Identification of Cryptosporidium spp. oocysts in fecal smears stained with Heidenhain's iron hematoxylin

There is no paucity of methods for diagnosing Cryptosporidium spp. infection. The merits of immunoassays notwithstanding, microscopic identification of Cryptosporidium spp. oocysts in fecal samples remains an important diagnostic procedure. It owes the persistence of its use to such characteristics as dispensing with expensive equipment and kits, requiring only basic laboratory facilities, and having a low probability of false positive results when permanent slides are prepared, which can be re-examined in case of doubt. Cryptosporidium spp. oocysts can be readily identified in fecal smears prepared according to a regressive iron hematoxylin staining technique. The number of steps and their duration, as well as costs, were reduced to a minimum without loss of image quality and permanence of the preparations.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.

Prevalence and genetic characterization of Cryptosporidium spp. and Cystoisospora belli in HIV-infected patients

Cryptosporidium spp. and Cystoisospora belli are monoxenic protozoa that have been recognized as the causative agents of chronic diarrhea in immunocompromised individuals, especially HIV-infected subjects. The objective of this study was to evaluate the frequency of these intestinal protozoa in HIV-positive patients in the Triângulo Mineiro region of Brazil and to correlate the presence of these infections with clinical, epidemiological and laboratory data of the patients. Oocysts were detected in stool samples of 10 (16.9%) of the 59 patients studied, while Cryptosporidium spp. were present in 10.1% (6/59) and C. belli in 6.7% (4/59). The frequency of these parasites was higher among patients with diarrheic syndrome and CD4+ T lymphocyte counts < 200 cells/mm 3 , demonstrating the opportunistic characteristic of these infections. A significant association was observed between the lack of adherence to antiretroviral therapy and the presence of Cryptosporidium spp. and/or C. belli. Parasitism with Cryptosporidium spp. was more frequent in February and April, the months following the period of high rainfall. The same was not observed for C. belli. Genetic characterization of two isolates led to the identification of Cryptosporidium parvum, one of the main species associated with the zoonotic transmission of cryptosporidiosis.

WATER FROM DIFFERENT SOURCES USED FOR THE IRRIGATION OF VEGETABLES TO BE MARKETED: RESEARCH ON Cryptosporidium spp., Giardia spp., AND COLIFORMS IN PARANA, BRAZIL

SUMMARY The aim of this work was to compare, from a parasitological ( Cryptosporidiumspp. and Giardia duodenalis), bacteriological (total and thermotolerants coliforms) and physicochemical perspective, water sources used for drinking and irrigation of vegetables intended to be sold for human consumption. From January 2010 to May 2011, samples of different water sources from vegetable producing properties were collected; 100 liters for parasitological analysis, 200 mL for bacteriological analysis, and five liters for physicochemical analysis. Water samples were filtered under vacuum with a kit containing a cellulose acetate membrane filter, 1.2 µm (Millipore(r), Barueri, SP, Brazil). The material retained on the membrane was mechanically extracted and analyzed by direct immunofluorescence (Merifluor(r)kit). From 20 rural properties investigated, 10 had artesian wells (40 samples), 10 had common wells (40 samples), and one had a mine (four samples), the latter contaminated by Cryptosporidiumspp. In samples from artesian wells, 90 to 130 meters depth, 42.5% were positive for total coliforms and 5.0% were identified to have abnormal coloration. From the samples of common wells, 14 to 37 meters depth, 87.5% were contaminated with total coliforms, 82.5% were positive for thermotolerant coliforms, and 12.5% had color abnormalities. We did not detect the presence of Giardiaspp. or Cryptosporidiumspp. in artesian and common wells. The use of artesian or common wells is an important step in the control of the spreading of zoonoses, particularly Cryptosporidiumspp. and Giardiaspp., as well as artesian wells for coliform control in local production of vegetables to be marketed.

Detection of Cryptosporidium spp and other intestinal parasites in children with acute diarrhea and severe dehydration in Rio de Janeiro

The objective of the present study was to estimate the frequency of infection by Cryptosporidium spp and other intestinal parasites in dehydrated children with gastroenteritis who were admitted to a pediatric hospital. Stool examinations from 218 children were performed. Cryptosporidium spp was identified in eighteen out of 193 stool samples (9.3%) subjected to safranin-methylene blue staining. Giardia lamblia was detected in ten out of 213 (4.7%) samples examined via the direct or Ritchie methods. Other parasites identified were Ascaris lumbricoides (4.2%), Blastocystis hominis (1.4%), Entamoeba coli (0.9%), Entamoeba histolytica/Entamoeba dispar (0.5%), Endolimax nana (0.5%), Trichuris trichiura (0.5%) and Enterobius vermicularis (0.5%).

Presença de Cryptosporidium spp em crianças com diarréia aguda em uma creche pública de Recife, Estado de Pernambuco

O objetivo do presente estudo foi analisar a freqüência de oocistos de Cryptosporidium spp em amostras fecais de crianças, de 1 a 14 anos, de uma creche pública localizada em uma comunidade carente da cidade do Recife, Pernambuco. A pesquisa foi realizada no período de 28 de junho de 2006 a 3 de abril de 2007, e envolveu 182 crianças. Das amostras analisadas 59 (32,4%) foram positivas quanto à presença de oocistos de Cryptosporidium spp, e a faixa etária mais acometida foi a de 3 a 5 anos de idade (54,2%). A alta freqüência de amostras positivas para Cryptosporidium spp obtidas neste estudo comprovam que creches são ambientes propícios a essa ocorrência devido ao contato direto entre criança-criança, crianças e funcionários. A maior via de infecção por Cryptosporidium spp é a transmissão interpessoal, que é bem ilustrada em creches. A imaturidade, deficiências do sistema imune e hábitos higiênicos inadequados são fatores que também contribuem para esse tipo de infecção.

Occurrence of Cryptosporidium spp., Giardia spp. and other pathogenic intestinal parasites in the Beberibe River in the State of Pernambuco, Brazil

INTRODUCTION: Transmission of pathogenic protozoa and helminths by water is a serious public health problem. In this study, we analyzed the presence of these organisms in the Beberibe River in Pernambuco, Brazil. METHODS: Parasite analysis was performed using the Hoffman, Pons, & Janer method followed by centrifugation and preparation of slides by staining with acetic acid and Lugol's solution. Protozoan oocysts were isolated by the modified Ziehl Neelsen method. RESULTS: Cryptosporidium spp., Giardia spp. and other parasites were found in the Beberibe River. CONCLUSIONS: Sanitation companies must assess pathogenic intestinal parasites in water basins providing public water and subsequently develop improved treatment systems for removal of such parasites.

Emergent and re-emergent parasites in HIV-infected children: immunological and socio-environmental conditions that are involved in the transmission of Giardia spp. and Cryptosporidium spp.

ABSTRACT INTRODUCTION: Emergent and re-emergent waterborne protozoans have become a worldwide public health problem, especially among vulnerable groups. METHODS: This cross-sectional study evaluated 17 HIV-infected children and their families. RESULTS: A high (76.5%) percentage of parasite-infected children was observed, even among children with CD4+ T-cell counts of >200 cells/mm3. Giardia spp., Cryptosporidium spp. and Cyclospora spp. were observed in 41.2% of these children Low income, poor hygiene practices, and co-infection in domestic, peridomestic and scholastic environments were significant sources of these intestinal infections. CONCLUSIONS: Early diagnosis, timely treatment, and socio-educational interventions may improve the health conditions of this vulnerable population.

Importância de Cryptosporidium spp. como causa de diarréia em bezerros

Avaliou-se a presença de oocistos de Cryptosporidium spp. em amostras de fezes de 14 bezerros e de suas mães até a oitava semana pós parição. A maior taxa de excreção de oocistos foi verificada em bezerros com sete dias de idade. Das vacas, 42,8% foram positivas para Cryptosporidium no período pós-parto. Em outra etapa deste estudo, foram acompanhados 57 bezerros positivos para Cryptosporidium, com até 30 dias de idade, provenientes de 32 propriedades leiteiras, e estudouse o grau de eliminação dos oocistos com a possível ocorrência de diarréia. Em todos os animais positivos para Cryptosporidium foi pesquisada a presença de bactérias enteropatogênicas, vírus (Rotavirus e Coronavirus) e protozoários (Eimeria spp.).

Fatores de risco associados à infecção por Cryptosporidium spp. e Giardia duodenalis em bovinos leiteiros na fase de cria e recria na mesorregião do Campo das Vertentes de Minas Gerais

Este estudo observacional do tipo transversal foi realizado com o objetivo avaliar os fatores de risco associados à infecção por Cryptosporidium spp. e Giardia duodenalis em bezerras provenientes de 20 propriedades leiteiras, localizadas na mesorregião do Campo das Vertentes de Minas Gerais. As propriedades foram divididas igualmente em dois grupos de acordo com o tipo de leite produzido: Grupo I = Leite B e Grupo II = Leite cru refrigerado. Amostras fecais de 356 bezerras foram coletadas no período de setembro de 2008 a agosto de 2009 e analisadas utilizando-se os métodos de Ziehl-Neelsen e flutuação em sulfato zinco a 33% para detecção, respectivamente, dos oocistos de Cryptosporidium spp. e cistos de G. duodenalis. Dados sobre práticas de manejo e condições sanitárias de criação dos bovinos foram obtidos por meio de entrevistas durante a visita a cada propriedade, no momento em que foi coletada uma única amostra de fezes de bezerras de 1 dia a 12 meses de idade. A frequência média global de bezerras infectadas por Cryptosporidium spp. foi de 21,62%, sendo a faixa etária de 7- 21 dias de idade a que apresentou o maior número de animais eliminando oocistos. Para G. duodenalis, a frequência média global foi de 25,56% e a faixa etária de 60-90 dias de idade foi a com maior número de animais com cistos nas fezes. Os resultados deste estudo indicam que infecções por Cryptosporidium spp. e G. duodenalis estão amplamente distribuídas entre fêmeas bovinas na fase de cria e recria provenientes de rebanhos leiteiros na mesorregião do Campo das Vertentes de Minas Gerais. Dentre os fatores associados a um maior risco de infecção por Cryptosporidium spp. e G. duodenalis em bezerras, discutidos neste estudo, se destacam os seguintes: a permanência no piquete maternidade por mais de 12h após o nascimento; o fornecimento de colostro a partir de 7h de vida; o primeiro fornecimento de água e concentrado entre 1 e 7 dias de idade; e a manutenção em instalação coletiva e/ou localizada próxima ao curral.

Ocorrência da infecção por Cryptosporidium spp. em cabritos (Capra hircus)

O presente estudo teve como objetivo determinar a ocorrência da infecção por Cryptosporidium spp. em cabritos de Quixadá, Ceará, Brasil. Participaram do estudo 400 cabritos, com idade entre três e 360 dias, de ambos os sexos, com e sem padrão racial definido, procedentes de 25 estabelecimentos rurais distribuídos em três circuitos. As fezes foram cadastradas de acordo com o aspecto e cor, distribuídas em tubos tipo "eppendorf®" e congeladas in natura a -20°C, até o momento das extrações de DNA genômico do parasito com auxílio de kit comercial. Para amplificação de fragmentos da subunidade 18S do RNA ribossômico (rRNA) foi utilizada a "Nested"-PCR. A ocorrência de Cryptosporidium spp em cabritos de Quixadá foi de 7,50% (30/400). A frequência no período seco e no chuvoso foi de 9,55% (19/199) e 5,47% (11/201), respectivamente (χ²=2,39 e P>0,05). Amostras positivas foram identificadas em 64,00% (16/25) das propriedades estudadas e dessas amostras 50,00% (15/30) e 70,00% (21/30) tinham as fezes com aspecto e cor normais, respectivamente, sugerindo que cabritos assintomáticos estão eliminando oocistos. Não foi observada positividade para Cryptosporidium spp. em animais com 301 a 360 dias, demonstrando que animais mais velhos apresentam menos possibilidade de se infectarem com o parasito.