Evaluation of DNA Extraction and PCR Methods for Detection of Enterocytozoon bienuesi in Stool Specimens

Abstract

An evaluation of the sensitivities of three DNA extraction methods, i.e., FTA filter paper, a QIAamp stool mini kit, and a conventional phenol-chloroform method, by using specimens with known concentrations of Enterocytozoon bieneusi spores was performed. FTA filter paper and the QIAamp stool mini kit were the most sensitive methods, which could detect E. bieneusi in specimens with a concentration of 800 spores/ml. We also compared five previously described PCR methods that use five different primer pairs for the detection of E. bieneusi and showed that MSP3-MSP4B and EBIEF1-EBIER1 were the most sensitive primers. Although both sets of primers showed the same sensitivity, using the MSP3-MSP4B primers can directly provide genotypic information by sequencing. A blinded diagnostic test to compare PCR and light microscopy methods for the detection of E. bieneusi in stool specimens was also conducted. The use of FTA filter paper for DNA extraction together with the PCR method using the primer pair MSP3-MSP4B showed 100% sensitivity and 100% specificity for the detection of E. bieneusi in stool specimens, while the light microscopy method gave a sensitivity of 86.7% and a specificity of 100%.

Frequency of Microsporidial Infection in Immunocompromised Patients with Staining and Molecular Methods Based on Internal Transcribed Spacer Region Gene in Two Cities of Southwest Iran during 2013-2014

“…Similarly, higher prevalence of E. intestinalis has been reported among HIV patients 9, 38,46 . In our study prevalence rate of microsporidia in cancer patients is 18% and this result is similar with 21 57,58 . Conversely, humans infected with E. intestinalis transmit this species 12 .…”

“…DNA extraction from microsporidia spores in stool has many complexities including very small size of spores, rigid doubled-layer wall and also lower counts of stool samples. Moreover, immune suppression, geographical and socioeconomic factors source of infection, and diagnostic tests used can impress the quality and quantity of molecular results 19,21,46 .In conclusion, the occurrence of microsporidia in patients with HIV + and cancer patients at the level of 27.9% and 18% respectively, suggests that these pathogens should be taken into account when other etiological agents cannot be found in those diarrheic patients. Moreover, this study shows that PCR is able to detect and identify species of intestinal microsporidia with the highest sensitivity than traditionally used staining methods.…”

“…Routine diagnosis is based on spore observation by light microscopy using modified trichrome staining, Uvitex 2B or Fungi flour, but unable to differentiate between the species. However, for the differentiation of the microsporidian species, PCR methods due to their sensitivity and specificity are recommended for this aim [19][20][21] .To our knowledge, there is no epidemiological study conducted on Microsporidiain Ahvaz area to date. This study determines the frequency of Microsporidia species in fecal isolates of patients with HIV + and cancer receiving chemotherapy in the southwest of Iran (Khuzestan) by using PCR method in comparison with modified trichrome blue (MTB) staining.…”

Exfoliated Cells in Stool: A Source for Reverse Transcription-PCR–Based Analysis of Biomarkers of Gastrointestinal Cancer

“…Whatman FTA cards have been designed to obtain both DNA and RNA. They are impregnated with a blend of chemicals that lyses cell membranes and denatures proteins on contact, leading to trapping, immobilization, and stabilization of nucleic acids (8,9). RNAlater was originally developed for RNA preservation but has been used successfully to stabilize and store RNA and DNA (10,11).…”

Coleta, transporte e armazenamento de amostras para diagnóstico molecular

“…Alguns protocolos de extração de DNA envolvem a diluição da amostra com solução tamponada (pH 7,4) e centrifugação para eliminação dos debris. A amostra passa por filtração para eliminação de restos celulares, sendo que a maioria dos microrganismos ficará no filtrado, que será submetido à extração de DNA (28,34) .…”