Evidence from Multiplex Molecular Assays for Complex Multipathogen Interactions in Acute Respiratory Infections

Abstract

While most diagnostic processes cease with the detection of the first relevant infectious agent, newer multiplexed molecular methods which provide simultaneous analysis of multiple agents may give a more accurate representation of the true pathogen spectrum in these samples. To examine this in the context of respiratory infections, acute-phase respiratory specimens submitted to our clinical diagnostic microbiology/ virology laboratory for our routine VIRAP diagnosis protocol during the spring 2006 peak respiratory infection season were processed in parallel by analysis with Genaco (QiaPlex) ResPlex I and ResPlex II molecular diagnostic panels. A total of 1,742 specimens were examined for 21 relevant targets each. The resulting data reveal that multiple infections are frequent and provide evidence for complex interactions between different infectious agents. Statistically relevant association patterns (both positive and negative) were observed between particular pathogens. While some interactions we observed are substantiated by prior reports in the literature, several specific patterns do not appear to have been reported previously. In addition, we report preliminary clinical evidence which supports a hypothesis that these coinfections are medically relevant and that effective treatment for severe respiratory tract infections will increasingly require diagnosis of all involved pathogens, as opposed to single-pathogen reporting.The majority of infection diagnoses proceed via an approach which assumes a single-agent etiology. This assumption is selfvalidating because diagnostic processes end with detection of the first relevant pathogen. While coinfections are more commonly accepted as occurring in respiratory infections than in many other clinical settings, this diagnostic bias towards singlepathogen detection and subsequent treatment is still prevalent. This is true in the case of our clinical diagnostic virology/ microbiology laboratory, which serves as a regional referral center for acute-phase respiratory specimen diagnosis through the VIRAP program (23). Based on a commercial direct fluorescence assay (DFA) (SimulFluor screen; Chemicon Inc., Temecula, CA) for seven pathogens (adenovirus, parainfluenza virus 1 [PIV-1], PIV-2, PIV-3, influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) A/B combined), with referral of DFA-negative specimens to viral cell culture, the VIRAP program processes several thousand specimens annually. Diagnosis proceeds via a flow approach tailored to seasonal and case parameters such that the most likely causal agents are assayed first, with the diagnostic process ending at the first positive result.While approximately 35% of all VIRAP specimens have a pathogen identified by DFA, the extended turn-around time and limited detection spectra of those ϳ65% of specimens referred to viral cell culture have led us to examine alternative second-line diagnostic methods. Our prior successful pilot study of the multiplex molecular assays sold by Genaco Biomedical Products ...

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