Cryptosporidiosis outbreak in a child day-care center caused by an unusual Cryptosporidium hominis subtype / Brote de cryptosporidiosis en una guardería causado por un subtipo inusual de Cryptosporidium hominis

INTRODUCTION: This work describes the genetic characterization of Cryptosporidium and Giardia involved in an outbreak in a nursery school in Granada, Spain, that affected seven children under the age of 4. METHODS: Nucleic acids were extracted from the seven stool samples positive to Cryptosporidium or Giardia by microscopy and/or immunochromatography. The species and subtypes of Cryptosporidium were identified by PCR-RFLP and PCR of the SSUrRNA and gp60 genes, respectively. The assemblages of Giardia duodenalis isolates were characterized by PCR of the tpi gene. PCR products were sequenced and analyzed. RESULTS: All of the isolates were positive for Cryptosporidium hominis. Five of them belonged to subtype IaA11R2, one to subtype IbA10G2R2, and the other could not be identified. Three of these samples were positive for G. duodenalis by PCR, two belonging to the assemblage A, and the other one to assemblage B. DISCUSSION: This is the first report of Cryptosporidium hominis subtype IaA11R2 as a cause of an outbreak in Europe where subtype IbA10G2R2 is the most frequently identified. In the case of Giardia, an outbreak could not be confirmed because of the low number of positive samples and the low genetic variability of the amplified fragments for assemblage A of tpi gene. CONCLUSIONS: A new subtype, of Cryptosporidium hominis named IaA11R2, has been described as a cause of an outbreak in a nursery school in Granada, Spain. However an outbreak of giardiasis could not be confirmed

INTRODUCCIÓN: Este trabajo describe la caracterización genética de Cryptosporidium y Giardia implicados en un brote en una guardería de Granada, España, que afectó a 7 niños menores de 4 años de edad. MÉTODOS: La extracción de ácidos nucleicos se realizó a partir de las 7 muestras de heces positivas para Cryptosporidium o Giardia mediante técnicas de microscopia y/o inmunocromatografía. Las especies y subtipos de Cryptosporidium fueron identificados por PCR-RFLP y PCR de los genes SSUrRNA ygp60, respectivamente. Los genotipos de los aislamientos Giardia duodenalis fueron caracterizados por PCR del gen tpi. Los productos de PCR obtenidos fueron secuenciados y analizados. RESULTADOS: Todos los aislamientos fueron positivos para Cryptosporidium hominis. Cinco de ellos correspondieron al subtipo IaA11R2, uno al subtipo IbA10G2R2 y otro no pudo ser identificado. Tres de las muestras resultaron positivas para G. duodenalis por PCR, 2 de ellas pertenecientes al genotipo A y la restante al genotipo B. DISCUSIÓN: Esta es la primera identificación del subtipo IaA11R2 de Cryptosporidium hominis como causa de un brote en Europa, donde el subtipo IbA10G2R2 es el más frecuentemente identificado. En el caso de Giardia, el bajo número de muestras positivas y la baja variabilidad genética del fragmento del gen tpiamplificado para el genotipo A no permitieron confirmar un brote. CONCLUSIONES: Un nuevo subtipo de Cryptosporidium hominis, denominado IaA11R2, ha sido descrito como causa de un brote en una guardería de Granada, España. Sin embargo, los resultados obtenidos no permiten confirmar un brote de giardiasis

Cryptosporidiosis outbreak in a child day-care center caused by an unusual Cryptosporidium hominis subtype.

INTRODUCTION: This work describes the genetic characterization of Cryptosporidium and Giardia involved in an outbreak in a nursery school in Granada, Spain, that affected seven children under the age of 4. METHODS: Nucleic acids were extracted from the seven stool samples positive to Cryptosporidium or Giardia by microscopy and/or immunochromatography. The species and subtypes of Cryptosporidium were identified by PCR-RFLP and PCR of the SSUrRNA and gp60 genes, respectively. The assemblages of Giardia duodenalis isolates were characterized by PCR of the tpi gene. PCR products were sequenced and analyzed. RESULTS: All of the isolates were positive for Cryptosporidium hominis. Five of them belonged to subtype IaA11R2, one to subtype IbA10G2R2, and the other could not be identified. Three of these samples were positive for G. duodenalis by PCR, two belonging to the assemblage A, and the other one to assemblage B. DISCUSSION: This is the first report of Cryptosporidium hominis subtype IaA11R2 as a cause of an outbreak in Europe where subtype IbA10G2R2 is the most frequently identified. In the case of Giardia, an outbreak could not be confirmed because of the low number of positive samples and the low genetic variability of the amplified fragments for assemblage A of tpi gene. CONCLUSIONS: A new subtype, of Cryptosporidium hominis named IaA11R2, has been described as a cause of an outbreak in a nursery school in Granada, Spain. However an outbreak of giardiasis could not be confirmed.

Identification of free-living amoebae and amoeba-associated bacteria from reservoirs and water treatment plants by molecular techniques.

The occurrence of free-living amoebae (FLA) was investigated in 83 water samples from reservoirs and water treatment plants, with culture positive in 64 of them (77.1%). Polymerase chain reaction (PCR) of partial 18S rRNA gene and ITS region was performed in order to identify amoeba isolates, and the presence of Legionella pneumophila , Mycobacterium spp., Pseudomonas spp., and Microcystis aeruginosa was investigated in 43 isolates of amoebae by multiplex PCR. Of the isolated amoebae, 31 were Acanthamoeba spp., 21 were Hartmannella vermiformis, 13 were Naegleria spp., and one was Vanella spp. T2, T4, and T5 genotypes of Acanthamoeba have been identified, and T4 isolates were grouped into five subgenotypes and graphically represented with a Weblog application. Inside amoebae, L. pneumophila was detected in 13.9% (6/43) of the isolates, and Pseudomonas spp. and Mycobacterium spp. were detected in 32.6% (14/43) and 41.9% (18/43), respectively. No statistical correlation was demonstrated between FLA isolation and seasonality, but the presence of intracellular bacteria was associated with warm water temperatures, and also the intracellular presence of Mycobacterium spp. and Pseudomonas spp. were associated. These results highlight the importance of amoebae in natural waters as reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Two cases of zoonotic cryptosporidiosis in Spain by the unusual species Cryptosporidium ubiquitum and Cryptosporidium felis

Introduction Two cases of infection by zoonotic transmission of unusual species of Cryptosporidium were detected in 2010-2011 in Spain (León and Zaragoza).Materials and methods Cryptosporidium spp. was detected by microscopic examination of modified Ziehl-Neelsen stained fecal smears. PCR-RFLP of the SSUr DNA gene and sequencing of the amplified fragment confirmed the species. Results C. ubiquitum and C. felis were identified in samples from an immunocompetent child and from a HIV-positive adult, respectively. Conclusions This is the first report of human infection by C. ubiquitum (cervine) and autochthonous C. felis, identified in Spain (AU)

Introducción: Se describen dos casos de infección por especies inusuales de Cryptosporidium de transmisión zoonótica detectados en España (León y Zaragoza) en 2010 y 2011. Material y métodos: Cryptosporidium se detectó por tinción Ziehl-Neelsen modificada de la concentración de heces Las especies se determinaron por PCR-RFLP del gen SSUr DNA y se confirmaron por secuenciación del fragmento amplificado. Resultados: C.ubiquitum y C. felis fueron identificados en muestras procedentes de un niño inmunocompetente y un adulto VIH-positivo respectivamente. Conclusiones: Este estudio es la primera comunicación de infecciones humanas por C. ubiquitum (cervine)y de Cryptosporidium felis de origen autóctono identificados en España (AU)

Two cases of zoonotic cryptosporidiosis in Spain by the unusual species Cryptosporidium ubiquitum and Cryptosporidium felis.

INTRODUCTION: Two cases of infection by zoonotic transmission of unusual species of Cryptosporidium were detected in 2010-2011 in Spain (León and Zaragoza). MATERIALS AND METHODS: Cryptosporidium spp. was detected by microscopic examination of modified Ziehl-Neelsen stained fecal smears. PCR-RFLP of the SSUrDNA gene and sequencing of the amplified fragment confirmed the species. RESULTS: C. ubiquitum and C. felis were identified in samples from an immunocompetent child and from a HIV-positive adult, respectively. CONCLUSIONS: This is the first report of human infection by C. ubiquitum (cervine) and autochthonous C. felis, identified in Spain.