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 ...

We report here on the results of a pilot study comparing our clinical diagnostic virology laboratory's current methods of respiratory pathogen detection against the Genaco Respiratory Infections Panels 1 and 2. These assays employ xMap (Luminex) liquid phase bead conjugated array technology to facilitate automated detection of PCR and RT-PCR products, which provides potential for levels of assay multiplexing above those currently practical with either conventional gel-resolved or real-time methods. In the study presented here we used the Genaco panels to simultaneously screen previously analyzed clinical specimens (nasopharyngeal washings) for twenty-one important pathogens. Our results indicate the Genaco panels met or exceeded our current methods' sensitivity and specificity although allowing for detection of a wider range of infectious agents than practical by current diagnostic laboratory practices. In addition, the Genaco panels provided data on the presence of multiple respiratory pathogens in single specimens, which would otherwise be missed in most instances. To our knowledge, this study represents the first trial of these panels on standard clinical specimens in a routine diagnostic setting.

Primary infection by human parvovirus B19 is often accompanied by arthropathy of varying duration, of which the most severe cases can be indistinguishable from rheumatoid arthritis (RA). While this might seem to imply a role in RA pathogenesis, recent studies have verified long-term persistence of B19 DNA in synovial tissue not only in patients with rheumatoid or juvenile arthritis, but also in immunocompetent, non-arthritic individuals with a history of prior B19 infection. However, the latter data are based on PCR amplification of short segments of DNA, with little sequence information. We determined the nucleotide sequence and examined the integrity of the protein-coding regions of B19 genomes persisting in synovial tissue and compared the results with data from synovial tissues of recently infected patients. In synovium of both previously and recently infected subjects, the viral coding regions were found to be present in an apparently continuous, intact DNA molecule. Comparison with sequences reported from blood or bone marrow showed that the synoviotropism or persistence of the B19 virus DNA was not due to exceptional mutations or particular genotype variants. The synovial retention of full-length viral genomes may represent a physiological process functioning in long-term storage of foreign macromolecules in this tissue.