Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China / Amplificação cruzada de gêneros e identificação de Colpodella sp. com iniciadores de Cryptosporidium em amostras fecais de zoofilia do nordeste da China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados ​​anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Prevalence and molecular characterization of Cryptosporidium in captive-bred Mustela putorius furo in Jiangsu Province / 中国血吸虫病防治杂志

Objective To investigate the prevalence and molecular features of Cryptosporidium in captive-bred Mustela putorius furo in Jiangsu Province.. Methods A total of 290 fresh stool samples were collected from a ferret farm in Jiangsu Province on May 2017, and the small subunit rRNA (SSU rRNA) gene of Cryptosporidium was amplified in stool samples using nested PCR assay. The actin, cowp and gp60 genes were amplified in positive samples and sequenced to characterize Cryptosporidium species/genotypes. Results A total of 18 stool samples were tested positive for Cryptosporidium SSU rRNA gene, with a detection rate of 6.2%. Sequence and phylogenetic analyses of SSU rRNA, actin and cowp genes characterized Cryptosporidium isolated from captive-bred ferrets as Cryptosporidium sp. ferret genotype. In addition, gp60 gene was amplified in 10 out of 18 stool samples tested positive for Cryptosporidium. Conclusions Cryptosporidium is widely prevalent in captive-bred ferrets in Jiangsu Province, and Cryptosporidium sp. ferret genotype is the only Cryptosporidium genotype in ferrets.

Myeloperoxidase as a biomarker for intestinal-brain axis dysfunction induced by malnutrition and Cryptosporidium infection in weanling mice

Abstract Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle is necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China / Amplificação cruzada de gêneros e identificação de Colpodella sp. com iniciadores de Cryptosporidium em amostras fecais de zoofilia do nordeste da China

The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Prevalence and genotyping of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality / 中国血吸虫病防治杂志

Objective To investigate the prevalence and genotypes of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality. Methods A total of 145 fresh fecal samples were collected from pet dogs and cats in a pet hospital in Shanghai Municipality during the period from November 2021 to June 2022, including 99 dog fecal samples and 46 cat fecal samples. The small subunit ribosomal ribonucleic acid (SSU rRNA) gene of Cryptosporidium and the triose phosphate isomerase (TPI) gene of G. lamblia were amplified using nested PCR assay, and the positive amplification products were sequenced from both directions. The sequence assembly was performed using the software Clustal X 2.1, and sequence alignment was conducted using BLAST. A phylogenetic tree was created with the Neighbor-Joining method using MEGA 11.0 to identify parasite species or genotype. Results The overall prevalence of Cryptosporidium and G. lamblia was 20.00% (29/145) in 145 pet dog and cat fecal samples, with the prevalence of 0.69% (1/145) and 19.31% (28/145) in Cryptosporidium and G. lamblia, respectively. G. lamblia was only detected in dog fecal samples, with prevalence of 18.18% (18/99), while the detection rates of Cryptosporidium and G. lamblia were 2.17% (1/46) and 21.74% (10/46) in cat fecal samples. Nucleotide sequence analysis showed that one Cryptosporidium positive sample was characterized as C. felis, and 28 G. lamblia positive samples were all characterized as Giardia assemblage A, which showed 100% sequence homology with human isolates of Giardia. Phylogenetic analysis revealed that the sequences obtained in this study belonged to the same branch with the reported Giardia assemblage A. Conclusions Cryptosporidium and G. lamblia infection was prevalent in pet dogs and cats from the study pet hospital in Shanghai Municipality, and there is a zoonotic risk for the species and genotype. Intensified surveillance of Cryptosporidium and Giardia infection is recommended in pets and their owners, and improved management of pet keeping is required.

Chlorogenic acid derived from Moringa oleifera leaf as a potential antiinflammatory agent against cryptosporidiosis in mice

@#Cryptosporidiosis is a serious illness in immunodeficient patients, and there is still no drug that can completely remove the parasite from the host. The present study represents the first report investigating the impact of the active molecule chlorogenic acid (CGA), naturally isolated from Moringa oleifera leaf extract (EMOLE), on immunosuppressed, Cryptosporidium parvum-infected BALB/c mice. Mice were divided into five groups: normal mice, infected immunosuppressed mice, and infected immunosuppressed mice treated with EMOLE, CGA, and nitazoxanide (NTZ) drugs. Parasitological, immunological, and histopathological investigations were recorded besides differences in the mice’ body weight. Infected control mice showed elevated levels of oocyst shedding throughout the study. The EMOLE- and CGA-treated groups showed 84.2% and 91.0% reductions in oocyst shedding, respectively, with no significant difference compared to the drug control. The inflammatory markers IFN-γ, IL-6, IL-1β, and TNF-α were significantly higher in the infected control group. Treatment with 300 mg/kg/day of EMOLE or 30 mg/kg/day of CGA significantly downregulated pro-inflammatory cytokine levels compared to the infected group, although they did not change significantly compared to the NTZ-treated group. Histopathology of intestinal sections showed inflammatory and pathological changes in the infected control group. Low-grade tissue changes and an obvious improvement in villi structure were seen in mice treated with CGA. This study highlighted the role of CGA, isolated and purified from EMOLE, as an effective anti-inflammatory agent in eradicating C. parvum infection.

Cryptosporidium spp. en muestras de lavado broncoalveolar de pacientes VIH/sida en un hospital de Guayaquil, Ecuador / Cryptosporidium spp. in bronchoalveolar lavage samples of HIV/AIDS patients in a hospital in Guayaquil, Ecuador

Introducción: Cryptosporidium spp. son parásitos que causan infecciones respiratorias principalmente en pacientes inmunocomprometidos. Objetivo: Detectar Cryptosporidium spp. en el lavado broncoalveolar (BAL) de pacientes VIH positivos y con síndrome respiratorio. Métodos: Se seleccionaron 60 muestras de BAL y se analizaron mediante microscopía óptica con tinción de Ziehl-Neelsen y PCR de punto final; esta última es una técnica eficiente para el diagnóstico de patógenos oportunistas. Se recolectaron datos clínicos y epidemiológicos de cada paciente. Resultados: La prevalencia hallada en este estudio mediante PCR de punto final fue del 5 %. Los signos y síntomas que se presentaron con mayor frecuencia, sobre todo en el grupo etario de 31 a 40 años, fueron fiebre, tos y disnea; sin embargo, no se obtuvieron asociaciones estadísticamente significativas a ninguna de las variables y no se pudo visualizar parásitos mediante la tinción de Ziehl-Neelsen. Conclusión: Cryptosporidium spp. puede causar infecciones pulmonares de difícil reconocimiento clínico, pues se confunde con otras infecciones oportunistas. En el presente estudio no puede establecerse si la detección del ADN parasitario correspondió a una verdadera infección o solamente a colonización, lo que es importante para implementar técnicas con mayor sensibilidad para el diagnóstico. Se debe considerar relevante la prevalencia encontrada en Ecuador, al ser inusualmente alta en comparación con países cercanos como Brasil(AU)

Introduction: Cryptosporidium spp. are parasites that cause respiratory infections mainly in immunocompromised patients. Objective: To detect Cryptosporidium spp. in bronchoalveolar lavage (BAL) of HIV-positive patients with respiratory syndrome. Methods: Sixty samples of BAL were selected and analyzed by optical microscopy with Ziehl-Neelsen staining and end-point PCR. The latter is an efficient technique for the diagnosis of opportunistic pathogens. Clinical and epidemiological data were collected from every patient. Results: In this study, the prevalence by end-point PCR was 5%. The most frequent signs and symptoms, mainly in the age group 31-40 years old, were fever, cough, and dyspnea. However, no significant statistical associations to any variable were obtained, and no parasites were observed with the Ziehl-Neelsen staining technique. Conclusions: Cryptosporidium spp. can cause pulmonary infections that are difficult to identify clinically, since they are confused with other opportunistic diseases. The current study could not establish whether the detection of parasitic DNA corresponded to a real infection or only to colonization, which is important to implement diagnostic techniques with greater sensitivity. The prevalence found in Ecuador should be considered relevant, as it is unusually high in comparison with nearby countries such as Brazil(AU)

Prevalência de la criptosporidiosis en seres humanos y terneros, y detección molecular del Cryptosporidium parvum / Prevalence of cryptosporidiosis in humans and calves and molecular detection of Cryptosporidium parvum

RESUMEN Objetivo. El objetivo de este estudio fue investigar la prevalencia de especies de Cryptosporidium en humanos y terneros en la provincia de Van, Turquía. Materiales y métodos. Se incluyeron en el estudio un total de 150 pacientes, incluidos 50 pacientes en hemodiálisis, 40 pacientes inmunosuprimidos con diarrea, 30 pacientes con diarrea solamente y 30 pacientes inmunocompetentes. Se recolectaron muestras de heces rectales de un total de 50 terneros alojados en establos y granjas en 10 aldeas centrales de Van, Turquía. Resultados. Se detectó Cryptosporidium parvum en el 17.3% de las 150 muestras de heces tomadas de seres humanos. C. parvum se observó en el 20% de los 50 pacientes en hemodiálisis, el 32.5% de los 40 pacientes inmunosuprimidos con diarrea y el 10% de los 30 pacientes con diarrea solamente, mientras que no hubo Cryptosporidium spp. detectado en los pacientes inmunocompetentes. C. parvum se observó en sólo el 6% de los 30 terneros diarreicos. Conclusiones. Claramente se entendio que la Criptosporidiosis fue detectada en una alta tasa en las muestras de los pacientes inmunosuprimidos sin y con sintomas de diarrea, y que además la especie activa que causó la enfermedad fue el agente etiologico Criptosporidium parvum. Por lo tanto, estos dos grupos de pacientes deben ser evaluados en lo que a términos de Criptosporidiosis se refiere.

ABSTRACT Objective. To investigate of the prevalence of Cryptosporidium species in humans and calves in the province of Van, Turkey. Materials and methods. Included in this research were 150 patients, comprising 50 hemodialysis patients, 40 immunosuppressed patients with diarrhea, 30 patients with diarrhea only, and 30 immunocompetent patients. Collected were stool rectal samples from 50 calves that were housed in stables and farms in 10 central villages of Van, Turkey. Results. Cryptosporidium parvum was detected in 17.3% of the 150 human stool samples. C. parvum was observed in 20% of the 50 samples from the hemodialysis patients, 32.5% of the 40 samples from the immunosuppressed patients with diarrhea, and 10% of the 30 samples from patients with diarrhea only, whereas no Cryptosporidium spp. was detected in the samples from the immunocompetent patients. C. parvum was observed in only 6% of the samples from the diarrheic 30 calves. Conclusions. It was clearly understood that cryptosporidiosis was detected at a high rate in the samples from the immunosuppressed patients and those who were immunosuppressed with diarrhea, and that the active and effective species that causes cryptosporidiosis in the Van region is C. parvum. Hence, these patient groups should be evaluated in terms of cryptosporidiosis.

Evaluación de pruebas inmunológicas en el diagnóstico de Giardia duodenalis y Cryptosporidium spp, Honduras / Evaluation of immunologic tests in the diagnosis of Giardia duodenalis and Cryptosporidium spp., Honduras

Antecedentes: No conocemos datos sobre evaluación de pruebas inmunológicas para mejorar el diagnóstico de Giardia duodenalis y Cryptosporidium spp., agentes etiológicos de diarrea de importancia mundial, en Honduras. Objetivos: Comparar dos pruebas inmunológicas para el diagnóstico de Giardia y Cryptosporidium spp. con microscopía de rutina y determinar su aplicabilidad local. Métodos: Estudio descriptivo transversal. En 2013, 134 muestras de heces recibidas en el Servicio de Parasitología del Hospital Escuela (HE) y 67 muestras del Centro de Salud Alonso Suazo (CSAS) se analizaron con una Prueba Rápida Inmunocromatográfica (PDR). En 2019-2020, 60 muestras de heces del HE se analizaron con una prueba inmunoenzimática ELISA. El protocolo de rutina incluyó examen directo en solución salina y solución de Lugol, coloración tricrómica y coloración ácido resistente modificada (ARM) (HE) y examen directo en solución salina y solución de Lugol (CSAS). Resultados: Cada prueba inmunológica mostró mayor positividad que la microscopía: en 134 muestras del HE para Giardia (6.7% vs 4.5%) y Cryptosporidium (3.7% vs 0.7%), similar en 67 muestras del CSAS (14.9% vs 7.5% para Giardia; 0.7% para Cryptosporidium con la prueba inmunológica). De 60 muestras analizadas por ELISA en HE, 31.7% fue positiva por Giardia vs 18.3% en examen directo y 23.3% en coloración tricrómica; 6.7% positiva por Cryptosporidium spp. vs 3.3% por coloración ARM. Discusión: Pruebas inmunológicas aumentaron significativamente el diagnóstico de ambas parasitosis; sin embargo, publicaciones sobre pruebas similares ofrecieron resultados no concluyentes. Por costo elevado podrían reservarse para pacientes pediátricos, pacientes inmunocomprometidos en hospitales, complementando microscopía. Los laboratorios de salud deben fortalecer capacidad diagnóstica...(AU)

Use of laboratory methodologies to confirm an outbreak of acute diarrheal disease caused by multiple pathogens / Uso de metodologias laboratoriais para confirmação de surto de doença diarreica aguda causada por múltiplos patógenos

The aim of this study was to investigate an outbreak caused by protozoa, which occurred in a municipality in the Brazil southern region. The investigations were carried out analyzing 47 fresh stool samples and 26 water samples by parasitological and molecular methods, as well as, direct immunofluorescence. After the filtrations of water samples and purification of stool samples, the concentrates were evaluated microscopically for presence of parasites. Molecular analyses were performed by polymerase chain reaction (PCR) for DNA detection of Giardia spp., Cryptosporidium parvum, C. hominis and Cyclospora cayetanensis. Out of 26 water samples, 30.8% (8/26) had waterborne protozoa and C. cayetanensis was the most prevalent (15.5%). Out of the 47 stool samples, 23.4% (11/47) were infected with C. cayetanensis and Giardia spp. The results showed that backwash water samples from filters of the Water Treatment Station were contaminated with C. cayetanensis, C. hominis and Giardia spp., suggesting the contamination of water sources with human waste brought by sewage. These results show the importance of protozoa investigation in water and stool samples by laboratory methodologies principally in outbreaks causing acute diarrheal disease (AU).

O objetivo do presente estudo foi investigar um surto causado por protozoários, ocorrido em um município da região sul do Brasil. As investigações foram realizadas analisando 47 amostras de fezes frescas e 26 amostras de água por métodos parasitológicos, moleculares e de imunofluorscência direta. Após as filtrações das amostras de água e purificação das amostras de fezes, os concentrados foram avaliados microscopicamente a procura de parasitas. A seguir, foram analisadas, pela reação em cadeia da polimerase (PCR), a detecção de DNA de Giardia spp., Cryptosporidium parvum, C. hominis e Cyclospora cayetanensis. Das 26 amostras de água, 30,8% (8/26) apresentaram protozoários de veiculação hídrica, sendo que, C. cayetanensis foi o mais prevalente (15,5%). Das 47 amostras de fezes, 23,4% (11/47) estavam infectadas por C. cayetanensis e Giardia spp. Os resultados mostraram que as águas de retrolavagem dos filtros da Estação de Tratamento de Água estavam contaminadas com C. cayetanensis, C. hominis e Giardia spp. sugerindo a contaminação dos mananciais com dejetos humanos trazidos pelo esgoto. Estes resultados mostram a importância da investigação de protozoários em água e fezes por metodologias laboratoriais, principalmente em surtos que causam doença diarreica aguda (AU).

Cryptosporidiosis, a public health challenge: A combined 3D shape-based virtual screening, docking study, and molecular dynamics simulation approach to identify inhibitors with novel scaffolds for the treatment of cryptosporidiosis

Cryptosporidiosis is a neglected tropical disease caused by the protozoan parasite Cryptosporidium parvum. Limited therapeutic options, limitation in in vitro parasite culture, and lack of a reliable animal model of parasite for replication of in vivo life cycle and drug testing demand alternative methods for drug development. The in silico methods of drug discovery prove a crucial process in such conditions.Recent research reported a limited number of small molecules for drug development. Purine nucleotide biosynthesis in Cryptosporidium species is dependent on the IMPDH (CpIMPDH) enzyme, so distortion of parasite IMPDH has been pursued as a compelling strategy for curbing Cryptosporidium infection due to its different kinetics from the host enzyme. Our study's primary aim was to discover novel ligand molecules with noticeable activity against Cryptosporidium parvum IMPDH. For this purpose, we selected 18 previously discovered ligands to understand the interaction feature between ligand and receptor, and their shape and electronic features are employed as a template for shape-based virtual screening of the ZINC database (drug-like subset) search approach via Schrodinger-2019 (Maestro 11.9). The obtained hits were subsequently subjected to structure-based screening, quantum polarized ligand docking (QPLD), and molecular dynamics simulations to fetch potential small molecules with the highest binding affinity for CpIMPDH protein. Further ligand binding energy and pharmacokinetic analysis were also taken into consideration as filtering criteria for selecting the most promising drug-like compounds. On this experimentation analysis, three top-ranked (ZINC24855054, ZINC58171263, and ZINC08000072) molecules were found to have appropriate pharmacokinetic properties along with surpassing in silico inhibitory potential towards the CpIMPDH compared to known inhibitors. The molecular docking and molecular dynamics simulation analysis results satisfactorily confirmed the inhibitory action. Therefore, these new scaffolds deduced by the presented computational methodology could recommend lead molecules for designing promising anti-cryptosporidial drugs targeting CpIMPDH protein.

Caracterización físico-química y microbiológica del agua de un municipio del departamento de Boyacá (Colombia) / Physical Chemical and Microbiological Characterization of Water of a Municipality of the Department of Boyacá-Colombia / Caracterização físico-química e microbiológica da água de um município do departamento de Boyacá (Colômbia)

Introducción: El agua salubre y fácilmente accesible es importante para la salud pública, si se utiliza para beber, uso doméstico, producir alimentos o fines recreativos. El agua contaminada y el saneamiento deficiente están re-lacionados con la transmisión de enfermedades. Objetivo: Evaluar las características físico-químicas y microbiológicas de la red de distribución del acueducto urbano de un municipio de Boyacá. Materiales y métodos: Estudio cuantitativo descriptivo transversal. Se tomaron muestras de agua en 13 puntos, 7 concertados y materializados de la red de distribución, uno en la bocatoma de entrada de la planta, dos pozos y tres nacimientos, de donde se abastece la población. Se realizaron análisis físicos, químicos y microbiológicos de cada una de las muestras. Resultados: En las muestras sin tratamiento hubo concentraciones altas de unidades platino, cobalto y turbiedad. El cloro residual libre en dos muestras de agua tratada estaba por debajo de los límites establecidos. Se detecta-ron microorganismos heterótrofos y coliformes en las muestras de agua obtenidas de fuentes de abastecimiento. En dos puntos se encontraron quistes de Giardia y ooquistes de Cryptosporidium. De las 13 muestras estudiadas, tres arrojaron un índice de riesgo por calidad del agua inviable sanitariamente. Conclusiones: Se evidenció que el agua se encuentra en un nivel de riesgo inviable sanitariamente, lo cual demuestra que el sistema de tratamiento es insuficiente para garantizar el suministro de agua apta para el consumo humano.

Introduction: Safe and easily accessible water is important to public health, whether it is used for drinking, domestic use, food production, or recreational purposes. Contaminated water and poor sanitation are linked to disease transmission. Objective: Evaluate the physical chemical and microbiological characteristics of the distribution network of the urban aqueduct of a municipality of Boyacá. Materials and methods: Cross-sectional descriptive quantitative study, water samples were taken at thirteen points, seven points agreed and materialized from the distribution network, one at the entrance of the plant, one in a deep well and in three births, where it is supplied the population. Phy-sical, chemical and microbiological analyzes were performed. Results: The untreated samples showed high levels of cobalt platinum units and turbidity. The free residual chlorine in two samples of treated water was below the established limits. Heterotrophic and Coliform microorganisms were detected in water samples obtained from sources of supply. At two points, Giardia cysts and Cryptosporidium oocysts were found. Of the Thirteen samples studied, three showed a Risk Index for sanitary water quality. Conclusions: It is evident that the water is at a level of sanitary unfeasible risk, which demonstrates that the treatment system is not sufficient to guarantee the supply of water suitable for human consumption.

Introdução: Água segura e de fácil acesso é importante para a saúde pública, seja ela usada para beber, uso doméstico, produção de alimentos ou para fins recreativos. A água contaminada e o sa-neamento precário estão ligados à transmissão de doenças. Objetivos: Avaliar as características físico-químicas e microbiológicas da rede de distribuição do aque-duto urbano de um município de Boyacá. Materiais e métodos: Estudo quantitativo descritivo transversal. Foram coletadas amostras de água em 13 pontos, 7 concertados e materializados da rede de distribuição, um na entrada da planta, dois poços e três nascentes, dos quais a população é abastecida. Foram realizadas analises físicas, químicas e microbiológicas em cada uma das amostras. Resultados: Nas amostras não tratadas havia concentrações altas de unidades platina, cobalto e turbidez. O cloro residual livre em duas amostras de água tratada estava abaixo dos limites estabele-cidos. Microrganismos heterótrofos e coliformes foram detectados em amostras de água obtidas de fontes de abastecimento. Os cistos de Giardia e os oocistos de Cryptosporidium foram encontrados em dois pontos. Das 13 amostras estudadas, três apresentaram um índice de risco para a qualidade não higiénica da água. Conclusões: Ficou evidente que a água está em um nível de risco sanitário inviável, o que mostra que o sistema de tratamento é insuficiente para garantir o abastecimento da água adequada para o consumo humano.

Cryptosporidium diagnosis in different groups of children and characterization of parasite species

ABSTRACT Background: Microscopy and enzyme-linked immunosorbent assay (ELISA) are routinely used for Cryptosporidium diagnosis, without differentiating the parasite species. Methods: Children's feces were analyzed by modified Ziehl-Neelsen (mZN) and ELISA for Cryptosporidium diagnosis and by polymerase chain reaction-restriction fragment length polymorphism for species identification. Results: Cryptosporidium frequency was 2.6%. The sensitivity and specificity of ELISA were 85.7% and 99.7%, respectively, with excellent concordance with mZN (kappa=0.854). Parasite species were characterized as Cryptosporidium hominis (78.3%), Cryptosporidium felis (17.4%), and Cryptosporidium parvum (4.3%). Conclusions: Coproantigen ELISA is as efficient as mZN for Cryptosporidium diagnosis. Cryptosporidium genotyping suggests anthroponotic and zoonotic transmission to children.

Cryptosporidium species in HIV patients in Alexandria, Egypt: distribution and associated clinical findings

@#Cryptosporidium sp. cause opportunistic infections in HIV patients. Molecular differentiation provides a better understanding of the epidemiology and clinical variations of cryptosporidiosis. The present work studied the species of Cryptosporidium in HIV patients and their associated demographic and clinical features. The study included 100 adult HIV patients receiving antiretroviral therapy in Egypt. Cryptosporidium infection was diagnosed by modified ZiehlNeelsen (MZN) stain and PCR amplification of COWP gene. The infecting species were molecularly identified by restriction fragment length polymorphism (RFLP) and DNA sequencing. Data were analyzed using Kappa (K) agreement, Mann–Whitney U, odds ratio and the 95% confidence interval, Chi-squared and Monte Carlo significance (MCp) tests. The statistical significance was judged at the 5% level. A total of 16 Cryptosporidium positive cases were detected (16%), with good agreement between PCR and MZN (K = 0.763). Among 11 PCR positive samples, RFLP identified C. hominis in five samples, C. parvum in three samples, C. meleagridis in two samples, and mixed C. hominis and C. meleagridis in one sample. Eight samples were successfully sequenced and the results confirmed the RFLP classification. C. hominis was found mainly in urban residents while C. parvum and C. meleagridis were significantly associated with rural areas (MCp =0.01). Diarrhoea and nausea/vomiting were recorded only in the presence of C. hominis infection while abdominal pain was the main symptom in C. parvum and C. meleagridis infections. Drinking water sources, contact with animals, and CD4+ count were not related to infection with a particular species. In conclusion, infection with Cryptosporidium sp. is common and frequently symptomatic in HIV patients in Egypt. The predominant species, C. hominis, C. parvum, and C. meleagridis show a distinct distribution in urban and rural residents.

Potential effect of methanol leaf extracts from three Podocarpus species on experimental cryptosporidiosis

@#Cryptosporidiosis causes diarrhea in both immunocompetent and immunocompromised individuals, with acute manifestations occurring particularly in children and the elderly. Up till now, there is no curative therapy for cryptosporidiosis, so discovery of new classes of drugs are of great importance. This study aimed to examine the effect of methanol leaves extracts of the three Podocarpus species; P. macrophyllus (Thunb.), P. gracilior (Pilg.) and P. elongatus (Aiton) L’ Hér. ex Pers and their combination on Cryptosporidium parvum (C. parvum) in experimentally infected mice in comparison with the commercially used drug, Nitazoxanide. As well as spectrophotometric estimation of the total phenolic and flavonoid content of these extracts was done. Results revealed that treatment with these three Podocarpus extracts and their combination showed a significant reduction of the number of C. parvum oocyst shed in the stool of infected mice compared to infected control group and Nitazoxanideinfected treated group at P < 0.001. The combination of the three Podocarpus extracts was the most effective treatment showing the lowest number of oocysts shedding in comparison with other used extracts and Nitazoxanide. Histopathological inspection of sections from ilium and colon displayed signs of improvement after treatment with P. macrophyllus and P. gracilior extracts and more remarkable improvement when the three extracts were combined. It was concluded that the three Podocarpus species extracts used in this study had a promising anti-Cryptosporidium activity especially when they were combined.

Risk to human health from protozoan (oo)cysts in water treatment plants / Risco à saúde humana por (oo)cistos de protozoários em estações de tratamento de água

ABSTRACT The presence of waterborne pathogens, when associated with the water supply system, poses risks to public health. This study investigated the occurrence of (oo)cysts of Cryptosporidium spp. and Giardia spp. in two Water Treatment Plants, with full-cycle technology, and assessed the microbiological risk to consumers' health. The membrane filtration technique was employed to identify the protozoan load in 24 samples of raw and filtered water. Physicochemical and bacteriological parameters were also analyzed. The Quantitative Microbial Risk Assessment model was used to determine the risk of daily and annual infection associated with the ingestion of filtered water. A total of 66.67% of raw water samples were positive for Cryptosporidium; and 33.33%, for Giardia. The maximum concentrations of 200 (oo)cysts/L and 50 cysts/L were detected in the Cerrado stream (located in Sanclerlândia, state of Goiás, Brazil), due to the predominance of grazing areas and intense agricultural activity. Water Treatment Plants did not completely remove the protozoa and the retention efficiency was lower than that recommended by the United States Environmental Protection Agency, with average values of 1.27 log for cysts and 1.04 log for oocysts. The probability of annual infection by cysts (100%) was higher than that of oocysts (86.61 - 98.32%) as for consumption of filtered water, and in the dry season, there was a higher risk of infection, due to the low performance of the Water Treatment Plants and higher concentration of pathogens. According to the results, the continuous intake of filtered water above the warning level can cause infectious diseases in the supplied population.

RESUMO A presença de patógenos de veiculação hídrica quando associados ao sistema de abastecimento de água causam risco à saúde pública. Esse estudo investigou a ocorrência de (oo)cistos de Cryptosporidium spp. e Giardia spp. em duas Estações de Tratamento de Água com tecnologia do tipo ciclo completo e avaliou o risco microbiológico à saúde dos consumidores. Utilizou-se a técnica de filtração em membranas para identificar a carga dos protozoários nas 24 amostras de água bruta e filtrada. Os parâmetros físico-químicos e bacteriológicos também foram analisados. O modelo de Avaliação Quantitativa de Risco Microbiológico foi empregado para determinar o risco de infecção diária e anual associada à ingestão de água filtrada. Um total de 66.67% das amostras de água bruta foram positivas para Cryptosporidium; e 33.33% para Giardia. As concentrações máximas de 200 oocistos/L e 50 cistos/L foram detectadas no ribeirão Cerrado (localizado em Sanclerlândia, Estado de Goiás, Brasil), devido ao predomínio de área de pastagens e à intensa atividade agropecuária. As Estações de Tratamento de Água não removeram completamente os protozoários e a eficiência de retenção foi inferior ao recomendado pela Agência de Proteção Ambiental dos Estados Unidos, com valores médios de 1,27 log para cistos e 1,04 log para oocistos. A probabilidade de infecção anual por cistos (100%) foi superior aos oocistos (86,61% a 98,32%) para o consumo da água filtrada, sendo que a estação seca, o risco de infecção foi maior, devido ao baixo desempenho das Estações de Tratamento de Água e à maior concentração de patógenos. Esses resultados indicam que a ingestão contínua de água filtrada acima do nível de alerta pode provocar doenças infecciosas na população abastecida.

Prevalence of infection by Cryptosporidium spp. in calves and associated risk factors in Northeastern Brazil / [Prevalência da infecção por Cryptosporidium spp. e fatores de risco associados em bezerros do Nordeste do Brasil]

Cryptosporidium spp. are zoonotic protozoa, frequently associated with diarrhea in calves, which are responsible for important economic losses. The aim of this study was to assess the prevalence of infection by Cryptosporidium spp. and its associated risk factors among calves raised in a milk production region of Northeastern Brazil. Fecal samples (n = 385) were obtained from young animals (up to ten months old) and evaluated by means of centrifugal fecal sedimentation in formalin-ether followed by the modified Ziehl-Neelsen staining technique. In addition, Odds Ratio (OR) was calculated to evaluate associations between variables and infection by these protozoa. Out of all samples analyzed, 25.7% (99/385) scored positive for the presence of Cryptosporidium spp. Contact with other species (goat and sheep) (OR = 3.33; p = 0.000), use of a semi-intensive rearing system (OR = 1.70; p = 0.024) and absence of hygienic conditions (fecal contamination of food and water) (OR = 1.64; p = 0.029) were considered to be risk factors. Data herein reported shows that the implementation of hygienic-sanitary measures on the farms studied, it is imperative to reduce Cryptosporidium spp. infection and consequently the economic impact caused by this pathogen.(AU)

Cryptosporidium spp. são protozoários zoonóticos frequentemente associados à diarreia em bezerros e responsáveis por importantes perdas econômicas. O objetivo deste estudo foi avaliar a prevalência e os fatores de risco associados à infecção por Cryptosporidium spp. em bezerros de propriedades leiteiras no Nordeste do Brasil. Amostras fecais (n = 385) foram obtidas de animais jovens (até 10 meses de idade) e avaliadas por centrífugo-sedimentação em formol éter, seguida da técnica de coloração de Ziehl-Neelsen modificada. A Odds Ratio (OR) foi calculada para avaliar a associação entre variáveis e infecção pelos protozoários. De todas as amostras analisadas, 25,7% (99/385) apresentaram oocistos de Cryptosporidium spp. Contato com outras espécies (caprino e ovino) (OR = 3,33; p = 0,000), sistema semi-intensivo de criação (OR = 1,70; p = 0,024) e ausência de condições higiênicas (contaminação fecal do alimento e da água) (OR = 1,64; p = 0,029) foram considerados fatores de risco. Com base nos resultados, é imprescindível a adoção de medidas higiênico-sanitárias nas fazendas estudadas, a fim de reduzir infecção por Cryptosporidium spp. e o impacto econômico causado por esse patógeno.(AU)

Antiparasitic and immunomodulating effects of nitazoxanide, ivermectin and selenium on Cryptosporidium infection in diabetic mice / Efeitos antiparasitários e imunomodulantes da nitazoxanida, ivermectina e selênio sobre a infecção por Cryptosporidium em camundongos diabéticos

Abstract The present work aims to investigate the antiparasitic and the immunomodulating effects of nitazoxanide (NTZ) and ivermectin (IVC) alone or combined together or combined with selenium (Se), on Cryptosporidium infection in diabetic mice. The results revealed that the combined NTZ and IVC therapy achieved the highest reduction of fecal oocysts (92%), whereas single NTZ showed the lowest reduction (63%). Also, adding Se to either NTZ or IVC resulted in elevation of oocyst reduction from 63% to 71% and from 82% to 84% respectively. All treatment regimens, with the exception of NTZ monotherapy, showed a significant improvement in the intestinal histopathology, the highest score was in combined NTZ and IVC therapy. The unique results of immunohistochemistry in this study showed reversal of the normal CD4/CD8 T cell ratio in the infected untreated mice, however, following therapy it reverts back to a normal balanced ratio. The combined (NTZ+ IVC) treatment demonstrated the highest level of CD4 T cell expression. Taken together, NTZ and IVC combined therapy showed remarkable anti-parasitic and immunostimulatory effects, specifically towards the CD4 population that seem to be promising in controlling cryptosporidiosis in diabetic individuals. Further research is required to explore other effective treatment strategies for those comorbid patients.

Resumo O presente trabalho tem como objetivo investigar os efeitos anti-parasitários e imunomodulantes da nitazoxanida (NTZ) e ivermectina (IVC), isoladas ou em associação, e do selênio (SE), associado à NTZ ou à IVC, sobre a infecção por Cryptosporidium em camundongos diabéticos. Os resultados revelaram que a terapia combinada com NTZ e IVC resultou em maior redução de oocistos fecais, enquanto a NTZ isolada mostrou a menor redução de oocistos fecais (63%). Além disso, a associação do SE com a NTZ ou IVC resultou em redução do número de oocistos fecais de 63% para 71% e de 82% para 84%, respectivamente. Todos os tratamentos, com exceção da monoterapia com NTZ, mostraram uma melhora significativa nos índices relacionados à histopatologia intestinal. Os resultados da imuno-histoquímica mostraram reversão da razão celular CD4/CD8 T normal nos camundongos infectados não tratados, no entanto, após a terapia, houve retorno à razão celular CD4/CD8 T normal. O tratamento combinado (NTZ+ IVC) demonstrou o mais alto nível de expressão celular CD4 T. Em conclusão, a terapia combinada com NTZ e IVC mostrou efeitos anti-parasitários e imunoestimuladores notáveis, especificamente para a população CD4, que parecem ser promissores para o controle da criptosporidiose em indivíduos diabéticos.

Identification, molecular characterization and factors associated with occurrences of Cryptosporidium spp. in calves on dairy farms in Brazil / Identificação, caracterização molecular e fatores associados à ocorrência de Cryptosporidium spp. em bezerros de propriedades leiteiras no Brasil

Abstract Cattle are an important source of zoonotic species of Cryptosporidium for humans. The aim of this study was to investigate the presence of Cryptosporidium, identify the species and determine the risk factors relating to environment, animals and management among dairy calves in eight Brazilian states. A total of 408 fecal samples from calves aged 1-60 days were analyzed. An epidemiological questionnaire was completed. Sample screening was performed using Ziehl-Neelsen technique and the positive samples were subjected to nested PCR. Cryptosporidium species were identified by means of the PCR-RFLP technique, using SSPI, ASEI and MBOII enzymes. The Ziehl-Neelsen technique showed that 89.7% (35/39) of the farms and 52.9% (216/408) of the samples were positive. Through nested PCR, these protozoa were detected in 54.6% of the samples. The 56 samples subjected to PCR-RFLP presented Cryptosporidium parvum. There was higher prevalence of the parasite in animals aged 7 to 28 days (62.6%). Diarrhea, ages between seven and 28 days and a spring water source were factors associated with the risk of infection. The calf hutch-type management system was associated with reduced infection. These findings demonstrate the high level of Cryptosporidium spp. circulation in cattle herds and the predominance of the species C. parvum.

Resumo O gado é uma fonte importante de espécies zoonóticas de Cryptosporidium para o homem. O objetivo deste estudo foi investigar a presença de Cryptosporidium, identificar a espécie e determinar os fatores de risco relacionados ao meio ambiente, aos animais e ao manejo em bezerros leiteiros em oito estados brasileiros. Um total de 408 amostras fecais de bezerros, com idade entre 1 e 60 dias, foram analisadas. Um questionário epidemiológico foi preenchido. A triagem das amostras foi realizada pela técnica de Ziehl-Neelsen, e as amostras positivas foram submetidas à "nested" PCR. As espécies de Cryptosporidium foram identificadas pela técnica de PCR-RFLP, utilizando-se as enzimas SSPI, ASEI e MBOII. A técnica de Ziehl-Neelsen mostrou que 89,7% (35/39) das fazendas e 52,9% (216/408) das amostras foram positivas. Por meio de nested PCR, esses protozoários foram detectados em 54,6% das amostras. As 56 amostras submetidas à PCR-RFLP apresentaram Cryptosporidium parvum. Houve maior prevalência do parasita em animais de 7 a 28 dias (62,6%). Diarreia, idade entre sete e 28 dias, e fonte de água mineral foram fatores associados ao risco de infecção. O sistema de manejo do tipo "casinha" para bezerros foi associado à redução da infecção. Esses achados demonstram o alto nível de Cryptosporidium spp. em circulação nos rebanhos bovinos e o predomínio da espécie C. parvum.