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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.

SUBMITTER: Wang Z

PROVIDER: S-EPMC1233985 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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

Wang Zheng Z Orlandi Palmer A PA Stenger David A DA

Journal of clinical microbiology 20050801 8

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 microarra ...[more]

PMID: 16081959

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Project description:BackgroundAvian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) are important avian pathogens that can cause enormous economic loss on the poultry industry. Different respiratory etiological agents may induce similar clinical signs that make differential diagnosis difficult. Importantly, AIV brings about severe threat to human public health. Therefore, a novel method that can distinguish these viruses quickly and simultaneously is urgently needed.ResultsIn this study, an oligonucleotide microarray system was developed. AIV, including H5, H7, and H9 subtypes; NDV; and IBV were simultaneously detected and differentiated on a microarray. Three probes specific for AIV, NDV, and IBV, as well as three other probes for differentiating H5, H7, and H9 of AIV, were first designed and jet-printed to predetermined locations of initiator-integrated poly(dimethylsiloxane) for the synchronous detection of the six pathogens. The marked multiplex reverse transcription polymerase chain reaction (PCR) products were hybridized with the specific probes, and the results of hybridization were read directly with the naked eyes. No cross-reaction was observed with 10 other subtypes of AIV and infectious bursal disease virus, indicating that the oligonucleotide microarray assay was highly specific. The sensitivity of the method was at least 100 times higher than that of the conventional PCR, and the detection limit of NDV, AIV, H5, H7, and H9 can reach 0.1 EID50 (50% egg infective dose), except that of IBV, which was 1 EID50 per reaction. In the validation of 93 field samples, AIV, IBV, and NDV were detected in 53 (56.99%) samples by oligonucleotide microarray and virus isolation and in 50 (53.76%) samples by conventional PCR.ConclusionsWe have successfully developed an approach to differentiate AIV, NDV, IBV, H5, H7, and H9 subtypes of AIV using oligonucleotide microarray. The microarray is an accurate, high-throughput, and relatively simple method for the rapid detection of avian respiratory viral diseases. It can be used for the epidemiological surveillance and diagnosis of AIV, IBV, and NDV.

Dataset Information

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

Publications

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

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