RESUMEN
PURPOSE: Cryptosporidium is an opportunistic parasite that manifests as chronic and severe diarrhea in the immune-compromised subject. We investigated the species of Cryptosporidium to understand the epidemiology, mode of transmission, response to treatment, and prevention. METHODS: Polymerase chain reaction/restriction fragment length polymorphism of the 18 S rRNA gene and sequencing were performed on 41 Cryptosporidium-positive stools from 36 patients with HIV AIDS, which comprised 36 pretreatment stools and 5 stools after treatment with Paromomycin. RESULTS: C. hominis, C. meleagridis, C. felis, and C. parvum were detected; 28 of 36 (77.7%) patients were infected with C. hominis and two (5.5%) patients with multiple species of Cryptosporidium. Treatment with Paromomycin resulted in different outcomes, perhaps because patients harbored other intestinal parasitic infections. CONCLUSIONS: Multiple infection with various Cryptosporidium species in the presence of other intestinal parasites occurs in patients with HIV AIDS suffering from chronic diarrhea who are severely immune-compromised. Common transmission of Cryptosporidium is anthroponotic.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/epidemiología , Criptosporidiosis/epidemiología , Cryptosporidium/clasificación , Cryptosporidium/aislamiento & purificación , Diarrea/diagnóstico , Infecciones por VIH/complicaciones , Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Infecciones Oportunistas Relacionadas con el SIDA/parasitología , Adolescente , Adulto , Animales , Antibacterianos/uso terapéutico , Niño , Preescolar , Estudios Transversales , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/parasitología , Criptosporidiosis/transmisión , Cryptosporidium/genética , Diarrea/complicaciones , Diarrea/epidemiología , Heces/parasitología , Femenino , Genes de ARNr , Infecciones por VIH/epidemiología , Humanos , Indonesia/epidemiología , Persona de Mediana Edad , Paromomicina/uso terapéutico , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia , Resultado del Tratamiento , Adulto JovenRESUMEN
A longitudinal study was conducted to determine the epidemiology of Cryptosporidium in 1,636 children in Nigeria. Oocyst prevalence ranged from 15.6% to 19.6% over one year. Cryptosporidium hominis (34), C. parvum (25), C. parvum/C. hominis (4), C. meleagridis (5), Cryptosporidium rabbit genotype (5), Cryptosporidium cervine genotype (3), and C. canis (1) were identified by polymerase chain reaction-restriction fragment length polymorphism analysis. Glycoprotein 60 subgenotyping showed that 28 amplifiable C. hominis isolates consisted of 12 subtypes that belonged to 5 subtype families (Ia, Ib, Id, Ie, and 1 novel subtype family, Ih) and 23 amplifiable C. parvum isolates consisted of 6 subtypes that belonged to 4 subtype families (IIa, IIc, Iii, and IIm). Three C. meleagridis isolates sub-genotyped by sequence analysis of the small subunit ribosomal RNA gene fragment were type 1. This study is the first one to genetically characterize Cryptosporidium species and subtypes in Nigeria and highlights the presence of a high Cryptosporidium diversity in this pediatric population.
Asunto(s)
Cryptosporidium/clasificación , Cryptosporidium/genética , Niño , Preescolar , Heces/parasitología , Femenino , Genotipo , Humanos , Lactante , Masculino , Nigeria/epidemiología , FilogeniaRESUMEN
Water and food are major environmental transmission routes for Cryptosporidium, but our ability to identify the spectrum of oocyst contributions in current performance-based methods is limited. Determining risks in water and foodstuffs, and the importance of zoonotic transmission, requires the use of molecular methods, which add value to performance-based morphologic methods. Multi-locus approaches increase the accuracy of identification, as many signatures detected in water originate from species/genotypes that are not infectious to humans. Method optimisation is necessary for detecting small numbers of oocysts in environmental samples consistently, and further work is required to (i) optimise IMS recovery efficiency, (ii) quality assure performance-based methods, (iii) maximise DNA extraction and purification, (iv) adopt standardised and validated loci and primers, (v) determine the species and subspecies range in samples containing mixtures, and standardising storage and transport matrices for validating genetic loci, primer sets and DNA sequences.
Asunto(s)
Criptosporidiosis/transmisión , Cryptosporidium/aislamiento & purificación , Parasitología de Alimentos , Enfermedades Transmitidas por los Alimentos/parasitología , Agua/parasitología , Zoonosis , Animales , Criptosporidiosis/epidemiología , Cryptosporidium/clasificación , Cryptosporidium/genética , ADN Protozoario/aislamiento & purificación , Brotes de Enfermedades/prevención & control , Heces/parasitología , Humanos , Oocistos/clasificación , Reacción en Cadena de la Polimerasa , Abastecimiento de Agua/normas , Zoonosis/parasitología , Zoonosis/transmisiónRESUMEN
Of 1346 faecal samples from the Chikwawa and Thyolo districts of Malawi, analysed for the presence of Cryptosporidium oocysts between October 2001 and May 2003, 61.3% were from cattle (29.8% of these were from calves <6 months old). Cryptosporidium oocysts were detected during all three seasons studied in Chikwawa and Thyolo. In Chikwawa, 13.6% of adult cattle and 11.7% of calves were infected, compared to 28.9% of adult cattle and 36.7% of calves in Thyolo. Dependent on season, between 7.8% and 37.7% (Chikwawa) and 16.7% and 39.3% (Thyolo) of cattle samples contained oocysts. In Chikwawa, the highest percentage of infections occurred in the cool season, whereas in Thyolo, the highest percentage of infections occurred in the dry season. Faecal samples from goats [n=225], pigs [n=92], sheep [n=6]), rabbits, guinea pigs, chickens, ducks, turkeys, doves and guinea fowls were also analysed. Up to 5.6% of goat samples contained oocysts in Chikwawa, compared to between 16.7% and 39.3% in Thyolo. Again, in Chikwawa, the highest percentage of infections occurred in the cool season and the lowest in the rainy season, whereas, in Thyolo, the highest percentage of infections occurred in the dry season and the lowest in the cool season. In pigs, more infections were detected in the dry season in Chikwawa, but infections in the cool season were similar (17.7%), whereas in Thyolo, infections occurred in all three seasons (17.9% in the rainy season, 25% in the cool season and 60% in the dry season). Of ten diarrhoeic, oocyst positive cattle faecal samples collected from Chikwawa and subjected to PCR-RFLP, four oocyst positive samples (two from heifers, one from a cow and one unknown) were amplified at an 18S rRNA and Cryptosporidium oocyst wall protein (COWP) loci. RFLP of the 18S rRNA locus indicated that Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium bovis and/or Cryptosporidium ryanae DNA, or a mixture of them was present. Cryptosporidium parvum DNA was identified in one sample that amplified at the COWP locus, indicating the presence of the major zoonotic Cryptosporidium species in Malawi.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Criptosporidiosis/veterinaria , Cryptosporidium/aislamiento & purificación , Heces/parasitología , Estaciones del Año , Animales , Animales Recién Nacidos , Bovinos , Enfermedades de los Bovinos/transmisión , Criptosporidiosis/epidemiología , Criptosporidiosis/transmisión , Cryptosporidium/clasificación , ADN Protozoario/análisis , Humanos , Malaui/epidemiología , Recuento de Huevos de Parásitos/veterinaria , Salud Pública , Especificidad de la Especie , ZoonosisRESUMEN
We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.
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Cryptosporidium/clasificación , Cryptosporidium/crecimiento & desarrollo , Dermatoglifia del ADN/métodos , Monitoreo del Ambiente/métodos , Oocistos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Abastecimiento de Agua , Animales , Gatos , Cryptosporidium/genética , Cryptosporidium/aislamiento & purificación , ADN Protozoario/análisis , ADN Protozoario/aislamiento & purificación , Genotipo , Humanos , Ratones , Oocistos/clasificación , Oocistos/genética , Sensibilidad y EspecificidadRESUMEN
We describe a rapid method for extracting and concentrating Cryptosporidium oocysts from human faecal samples with subsequent DNA preparation for mainstream PCR applications. This method consists of extracting faecal lipids using a modified water-ether treatment and releasing DNA from semi-purified oocysts by freeze thawing in lysis buffer. Following immunomagnetisable separation (IMS), recovery rates of 29.5%, 43.2% and 49.8% were obtained from oocyst-negative solid, semi-solid and liquid faeces, respectively, seeded with 100 +/- 2 C. parvum oocysts, which were enumerated by flow cytometry. A retrospective analysis was conducted on 92 positive human faecal samples including 78 oocyst-positive cases from 2 UK cryptosporidiosis outbreaks (outbreak A = 34 samples, outbreak B = 44 samples) and 14 oocyst-positive, sporadic cases. We used primers targeting the Cryptosporidium oocyst wall protein gene (COWP; STN-COWP), the 18S rRNA (direct PCR) and the dihydrofolate reductase gene (dhfr, MAS-PCR) fragments to evaluate extracted DNA by PCR. PCR inhibitors were present in 20 samples when template was co-amplified with the 18S rRNA gene primers and an internal control. Template dilution (1/5) in polyvinylpyrrolidone (10 mg ml(-1), pH 8.0) transformed four PCR-negative samples to PCR-positive and increased amplicon intensity in previously positive samples. Eighteen of 20 PCR-negative samples produced visible amplicons when Taq polymerase concentration in the STN-COWP PCR was increased from 2.5 to 5 U. The STN-COWP PCR assay amplified 90 of 92 samples (97.8%) and the MAS-PCR assay amplified 70 of 92 samples (76.1%) tested. In the absence of inhibitors, DNA equivalent to 3 C. parvum oocysts was amplified.
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Criptosporidiosis/epidemiología , Cryptosporidium/aislamiento & purificación , ADN Protozoario/aislamiento & purificación , Brotes de Enfermedades , Heces/parasitología , Oocistos/aislamiento & purificación , Animales , Criptosporidiosis/parasitología , Cryptosporidium/genética , Cryptosporidium/crecimiento & desarrollo , ADN Protozoario/análisis , Humanos , Oocistos/genética , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Cryptosporidium parvum excystation and host cell invasion have been characterized in some detail ultrastructurally. However, until recently, the biochemical and molecular basis of host-parasite interactions and parasite- and host-specific molecules involved in excystation, motility and host cell invasion have been poorly understood. This article describes our understanding of Cryptosporidium excystation and the events leading to host cell invasion, and draws from information available about these processes in other apicomplexans. Many questions remain but, once the specific mechanisms are identified, they could prove to be novel targets for drug delivery.
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Criptosporidiosis/parasitología , Cryptosporidium/fisiología , Animales , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/fisiología , Interacciones Huésped-Parásitos/fisiología , Humanos , Estadios del Ciclo de Vida , Oocistos/fisiologíaRESUMEN
The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65 degrees C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65 degrees C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.