Detection and molecular characterization of Cryptosporidium bovis-like isolate from a newborn lamb in Spain.

Species of Cryptosporidium infect a broad variety of animals. Because morphological features of the secreted oocysts are not useful in identifying the parasite at the species level, molecular tools were used to accomplish this task, leading to discovery of new Cryptosporidium species. With the use of this approach, Cryptosporidium bovis has recently been described as a new species infecting bovines and several other hosts, but clearly distinct from C. parvum. In this report, we present a description of a Cryptosporidium sp. isolate from a newborn lamb from a farm in Spain. The isolate seemed to be very similar to C. bovis based on the analysis of the gene that codes for the 18S rRNA.

Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa.

The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I-75E) pfcrt point mutation as molecular marker for CQR.

Multilocus genotyping of Cryptosporidium hominis associated with diarrhea outbreak in a day care unit in São Paulo.

UNLABELLED: A number of species of Cryptosporidium are associated with diarrhea worldwide. Little data exists regarding the genotypes and species of Cryptosporidium associated with cases of infections in Brazil. PURPOSE: In the present study, we ascertained by molecular methods the species and the genotype of Cryptosporidium sp from a diarrhea outbreak diagnosed in a day care at the Hospital Clínicas, São Paulo University Medical School. MATERIALS AND METHODS: Specific identification and typing of the isolates associated with the outbreak was done by DNA sequencing analysis of fragments amplified by polymerase chain reaction (PCR) from 3 different Cryptosporidium loci: the SSUrRNA coding region, the Cryptosporidium oocyst wall protein (COWP) gene, and the microsatellite locus 1 (ML1), a tandem GAG-trinucleotide repeat containing substitutions that differentiate the genotypes of Cryptosporidium parvum and Cryptosporidium hominis. RESULTS: A total of 29 positive samples from the outbreak were studied by the molecular methods described. Our study revealed the presence of a single genotype of Cryptosporidium hominis in all samples. CONCLUSION: The molecular analysis reinforced the hypothesis that the transmission of Cryptosporidium hominis during the period the samples were collected occurred in an outbreak pattern, possibly by person-to-person contact through the fecal-oral route. As far as we know, this is the first time that molecular tools have been used to identify the species and the genotype of isolates showing the presence of the ML1 genotype in samples from Brazilian patients.

Urban feral pigeons (Columba livia) as a source for air- and waterborne contamination with Enterocytozoon bieneusi spores.

This study demonstrated that a person with 30 min of occupational or nonoccupational exposure to urban feral pigeons, such as exposure through the cleaning of surfaces contaminated with pigeon excrement, could inhale approximately 3.5 x 10(3) Enterocytozoon bieneusi spores and that 1.3 x 10(3) spores could be inhaled by a nearby person.

Myosporidium merluccius n. g., n. sp. infecting muscle of commercial hake (Merluccius sp.) from fisheries near Namibia.

A new species of Microsporidia classified to a new genus was observed in the trunk muscle of commercial hake (Merluccius capensis/paradoxus complex) from Namibian fisheries. Macroscopic examination revealed thin and dark filaments inserted among muscle fibers. Inside the filaments were many sporophorous vesicles with about 30-50 spores per vesicle. The shape of the spore was pyriform and the extruded polar filament was of moderate length (up to 4.29 microm, n=12). This new species of Microsporidia is described using macrophotography, microphotography, staining, and transmission electron microscopy (TEM), as well as molecular methods. Its 16S rRNA was found to be similar to that of Microsporidium prosopium Kent et al., 1999, while both sequences were quite different from 16S rRNA sequences known for other Microsporidia. Nevertheless, this new species is separated morphologically from M. prosopium by the presence of 11-12 anisofilar coils and the formation of the xenoma at the site of infection. Type species.

A single genotype of Encephalitozoon intestinalis infects free-ranging gorillas and people sharing their habitats in Uganda.

Microsporidian spores have been detected by Chromotrope 2R and calcofluor stains in fecal samples of three free-ranging human-habituated mountain gorillas in Uganda and in two people who share gorilla habitats. All spore isolates have been identified by PCR with species-specific primers and fluorescent in situ hybridization with a species-specific oligonucleotide probe to be Encephalitozoon intestinalis. Sequencing analyses of the full length SSUrRNA amplified from all spore isolates were identical with Enc. intestinalis SSUrRNA GenBank SIU09929. Sequences generated from a fragment containing the internal transcribed spacer of these isolates were identical to GenBank sequence Y11611, i.e., Enc. intestinalis of anthroponotic origin. A single pathogen genotype in two genetically distant but geographically united host groups indicates anthropozoonotic transmission of Enc. intestinalis. It is highly unlikely that these two identical Enc. intestinalis genotypes were acquired independently by gorillas and people; it is much more probable that one group initiated infection of the other.

Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes.

A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.

Epidemiology of Cyclospora cayetanensis and other intestinal parasites in a community in Haiti.

We conducted an exploratory investigation in a community in Haiti to determine the prevalence of Cyclospora cayetanensis infection and to identify potential risk factors for C. cayetanensis infection. In 2001, two cross-sectional stool surveys and a nested case-control study were conducted. In 2002, a follow-up cross-sectional stool survey was conducted among children < or =10 years of age. Stool specimens from study participants and water samples from their wells were examined for Cyclospora and other intestinal parasites. In stools, the prevalence of infection with Cyclospora in persons of all ages decreased from 12% (20 of 167 persons) in February 2001 to 1.1% (4 of 352 persons) in April 2001, a 90.8% decrease. For children < or =10 years of age, the prevalence rates were 22.5% (16 of 71 children) in February 2001, 3.0% (4 of 135 children) in April 2001, and 2.5% (2 of 81 children) in January 2002. Use of the water from the artesian well in the northern region of the community versus the one in the south was the only risk factor associated with Cyclospora infection in multivariate analyses (odds ratio, 18.5; 95% confidence interval, 2.4 to 143.1). The water sample from one of the nine wells or water sources tested (one sample per source) in January 2001, shortly before the investigation began, was positive for Cyclospora by UV fluorescence microscopy and PCR. None of the water samples from the 46 wells or water sources tested during the investigation (one sample per source per testing period, including the artesian wells) were positive for Cyclospora. Further studies are needed to assess the role of water as a possible risk factor for Cyclospora infection in Haiti and other developing countries.

Multilocus genotyping of Cryptosporidium hominis associated with diarrhea outbreak in a day care unit in São Paulo

Mundialmente, diferentes espécies de Cryptosporidium estão relacionadas com doenças diarréicas. No Brasil há poucos dados sobre os genótipos das espécies de Cryptosporidium associadas a infecções. OBJETIVO: No presente estudo, caracterizamos, por métodos moleculares, a espécie e o genótipo de Cryptosporidium sp diagnosticado em surto diarréico ocorrido na creche do Hospital das Clínicas, São Paulo, Brasil. MATERIAL E MÉTODOS: Identificação específica e tipagem dos isolados associados ao surto foram feitos a partir do seqüenciamento de fragmentos de DNA amplificados por PCR dos seguintes loci: a região que codifica o SSUrRNA, o gene que codifica uma proteína do envoltório dos oocistos de Cryptosporidium (COWP), e o locus de microsatélite ML1, representado por seqüências repetitiva de três nucleotídeos GAG contendo substituições que diferem entre os genótipos de Cryptosporidium parvum e Cryptosporidium hominis. RESULTADOS: Um total de 29 amostras positivas para Cryptosporidium associadas ao surto diarréico foi analisado com base nos métodos moleculares acima descritos. O estudo revelou a presença do genótipo ML1 de Cryptosporidium hominis. DISCUSSÃO: A análise molecular reforçou a hipótese de que a transmissão de Cryptosporidium hominis durante o surto diarréico ocorreu de pessoa a pessoa através da rota fecal oral. Esta é a primeira vez que ferramentas moleculares são utilizadas para identificação de espécies e genótipos de isolados acusando a presença do genótipo ML1 em pacientes brasileiros.