Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays

Abstract

The exponential growth of pathogen nucleic acid sequences available in public domain databases has invited their direct use in pathogen detection, identification, and surveillance strategies. DNA microarray technology has offered the potential for the direct DNA sequence analysis of a broad spectrum of pathogens of interest. However, to achieve the practical attainment of this potential, numerous technical issues, especially nucleic acid amplification, probe specificity, and interpretation strategies of sequence detection, need to be addressed. In this report, we demonstrate an approach that combines the use of a custom-designed Affymetrix resequencing Respiratory Pathogen Microarray (RPM v.1) with methods for microbial nucleic acid enrichment, random nucleic acid amplification, and automated sequence similarity searching for broad-spectrum respiratory pathogen surveillance. Successful proof-of-concept experiments, utilizing clinical samples obtained from patients presenting adenovirus or influenza virus-induced febrile respiratory illness (FRI), demonstrate the ability of this approach for correct species- and strain-level identification with unambiguous statistical interpretation at clinically relevant sensitivity levels. Our results underscore the feasibility of using this approach to expedite the early surveillance of diseases, and provide new information on the incidence of multiple pathogens.

Figure 1.

RPM v.1 process diagram. This diagram shows the process for each step when using RPM v.1 with a random amplification strategy for clinical samples.

Figure 2.

Random amplification and resequencing microarray-based identification of two common respiratory tract viral pathogens. (A) RPM v.1 design overview. The tiled sequence regions for each of the targeted respiratory tract pathogens have been color-coded (left). (B) Hybridization profile of the HAdV-4 prototype strain RI-67. (C) Identification of an H3N2 influenza A virus (A/Fujian/411/2002) from Lackland AFB clinical nasal wash sample #NW20031114–03–7. (Upper arrow) Tile region for hemagglutinin (H3), (lower arrow) tile regions for neuraminidase (N2) and matrix. The black region interspersed between the two arrows constitutes the tile regions for hemagglutinin (H5) and neuraminidase (N1). (D) Identification of an HAdV-5/HAdV-21 coinfection in NHRC clinical throat swab sample #7151. The arrows on the right of each image are color coded according to the legend on the far left.

Figure 3.

Examples of primary sequence data generated by the hybridization of randomly amplified targets to RPM v.1 tiled probe sets. Clinical throat swab sample NHRC #7151 was found to contain an adenoviral coinfection (Fig. 2D), as hybridization to the HAdV-5 E1A tiled prototype sequence region resulted in the identification of HAdV-5 E1A (A), whereas the sequence generated via hybridization to the HAdV-7 E1A tiled prototype sequence region suggested the presence of HAdV-21 (B). (C) Amplicons generated from clinical throat swab sample NHRC #49110 hybridized to the influenza A virus (A/New Caledonia/20/99) hemagglutinin (H1) gene prototype sequence but were identified (via REPI analysis) as influenza A virus (A/Madrid/1082/2001) H1N1. (*) Single nucleotides that differentiate the experimental sequence from the tiled prototype sequence.

Figure 4.

(A) REPI logic diagram. The algorithm expands subsequences to the maximum length within the constraints of the allowable no-call (N) percentage (here, 25%). Each subsequence was submitted for BLAST analysis. (B) A sample REPI output (truncated) for Lackland AFB clinical nasal wash sample #NW20031114 that unambiguously identified influenza A virus type H3N2.