Massively parallel pathogen identification using high‐density microarrays

Abstract

SummaryIdentification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole‐genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1–4 h. Using this method, we identified Monkeypox virus and drug‐resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner.

Application of Broad-Spectrum Resequencing Microarray for Genotyping Rhabdoviruses

“…This technology also offers the potential for a single test that detects and discriminates between a target pathogen and its closest phylogenetic neighbors, which expands the repertoire of identifiable organisms far beyond those that are initially included in the array. Successful results have been obtained using this tech-nology, especially for the detection of broad-spectrum respiratory tract pathogens using respiratory pathogen microarrays (2,25,26) or the detection of a broad range of biothreat agents (1,23,36,45). The amplification step, which is more often limiting for this technology, has also benefited from recent developments.…”

“…The microarray hybridization process was carried out according to the protocol recommended by the manufacturer (Affymetrix). All of the details and parameter settings for the data analysis (essentially conversion of raw image files obtained from scanning of the microarrays into FASTA files containing the sequences of base calls made for each tiled region of the microarray) have been described previously (1). The base call rate refers to the percentage of base calls generated from the full-length tiled sequence.…”

“…A direct application using this sequence region was recently described for lyssavirus RNA detection in human rabies diagnosis (13). Taking advantage of these characteristics, this polymerase region was also used to design probes for high-density RMAs, also called PathogenID arrays (Affymetrix), which are optimized for the detection and sequence determination of several RNA viruses, particularly rhabdoviruses (1).…”

Global Sequencing: A Review of Current Molecular Data and New Methods Available to Assess Microbial Diversity

“…DNA arrays have two main uses in microbiology: Detection of known pathogens, and assessment of diversity 113) . In general, a PCR amplification step using specific primer sets is used to generated labeled DNA 9,11,27,58,79,95,96,100,105,136,156,173,182) . This requirement of predesigned oligomers for amplification and hybridization opens the possibility that many unknown organisms remain undetected (especially viruses which have no universal homologous gene).…”

Maculopapular lesions in the Central African Republic

“…The 2001 and 2010 strains were also tested by a resequencing microarray for detection and characterisation of a large panel of viruses and bacteria. 2,3 We were able to detect, in one step (48 h), two sequences of haemagglutinin and DNA polymerase DNA-dependent genes of the viral genome in both samples and to confi rm the genotype (DRC clade). Our patients were admitted to the local health centre and kept in isolation until they were no longer infectious.…”