Cyclosporiasis outbreak, Indonesia.

We describe an outbreak of Cyclospora cayetanensis infection among Dutch participants at a scientific meeting in September 2001 in Bogor, Indonesia. Fifty percent of the investigated participants were positive for C. cayetanensis. To our knowledge, this outbreak is the first caused by C. cayetanensis among susceptible persons in a disease-endemic area.

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Cyclospora cayetanensis is a newly recognized protozoan parasite that causes gastrointestinal illness. C. cayetanensis infection is mostly characterized by a gradual onset of watery diarrhea, sometimes with explosive diarrhea, nausea, and abdominal cramping. Symptoms are often prolonged and can relapse after months (1). Oocysts of C. cayetanensis are, in comparison with those of other coccidian parasites, noninfectious in freshly excreted stool. Therefore, direct person-to-person transmission through fecal exposure is unlikely. Food and water contaminated with sporulated oocysts are the primary modes of transmission (1). Infections have been associated mainly with outbreaks from eating food such as raspberries, salads, and basil (1-3). Infections with C. cayetanensis are seasonal; in the tropics, the wet and cooler seasons provide conditions more favorable for sporulation than do the dry and warmer seasons (1,3).

A scientific meeting involving Dutch and Indonesian microbiologists was held September 2-6, 2001, in Indonesia. Immediately after the meeting, several participants reported mild-to-severe gastrointestinal symptoms, and C. cayetanensis infection was diagnosed in 4 participants. We investigated the extent of the outbreak among participants from our institute and identified C. cayetanensis-specific symptoms.

The Study

The meeting was held in a hotel near Bogor, Indonesia. All Dutch participants stayed in this hotel during the meeting and consumed the same meals (buffet). After the meet approximately half of the participants went home, while others took the opportunity to travel further.

Six weeks after the meeting, all members of our institute who visited the Bogor meeting were asked to participate in a cohort study. Participants were asked to complete a questionnaire and deliver 2 fecal samples. The questionnaire was set up to collect information about gastrointestinal symptoms, duration of stay, and results of earlier fecal diagnostic examination.

One of the 2 fecal samples was directly examined for C. cayetanensis after Ridley concentration by 2 microscopy methods. Presence of C. cayetanensis was demonstrated by nonrefractile spheres seen in a direct saline wet mount or by light pink-to-deep red, 8- to 10-[micro]m long oocysts seen on modified acid-stained smears. Because the aim of the study was to determine the extent and duration of C. cayetanensis infection in our cohort, no special efforts were made to detect other possible pathogens. Another fecal sample was stored at -20[degrees]C until DNA was isolated for polymerase chain reaction (PCR). After DNA isolation, C. cayetanensis specific real-time PCR was performed as described previously (4). The specific primers and probe were based on the known small ribosomal subunit RNA gene sequence for C. cayetanensis. This real-time PCR was specific when tested with a range of other intestinal parasites, and the DNA of [greater than or equal to] 0.5 oocysts was estimated as the detection limit. Names and clinical conditions of the participants were blinded to laboratory technicians.

A case-control study was set up to investigate whether certain gastrointestinal symptoms were associated with C. cayetanensis infection. A case was defined as C. cayetanensis-positive microscopic result, positive PCR result, positive C. cayetanensis diagnosis 1-6 weeks before entering the study, or any combination. Only data from participants who submitted completed questionnaires were used for statistical analyses.

Analyses were performed by using the [chi square] test if variables were categorical. Continuous variables were not normally distributed; therefore, they were compared and tested with the Mann-Whitney U test. The ethics committee of the University Medical Center Utrecht approved the project; written informed consent was obtained from all participants before participation.

Thirty-two (94%) of the 34 attendees of our institute responded, and 29 completed the questionnaire; 3 participants delivered only fecal samples. Fourteen (48%) of the 29 attendees had cases that met the definition: 10 case-patients had a positive PCR result, 5 of which were also positive on microscopic analysis, and 4 case-patients had a positive diagnosis 1-6 weeks before entering the study. Fecal samples of these 4 case-patients were all negative on microscopic analysis; 2 were also negative on PCR analysis. Two samples were lost before PCR could be performed. Fecal samples from the 3 participants who did not return the questionnaire were all positive by PCR and negative by microscopic analysis. They were not included in the case-control study.

Symptoms of the 29 participants are listed in the Table. More women had cyclosporiasis than men (71% vs. 40%). Case-patients mentioned bowel disorders significantly more than noncase-patients: stomach cramps, nausea, and flatulence were common symptoms among the case-patients (71% vs. 40%, 93% vs. 27%, and 93% vs. 33%, respectively). Duration of symptoms was significantly longer for case-patients than for noncase-patients.

Conclusions

We showed that approximately half of the investigated meeting attendees were positive for C. cayetanensis. The number of proven infected persons would be higher if the investigations had started directly after the first symptoms appeared. The fact that 6 weeks after the probable exposure, C. cayetanensis DNA was still detectable in 13 persons corresponds to the known persistence of the parasite. Diagnosis in our study was primarily based on PCR-positive results. PCR is a much more reliable method to detect C. cayetanensis than diagnostic microscopy (4,5); however, ultraviolet fluorescence microscopy may be as sensitive as PCR (4).

An outbreak with a common source of infection has not been proven. We could not investigate potential food sources because we started surveillance 6 weeks after the assumed exposure in Indonesia, when participants had returned to the Netherlands. Only 2 of the participants could recall a meal that might have been a source of infection, and each suggested a different meal. Participants may have acquired C. cayetanensis infection on other occasions during their stay in Indonesia. Genotyping the different isolates to connect cases on a molecular basis was not possible because the number of oocysts detected in stools was small. However, assuming a joint exposure to C. cayetanensis is not unreasonable. First and most important, 3 of the participants in this study went to the conference and returned immediately to the Netherlands, and the conference was the only place where infection may have been acquired. Secondly, infections were acquired during the season with low incidence of transmission, so a common source is a more obvious route of infection than cases acquired at separate occasions. In our study, the main symptoms of cyclosporiasis were stomach cramps, nausea, and flatulence, without bloody diarrhea. The prolonged and relapsing character of the symptoms, especially diarrhea and abdominal cramps, was striking. This pattern of symptoms is mentioned in other outbreaks as well and seems to be characteristic for cyclosporiasis (1-3). Although nonindigenous persons are at risk for travelers' diarrhea in Indonesia, we believe that the gastrointestinal problems in the patients in our study were caused by cyclosporiasis. All 4 patients with an earlier diagnosis of C. cayetanensis infection were successfully treated with cotrimoxazole.

In conclusion, we report a possible outbreak of C. cayetanensis among Dutch microbiologists attending a meeting in Indonesia. At least 50% of participants were infected. To our knowledge, this outbreak is the first of C. cayetanensis among susceptible persons in a disease-endemic area.

Dr Blans is a medical microbiologist in the Gelre Ziekenhuizen, Apeldoorn, the Netherlands. Her research interests are in diagnostics and management of diarrheal diseases.

References

(1.) Herwaldt BL. Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s. Clin Infect Dis. 2000;31 : 1040-57.

(2.) Ho AY, Lopez AS, Eberhart MG, Levenson R, Finkel BS, da Silva AJ, et al. Outbreak of cyclosporiasis associated with imported raspberries, Philadelphia, Pennsylvania, 2000. Emerg Infect Dis. 2002;8:783-8.

(3.) Fryauff DJ, Krippner R, Prodjodipuro P, Ewald C, Kawengian S, Pegelow K, et al. Cyclospora cayetanensis among expatriate and indigenous populations of West Java, Indonesia. Emerg Infect Dis. 1999;5:585-8.

(4.) Verweij JJ, Laeijendecker D, Brienen EAT, van Lieshout L, Polderman AM. Detection of Cyclospora cavetanensis in travellers returning from the tropics and subtropics using microscopy and real-time PCR. Int J Med Microbiol. 2003;293:199-202.

(5.) Varma M, Hester JD, Schaefer FW, Ware MW, Lindquist HD. Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay. J Microbiol Methods. 2003;53:27-36.

All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required.

Marjolijn C.A. Blahs,* Ben U. Ridwan, ([dagger]) Jaco J. Verweij, ([double dagger]) Maja Rozenberg-Arska, ([dagger]) and Jan Verhoef ([dagger])

* Gelre Ziekenhuizen, Apeldoorn, the Netherlands; ([dagger]) University Medical Center Utrecht, Utrecht, the Netherlands; and ([double dagger]) Leiden University Medical Center, Leiden, the Netherlands

Address for correspondence: M.C.A. Blans, Department of Medical Microbiology, Gelre Ziekenhuizen, Postbus 9014, 7300 DS Apeldoorn, the Netherlands; fax: [phone]; email: [e-mail]

Table. Characteristics of meeting members exposed to Cyclospora
cayetanensis, Bogor, Indonesia, 2001 *

                                                         Noncases (%),
Variable                           Cases (%), n = 14        n = 15

Female sex                              10 (71)             6 (40)
Symptoms
  Diarrhea ([double dagger])            11 (79)             9 (60)
  Obstipation                            3 (21)                0
  Stomach and abdominal cramps          10 (71)             6 (40)
  Flatulence                            13 (93)             5 (33)
  Fever                                  2 (13)              1 (7)
  Nausea/appetite loss                  13 (93)             4 (27)
Median symptom duration (IQR),
  days ([section])                     41 (29-42)           1 (0-6)
Median stay in Indonesia (IQR),
  days                               14 (10.5-18.5)        16(11-23)

Variable                           p value ([dagger])

Female sex                                0.09
Symptoms
  Diarrhea ([double dagger])               NS
  Obstipation                              NS
  Stomach and abdominal cramps            0.09
  Flatulence                             0.001
  Fever                                    NS
  Nausea/appetite loss                   <0.001
Median symptom duration (IQR),
  days ([section])                       <0.001
Median stay in Indonesia (IQR),
  days                                     NS

* N5, nonsignificant; IQR, interquartile range.

([dagger]) Determined by [chi square] test for categorical variables
and by Mann-Whitney U test for continuous variables.

([double dagger]) Diarrhea was defined as [greater than or equal to] 3
loose stools in 24 hours.

([section]) If patients had symptoms when entering the study, the
duration was fixed at 42 days.