A Survey of Cryptosporidium Oocysts in Water Supplies during a 10-Year Period (2000-2009) in Seoul

This study has been conducted to estimate the occurrence of Cryptosporidium oocysts in water supplies in the Metropolitan area of Seoul, South Korea, for 10 years from 2000 to 2009. Water samples were collected quarterly at 6 intakes in the Han River and its largest stream and 6 conventional Water Treatment Plants (WTPs) serving drinking water for 10 million people of Seoul. Cryptosporidium oocysts were found in 22.5% of intake water samples and arithmetic mean was 0.65 oocysts/10 L (range 0-22 oocysts/10 L). Although the annual mean of oocyst number was as low as 0.04-1.90 oocysts/10 L, 3 peaks in 2004 and 2007 were observed and the pollution level was a little higher in winter. The lowest density was observed at Paldang intake and the pollution level increased at Kuui and Jayang intakes. At the end of the largest stream, oocysts were found in 70% of collected samples (mean 5.71 oocysts/10 L) and it seemed that its joining the Han River resulted in the increase at Kuui intake and downstream. Oocyst removal by physical process exceeded 2.0-2.3 log and then all finished water samples collected at 6 WTPs were negative for Cryptosporidium in each 100 L sample for 10 years. These results suggested that domestic wastewater from the urban region could be a source of Cryptosporidium pollution and separating sewage systems adjacent to the intakes could be meaningful for some intakes having weakness related to parasitological water quality.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10 percent IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71 percent at 48 h vs 14.5 percent), even after two weeks (47 percent vs 1.9 percent). Also, the percentage of extracellular stages augmented (25.3 percent vs 1.1 percent at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

In vivo culture of Cryptosporidium oocysts for laboratory use.

In the present study, we describe in vivo cultivation to produce oocysts. Seven-day-old mice were orally infected with 100,000-120,000 Cryptosporidium oocysts. On day 8 post-infection, the mice were killed by ether, and the small and large intestines collected. A simple extraction procedure was used and purified using Ficoll gradient centrifugation. After purification, the oocysts were preserved in phosphate buffered saline with antibiotic at 4 degrees C before use.

A survey of cryptosporidiosis among 2,541 residents of 25 coastal islands in Jeollanam-do (Province), Republic of Korea

In order to determine the distribution and prevalence of human cryptosporidiosis on western and southern coastal islands of Jeollanam-do (Province), fecal samples were collected from 2,541 people residing on 25 islands, 13 in the western coasts and 12 in the southern coasts, during July and August 2000. Fecal smears were prepared following formalin-ether sedimentation of the samples and stained by a modified acid-fast procedure. The presence of Cryptosporidium oocysts was determined by light microscopy. Cryptosporidium oocysts were detected in 38 specimens (1.5%). The oocyst positive rate varied (0-6.0%) according to island; the highest was detected on Oenarodo (6.0%), followed by Naenarodo (5.6%) and Nakwoldo (5.4%). The majority (35 persons, 94.6%) of Cryptosporidium-infected individuals were older than 50 years of age. Men (22/1,159; 1.9%) were infected at a higher rate than women (16/1,382; 1.2%). The results of the present survey indicate that human Cryptosporidium infections (due to Cryptosporidium hominis and/or C. parvum) are maintained at a relatively low prevalence on coastal islands of Jeollanam-do, Republic of Korea.

Parásitos en aguas del arroyo Napostá, aguas de recreación y de consumo en la ciudad de Bahia Blanca (Provincia de Buenos Aires, Argentina) / Parasites in water used for consumption and for recreational purposes as well as in the water from arroyo Naposta in city of Bahia Blanca, Province of Buenos Aires, Argentina

The aim of the present study was to detect the presence of sanitarily important parasit es in the water from Arroyo Napostá as well as in the water used for recreational purposes (public swimming-pools) and for consumption in the city of Bahía Blanca, province of Buenos Aires, Argentina. To this end, 24,000 liters of water from different sectors of Arroyo Napostá; 2,000 liters of water from each of the three public swimming-pools of Bahía Blanca; and 8,000 liters of water for consumption were collected using polypropylene spun cartridge filters with a 1 mm porosity (Cuno Micro Wind II). Further processing was conducted following Madore´s technique (1987) modified by Pezzani (2000). Detection of Cryptosporidium sp. Giardia sp. was carried out via direct immunofluorescence. Our study confirmed the presence of the following parasites in the areas of study: Hymenolepis diminuta eggs, Giardia sp cysts, Toxocara sp eggs, Entamoeba sp cysts, Endolimax sp cysts, Nematode larvae, Trichostrongylus sp eggs, Ascaris sp eggs, and Cryptosporidium sp cysts in the water from Arroyo Napostá; Hymenolepis diminuta eggs, Giardia sp cysts, Toxocara sp eggs, Entamoeba sp cysts, Endolimax sp cysts, Nematode larvae, Trichostrongylus sp eggs, Ascaris sp in the water from swimming-pools; and Cryptosporidium sp in the water for consumption. The results from the present study urge sanitary authorities from the city of Bahía Blanca to take measures in order to prevent not only humans but also animals from getting in contact with these waters which are contaminated and dangerous to health.

El objetivo del presente trabajo fue detectar la presencia de parásitos de importancia sanitaria para el hombre, en el arroyo Napostá, en aguas recreacionales (piscinas públicas) y de consumo en Bahía Blanca (Provincia de Buenos Aires) Argentina. Se recolectaron 24.000 litros de agua de diferentes sectores del arroyo Napostá, 2.000 litros de cada una de las tres piscinas públicas y 8.000 litros de agua de consumo, mediante filtros de poro de una micra de diámetro (Cuno Micro Wind). El procesamiento posterior se efectuó siguiendo la técnica de Madore (1) modificada por Pezzani (2). La detección de Cryptosporidium sp. Giardia sp. se realizó mediante inmunofluo-rescencia directa. Las formas parasitarias observadas, fueron: huevos de Hymenolepis diminuta, quistes de Giardia sp, huevos de Toxocara sp, quistes de Entamoeba sp. Quistes de Endolimax sp, larvas de Nematodos, huevos de Trichostrongylus sp, huevos de Ascaris sp y quistes de Cryptosporidium sp. en las aguas del Arroyo Napostá; huevos de Hymenolepis diminuta, quistes de Giardia sp, huevos de Toxocara sp, quistes de Entamoeba sp. quistes de Endolimax sp, larvas de Nematodos, huevos de Trichostrongylus sp, huevos de Ascaris sp. en agua de piscina; y quistes de Cryptosporidium sp. en agua de consumo.En conclusión es necesario que las autoridades sanitarias adopten medidas tendientes a evitar que el hombre y los animales tomen contacto con estas aguas contaminadas y peligrosas para la salud.

In vitro culture of Cryptosporidium muris in a human stomach adenocarcinoma cell line

We investigated the optimal culture conditions for Cryptosporidium muris in a human stomach adenocarcinoma (AGS) cell line by determining the effects of medium pH and of selected supplements on the development of C. muris. The optimum pH of the culture medium required for the development of C. muris was determined to be 6.6. The number of parasites significantly increased during cultivation for 72 hr (p < 0.05) at this level. On the other hand, numbers decreased linearly after 24 hr of incubation at pH 7.5. When cultured in different concentrations of serum, C. muris in media containing 5% FBS induced 4-7 times more parasites than in 1% or 10% serum. Of the six medium supplements examined, only 1 mM pyruvate enhanced the number of C. muris in vitro. Transmission electron microscopic observation showed the developmental stages of C. muris in the cytoplasm of the cells, not in an extracytoplasmic location. The growth of C. muris in AGS cells provides a means of investigating its biological characteristics and of testing its response to therapeutic agents. However, a more optimized culture system is needed for the recovery of oocysts on a large scale in vitro.

Testing methods for detection of Cryptosporidium spp in water samples.

A large waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, USA in 1993 prompted a search for ways to prevent large-scale waterborne outbreaks of protozoan parasitoses. Methods for detecting Cryptosporidium parvum play an integral role in strategies that lead to appropriate treatment of surface water, but are criticized because they produce results that are highly variable. The US Environmental Protection Agency developed a set of criteria to evaluate detection methods for protozoan parasites in water. As a consequence, the Agency has had to develop approaches to reducing uncertainty of evaluations. The variability and accuracy of various methods of producing small numbers of Cryptosporidium spp oocysts were tested. The least variable and most accurate method was used to spike seven surface water, and one tap water sample to compare 4 detection methods that had been reported in the literature. The least variable and most accurate method for spiking specified numbers of oocysts into samples was found to be flow cytometry. The most effective of the methods tested for detection in surface, tap and reagent water was solid phase cytometry.

Factores que influyen en el desenquistamiento in vitro de Cryptosporidium sp / Factors affecting the in vitro excystation of Cryptosporidium sp

Para evaluar la viabilidad de los ooquistes de Cryptosporidium sp se desarrolló y optimizó una técnica de desenquistamiento in vitro. Los ooquistes de Cryptosporidium sp se obtuvieron de una muestra de materia fecal de un niño con cryptosporidiosis sintomática. La muestra fue procesada por la técnica de gradiente discontinuo de sacarosa hasta obtener una suspensión de ooquistes purificados y concentrados (OPC). Los ooquistes se cuantificaron en cámara de Neubauer y preservaron en PBS pH 7, 2 a 4§C. Los dos protocolos ensayados a partir de alícuotas de 100µl de OPC con 178.000 ooquistes/mm3 fueron: I. Tratamiento con bilis 1 por ciento y bicarbonato de sodio al 0,44 por ciento a 37§C durante 24 horas. II Tratamiento con bilis 1 por ciento con pH 6 y pH 8, con y sin bicarbonato de sodio al 0,44 por ciento, con y sin incubación en atmósfera de 10 por ciento de CO2 durante 24 h a 37§C. Se evaluó el grado de desenquistamiento en todos los grupos efectuando el conteo de ooquistes intactos en cámara de Neubauer. La preincubación con tripsina no incrementó el desenquistamiento de los ooquistes de Cryptosporidium sp. Las mejores condiciones de desenquistamiento fueron utilizando bilis al 1 por ciento pH 8, incubado con 10 por ciento de CO2, a 37§C durante 24 h.

Cryptosporidiosis in patients with diarrhoea in five hospitals in Nigeria.

A total of 413 soft, loose or watery stool specimens from patients with acute diarrhoea were screened for presence of Cryptosporidium oocysts. Safranin-methylene blue staining technique was used for detecting Cryptosporidium oocysts in the stool samples. The oocysts were identified in 52 (12.5 per cent) of the samples, while 69 samples (16.7 per cent) showed mixed infections. Children between the ages of 2 and 15 years were most infected by this parasite while infection occurred more in females than males for all age groups. These findings indicate that Cryptosporidium is an important etiologic agent of diarrhoea. Hence there is need to include its identification in laboratory analysis of diarrhoeal stool samples.

Criptosporidiosis en preescolares de tres guarderias del área metropolitana de Caracas / Criptosporidiosis in preeshool of three, keepership the urban area of Caracas

Para el presente trabajo se seleccionaron 268 niños quienes provienen de diferentes zonas marginales de la ciudad de Caracas y acuden diariamenme a tres instituciones educativas subsidiadas por la Alcaldía de Caracas. Entre los meses de julio, agosto y septiembre se tomó de cada niño una muestra de heces, la cual fue fijada con Metanol y coloreada con la técnica de Kinyoun, para luego identificar las diferentes formas evolutivas de Cryptosporidium sp. De las muestras examinadas se constató la presencia de Cryptosporidium sp. en 128 (47,8 por ciento) en tanto que en 140 (52,2 por ciento) resultaron negativas para este mismo protozoario. La alta incidencia de Cryptosporidiosis puede explicarse por la época del año en que se recolectaron las muestras y las inadecuadas condiciones higiénicas en las que se desenvuelve esta población