Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals

ABSTRACT The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN® Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA®QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water / Produção de anticorpos policlonais anti-Cryptosporidium e padronização da imunofluorescência direta para a detecção de oocistos na água

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20 percent of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

INTRODUÇÃO: A produção de anticorpos policlonais anti-Cryptosporidium e sua utilização na imunofluorescência para determinar a presença de Cryptosporidium em água são descritas no presente trabalho. MÉTODOS: Dois coelhos foram imunizados com antígeno solúvel e particulado provenientes de oocistos purificados de Cryptosporidium. O soro produzido foi preparado para a extração de imunoglobulinas G, que foram purificadas e conjugadas com isotiocianato de fluoresceína (FITC). Lâminas contendo quantidades conhecidas de oocistos foram preparadas para determinar a sensibilidade da técnica. Para testar a especificidade foram preparadas lâminas contendo cistos de Giardia duodenalis. RESULTADOS: O conjugado foi usado com sucesso em amostras de água contaminadas experimentalmente com oocistos de Cryptosporidium, sendo capaz de detectar até cinco oocistos/spots que corresponde a uma contaminação de 250 oocistos/mL. CONCLUSÕES: As três imunizações realizadas nos coelhos foram suficientes para produção de anticorpos contra Cryptosporidium; a reação de imunofluorescência direta padronizada permitiu a detecção de cinco oocistos em 20 por cento das amostras; não houve reação cruzada com cistos de Giardia duodenalis.

Infestación por Cryptosporidium Spp e Isospora Belli en preescolar inmunocompetente: a propósito de un caso / Infestation by Crytosporidium Spp and Isospora belli in immunocompetent preschool: a report of a case

El Cryptosporidium spp e Isospora belli son parásitos emergentes, que representan la cuarta causa de diarrea a nivel mundial, principalmente en niños y en pacientes inmunocomprometidos. Producen diarrea aguda o crónica dependiendo de la edad del paciente, estado nutricional e inmunológico asociado a factores sanitarios desfavorables. El diagnostico se realiza por visualización directa en heces con tinción de Zelh Neelsen modificado o Kinyou y biopsia intestinal con presencia de protozoos en las criptas y atrofia vellositaria de acuerdo al grado de infestación. Se reporta el caso de preescolar de 2 años de edad, eutrófico e inmunocompetente, perteneciente a estrato social bajo; con episodios de diarreas acuosas autolimitadas, dolor y distensión abdominal frecuentes. La biopsia intestinal revelo atrofia vellositaria e infestación simultanea por Cryptosporidium spp e Isospora belli corroborado por Tinción de Kinyou en heces; se descarto además Alergia Alimentaria, Enfermedad Celiaca e Inmunodeficiencias. El propósito de este caso clínico es alertar sobre la necesidad de incluir dentro del protocolo de estudio de diarrea crónica, la búsqueda de protozoarios formadores de esporas, mediante tinción especial en heces; un método no invasivo y sencillo, no solicitado en forma rutinaria.

Cryptosporidium spp and Isospora belli parasites are emerging that represent the fourth leading cause of diarrhea worldwide, mainly in children and in immunocompromised patients. Acute or chronic diarrhea occur depending on the patient's age, nutritional status and immunological factors associated with adverse health. The diagnosis is made by direct visualization in feces Neelsen stain Zelh Kinyou modified or intestinal biopsy and the presence of protozoa in the crypts and villous atrophy according to the degree of infestation. We report the case of preschool age 2, eutrophic immunocompetent, belonging to low socioeconomic levels, with self-limiting episodes of acute watery diarrhea, frequent abdominal pain and bloating. The intestinal biopsy revealed villous atrophy and simultaneous infestation by Cryptosporidium spp and Isospora belli Kinyou confirmed by staining in feces, while discarding also Food Allergy, Celiac disease and immunodeficiencies. The purpose of this case to alert about the need to include in the study protocol of chronic diarrhea, the search for spore-forming protozoa by special staining in feces, a noninvasive and simple method, not routinely requested.

Cryptosporidium SPP: en niños sanos menores de 10 años de la comuna 18 de Cali, Colombia / Cryptosporidium spp. in healthy children under 10 years of the Commune 18, Cali, Colombia

Introducción: El Cryptosporidium spp. (C. spp.) es uno de los principales agentes causales de diarrea enniños. En Colombia, hay pocos estudios de prevalencia en niños sanos. Objetivo: Determinar la prevalencia de C. spp. mediante ELISA en niños de Cali, Colombia. Materiales y métodos: Estudio descriptivo observacional de corte transversal en 107 niños sanos < 10 años, de la Comuna 18 de Cali, Colombia, quienes consultaron al Programa de Crecimiento y Desarrollo del Centro de Salud Lourdes. El análisis estadístico incluyó medidas de tendencia central, de dispersión y pruebas de asociación. Se incluyeron datos como edad, género, signos y síntomas, peso (P) y talla (T), y condiciones ambientales. Según las tablas de la NCHS fueron clasificados según grado de desnutrición (DNT). La identificación del C. spp., se realizó en materia fecal mediante la técnica de ELISA. Resultados: Fueron analizados 100 niños, 66% estrato 1, con edad = 4 años ± 2 meses, 50 niños, 30% en hacinamiento, 95% con agua potable, 85% con disposición de excretas, 52% con animales intradomiciliares, y 63% asisten a guardería o colegio. Ninguno de los niños C. spp. positivos, presentó síntomas. Tuvieron P = 16±6 kg y T = 97±18 cm, 40% con algún tipo de DNT. La prevalencia fue del 4% (IC 95% 0,1-7,89) sin diferencias significativas (p>0.05). Conclusiones: La prevalencia para C. spp. fue del 4% sin posibles asociaciones demográficas, ambientales o clínicas.

Introduction: Cryptosporidium spp. (C. spp.) is a major causative agents of diarrea in children. In Colombia, there are few prevalence studies in healthy children. Objective: To determine the prevalence ofC. spp. by ELISA in children of Cali, Colombia. Materials and methods: A cross-sectional observational in 107 healthy children < 10 years from the Commune 18 in Cali, Colombia, who consulted the Growth and Development Program of Centro de Salud Lourdes. Statistical analysis included measures of central tendency, dispersión and association tests. Data included age, gender,signs and symptoms, weight (W) and height (H), and environmental conditions. According to the NCHS tables were classified by degree of malnutrition (MNT). The identification of C. spp. was held in stool by ELISA. Results: 100 children were analyzed, 66% stratum 1, age = 4 years ± 2 months, 50 boys, 30% overcrowding, 95% with wáter, 85% with waste disposal, 52% with home animals, and 63% attend daycare or school. None of the children C. spp. positive had symptoms. The W were 16±6 kg and H were 97±18 cm, 40% were some form of MNT. The prevalence was 4% (95% CI 0.1 to 7,89) without significant differences (p>0.05). Conclusions: The prevalence of C. spp. was 4% without any demographic, environmental or clinics associations.

Performance of an immunoenzymatic assay for Cryptosporidium diagnosis of fecal samples

We evaluated the diagnostic performance of a Cryptosporidium immunoenzymatic assay (ELISA). Fecal samples were collected from 94 HIV-seropositive patients. All specimens were processed with a commercially-available ELISA to detect C. parvum specific coproantigen and with a modified Ziehl-Neelsen stain (ZNm) microscope exam. Overall, sensitivity of the immunoenzymatic test was 100 percent, with a specificity of 96 percent; positive and negative predictive values were 89 percent and 100 percent, respectively. The commercial ELISA and ZNm proved to be valuable diagnostic tools for Cryptosporidium infection.

Human cryptosporidiosis: detection of specific antibodies in the serum by an indirect immunofluorescence

Utilizando a técnica de imunofluorescência indireta, foram demonstradas imunoglobulinas G e M, no soro humano, contra o Cryptosporidium, coccídeo implicado recentemente como agente de doença intestinal humana, principalmente em pacientes imunocomprometidos. Foi obtida positividade de 62 por cento para imunoglobulinas G e M nos soros das crianças imunocompetentes com oocistos nas fezes, e, respectivamente, 20 por cento e 40 por cento, nos soros das crianças sem oocistos. Nos pacientes adultos, com o vírus da imunodeficiência humana e excreçäo fecal do parasita, foram encontrados índices de positividade de 57 por cento da IgG mas apenas 2 por cento para IgM e aqueles com excreçäo näo determinada apresentaram 23 por cento da IgG. Crianças com o vírus da imunodeficiência humana, apresentaram apenas 14 por cento da IgG e foram negativas quanto à IgM. Os resultados apontaram para a utilidade do teste, associado a outras técnicas parasitológicas, em estudos populacionais retrospectivos ou diagnósticos na infecçäo aguda e, ainda, que a resposta imune humoral a este protozoário necessita de maiores investigaçöes, nos pacientes imunocomprometidos, principalmente crianças

Statistical analysis of clinical, immunological and nutritional factors in pediatric cryptosporidiosis in the Philippines.

A statistical analysis of clinical, nutritional, and immunological data gathered in a previous study suggest that nutritional factors, and in particular, iron status, appeared to be of significance in mounting an effective immune response to Cryptosporidium infection in young children. The primary protective mechanism seemed to be cell-mediated; humoral immunity was intact in all the study subjects, however, CMI was initially impaired but improved over six weeks.