Diagnostic microbiologic methods in the GEMS-1 case/control study.

To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged <5 years at 7 sites. Each site employed an identical case/control study design and each utilized a uniform comprehensive set of microbiological assays to identify the likely bacterial, viral and protozoal etiologies. The selected assays effected a balanced consideration of cost, robustness and performance, and all assays were performed at the study sites. Identification of bacterial pathogens employed streamlined conventional bacteriologic biochemical and serological algorithms. Diarrheagenic Escherichia coli were identified by application of a multiplex polymerase chain reaction assay for enterotoxigenic, enteroaggregative, and enteropathogenic E. coli. Rotavirus, adenovirus, Entamoeba histolytica, Giardia enterica, and Cryptosporidium species were detected by commercially available enzyme immunoassays on stool samples. Samples positive for adenovirus were further evaluated for adenovirus serotypes 40 and 41. We developed a novel multiplex assay to detect norovirus (types 1 and 2), astrovirus, and sapovirus. The portfolio of diagnostic assays used in the GEMS study can be broadly applied in developing countries seeking robust cost-effective methods for enteric pathogen detection

Validation Of A New Technique To Detect Crytosporidium Spp. Oocysts In Bovine Feces

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Due to its important zoonotic potential, cryptosporidiosis arouses strong interest in the scientific community, because, it was initially considered a rare and opportunistic disease. The parasitological diagnosis of the causative agent of this disease, the protozoan Cryptosporidium spp., requires the use of specific techniques of concentration and permanent staining, which are laborious and costly, and are difficult to use in routine laboratory tests. In view of the above, we conducted the feasibility, development, evaluation and intralaboratory validation of a new parasitological technique for analysis in optical microscopy of Cryptosporidium spp. oocysts, called TF-Test Coccidia, using fecal samples from calves from the city of Aragatuba, Sao Paulo. To confirm the aforementioned parasite and prove the diagnostic efficiency of the new technique, we used two established methodologies in the scientific literature: parasite concentration by centrifugal sedimentation and negative staining with malachite green (CSN-Malachite) and Nested-PCR. We observed good effectiveness of the TF-Test Coccidia technique, being statistically equivalent to CSN-Malachite. Thus, we verified the effectiveness of the TF-Test Coccidia parasitological technique for the detection of Cryptosporidium spp. oocysts and observed good concentration and morphology of the parasite, with a low amount of debris in the fecal smear. (C) 2016 Elsevier B.V. All rights reserved.13415Agenda de Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - CAPESSao Paulo Research Foundation (FAPESP) [99/06228-4]ImmunoCamp Science and TechnologyCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

First Description Of Cryptosporidium Hominis Gp60 Genotype Ika20g1 And Cryptosporidium Parvum Gp60 Genotypes Iiaa18g3r1 And Iiaa15g2r1 In Foals In Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The present study focuses on Cryptosporidium infections of foals in Brazil. A total of 92 animals of different breeds from 11 farms in the vicinity of Aracatuba in the state of Sao Paulo, were examined. According to PCR targeting the 18S rRNA gene, Cryptosporidium sp. DNA was detected in 21.7% (20/92) of foals. Good quality 18S rRNA, actin, HSP70 and gp60 genes nPCR amplicons were obtained from five fecal samples. PCR amplification and sequencing of a fragment of the GP60 sporozoite surface glycoprotein gene revealed C parvum genotypes IIaAl8G3R1, lIaAl5G2R1. Interestingly, we also detected in two foals a GP60 genotype related to the human parasite C. hominis. (C) 2016 Elsevier B.V. All rights reserved.2334851FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo) [2010/52542-3]NIAID award [1R15AI122152]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Use of the aqueous biphasic system as an alternative for concentration of ascaris lumbricoides eggs, with non-toxic separation of faecal residues and fats

Human enteroparasites are considered a serious public health problem in underdeveloped countries located in world regions with tropical, subtropical and equatorial climates. These parasites are commonly diagnosed by the Parasitological Examination of Faeces (PEF), performed by conventional techniques and/or commercial kits that result in tests with low-to-moderate sensitivity, due to the use of destructive chemical solvents to parasite structures, and to present excess adipose substance and digestive residues in their microscopic slides. In order to improve the efficacy of these tests/examinations, this study aimed to investigate a new alternative for the PEF, with the use of Aqueous Biphasic System (ABS). For this, four ABSs containing poly (ethylene glycol), PEG (PEG-4000 and PEG-6000), dipotassium phosphate and sodium citrate at different concentrations in the biphasic systems were evaluated with faecal samples containing eggs of Ascaris lumbricoides. The ABS consisting of PEG-4000 and dipotassium phosphate, at concentrations of 55% w/w and 20% w/w, respectively, achieved 100% satisfactory results compared to the conventional TF-Test technique in terms of preservation and concentration of A. lumbricoides eggs, with adequate separation of digestive residues, without using a centrifuge or chemical solvents that may cause harm to the parasites. This study presents ABS as a new low-cost technical principle for the detection of parasite eggs in PEF. The new technique is simple, fast, non-toxic, not harmful to the parasite and does not require a centrifuge. Human enteroparasites are considered a serious public health problem in underdeveloped countries located in world regions with tropical, subtropical and equatorial climates. These parasites are commonly diagnosed by the Parasitological Examination of Faeces (PEF), performed by conventional techniques and/or commercial kits that result in tests with low-to-moderate sensitivity, due to the use of destructive chemical solvents to parasite structures, and to present excess adipose substance and digestive residues in their microscopic slides. In order to improve the efficacy of these tests/examinations, this study aimed to investigate a new alternative for the PEF, with the use of Aqueous Biphasic System (ABS). For this, four ABSs containing poly (ethylene glycol), PEG (PEG-4000 and PEG-6000), dipotassium phosphate and sodium citrate at different concentrations in the biphasic systems were evaluated with faecal samples containing eggs of Ascaris lumbricoides. The ABS consisting of PEG-4000 and dipotassium phosphate, at concentrations of 55% w/w and 20% w/w, respectively, achieved 100% satisfactory results compared to the conventional TF-Test technique in terms of preservation and concentration of A. lumbricoides eggs, with adequate separation of digestive residues, without using a centrifuge or chemical solvents that may cause harm to the parasites. This study presents ABS as a new low-cost technical principle for the detection of parasite eggs in PEF. The new technique is simple, fast, non-toxic, not harmful to the parasite and does not require a centrifuge24111320132

Prevalence of ehrlichia canis (rickettsiales: ehrlichieae) DNA in tissues from rhipicephalus sanguineus (acari: ixodidae) ticks in areas endemic for canine monocytic ehrlichiosis in Brazil

Canine monocytic ehrlichiosis (CME) is a disease caused by the obligate intracellular bacterium Ehrlichia canis. Tropical lineages of Rhipicephalus sanguineus ticks play an essential role in the transmission of this pathogen. The aim of the present study was to evaluate the prevalence of E. canis DNA in tissue from R. sanguineus ticks in areas endemic for CME in Brazil and quantify levels of E. canis DNA in dissected tissues from these samples. A total of 720 ticks were collected from 72 dogs (36 dogs from the city Aracatuba in Sao Paulo state and 36 from Campo Grande in the state of Mato Grosso do Sul). Ticks were dissected to collect the guts, ovaries and salivary gland. A quantitative polymerase chain reaction (qPCR) targeting the disulphide bond formation (dsb) protein gene was performed to quantify the level of E. canis infection. The E. canis dsb-qPCR assay was positive for 31.9, 10, and 15.2% of the gut, ovary, and salivary glands, respectively. The average gut, ovary, and salivary gland bacterial load estimated by qPCR was 1.21 x 10(3), 2.60 x 10(3), and 4.92 x 10(3) gene copies/mu l, respectively. This is the first report of E. canis DNA in ovaries of R. sanguineus ticks parasitizing dogs in these CME-endemic areas. These observations raise the possibility of E. canis trans-ovarial transmission563828831FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2014/26461-

Diagnostic microbiologic methods in the GEMS-1 case/control study

To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged &lt;5 years at 7 sites. Each site employed an identical case/control study design and each utilized a uniform comprehensive set of microbiological assays to identify the likely bacterial, viral and protozoal etiologies. The selected assays effected a balanced consideration of cost, robustness and performance, and all assays were performed at the study sites. Identification of bacterial pathogens employed streamlined conventional bacteriologic biochemical and serological algorithms. Diarrheagenic Escherichia coli were identified by application of a multiplex polymerase chain reaction assay for enterotoxigenic, enteroaggregative, and enteropathogenic E. coli. Rotavirus, adenovirus, Entamoeba histolytica, Giardia enterica, and Cryptosporidium species were detected by commercially available enzy

Criptosporidiose em animais domésticos: aspectos epidemiológicos

Studies related cryptosporidiosis will be essential, due to its relevance in public health and pathogenicity in pets and production animals. Over the past 20 years, there has been a rapid expansion of research involving the Cryptosporidium genus, largely related to molecular studies, providing a description of various species, genotypes and subtypes of the parasite. The molecular characterization of isolates from different sources (human, animal and environmental) has been widely used in order to investigate the potential zoonotic of this protozoa. The documented transmission forms from animals to humans, from person to person, through water intake or water for the leisure that are directly or indirectly contaminated with sporulated oocysts. The high rate of animals naturally infected and the susceptibility by protozoan, justify the importance of attending to the occurrence of this disease. So are demonstrated epidemiological aspects of this zoonotic disease in domestic animals

First description of Cryptosporidium hominis GP60 genotype IkA20G1 and Cryptosporidium parvum GP60 genotypes IIaA18G3R1 and IIaA15G2R1 in foals in Brazil

The present study focuses on Cryptosporidium infections of foals in Brazil. A total of 92 animals of different breeds from 11 farms in the vicinity of Aracatuba in the state of Sao Paulo, were examined. According to PCR targeting the 18S rRNA gene, Cryptosporidium sp. DNA was detected in 21.7% (20/92) of foals. Good quality 18S rRNA, actin, HSP70 and gp60 genes nPCR amplicons were obtained from five fecal samples. PCR amplification and sequencing of a fragment of the GP60 sporozoite surface glycoprotein gene revealed C parvum genotypes IIaAl8G3R1, lIaAl5G2R1. Interestingly, we also detected in two foals a GP60 genotype related to the human parasite C. hominis2334851FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2010/52542-