cell adherence assay or by colony PCR. Stool samples were examined for the presence of cryptosporidiosis by a commercially available microplate ELISA kit (Remel ProSpecT, Lenexa, KS) following the manufacturer’s instructions.

PCR for cryptosporidiosis.

An aliquot of stools was frozen for PCR studies. DNA extraction from stools (2 mg of frozen stools or 200 μL of liquid stools) was carried out with QIA amp DNA stool mini kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The small subunit of the C. parvum 18S rRNA gene was amplified by a two-step nested PCR protocol. Amplification of a PCR product of 833 bp (GenBank accession no. X64341) was done with the forward primer CryrRNAF3 (5′-GGAAGGGTTGTATTTATTA-GATAAAG-3′), and the reverse primer CryrRNAR3 (5′-AAGGAGTAAGGAACAACCTCCA-3′) for the first PCR.13 The first PCR was performed in 1:10 reaction volume of 1× Hot Star Taq master mix buffer using 0.5 μmol/L of each primer, 1:2 dilution of 0.05 μg/μL bovine serum albumin, and 2 μL of 1:2 dilution of stool DNA as a template. The first PCR cycling steps consisted 35 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, and a final extension step at 72°C for 4 minutes. For the second amplification step, the PCR nested primers CryNestF1 (5′-TTCCAGCTCCAATAGCGTATA-3′), and CryNestR1 (5′-TCACCTCTGACTGTTAAATAC-3′) were used. An amplicon of 308 bp was produced in this reaction. The same quantities of reagents were used during the second PCR as were used in the first PCR reaction, except that a 1:2 dilution of the primary PCR mixture was used as a template. The nested reaction consisted of 35 cycles that were run after an initial denaturation step at 94°C for 1 minute, primer annealing at 58°C for 1 minute, and DNA extension at 72°C for 1 minute; these cycles were followed by a final incubation at 72°C for 4 minutes. The amplified product was separated by electrophoresis on a 1.5% agarose gel and visualized under a UV transilluminator after incubation with ethidium bromide. PCR products from the nested reaction used CryNestF1 (5′-TTCCAGCTCCAATAGCGTATA-3′) and CryNestR1 (5′-TCACCTCTGACTGTTAAATAC-3′) primers, which allow for the amplification of polymorphic regions at nucleotide position 639–656, and 689–699. An ABI 3130×1 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for sequencing.

Statistical analysis.

Univariate analyses were performed using SPSS software, version 15 (SPSS, Chicago, IL). The χ2 test was used to determine association between socio-demographic, clinical, and microbiological data. The cases of cryptosporidiosis with bacterial co-infection were analyzed as cryptosporidiosis. Sensitivity and specificity were used to determine the diagnostic performance of ELISA using PCR followed by sequencing as the gold standard.

RESULTS

Among the 1,179 eligible students who traveled to Mexico between June 2005 to January 2007, 521 (44.19%) developed TD (Table 1). There was no difference in the proportion of TD by sex. US travelers with Asian ancestry displayed a lesser predisposition to develop diarrhea in Mexico. The risk for TD was associated to traveler’s age, length of stay, and season of travel. There was a 2% decrease in risk of diarrhea for every 1-year increase in age (odds ratio [OR], 0.98; 95% confidence interval [CI], 0.97–0.99; P < 0.001) and a 3% increase in risk for diarrhea with every additional day of stay in Mexico (OR, 1.03; 95% CI, 1.02–1.05; P < 0.001). We also noted that travel during months of March to September was associated with an increased frequency of diarrhea compared with travel during October to February (OR, 2.19; 95% CI, 1.55–3.07; P < 0.001).

A subset of 218 travelers with TD, in whom Cryptosporidium ELISA and fecal PCR for Cryptosporidium were done, was analyzed further in this study. Overall, Cryptosporidium was detected in 14 (6%) stools examined. ELISA detected seven (3%) cases of cryptosporidiosis. In contrast, an additional seven cases were identified by PCR (P = 0.12; Table 2). Bacterial pathogens were isolated from 141 (66%) cases. Enteroaggregative E. coli (N = 102; 47%) was the predominant pathogen, followed by enterotoxigenic E. coli (N = 62; 28%). Other bacterial pathogens isolated were Salmonella spp. (N = 7; 3%), Campylobacter spp. (N = 2; < 1%), and Pleasiomonas spp. (N = 1; 0.5%). Multiple bacterial pathogens were identified from 33 (15.1%) of stools examined.

No significant differences were noted in terms of age, race, ethnicity, or sex between bacterial cases and Cryptosporidium diarrhea. Of interest, the proportion of men with cryptosporidiosis was higher in the bacterial subgroup (43% versus 28%; P = 0.23). A longer period of stay in Mexico was significantly associated with an increased risk of TD caused by Cryptosporidium (OR, 1.07; 95% CI, 1.03–1.13; P = 0.008) compared with bacterial diarrhea. Most cases of cryptosporidiosis and bacterial TD occurred between June and August.

The clinical characteristics of the TD cases categorized by cause of diarrhea are depicted in Table 3. Students infected with Cryptosporidium had greater numbers of episodes (P = 0.05) and passed more unformed (P = 0.07) and watery stools (P = 0.02) than TD caused by bacterial agents. The time from day of arrival in Mexico to the onset of diarrhea after arrival in Mexico was similar in both groups. Among cryptosporidiosis cases, abdominal pain (N = 14; 100%) was the most common complaint. Other frequently experienced clinical features were flatulence (N = 12; 86%), urgency (N = 11; 79%), nausea (N = 8; 57%), and tenesmus (N = 7; 50%). Vomiting (N = 2; 14%) and fever (N = 3; 21%) were relatively infrequent symptoms. Presenting symptoms were similar for cases with Cryptosporidium and bacterial infection except for tenesmus (P = 0.05), which was more common in cases of Cryptosporidium.

Results of the DNA sequencing of the 18SrRNA gene showed that 13 of 14 (93%) cases were infected with Cryptosporidium parvum and only one with Cryptosporidium hominis. Using PCR followed by genetic sequencing as the gold standard, the sensitivity of ELISA was 50% and specificity was 100% (Table 3). The positive predictive value of ELISA was 100%, and the negative predictive value was 96.7%. Thirty-one matched controls were studied by ELISA and PCR; none of the control samples were positive by ELISA. Three C. parvum cases were identified among healthy controls by PCR.

DISCUSSION

In this prospective study done in US travelers to Mexico, the incidence of cryptosporidiosis was found to be 6.4%, which was similar to rate of cryptosporidiosis in Mexican children1416 and comparable to the previous series of travelers with similar characteristics.17,18 Consistent with prior observations, we found that TD was more frequent among subjects who had a longer length of stay, were younger in age, and traveled during the summer season. Our data are consistent with existing literature that describes the above factors as known risk factors for TD.1,2,21

Cryptosporidiosis manifested with greater numbers of episodes and watery stools compared with bacterial diarrhea. This may be explained by the use of empiric antibiotics to treat travelers with diarrhea. Symptoms experienced included abdominal pain, flatulence, and nausea in addition to diarrhea in frequencies that correspond with observations in other studies.20,23 In our study, 10 of the 14 cases of Cryptosporidium diarrhea (71.4%) presented with bacterial co-infection. The common bacterial agents co-infecting being enteroaggregative E. coli and enterotoxigenic E. coli.

Our findings are also consistent with a previous report on travel-associated cryptosporidiosis in German travelers, which found cryptosporidiosis to be associated with longer length of stay abroad.20 In our study, all cases of Cryptosporidium occurred in travelers who remained in Mexico for at least 2 weeks (P = 0.02). This likely reflects the longer incubation period needed for Cryptosporidium compared with bacterial causes of diarrhea. However, we also found that the mean time to onset for diarrhea after arrival in Mexico was comparable in both groups. A possible reason could be the high proportion of concomitant bacterial infections among the Cryptosporidium cases. The mean incubation period of Cryptosporidium is ~9 days,2224 and some participants may have acquired the parasite on their last days of the visit. Hence, our study may underestimate the true number of cases of travel related cryptosporidiosis.

The distribution of the infecting species of Cryptosporidium varies according to the type of population studied and the geographic location.2540 Studies have noted that differences in predominant infecting Cryptosporidium species relate to the level of urbanization, season studied, the immune status, and age of subjects considered.26,39,40 Studies in Europe and New Zealand have reported a predominance of the C. parvum species among human infections.2617,33 In other series, a greater proportion of C. hominis has been reported from Thailand, the United States, and Japan.26,2832 C. hominis has been frequently detected among children and HIV-positive adults in other Latin American studies.3438 The high proportion of C. parvum compared with C. hominis seen in our study suggests that the source of infection may have been zoonotic in origin, although the possibility of person to person transmission cannot be ruled out. As far as we know, this is the first study on the speciation of infecting Cryptosporidium species acquired by US travelers to Mexico. Our subjects were healthy adult travelers, whereas previous Latin American studies focused on children and HIV-positive men.26,34,36 It is possible that the risk factors and modes of transmission are different among these study populations. There is a need for more extensive molecular studies with a larger number of cases to unravel the relationship between the distribution, epidemiology, and transmission routes of Cryptosporidium in travelers.

Cryptosporidium ELISA showed a higher specificity and sensitivity compared with conventional staining techniques.6,7,10 Because oocyst wall antigens are well conserved among most species in the Cryptosporidium genus, a limitation of the ELISA is its inability to identify the different infecting species.13,40 Although previous studies have noted high sensitivity rates for fecal PCR,1316 the detection of Cryptosporidium spp. in environmental samples has a low sensitivity.4143 The presence of bilirubin, bile salts, and other contaminants in fecal samples can inhibit the DNA extension in PCR reactions, making the detection less accurate.13 We minimized this by adopting a DNA extraction protocol that remove DNases and facilitates the downstream enzymatic polymerase reaction (QIAamp; Qiagen). We also negated the likelihood of contamination in our study by the use of multiple negative controls during the reactions. The probability of underdetection was also decreased by the use of nested PCR, a method sensitive enough to detect < 1 pg of C. parvum DNA in human stool.12

Our study confirmed that the infection rates of cryptosporidiosis are underestimated by Cryptosporidium fecal ELISA. ELISA detected only 50% of the positive cases that were identified by PCR followed by genetic sequencing. A possible explanation could be that the parasite burden was lower in PCR-only positive cases, because the threshold for detection by PCR is thought to be ~10–50 oocysts/g of stool in contrast to the 103–104 parasites needed for ELISA.710,44

Being a highly infectious and ubiquitous parasite that can cause large outbreaks in healthy, immunocompromised persons and the elderly, a quick and reliable diagnosis along with knowledge of the infecting species are of great importance in the management of cryptosporidiosis. The use of molecular tools for the diagnosis of cryptosporidiosis coupled with the knowledge of the parasite’s epidemiology in travelers will promote the understanding of the factors influencing species transmission pathways and host susceptibility factors, which can aid in more targeted and successful interventions for prevention and control of cryptosporidiosis.

Table 1.

Demographic features of US travelers to Mexico and risk factors for acquiring travelers’ diarrhea

Healthy ( N = 658)Diarrhea (N = 521)
CharacteristicsNPercentNPercentOR (95% CI)P value
* Student t test: means ± SD.
Sex
    Females (N = 861)49174.637071.0(Ref)
    Males (N = 318)16725.415129.01.2 (0.9–1.6)0.19
Ethnicity
    Hispanics (N = 133)7611.65710.9(Ref)
    Non-Hispanics (N = 1046)58288.546489.11.1 (0.7–1.5)0.74
Race
    Whites (N = 1056)58188.347591.2(Ref)
    African Americans (N = 71)446.7275.20.8 (0.5–1.2)0.26
    Asians (N = 29)213.281.50.5 (0.2–1.1)0.07
    Others (N = 23)121.8112.11.1 (0.5–2.5)0.78
Season
    October–February (N = 187)13320.25410.4(Ref)
    March–September (N = 992)52579.846789.62.2 (1.6–3.1)< 0.001
Length of stay (days)21.4 ± 9.1*24.3 ± 9.6*1.03 (1.02–1.05)< 0.001
Age (years)32.5 ± 14.3*29.6 ± 12.8*0.98 (0.97–0.99)< 0.001
Table 2.

Comparison of PCR followed by genotyping versus ELISA for Cryptosporidium detection

MethodNo of Samples ExaminedNo of positives detectedSensitivity(%)†Specificity(%)‡Positive predictive value (%)§Negative predictive value (%)¶
* PCR followed by genotype sequencing.
† Calculated as [number of true positives/(number of true positives + number of false negatives)].
‡ Calculated as [number of true negatives/(number of true negatives + number of false positives)].
§ Calculated as [number of true positives/(number of true positives + number of false positives)].
¶ Calculated as [number of true negatives/(number of true negatives + number of false negatives)].
PCR*21814100100100100
ELISA21875010010097.6
Table 3.

Epidemiologic and clinical features of bacterial TD compared with Cryptosporidium TD

Bacterial diarrhea (N = 141)Cryptosporidiosis* ( N = 14)
VariableNPercentNPercentP value
* Includes the cases of cryptosporidiosis only and cryptosporidiosis with bacterial co-infection. Cryptosporidiosis cases in which bacterial entero-pathogens were identified were counted once in the cryptosporidiosis group for analysis and were not included in the bacterial infection subgroup.
†Student t test: mean (95% CI).
Clinical features
    Episodes
        Single (N = 114)10776.4757.10.23
        Multiple (N = 41)3423.6742.9
    Total unformed stools/100 days of stay84.3 (72.2–96.4)†121.1 (86.2–155.9)†0.07
    Total watery stools/100 days of stay40.8 (31.6–50.0)†76.4 (39.4–113.3)†0.02
    Onset after arrival in Mexico (days)10.6 (9.3–11.8)†9.3 (5.4–13.16)†0.53
Symptoms
    Abdominal pain (N = 130)11682.314100.0
    Flatulence ( N = 118)10675.21285.70.75
    Nausea (N = 92)8459.6857.11
    Vomiting (N =33)3122.0214.30.73
    Urgency (N = 116)10574.51178.61
    Tenesmus (N = 40)3323.4750.00.05
    Fever (N = 15)128.537.11

*

Address correspondence to Pablo C. Okhuysen, Division of Infectious Diseases, The University of Texas Medical School, 6431 Fannin Street, MSB 2.112, Houston, TX 77030. E-mail: Pablo.c.okhuysen@uth.tmc.edu

Presented in part at the Annual Meeting of the Infectious Diseases Society of America (IDSA), October 5, 2007, San Diego, CA.

Authors’ addresses: Parvathy Nair, Jamal A. Mohamed, Jose Flores Figueroa, and Lily G. Carlin, 6431 Fannin, MSB 7.510, Houston, TX 77030. Herbert L. DuPont and Zhi-Dong Jiang, 1200 Hermann Pressler Dr., Houston, TX 77030. Jaime Belkind-Gerson, Av. Universidad #655, Col. Santa maria Ahauacatitlan CP 62508, Cuernavaca, Morelos, Mexico. Francisco G. Martinez-Sandoval, Av. Patria 1201, Lomas del Valle, 3a seccion, Apartado postal 1 440, CP 44100, Guadalajara, Jalisco, Mexico. Pablo C. Okhuysen, Division of Infectious Diseases, The University of Texas Medical School, 6431 Fannin Street, MSB 2.112, Houston, TX 77030, Tel: 713-500-6736, Fax: 713-500-5495, E-mail: Pablo.c.okhuysen@uth.tmc.edu.

Acknowledgments: We thank Dorothy Ruelas, RN, Judy Guillen, RN, Margaret DuPont, MS, Jackie Vaca, RN, and the administration and staff of Universidad Internacional in Cuernavaca, Morelos, for assistance with this project.

Financial support: This work was supported by the following sources: NIHDMID R01 AI54948–01 (PCO), to the Center for Clinical and Translational Sciences of the University of Texas Health Science Center at Houston, NIHNCRR ULIRR024148 (CTSA), of the University of Texas Medical School at Houston, and DK56338, which funds the Texas Gulf Coast Digestive Diseases Center.

Disclosure: The authors report that there are no conflicts of interest related to this work.

REFERENCES

  • 1

    Wolfe MS, 1990. Acute diarrhea associated with travel. Am J Med 88 :34S–37S.

  • 2

    Hill DR, 2000. Occurrence, and self-treatment of diarrhea in a large cohort of Americans traveling to developing countries. Am J Trop Med Hyg 62: 585–589.

    • Search Google Scholar
    • Export Citation
  • 3

    Yoder JS, Beach MJ, Centers for Disease Control, and Prevention, 2007. Cryptosporidiosis surveillance—United states, 2003–2005. MMWR 56: 1–10.

    • Search Google Scholar
    • Export Citation
  • 4

    Chappell CL, Okhuysen PC, 2002. Cryptosporidiosis. Curr Opin Infect Dis 15: 523–527.

  • 5

    Hashmey R, Genta RM, White AC Jr, 1997. Parasites and diarrhea. I: Protozoans and diarrhea. J Travel Med 4: 17–31.

  • 6

    Parisi MT, Tierno PT, 1995. Evaluation of new rapid commercial enzyme immunoassay for detection of Cryptosporidium oocysts in untreated stool specimens. J Clin Microbiol 33: 1963–1965.

    • Search Google Scholar
    • Export Citation
  • 7

    Marques FR, Cardoso LV, Cavasini CE, Almeida MC, Bassi NA, Almeida MT, Rossit AR, Machado RL, 2005. Performance of an immunoenzymatic assay for Cryptosporidium diagnosis of fecal samples. Braz J Infect Dis 9: 3–5.

    • Search Google Scholar
    • Export Citation
  • 8

    Morgan UM, Pallant L, Dwyer BW, Forbes DA, Rich G, Thompson RC, 1998. Comparison of PCR, and microscopy for detection of Cryptosporidium parvum in human fecal specimens: Clinical trial. J Clin Microbiol 36: 995–998.

    • Search Google Scholar
    • Export Citation
  • 9

    Balatbat AB, Jordan GW, Tang YJ, Silva J Jr, 1996. Detection of Cryptosporidium parvum DNA in human feces by nested PCR. J Clin Microbiol 34: 1769–1772.

    • Search Google Scholar
    • Export Citation
  • 10

    Kehl KS, Cicirello H, Havens PL, 1995. Comparison of four different methods for detection of Cryptosporidium species. J Clin Microbiol 33: 416–418.

    • Search Google Scholar
    • Export Citation
  • 11

    Bialek R, Binder N, Dietz K, Joachim A, Knobloch J, Zelck UE, 2002. Comparison of fluorescence, antigen, and PCR assays to detect Cryptosporidium parvum in fecal specimens. Diag Microbiol Infect Dis 43: 283–288.

    • Search Google Scholar
    • Export Citation
  • 12

    Zhu G, Marchewka MJ, Ennis JG, Keithly JS, 1998. Direct isolation of DNA from patient stools for polymerase chain reaction detection of Cryptosporidium parvum. J Infect Dis 177: 1443–1446.

    • Search Google Scholar
    • Export Citation
  • 13

    Miller WA, Gardner IA, Atwill ER, Leutenegger CM, Miller MA, Hedrick RP, Melli AC, Barnes NM, Conrad PA, 2006. Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus). J Microbiol Methods 65: 367–379.

    • Search Google Scholar
    • Export Citation
  • 14

    Sanchez-Vega JT, Tay-Zavala J, Aguilar-Chiu A, Ruiz-Sanchez D, Malagon F, Rodriguez-Covarrubias JA, Ordonez-Martinez J, Calderon-Romero L, 2006. cryptosporidiosis and other intestinal protozoan infections in children less than one year of age in Mexico City. Am J Trop Med Hyg 75: 1095–1098.

    • Search Google Scholar
    • Export Citation
  • 15

    Javier Enriquez F, Avila CR, Ignacio Santos J, Tanaka-Kido J, Vallejo O, Sterling CR, 1997. Cryptosporidium infections in Mexican children: Clinical, nutritional, enteropathogenic, and diagnostic evaluations. Am J Trop Med Hyg 56: 254–257.

    • Search Google Scholar
    • Export Citation
  • 16

    Solorzano-Santos F, Penagos-Paniagua M, Meneses-Esquivel R, Miranda-Novales MG, Leanos-Miranda B, Angulo-Gonzalez D, Fajardo-Gutierrez A, 2000. Cryptosporidium parvum infection in malnourished and non malnourished children without diarrhea in a Mexican rural population. Rev Invest Clin 52: 625–631.

    • Search Google Scholar
    • Export Citation
  • 17

    Bouckenooghe AR, Jiang ZD, De La Cabada FJ, Ericsson CD, DuPont HL, 2002. Enterotoxigenic Escherichia coli as cause of diarrhea among Mexican adults and US travelers in Mexico. J Travel Med 9: 137–140.

    • Search Google Scholar
    • Export Citation
  • 18

    Taylor DN, Bourgeois AL, Ericsson CD, Steffen R, Jiang ZD, Halpern J, Haake R, Dupont HL, 2006. A randomized, double-blind, multicenter study of rifaximin compared with placebo and with ciprofloxacin in the treatment of travelers’ diarrhea. Am J Trop Med Hyg 74: 1060–1066.

    • Search Google Scholar
    • Export Citation
  • 19

    Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, Kazmierczak JJ, Addiss DG, Fox KR, Rose JB, 1994. A massive out break in Milwaukee of Cryptospridium infection transmitted through the public water supply. N Engl J Med 331: 161–167.

    • Search Google Scholar
    • Export Citation
  • 20

    Weitzel T, Wichmann O, Muhlberger N, Reuter B, Hoof HD, Jelinek T, 2006. Epidemiological, and clinical features of travel-associated cryptosporidiosis. Clin Microbiol Inf 12: 921–924.

    • Search Google Scholar
    • Export Citation
  • 21

    Mohamed JA, DuPont HL, Jiang ZD, Belkind–Gerson J, Figueroa JF, Armitige LY, Tsai A, Nair P, Martinez–Sandoval FJ, Guo DC, Hayes P, Okhuysen PC, 2007. A novel single–nucleotide polymorphism in the lactoferrin gene is associated with susceptibility to diarrhea in North American travelers to Mexico. Clin Infect Dis 44: 945–952.

    • Search Google Scholar
    • Export Citation
  • 22

    DuPont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski W, 1995. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 332: 855–859.

    • Search Google Scholar
    • Export Citation
  • 23

    Chappell CL, Okhuysen PC, Sterling CR, DuPont HL, 1996. Cryptosporidium parvum: Intensity of infection, and oocyst excretion patterns in healthy volunteers. J Infect Dis 173: 232–236.

    • Search Google Scholar
    • Export Citation
  • 24

    Okhuysen PC, Chappell CL, Crabb JH, Sterling CR, DuPont HL, 1999. Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J Infect Dis 180: 1275–1281.

    • Search Google Scholar
    • Export Citation
  • 25

    Llorente MT, Clavel A, Goñi MP, Varea M, Seral C, Becerril R, Suarez L, Gómez–Lus R, 2007. Genetic characterization of Cryptosporidium species from humans in Spain. Parasitol Int 56: 201–205.

    • Search Google Scholar
    • Export Citation
  • 26

    Xiao L, Ryan UM, 2004. Cryptosporidiosis: an update in molecular epidemiology. Curr Opin Infect Dis 17: 483–490.

  • 27

    Alves M, Xiao L, Sulaiman I, 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41: 2744–2747.

    • Search Google Scholar
    • Export Citation
  • 28

    McLauchlin J, Amar C, Pedraza–Diaz S, Nichols GL, 2000. Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping Cryptosporidium spp. in 1,705 fecal samples from humans, and 105 fecal samples from livestock animals. J Clin Microbiol 38: 3984–3990.

    • Search Google Scholar
    • Export Citation
  • 29

    Fretz R, Svoboda P, Ryan UM, 2003. Genotyping of Cryptosporidium spp. isolated from human stool samples in Switzerland. Epidemiol Infect 131: 663–667.

    • Search Google Scholar
    • Export Citation
  • 30

    Tiangtip R, Jongwutiwes S, 2002. Molecular analysis of Cryptosporidium species isolated from HIV–infected patients in Thailand. Trop Med Int Health 7: 357–364.

    • Search Google Scholar
    • Export Citation
  • 31

    Yagita K, Izumiyama S, Tachibana H, Masuda G, Iseki M, Furuya K, Kameoka Y, Kuroki T, Itagaki T, Endo T, 2001. Molecular characterization of Cryptosporidium isolates obtained from human, and bovine infections in Japan. Parasitol Res 87: 950–955.

    • Search Google Scholar
    • Export Citation
  • 32

    Nichols RA, Campbell BM, Smith HV, 2003. Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters, and drinking waters by using a modified nested PCR–restriction fragment length polymorphism assay. Appl Environ Microbiol 69: 4183–4189.

    • Search Google Scholar
    • Export Citation
  • 33

    Learmonth JJ, Ionas G, Pita AB, Cowie RS, 2003. Identification and genetic characterization of Giardia, and Cryptosporidium strains in humans, and dairy cattle in the Waikato Region of New Zealand. Water Sci Technol 47: 21–26.

    • Search Google Scholar
    • Export Citation
  • 34

    Cama VA, Bern C, Sulaiman IM, Gilman RH, Ticona E, Vivar A, Kawai V, Vargas D, Zhou L, Xiao L, 2003. Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru. J Eukaryot Microbiol 50: 531–533.

    • Search Google Scholar
    • Export Citation
  • 35

    Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W, Cabrera L, Gilman RH, Lal AA, 2001. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis 183: 492–497.

    • Search Google Scholar
    • Export Citation
  • 36

    Bushen OY, Kohli A, Pinkerton RC, Dupnik K, Newman RD, Sears CL, Fayer R, Lima AA, Guerrant RL, 2007. Heavy cryptosporidial infections in children in northeast Brazil: Comparison of Cryptosporidium hominis, and Cryptosporidium parvum. Trans R Soc Trop Med Hyg 101: 378–384.

    • Search Google Scholar
    • Export Citation
  • 37

    Gatei W, Greensill J, Ashford RW, Cuevas LE, Parry CM, Cunliffe NA, Beeching NJ, Hart CA, 2003. Molecular analysis of the 18S rRNA gene of Cryptosporidium parasites from patients with or without human immunodeficiency virus infections living in Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. J Clin Microbiol 41: 1458–1462.

    • Search Google Scholar
    • Export Citation
  • 38

    Di Giovanni GD, Betancourt WQ, Hernandez J, Assadian NW, Flores Margez JP, Lopez EJ, 2006. Investigation of potential zooanthroponotic transmission of cryptosporidiosis, and giardiasis through agricultural use of reclaimed wastewater. Int J Environ Health Res 16: 405–418.

    • Search Google Scholar
    • Export Citation
  • 39

    Gasser RB, El–Osta YG, Chalmers RM, 2003. Electrophoretic analysis of genetic variability within Cryptosporidium parvum from imported, and autochthonous cases of human cryptosporidiosis in the United Kingdom. Appl Environ Microbiol 69: 2719–2730.

    • Search Google Scholar
    • Export Citation
  • 40

    Warren KS, Swan RA, Morgan–Ryan UM, 2003. Cryptosporidium muris infection in bilbies (Macrotis lagotis). Aust Vet J 81: 739–741.

  • 41

    Fayer R, Morgan U, Upton SJ, 2000. Epidemiology of Cryptosporidium: Transmission, detection, and identification. Int J Parasitol 30: 1305–1322.

    • Search Google Scholar
    • Export Citation
  • 42

    Kostrzynska M, Sankey M, Haack E, Power C, Aldom JE, Chagla AH, Unger S, Palmateer G, Lee H, Trevors JT, De Grandis SA, 1999. Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples. J Microbiol Methods 35: 65–71.

    • Search Google Scholar
    • Export Citation
  • 43

    Mayer CL, Palmer CJ, 1996. Evaluation of PCR, nested PCR and fluorescent antibodies for detection of Giardia, and Cryptosporidium species in wastewater. Appl Environ Microbiol 62: 2081–2085.

    • Search Google Scholar
    • Export Citation
  • 44

    Sturbaum GD, Reed C, Hoover PJ, Jost BH, Marshall MM, Sterling CR, 2001. Species–specific, nested PCR–restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts. Appl Environ Microbiol 67: 2665–2668.

    • Search Google Scholar
    • Export Citation
Save