Ocorrência e identificação de Cryptosporidium e Giardia em amostras de água superficial destinada ao abastecimento público do estado de São Paulo / Occurrence and identification of Cryptosporidium and Giardia from surface water catchment in Sao Paulo, Brazil

Estudos de revisão sobre surtos associados à transmissão hídrica revelaram que os protozoários parasitas Cryptosporidium parvum e Giardia duodenalis (sinonímia: G. lamblia e G. intestinalis) são os principais responsáveis pelo maior número de casos registrados em todo o mundo. A contaminação das águas superficiais que servem ao abastecimento público por estes protozoários representa risco à saúde humana e animal, pois ambos parasitas apresentam resistência à cloração, processo convencional utilizado para desinfecção em Estações de Tratamento de Água (ETAs).Em vista desta lacuna, o presente estudo propõe identificar espécies e genótipos de Cryptosporidium e Giardia a partir de 128 amostras de águas superficiais de 11 mananciais do estado de São Paulo, de acordo com o Método 1623.1 (USEPA, 2012). Para identificar estes parasitas, foi realizada a recuperação dos (oo) cistos a partir de lâminas raspadas, seguindo o protocolo adaptado da USEPA, em seguida, utilizou-se o PCR em tempo real para identificar os genes 18S rRNA para Cryptosporidium e SSU para Giardia. Os resultados mostraram que a frequência de ocorrência desses protozoários nos pontos de captação foi de 29,7% para Giardia e 30,4% para Cryptosporidium. Os cistos estavam presentes em 10 dos 11 pontos de captação com frequências que variaram de 17 a 100%, e concentrações que variaram de

Review studies on waterborne outbreaks have been showing that Cryptosporidium parvum and Giardia duodenalis (synonym: G. lamblia and G. intestinalis) are the primarily responsible for the highest number of the cases recorded worldwide. Contamination of surface waters catchments by these protozoa is a risk factor to human health because both parasites are resistant to chlorination, which is a conventional process used for disinfection in water treatment plants (WTP). The present study aimed to identify species of Cryptosporidium and Giardia recovered from surface water catchment samples from 11 municipalities from the State of São Paulo, totalizing 128 samples. Quantification of both parasites was carried out according to method 1623.1 (USEPA, 2012). In order to identify parasites, the recovering of (oo)cysts from slides followed USEPA´s protocol by scraping slides, then Real Time PCR using the 18S rRNA genes for Cryptosporidium and SSU for Giardia were carried out. Results showed that the frequency of occurrence of these protozoa at the catchment points was 29,7% for Giardia and 30,4% for Cryptosporidium. Cysts were present in 10 of 11 catchments points with frequencies varying from 17 to 100% with concentrations ranging from

Detection of Cryptosporidium parvum in Environmental Soil and Vegetables

Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidial enteritis. Numerous outbreaks of cryptosporidiosis have been reported worldwide. Cryptosporidium is transmitted to hosts via consumption of contaminated water and food but also by direct contact with contaminated soil or infected hosts. The present study investigated farm soil collected from 34 locations along the western Korean peninsula and 24 vegetables purchased from local grocery markets in Seoul. The soil and vegetable samples were examined by real-time polymerase chain reaction (qPCR) to estimate the risk of infection. Eleven of 34 locations (32.4%) and 3 of 24 vegetable samples (12.5%) were contaminated with Cryptosporidium parvum, as confirmed by TaqI enzyme digestion of qPCR products and DNA sequencing. It is suggested that Cryptosporidium infection can be mediated via farm soil and vegetables. Therefore, it is necessary to reduce contamination of this organism in view of public health.

A Waterborne Outbreak and Detection of Cryptosporidium Oocysts in Drinking Water of an Older High-Rise Apartment Complex in Seoul

From May to June 2012, a waterborne outbreak of 124 cases of cryptosporidiosis occurred in the plumbing systems of an older high-rise apartment complex in Seoul, Republic of Korea. The residents of this apartment complex had symptoms of watery diarrhea and vomiting. Tap water samples in the apartment complex and its adjacent buildings were collected and tested for 57 parameters under the Korean Drinking Water Standards and for additional 11 microbiological parameters. The microbiological parameters included total colony counts, Clostridium perfringens, Enterococcus, fecal streptococcus, Salmonella, Shigella, Pseudomonas aeruginosa, Cryptosporidium oocysts, Giardia cysts, total culturable viruses, and Norovirus. While the tap water samples of the adjacent buildings complied with the Korean Drinking Water Standards for all parameters, fecal bacteria and Cryptosporidium oocysts were detected in the tap water samples of the outbreak apartment complex. It turned out that the agent of the disease was Cryptosporidium parvum. The drinking water was polluted with sewage from a septic tank in the apartment complex. To remove C. parvum oocysts, we conducted physical processes of cleaning the water storage tanks, flushing the indoor pipes, and replacing old pipes with new ones. Finally we restored the clean drinking water to the apartment complex after identification of no oocysts.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Infección por Cryptosporidium parvum en una mujer embarazada, inmunocompetente, con riesgo ocupacional / Cryptosporidium parvum infection in a pregnant inmunocompetent woman with occupational risk

La criptosporid iosis es una zoonosis parasitaria provocada por diversas especies de Cryptosporidium. Esta coccidiosis afecta a múltiples vertebrados, incluido el ser humano. En Chile, al igual que en otros países, es una infección poco frecuente en inmunocompetentes y adquiere gran relevancia en pacientes inmunocom-prometidos. Se presenta el caso de una egresada de la carrera de Medicina Veterinaria, embarazada, con 20 semanas de gestación, procedente del sector de Laguna Verde, Región de Valparaíso, que fuera infectada por Cryptosporidium sp. El diagnostico etiológico se realizó con tinción de Ziehl Neelsen, RPC anidada y posterior secuenciación. En el mismo periodo se detectó la infección en sus gatos asintomáticos. En ella y los animales se identificó a C. parvum. Su cónyuge así como sus otras mascotas no estaban infectados. Este corresponde al primer reporte de una posible transmisión de criptos-poridiosis entre ser humano y gato.

Cryptosporidioses is a parasitic zoonoses generated by diverse Cryptosporidium species. This coccidiosis affects multiple vertebrate species, including human beings. In Chile, as it happens in other countries, cryptosporidioses is a low frequency infection in immunocompetent individuals, acquiring a big relevance in immunocompromised ones. We present the following case: a recently graduated student from Veterinary medical school, with a 20 week pregnancy, living in “Laguna Verde” area in the Region of Valparaíso and who was infected with Cryptosporidium sp. Etiologic diagnosis was made by Ziehl Neelsen, and nested PCR followed by PCR product sequencing. During the same period, the infection was detected in her cats which were asymptomatic. In all of them, her and the cats, the species identified was Cryptosporidium parvum. Her husband and her other pets were all asymptomatic and non infected. This is the first report of a possible cryptosporidioses transmission between humans and cat.

Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10(3) to 10(4) oocysts, and the nested PCR method was able to detect 10(0) to 10(2) oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.

Quantitative Evaluation of Infectivity Change of Cryptosporidium parvum after Gamma Irradiation

Cryptosporidium parvum is a well-known waterborne and opportunistic intracellular protozoan parasite that causes diarrheal illness. In this study, we quantitatively investigated reduction of the infectivity of C. parvum after gamma irradiation and repair of the infectivity during incubation time after irradiation. C. parvum oocysts were subjected to gamma irradiation at various doses (1, 5, 10, and 25 kGy), and the in vitro infectivity was measured by real-time PCR every day up to 7 days after irradiation. The in vitro infectivity of C. parvum on human ileocecal adenocarcinoma cells (HCT-8) was effectively reduced (> 2 log(10)) by irradiation at 10 kGy or more. However, in the experiment to find out repair of the infectivity, recovery was not noted until day 7 post-incubation.

Detection of Cryptosporidium parvum in HIV-infected patients in Malaysia using a molecular approach.

This cross-sectional study determined the prevalence of cryptosporidiosis in HIV-infected patients using polymerase chain reaction (PCR). Stool specimens were collected from HIV infected patients who were admitted to Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia, for various indications from December 2004 to December 2005. A modified acid-fast stain was performed on the direct stool smears, then the stool specimens were further tested using nested PCR targeting the 18S rRNA gene of Cryptosporidium parvum, with a built-in internal control (IC). Out of 59 samples, 11 were positives. Nested PCR identified a total of nine samples (16%) compared to microscopy, which identified only three samples. All PCR negative results showed IC amplicons, suggesting that these samples were true negatives and were not due to inhibition of PCR. This study highlights the importance of molecular diagnosis in determining the true prevalence and epidemiology of C. parvum.

Genotype analysis of Cryptosporidium spp. prevalent in a rural village in Hwasun-gun, Republic of Korea

Two species of Cryptosporidium are known to infect man; C. hominis which shows anthroponotic transmission between humans, and C. parvum which shows zoonotic transmission between animals or between animals and man. In this study, we focused on identifying genotypes of Cryptosporidium prevalent among inhabitants and domestic animals (cattle and goats), to elucidate transmittal routes in a known endemic area in Hwasun-gun, Jeollanam-do, Republic of Korea. The existence of Cryptosporidium oocysts was confirmed using a modified Ziehl- Neelsen stain. Human infections were found in 7 (25.9%) of 27 people examined. Cattle cryptosporidiosis cases constituted 7 (41.2%) of 17 examined, and goat cases 3 (42.9%) of 7 examined. Species characterizations were performed on the small subunit of the rRNA gene using both PCR-RFLP and sequence analysis. Most of the human isolates were mixtures of C. hominis and C. parvum genotypes and similar PCR-RFLP patterns were observed in cattle and goat isolates. However, sequence analyses identified only C. hominis in all isolates examined. The natural infection of cattle and goats with C. hominis is a new and unique finding in the present study. It is suggested that human cryptosporidiosis in the studied area is caused by mixtures of C. hominis and C. parvum oocysts originating from both inhabitants and domestic animals.

Genotype and animal infectivity of a human isolate of Cryptosporidium parvum in the Republic of Korea

Cryptosporidium parvum oocysts were isolated from a child suffering from acute gastroenteritis and successfully passaged in a calf and mice (designated hereafter SNU-H1) in the Republic of Korea; its molecular genotype has been analyzed. The GAG microsatellite region was amplified by a polymerase chain reaction (PCR), with a 238 base pair product, which is commonly displayed in C. parvum. The isolate was shown to be a mixture of the genotypes 1 (anthroponotic) and 2 (zoonotic). To study its infectivity in animals, 2 calves and 3 strains of mice were infected with the SNU-H1; in these animals, the propagation of both genotypes was successful. In immunosuppressed (ImSP) BALB/c and C57BL/6 mice the number of oocysts decreased after day 10 post-infection (PI) ; but in ImSP ICR mice, they remained constant until day 27 PI. The results show that both the C. parvum genotypes 1 and 2 can be propagated in calves and ImSP mice.