Retrospective study of the occurrence of Cyclospora cayetanensis at Clinical Hospital of the University of São Paulo Medical School , SP

Cyclospora cayetanensis causes watery diarrhea in tropical countries, among travelers and after ingestion of contaminated water and food. Very little is known about its epidemiology, pathogenic aspects and reservoirs. In Brazil, its prevalence is unknown and to date there have been reports of three outbreaks. We report here a retrospective study of 5,015 stool samples from 4,869 patients attended at Clinical Hospital of the University of São Paulo Medical School, SP, Brazil between April 1996 and January 2002, with 14 cases of Cyclospora cayetanensis being detected there was a prevalence of 0.3%. Of the 14 infected patients, the mean age was 38 years and 71.4% were female. Ten patients presented symptoms; six presented levels of immunological markers and five patients were immunodeficient. Key-words: Cyclospora cayetanensis. Prevalence. Cyclosporiasis. Parasitology. Pathology. Brazil. RESUMO Cyclospora cayetanensis causa diarréia líquida em países tropicais, viajantes e após ingestão de água e alimentos contaminados. Muito pouco é conhecido sobre sua epidemiologia, aspectos patogênicos e reservatórios. No Brasil, sua prevalência é desconhecida com relato de três surtos. Nós relatamos um estudo retrospectivo de 5.015 amostras fecais provenientes de 4.869 pacientes atendidos no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, SP, Brasil entre abril de 1996 e janeiro de 2002 com detecção de 14 casos de ciclosporíase e prevalência de 0,3%. Deste total 71,4% eram do sexo feminino com idade média de 38 anos. Dez apresentaram sintomatologia, seis tinham níveis de marcadores imunológicos e cinco imunodeficiência. Palavras-chaves: Cyclospora cayetanensis. Prevalência. Ciclosporíase. Parasitologia. Patologia. Brasil. 1. Central Laboratory Division. Clinical Hospital. University of São Paulo Medical School, São Paulo, SP. 2. Laboratory of Medical Investigation in Infectious Disease Pathology. University of São Paulo Medical School, São Paulo, SP. Supported by DLC – HCFMUSP, LIM 03, LIM 50 & CNPq. Address to: Dr. Carlos Eduardo Pereira Corbett. Dept de Patologia/FM/USP. Av. Dr. Arnaldo 455/1o andar, sala 1215, 01246-903 São Paulo, SP, Brasil. Telefax: 55 11 3081-7799 e-mail: ccorbett@usp.br Recebido para publicação em 4/2/2004 Aceito em 8/5/2005 Cyclospora cayetanensis is an emerging and opportunistic coccidium, first described as a causal agent of human infection in 1979; it was included in the Cyclospora genus in 1993 with referenced species in 1994. To date, 17 species of Cyclospora have been identified through the use of molecular methods, although humans are the only host of Cyclospora cayetanensis and no animal model has been identified . The exact means of transmission are still unknown; but it is probably by fecal-oral route, through the ingestion of sporulated oocysts, which are resistant to routine water treatment and pesticide use, since contaminated water and food account for the outbreaks 22 23 . It has, however, been detected in sputum, suggesting oocyst ingestion by aerosol. The infection is characterized by watery diarrhea, weight loss, abdominal distension, flatulence, nausea, fatigue and low-grade fever. These symptoms can disappear in a few weeks or persist for months, or even be absent . The diagnostic routine of Cyclospora cayetanensis is based on the observation of non-sporulated oocysts measuring from 8 to

Cyclospora cayetanensis is an emerging and opportunistic coccidium, first described as a causal agent of human infection in 1979 4 ; it was included in the Cyclospora genus in 1993 20 with referenced species in 1994 19 .To date, 17 species of Cyclospora have been identified through the use of molecular methods, although humans are the only host of Cyclospora cayetanensis and no animal model has been identified 9 27 .The exact means of transmission are still unknown; but it is probably by fecal-oral route, through the ingestion of sporulated oocysts, which are resistant to routine water treatment and pesticide use, since contaminated water and food account for the outbreaks 17 22 23 29 .It has, however, been detected in sputum, suggesting oocyst ingestion by aerosol 8 .The infection is characterized by watery diarrhea, weight loss, abdominal distension, flatulence, nausea, fatigue and low-grade fever.These symptoms can disappear in a few weeks or persist for months, or even be absent 27 29 .The diagnostic routine of Cyclospora cayetanensis is based on the observation of non-sporulated oocysts measuring from 8 to 10µm in stool smears stained by Kinyoun´s modified method, also used for the detection of other coccidian parasites.It is necessary to measure these oocysts through a micrometer, since Cyclospora cayetanensis oocysts can be easily seen in phase contrast and fluorescence microscopes 12 19 20 29 .Oocyst sporulation in 2.5% potassium dichromate can also be used 29 .Cyclosporiasis has been reported in many countries, but it seems to be more common in tropical and subtropical regions 27 28 .Its prevalence in Brazil is still unknown, with a case reported in Rio de Janeiro (RJ) 24 , a case in Brasilia (DF) 3 , 10 cases in the State of Goiás (GO) 13 14 , 3 cases in Juiz de Fora (MG) 6 and a case in São Paulo (SP) 10 .Three outbreaks have been registered: two in General Salgado (SP), one in November 2000 and another in January 2001, and the third in Antonina (PR), in November 2001 11 25 27 .The objective was to perform a retrospective study of 5,015 stool samples, collected from 4,869 patients attended at Clinical Hospital of the University of São Paulo Medical School (HC-FMUSP) between April 1996 and January 2002, with 14 detected cases of Cyclospora cayetanensis.

MATERIAL AND METHODS
Clinical Hospital of the University of São Paulo Medical School (HCFMUSP), located in São Paulo, Brazil.This is a highly specialized public hospital, associated to the University of Sao Paulo with 3,271 beds and 9,888 employees and attends patients referred from the Public Health System and a small number of private patients as well as those with healthcare insurance.The Parasitology Service of the Division of the Central Laboratory of HC-FMUSP performs a monthly average of 10,000 parasitologic tests.
A retrospective study of 5,015 stool samples derived from 4,869 patients was conducted.The samples were sent to our Service for Cyclospora cayetanensis investigation, from April 1996 to January 2002.All samples were submitted to standard concentration methods: Hoffman, Pons and Janer, Faust and cols and modified Rugai method; they were stained with an iodine solution for the diagnosis of helminthes and protozoa (PPF).For the identification of Cyclospora cayetanensis, the Kinyoun´s modified method 12 was used in all samples, followed by oocyst sporulation in 2.5% potassium dichromate, whenever samples were positive.All oocysts were measured through an ocular micrometer.Samples were considered positive for Cyclospora cayetanensis when they presented spherical structures that stained in different shades of red, or colorless, with size varying from 8-10µm.
Statistical analysis.For the positive results, data such as age, sex, symptoms, diseases and immunological markers were collected from the hospital records using MS Office Excel version 2000.In order to characterize positive samples for Cyclospora cayetanensis, we present the relative and absolute frequencies of each qualitative variable.Regarding the quantitative variables, means and medians were used to summarize the data and standard errors, standard deviations, and minimum and maximum values were used to indicate data variability.To determine risk factors for Cyclospora cayetanensis, we compared the positive patients with 60 randomly selected patients, from the general population, with no infection.For the qualitative variables such as sex, diarrhea, disease and symptoms, we compared the frequency distribution between the positive and negative groups through Pearson's Chi-square test 31 .Fisher's exact test was used in situations where the expected values were lower than 5.For p values lower than 0.05, we considered the association among the variables to be statistically significant.To compare the groups regarding the quantitative variables such as age, CD3, CD4, CD8 and CD4/CD8 ratio, we applied Student's t-test 31 , in order to compare the means of the independent populations.For p values lower than 0.05, we considered that the two groups are different regarding patients' age.

RESULTS
Spherical organisms with a mean diameter of 9µm, showing an internal granular structure and great acid-alcohol resistant staining variability were identified in 14 stool samples, diagnosed as Cyclospora cayetanensis (Figure 1) and listed as follows:  Paulo.In May 2001 she came to the hospital and was attended at the Gastroenterology Service of HCFMUSP, for a clinical investigation of chronic constipation.The stool sample analysis was positive for Blastocystis hominis and Cyclospora cayetanensis.She did not return to the Service.
When an inferential analysis was performed in order to determine the risk factors for Cyclospora cayetanensis, using     Regarding sex, Chi-square test results did not present statistically significant values at the usual level of 5% (p = 0.058) but this test showed a slight concentration of females (71%) in the infected group in comparison to 43.3% in the healthy group, as shown in Table 6 and Figure 2.
In relation to the quantitative variables: age, CD3, CD4, CD8 and CD4/CD8 ratio, we applied Student's T-test and observed a statistically significant difference regarding CD4 only (p<0.001), with a mean of 141 cells/µl, as depicted in the Table 7 and Figure 3.

Figure 2 -
Figure 2 -Concentrations of positive and negative results forCyclospora cayetanensis, according to sex.

Figure 3 -
Figure 3 -Confidence Interval of 95% for CD4 Means Analyzed Separately According to the Positive Result for Cyclospora cayetanensis.
concentration and staining methods, and was positive for Cyclospora cayetanensis; his CD4 T cell count was 162/µl.4. LCS, 47-year-old widower, born in the State of Bahia and residing in São Paulo, with AIDS; she was attended at the Extended Care Center for the HIV/AIDS patient.In July 1999, she presented anorexia, loss of 20kg of body weight in a 3month period, fever accompanied by productive cough with yellowish sputum, bloody-mucous diarrhea for a period of one year, with an average of ten bowel movements a day and swallowing difficulty.Her skin was dry and pale.CD4 T-cell count was 84 cells/µl; her stool sample was submitted to concentration and staining methods, and was positive for Entamoeba coli, Entamoeba histolytica/dispar, Schistosoma mansoni and Cyclospora cayetanensis.5. LFNS, 50-year-old single male, homosexual, born in Rio de Janeiro, has been residing in São Paulo for 22 years.The patient was a smoker, but denied using illicit drugs.Patient had AIDS and came to the hospital for management of the disease, presenting good general health status and no gastrointestinal alterations.His CD4 T cell count was 126 cells/µl, and his stool sample was submitted to concentration and staining methods, and was positive for Cyclospora cayetanensis.6. MSS, 57-year-old married female, born and residing in São Paulo.She denied using tobacco, alcohol or illicit drugs.Cyclospora cayetanensis.10.OM, 30-year-old male, born and residing in General Salgado, SP.He presented persistent diarrhea, with an average of 10 bowel movements a day.His stool sample was submitted to laboratory investigation for the presence of probable etiologic agent, which was the cause of the diarrhea that affected inhabitants of that town in November 2000.The parasitologic analysis through concentration and staining methods was positive for Cyclospora cayetanensis.11.MJCL, 40-year-old, married female, born and residing in São Paulo.The patient had hepatitis C and schizophrenia, with no gastrointestinal symptoms.She came to the hospital for routine management, and presented good general health status in January 2001.The parasitologic analysis was positive for Cyclospora cayetanensis.12. ESS, 19-year-old, single female, born and residing in São Paulo.The patient had had Hodgkin's disease for 7 years.She denied tobacco, alcohol and illicit drug use.In February 2001, she came to hospital ER with fever, vomiting and diarrhea for a period of 3 days, with an average of 5 bowel movements a day.The stool sample analysis was positive for Cyclospora cayetanensis.13.SGV, 20-year-old, single female, born and residing in São Paulo.She denied tobacco, alcohol and illicit drug use.The patient had sickle-cell anemia.In May 2001 she came to the hospital for a clinical investigation at the Hematology Service, presenting nausea, vomiting, abdominal pain and sporadic diarrhea.The parasitologic analysis through concentration and staining methods was positive for Cyclospora cayetanensis.14.MJA, 53-year-old, married female, born and residing in São