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High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis ( n = 29), giardiasis ( n = 47) and amoebiasis by Entamoeba histolytica ( n = 3) or E. dispar ( n = 10) and apparently healthy subjects ( n = 24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56 °C were proven more efficient for the release of DNA from Cryptosporidium oocysts. [ABSTRACT FROM AUTHOR]

Infections by members of the protozoan genus Cryptosporidium are among the most common causes of human gastrointestinal illness worldwide. In Spain cryptosporidiosis is not a compulsory notifiable disease, so the actual burden of the infection in both clinical and general populations remains largely unknown. We present here data on the diversity and frequency of the Cryptosporidium species and sub-genotypes identified in symptomatic individuals seeking medical care in two major hospitals in Madrid, Spain, between December 2013 and January 2015. Initial detection of the parasite was conducted on a total of 122 stool samples collected from 120 patients by microscopy with modified Ziehl-Neelsen and/or immunochromatographic tests. We used immunofluorescence, PCR-based methods and sequence analyses of the 60-kDa ( GP60 ) glycoprotein and the small subunit ribosomal RNA ( SSU rRNA) genes for confirmatory purposes and to characterize Cryptosporidium isolates. A total of 110 patients were confirmed with cryptosporidiosis. Overall, 101 isolates were successfully sub-genotyped at the GP60 locus, and an additional seven at the SSU rRNA locus. The analyses of all amplicons defined 10 distinct sequence types representing the GP60 family sub-genotypes IbA10G2 (78.7%), IeA11G3T3 (3.7%) of C. hominis , and the GP60 family sub-types IIaA15G2R1 (5.6%), IIaA18G6R1 (0.9%), IIcA5G3a (0.9%), IIdA18G1 (0.9%), IIdA19G1 (0.9%), IIdA21G1 (0.9%), and IIdA22G1 (0.9%) of C. parvum . A single isolate was assigned to C. felis (0.9%), two C. parvum isolates (1.9%) could not be characterized at the sub-genotype level and an additional four isolates (3.7%) were not typable. These results strongly suggest that transmission of cryptosporidiosis is mostly anthroponotic in origin in the clinical sample under study. We expect that our molecular epidemiological data will make a significant contribution to unravel the actual epidemiological situation of cryptosporidiosis in Spain, providing health care and policy makers with solid baseline information to unavoidably improve the national surveillance system and allocate additional resources to research, diagnosis, and treatment of cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Abstract: Feline toxoplasmosis can usually be diagnosed by serological and coprological methods. However, when the interpretation of results is difficult, it becomes necessary to rely on the direct detection of the parasite. In this study, samples of brain from 64 Toxoplasma-seropositive cats were subjected to direct detection of Toxoplasma gondii by bioassay in mice and nested-PCR. T. gondii was isolated by bioassay in mice in 41 (64%) cases, and nested-PCR was positive in 37 (57.8%) cases (p >0.05). The results therefore suggest that nested-PCR could be a rapid technique for diagnosing toxoplasmosis in cats. [Copyright &y& Elsevier]

Abstract: Seroprevalence of Toxoplasma gondii was tested for in 585 cats in different regions of Spain. Sera were tested by the indirect immunofluorescence antibody test (IFAT). Specific antitoxoplasma IgG (IFAT titer ≥ 1/80) were found in 189 of 585 (32.3%): 117 of 317 (36.9%) stray cats, 16 of 48 (33.3%) farm cats and 56 of 220 (25.5%) household cats. The overall prevalence was significantly higher in stray groups (36.4% of 365) than in household cats (25.5% of 220), higher in adult cats (>6 months old, 36.8% of 443) than in juvenile cats (<6 months old, 13.9% of 101), and higher in male stray cats (45.3% of 128) than in female stray cats (32% of 153). Prevalence of intestinal parasites was also analysed by a routine coprological method in 382 of the 585 cats. Intestinal parasites were found in 107 faecal samples (28%): 76 of 231 (32.9%) stray cats, 14 of 48 (29.2%) farm cats and 17 of 103 (16.5%) household cats. T. gondii oocysts were not found in any faecal samples analysed. The following prevalences of other intestinal parasites were found: Toxocara cati (18.3%), Toxascaris leonina (1.3%), Ancylostoma sp. (1%), Capillaria spp. (1.3%), Aelurostrongylus abstrusus (1%), Taenia like (3.7%), Dipylidium caninum (2.6%) and Cystoisospora spp. (6.3%). [Copyright &y& Elsevier]

Abstract: There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA – extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method – using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results. [ABSTRACT FROM AUTHOR]