Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray

Abstract

Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microarray assay for the simultaneous detection and identification to the species level of four major microsporidian species: Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis. The 18S small-subunit rRNA gene was chosen as the amplification target, labeled with fluorescence dye, and hybridized to a series of species-specific oligonucleotide probes immobilized on a microchip. The specificity and sensitivity of the microarray were clearly demonstrated by the unique hybridization profiles exhibited by each species of microsporidian tested and its ability to detect as few as 10 spores. In order to assess the applicability of this microarray in a clinical setting, we conducted microarray assays of 20 fecal samples from AIDS patients. Twelve of these samples were positive for the presence of microsporidia and could be confidently identified; 11 of them were positive for more than one species. Our results suggested that this microarray-based approach represents an attractive diagnostic tool for high-throughput detection and identification of microsporidian species in clinical and epidemiological investigations.

FIG. 1.

Specificity and sensitivity of the microarray for four microsporidian species. (A) Microarray template showing the identity of each probe (Table 1). The spots at the left and right ends of the array represent internal positive controls. (B) Hybridization analysis of amplified SSU rRNA sequences from four reference species. (C and D)Sensitivity testing of the microarray with 10 pure spores of E. intestinalis and 100 spores spiked into 100 μl fecal material. (E) Hybridization of mixed amplified SSU rRNA fragments from all four reference species. (F) Hybridization of SSU rRNA PCR products amplified from the mixture of E. cuniculi (10⁵ spores) and E. intestinalis (100 spores).

FIG. 2.

Microarray hybridization results from clinical samples. SSU rRNA PCR products from 20 clinical samples were hybridized to microarrays. Twelve samples were positive, and eight were negative, which are represented by six hybridization profiles. Panels: A, E. cuniculi and E. intestinalis, two samples; B, E. cuniculi and E. hellem, one sample; C, E. intestinalis, one sample; D, E. cuniculi, E. hellem, and E. intestinalis, one sample; E, E. bieneusi and E. intestinalis, seven samples; F, negative, eight samples; G, species-specific PCR analysis of six representative clinical samples. Groups A to F in panel G correspond to the six hybridization profiles shown above. Lanes b, c, h, and i show designated amplification of clinical samples with primer pairs specific for E. bieneusi, E. cuniculi, E. hellem, and E. intestinalis, respectively. The expected amplicon sizes are 607 bp (b), 556 bp (c), 554 bp (h), and 528 bp (i).