RESUMO
Detection of Blastocystis is routinely performed by microscopy, culture, and formyl-ether (ethyl acetate) concentration technique (FECT). Yet, these methods require special skilled personnel, are time consuming, and often involve processing that may cause misdiagnosis. The aim of this work is to demonstrate the usefulness of a newly introduced ELISA test for the detection of Blastocystis antigens in stool samples (CoproELISA(TM) Blastocystis, Savyon Diagnostics) as a proper alternative to currently used methods, especially microscopy. A cohort of 179 fresh/frozen clinical stool samples was tested by the ELISA test, and results were compared to consensus methods comprised of microscopic examination of Lugol's iodine staining, culture, and immunofluorescence assay (IFA). The new ELISA test was able to detect fewer than 10(3) cells, recognized subtypes 1, 2, 3, and 5 (comprising >95 % of human Blastocystis infections), and exhibited similar reactivity when comparing formalin-preserved samples to fresh/frozen samples. The test demonstrated 92 % sensitivity, 87 % specificity, and 89 % accuracy when culture, and IFA or microscopy consensus results were taken as reference. When the consensus was comprised of culture and IFA, the test demonstrated sensitivity, specificity, and accuracy of 82, 86, and 84 %, respectively. In contrast, the sensitivity of Lugol staining microscopy was only 18 %. This work presents a unique ELISA test that provides an alternative to the use of microscopy, currently most widely used method. The test enables high-throughput screening and diagnosis of Blastocystis, adaptation to automatic procedures.
Assuntos
Infecções por Blastocystis/diagnóstico , Blastocystis/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/parasitologia , Antígenos de Protozoários/isolamento & purificação , Blastocystis/imunologia , Infecções por Blastocystis/parasitologia , Estudos de Coortes , Imunofluorescência , Humanos , Microscopia , Sensibilidade e EspecificidadeRESUMO
Microsporidia, depending on their different species, generally lead to self-limited, sporadic and mild infections such as diarrhea, corneal ulcer and myositis. They are considered as opportunistic pathogens in HIV-positive patients however in recent years Microsporidia have been detected also in immunocompetent individuals as a cause of diarrhea. Diagnosis of Microsporidia depends on the detection of spores or different developmental stages of protozoon in stool, urine, sinus aspirates, nasal discharge, bronchoalveolar lavage or tissue biopsies. Diagnosis of Microsporidia infections is usually achieved by the use of different staining methods, serological tests, polymerase chain reaction, and electron microscopic methods. The aims of this study were to detect the incidence of microsporidia in patients with diarrhea by using three different staining methods and to compare the performance of these methods. A total of 225 stool samples from diarrheal patients (84 were children, 141 were adults; 103 were female, 122 were male) admitted to Gazi University Medical Faculty Hospital between March-June 2009, have been evaluated in the laboratory of Medical Microbiology Department. Stool samples were examined in terms of the presence of Microsporidia spores by Weber's modified trichrom staining (MTS), calcofluor (CF) and acridine orange (AO) staining methods. Microsporidia positivity rate was 9.8% (22/225) in the diarrheal patients, the rate being 9.5% (8/84) in children and 9.9% (14/141) in adults. There was no statistically significant difference between age and gender groups (p> 0.05) regarding Microsporidia detection. When MTS was considered as the reference method, sensitivity, specifity and consistency of AO staining were estimated as 100%, 91.6% and 92%, respectively, while those rates for CF staining were 95.4%, 99.5% and 99%, respectively. There was very strong and significant correlation (r= 0.950, p< 0.001) between CF staining and MTS, while there was strong and significant (r= 0.719, p< 0.001) correlation between AO staining and MTS. Although AO staining is rapid and convenient, the positive predictive value was measured very low (56.4%) and the interpretation of stained slides was very difficult since background of the slides was stained orange and there were a lot of dye artefacts. In conclusion, screening Microsporidia in all diarrheal stool samples is of diagnostic value. To increase sensitivity and reliability in the detection of Microsporidia spores in diarrheal samples, initial application of calcofluor staining should be followed by the confirmatory MTS method.
Assuntos
Diarreia/microbiologia , Microsporídios/isolamento & purificação , Microsporidiose/microbiologia , Adolescente , Adulto , Criança , Pré-Escolar , Diarreia/epidemiologia , Fezes/microbiologia , Feminino , Humanos , Incidência , Lactente , Masculino , Microsporidiose/epidemiologia , Esporos Fúngicos/classificação , Esporos Fúngicos/isolamento & purificação , Turquia/epidemiologia , Adulto JovemRESUMO
Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial kits. In this study, we compared the detective sensitivity among five commercially available kits (MagNA Pure LC DNA Isolation Kit I, Roche; QuickGene SP Kit DNA, FujiFilm; NucleoSpin Plant II, Macherey-Nagel; QIAamp DNA Stool Mini Kit, Qiagen; ZR Fecal DNA Kit, Zymo Research) and fecal culture method. In a preliminary test, the DNA isolated with two kits (FujiFilm and Macherey-Nagel) showed negative PCR, while the other three kits showed positive PCR. Then, DNA from 50 clinical samples that was Blastocystis-positive in the examination of fecal culture method were isolated with the three kits and 1.1 kbp SSU rRNA gene was detected with PCR. The positive rates of the three kits (Roche, Qiagen, and Zymo Research) were 10, 48 and 94%, respectively. The present study indicated that there is different detective sensitivity among the commercial kits, and fecal culture method is superior in detection rate and cost performance than DNA-elution kits for diagnosis of Blastocystis sp. subtypes.
Assuntos
Infecções por Blastocystis , Blastocystis/genética , DNA de Protozoário/análise , Fezes/parasitologia , Kit de Reagentes para Diagnóstico/normas , Blastocystis/classificação , Blastocystis/isolamento & purificação , Infecções por Blastocystis/diagnóstico , Infecções por Blastocystis/parasitologia , Técnicas de Cultura de Células , Impressões Digitais de DNA , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Genes de RNAr , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sensibilidade e EspecificidadeRESUMO
Objective. Schizophrenia is a pervasive neuropsychiatric disease of uncertain etiology. We aimed to investigate a possible association between Toxoplasma gondii infection and schizophrenia in this study. Method. We selected individuals with schizophrenia (n=88) and tested them with commercially available enzyme-linked immunosorbent assay (ELISA) for anti-Toxoplasma IgG and IgM antibodies and compared these seropositivity rate to those of controls without psychiatric disease (n=88). Results. The rate of IgG antibody in the schizophrenia patients (47.7%) was higher than the control groups (20.4%) (P<0.001). We did not find any anti-Toxoplasma IgM seropositivity in both schizophrenia patients and control group. In schizophrenic patients with and without anti-Toxoplasma IgG groups statistical analysis did not reveal any correlation between demographic variables and Toxoplasma infection. Conclusion. Our findings supported previous studies indicate that T.gondii may play a role in etiopathogenesis in some cases of schizophrenia.