A powerful DNA extraction method and PCR for detection of microsporidia inclinical stool specimens

The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biops y samples by light and/or electron microscopy, Limited information about th e specificity and sensitivity of PCR for the detection microsporidia in cli nical stool specimens is available. To establish a sensitive and specific m ethod for the detection of microsporidia in clinical samples,,ve studied cl inical stool specimens of 104 randomly selected human immunodeficiency viru s-infected patients with diarrhea to compare light microscopy and PCR, Fluo rochrome Uvitex 2B staining was used for light microscopy, To raise the sen sitivity of PCR, we used a powerful and fast DNA extraction method includin g stool sedimentation, glass bead disruption, and proteinase K and chitinas e digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1- SI500, and the nature of the PCR products was confirmed by Southern blot hy bridization, Microsporidiosis was diagnosed by light microscopy in eight pa tients, Ten patients tested positive for microsporidiosis by PCR, Enterocyt ozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis w as found in four cases. In one case a double infection with E. bieneusi and E, intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagno sis and species differentiation of intestinal microsporidiosis in HIV patie nts.