Comparison of polymerase chain reaction with light microscopy for detection of microsporidia in clinical specimens

Abstract

The detection of microsporidial DNA by the polymerase chain reaction (PCR) has been suggested as an alternative or supplement to conventional microscopic methods. However, the relative merits of these techniques remain uncertain. In the present study, clinical specimens of different origin (stool, urine, sputum, nasal discharge, and cerebrospinal fluid) containing four different microsporidial species were blinded after microscopic examination and analyzed by PCR and subsequent restriction fragment length polymorphism (RFLP) to determine the respective species. Thirty-four specimens from 31 patients were evaluated, 16 of which were positive and 18 negative by microscopic examination; PCR detection of microsporidia produced identical results in 82% (28/34) of these specimens. Four samples were microscopically negative, PCR-positive, and two were microscopically positive, PCR-negative. Species determination by RFLP analysis of the amplified product was accurate for all isolates containing Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi compared with microscopic identification of the genus Enterocytozoon or molecular analysis of Encephalitozoon species after in vitro culture. Therefore, PCR-RFLP is useful for the rapid detection and differentiation of microsporidian spores in clinical specimens.

Phacoemulsification for the management ofEncephalitozoon cuniculi‐induced phacoclastic uveitis in a rabbit

“…Gram stain, Weber's chromotrope‐based stain, Ziehl‐Neelsen acid‐fast stain and Gomori's methenamine silver stain may all be used for the identification of microsporidial spores 10,31,32 . Because E. cuniculi and E. hellem are morphologically identical, stain and culture methods do not allow for their differentiation; further diagnostic testing is required for species identification 31 .…”

Encephalitozoonosis in rabbits

“…Electron microscopy allows differentiation of most but not all species of microsporidia and usually requires multiplication of spores in cell culture (De Groote et al 1995;Deplazes et al 1996;Weber et al 1999). For clinical applications, direct PCR amplification either with species-specific primers (Weber et al 1999) or with conserved primers in combination with sequencing or restriction fragment length-polymorphism analysis is applied (Katzwinkel-Wladarsch et al 1997). Alternatively, nested PCR is carried out to increase the sensitivity of detection (Katzwinkel-Wladarsch et al 1996;Kock et al 1997).…”

Zoonotic Potential of the Microsporidia

“…Diagnosis to the species level is achieved by using antibodies (polyclonal or monoclonal) and by molecular methods based on the PCR (reviewed in references 116, 322, and 327). This latter sensitive and specific method has, in addition, the intrinsic advantage that upon further analysis of the PCR products with various methods (restriction fragment length polymorphism, SSCP, or sequencing), identification at the subspecies level (strains or genotypes) can be achieved (86,154).…”