Detection of some intestinal protozoa in commercial fresh juices

Fresh fruit juices are popular, but not always safe. For assessing the likelihood of infection with newly emerging intestinal protozoa, commercial fresh orange, lemon, sugar cane, strawberry, and mango juices were screened by wet mounts, Webers modified trichrome and modified Ziehl-Neelsen stains. Protozoa viability was done by fluorescein-diacetate/propidium-iodide staining, and infectivity was performed in Swiss albino mice. Results showed that 35.43% were contaminated with one or more of Cryptosporidia, Microsporidia, and Cyclospora, as well as Giardia spp. Strawberry was the most contaminated juice [54.28%], while orange was the slightest [22.86%]. Cryptosporidia was the highest contaminant [61.29%], and Cyclospora was the least [14.52%]. Microsporidia spp. was the most robust contaminant which retained its viability and infectivity in juices in which it was detected. Moderately acidic strawberry and mango juices and alkaline sugar cane juice pose a possible threat, due to harboring the highest viable and infectious protozoa. Regarding highly acidic juices, viability and infectivity decreased in lemon, yet was not still risk free. Orange juice was comparatively safe, as viability dramatically declined, while infectivity was completely abolished. Hence consumers, especially high risk group, are placed at hazard of contracting intestinal protozoa infections, especially through moderately acidic and alkaline juices

Multiplex PCR Detection of Waterborne Intestinal Protozoa; Microsporidia, Cyclospora, and Cryptosporidium

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 101 to 102 oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

Infección por Cyclospora cayetanensis: Revisión a propósito de tres casos de diarrea del viajero / Cyclospora cayetanensis infection: Updated review a propos of three cases of traveler's diarrhea

Human infection by Cyclospora cayetanensis, namely cyclosporiasis, can cause a wide range of symptoms in immunocompetent patients, from mild to severe diarrhea. Immunocompromised patients can present with chronic diarrhea and it has been recognized as a cause of traveler's diarrhea. We report three patients who traveled from Chile to Peru, who presented upon returning with prolonged traveler's diarrhea. A literature review about cyclosporiasis is presented, with emphasis on the clinical, epidemiological, diagnostic and therapeutic aspects of this disease.

Coloraciones Ziehl-Neelsen y Safranina modificadas para el diagnóstico de Cyclospora cayetanensis / Modified Ziehl-Neelsen and modified Safranin staining for diagnosing Cyclospora cayetanensis

Objetivo Comparar las coloraciones Ziehl-Neelsen modificada (ZNm) y Safranina modificada (Sm) en la detección de ooquistes de C. cayetanensis en muestras de materia fecal . Metodología Se analizaron 100 muestras de materia fecal que fueron evaluadas previamente por coprológico directo y por concentración, posteriormente se realizaron las coloraciones ZNm y Sm para cada una de las muestras. Para la comparación de las dos técnicas se hicieron análisis estadísticos tomando como prueba oro el examen directo. Resultados Se encontró una sensibilidad y especificidad del 95 por ciento y 98 por ciento para ZNm y del 90 por ciento y 100 por ciento para Sm respectivamente. El índice Kappa fue de 0,93, correspondiendo a un muy buen grado de concordancia entre las dos coloraciones. Conclusiones Debido a la alta sensibilidad y especificidad encontradas en este estudio para Zm y Sm y al alto grado de concordancia entre ellas, cualquiera de las dos coloraciones puede ser utilizada para el diagnóstico de rutina de C. cayetanensis.

Objective Comparing modified Ziehl-Neelsen (ZNm) and modified Safranin (Sm) staining for detecting C. cayetanensis oocysts in stool samples. Methods The sample to be analysed consisted of 100 stool samples which had been previously evaluated by direct microscopic examination and concentration. Each sample was then ZNm and Sm stained. Microscope examination was used as gold standard for comparing both techniques by statistical analysis. Results There was 95 percent sensitivity and 90 percent specificity for ZNm and 98 percent sensitivity and 100 percent specificity for Sm. The Kappa index was 0,93, signifying a very good degree of agreement between the two techniques. Conclusions The use of either of the two stains for diagnosing C. cayetanensis can be recommended due to the high sensitivity and specificity for Zm and Sm found in this study and to the high degree of agreement between them.

Chronic diarrhoea in HIV patients: prevalence of coccidian parasites.

The purpose of this study was to determine the prevalence of intestinal parasites in HIV patients with or without diarrhoea and to see an association between diarrhoea and the coccidian parasites in our setting. Stool samples from 113 HIV patients, 34 chronic diarrhoea and 79 without any history of diarrhoea were collected and examined for enteric parasites by microscopy. One hundred and thirteen control samples from HIV negative patients complaining of prolonged diarrhoea were also collected and analysed. Prevalence of coccidian parasites in HIV and non-HIV patients; with and without diarrhoea was compared using chi-square tests. Enteric parasites were detected in 55.8% HIV patients with diarrhoea compared to 16.4% in patients without diarrhoea (P<0.001). Isospora belli was found in 41.1% (14/34) of chronic diarrhoea and 6.3% (5/79) in non-diarrhoeal cases (P<0.001). Cryptosporidium was detected in 20.6% (7/34) of chronic diarrhoea and 2.5% (2/79) in non-diarrhoeal cases (P<0.001). Cyclospora cayetanensis associated diarrhoea was detected in only one case of chronic diarrhoea (2.9%). CD4+ T-cell count was lower (180 cells/microL) in diarrhoeal HIV patients as compared to non-diarrhoeal patients. Coccidian parasites were seen at a mean CD4+ T-cell count of 186.3 cells/microL. This study concluded that Isospora belli was the predominant parasite followed by Cryptosporidium spp. and both were strongly associated with diarrhoea among HIV patients.

Observações sobre Blastocystis hominis e Cyclospora cayetanensis em exames parasitológicos efetuados rotineiramente / Observations on Blastocystis hominis and Cyclospora cayetanensis in routine parasitological examinations

Relatamos algumas observações, efetuadas com exames parasitológicos de fezes, em atividades rotineiras: os métodos de Faust e cols e de sedimentação espontânea em água não servem para evidenciação de Blastocystis hominis; foram encontradas expressivas porcentagens de presença desse protozoário, sobretudo quando realizada coloração pela hematoxilina férrica; houve 0,7 por cento de registro de positividade para Cyclospora cayetanensis, sugerindo inclusão habitual de pesquisa, por técnicas apropriadas, de tal parasita.

We report some observations made from routine parasitological examinations on feces. The methods of Faust et al. and of spontaneous sedimentation in water are not enough to identify Blastocystis hominis. Significant percentage presence of this protozoan was found, especially when staining with iron hematoxylin was performed. Cyclospora cayetanensis was found in 0.7 percent of the cases, which suggests that this parasite should also routinely be investigated by appropriate techniques.

Brote epidémico por Cyclospora cayetanensis en Medellín, Colombia / An outbreak of Cyclospora cayetanensis in Medellín, Colombia

Objetivo: Describir un brote epidémico de enfermedad gastrointestinal en un grupo de 56 pacientes de Medellín, Colombia. Metodología En abril de 2002 se presentó un número inusitado de casos de diarrea y a partir del hallazgo de Cyclospora cayetanensis en uno de los pacientes, se recolectaron 56 muestras de materia fecal de igual número de individuos. Se realizó una encuesta clínico-epidemiológica, coprológico directo, concentración con formol-éter y coloración de Zielh Neelsen modificada. Además, se estudiaron por directo y Zielh Neelsen muestras de algunos alimentos para la búsqueda del parásito. Resultados Del total de pacientes evaluados, 55,4 por ciento (31/56) fueron positivos para C. cayetanensis, de éstos, el 77,4 por ciento (24/31) fueron mujeres, y el 83,9 por ciento (26/31) pertenecía a la Universidad de Antioquia, de los cuales el 88,6 por ciento eran empleados no docentes. No se encontraron diferencias significativas entre pacientes positivos y negativos para Cyclospora con respecto a las manifestaciones clínicas, excepto en la deshidratación que fue mayor en los pacientes con ciclosporidiasis. Sin embargo, se encontró una diferencia significativa con relación al consumo de ensaladas y jugos, la cual fue mayor entre los pacientes positivos que los negativos. Conclusión Se presentó un brote epidémico de C. cayetanensis en pacientes sintomáticos atendidos por el grupo GIEPI en abril de 2002.

Objective: Describing an outbreak of gastrointestinal disease in a group of 56 patients from Medellín, Colombia. Methods An unusual number of cases of diarrhoea appeared in April 2002 and 56 samples of stool from the same number of individuals were collected because a patient proved Cyclospora cayetanensis positive. A clinical-epidemiological survey, direct coprology, formol-ether concentration and modified Zielhs Neelsen staining were then carried out. Some food samples were also studied for parasites by direct and modified Zielh Neelsen staining. Results 55,4 percent (31/56) of the patients being evaluated proved positive for C. cayetanensis. 77,4 percent (24/31) were women and 83,9 percent (26/31) belonged to the University of Antioquia, 88,6 percent of whom were not teachers. There was no statistically significant difference between positive and negative patients for Cyclospora regarding clinical manifestations, except for dehydration which was greater in patients having cyclosporiasis. However, there was a significant difference regarding consuming salads and juice, this being greater amongst positive patients than negative ones. Conclusion An outbreak of C. cayetanensis appeared in symptomatic patients being taken care of by the GIEPI group during April 2002.

Cyclosporiasis in an infant.

This report describes cyclosporiasis in a seven month old infant who presented with incessant crying and refusal of feeds. The routine modified ZN stained smears showed the oocysts of Cyclospora when all other tests failed to reveal enteric pathogens. The need for the clinical laboratory to screen faeces samples for all possible pathogens in a given clinical situation needs to be emphasized.

Elevada prevalencia de Cyclospora cayetanensis en indígenas del estado Bolívar, Venezuela / High prevalence of Cyclospora cayetanensis among indigenous people in Bolivar State, Venezuela

En la actualidad las coccidiosis intestinales causadas por Cryptosporidium parvum, Isospora belli y Cyclospora cayetanensis constituyen parasitosis de gran importancia médica, sin embargo, en el Estado Bolívar, Venezuela, se desconocen muchos aspectos epidemiológicos de estas infecciones. Entre julio de 2003 y abril de 2004 fue evaluada la población indígena San Antonio de Morichal con el objetivo de determinar la prevalencia de coccidiosis intestinales. Las muestras fecales obtenidas por evacuación espontánea fueron preservadas en dicromato de potasio 2,5 por ciento y posteriormente examinadas mediante la técnica de concentración con formol-éter y coloración de Kinyoun. De los 160 indígenas evaluados, el 92,5 por ciento (148 casos) estaba parasitado. Entamoeba coli (58,8 por ciento), Ascaris lumbricoides (38,8 por ciento) y Giardia lamblia (18,8 por ciento) fueron los más comunes. La prevalencia de coccidiosis intestinal fue de 13,1 por ciento, siendo la cyclosporiosis la más importante con 11,9 por ciento (19 casos). Solamente se diagnosticó un caso con ooquistes de C. parvum (0,6 por ciento) y otro con I. belli (0,6 por ciento). No hubo diferencias en cuanto al sexo (p > 0,05) y la edad (c2 = 5,6; g.l: 6) de los habitantes infectados con C. cayetanensis. De los infectados con C. cayetanensis únicamente dos tenían diarrea. En conclusión, se determinó una prevalencia elevada de coccidiosis intestinal en habitantes de la comunidad indígena evaluada, siendo la infección por C. cayetanensis las más prevalente.

Study on cyclospora cayetanensis associated with diarrheal disease in Nepal and Loa PDR.

A study of diarrheal diseases associated with Cyclospora cayetanensis was conducted in Nepal and Lao PDR. A total of 2083 samples were included in this study. Samples in Nepal were collected from October 1999 to August 2002 whereas samples in Lao PDR were collected from February 2002 to June 2003. C. cayetanensis was detected by direct microscopy using ultraviolet and differential interference contrast microscopy. The overall positive rate in Nepal was 9.2% (128/ 1397). A higher positive rate was observed in children aged 10 years and under (11.1%) and was lowest in the age group of 51-60 years (3.1%). A significantly higher positive rate was observed in the summer (rainy season) (12.6%) with the lowest prevalence in the spring (dry season) (1.8%) (p < 0.05). The positive rate was closely associated with rainfall (ml/month). Interestingly, only one of the total 686 samples (0.1%) from Lao PDR was found to be positive for Cyclospora oocysts.

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

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.

The Genus Cyclospora (Apicomplexa: Eimeriidae), with a description of Cyclospora schneideri n.sp. in the snake Anilius scytale scytale (Aniliidae) from Amazonian Brazil: a review

A review is made of the recorded species of the coccidian genus Cyclospora and major events leading up to the discovery of C. cayetanensis, which is responsible for serious outbreaks of diarrhoea in man and is one of the aetiological agents of "traveller's diarrhoea". Humans appear to be the specific hosts, with the entire life-cycle in the intestine: to date there is no convincing evidence that the disease is a zoonosis. A description is given of oocysts and endogenous stages of C. schneideri n.sp., in the snake Anilius scytale scytale. Sporulation is exogenous and completed after about one week at 24-26°. Mature oocysts 19.8 Î 16.6 (15.1 Î 13.8-25.7 Î 20.1), shape-index 1.2 (1.0-1.3): no oocyst residuum or polar bodies. Oocyst wall a single colourless, smooth layer with no micropyle: it is rapidly deformed or broken. Sporocysts 13.6 Î 9.4 (11.3 Î 8.3-15.1 Î 9.9), shape-index 1.4 (1.2-1.5) with an inconspicuous Stieda body. Sporozoites 11-13 Î 2.5-3. Endogenous stages are intracytoplasmic in the epithelial cells of the small intestine and with the characters of the Eimeriorina.

Cyclospora cayatanensis en pacientes con SIDA y diarrea crónica / Cyclospora cayetanensis in patients with AIDS and chronic diarrhea

Cyclospora spp. is a protozoan parasite responsible for significant gastrointestinal disease in patients infected with the human immunodeficiency virus. We report the clinical features of two patients with chronic diarrhea and intestinal cyclosporosis caused by Cyclospora cayetanensis. The average value for CD4 count in these patients was lower than or equal to 100 cells/mm3. The oocysts were detected in smears from stool samples stained with modified acid-fast or safranin technique. Light microscopy revealed parasites in the enterocytes and these parasites were associated with villous atrophy. Cyclospora cayetanensis infection might be an important cause of diarrhea in patients with AIDS in Argentina.

A report case of Cyclospora and Cryptosporidium mixed infection in a HIV-negative child in Thailand.

The first case of cyclosporosis in a non HIV-infected child in Thailand, co-infected with Cryptosporidium, was reported. The patient was a 3 year-old malnourished orphan who presented with fever, abdominal distension and relapsing diarrhea. There was no leukocyte in her stool, however, numerous Cyclospora and Cryptosporidium oocysts were identified by modified acid-fast staining. The illness was cured by co-trimoxazole and fluid therapy. More coccidial infections in Thailand may be detected if modified acid-fast staining is routinely performed.