Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P < 0.05) and improved the sensitivity of pathogen detection (P < 0.01), with a 20-to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples. Clinical microbiology is one of the most rapidly changing areas of laboratory medicine today due to the introduction of new technologies and automation (1). Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3-6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays. These less common organisms, for which tests are not readily available, are likely responsible for many cases of undiagnosed illness, particularly in critically ill patients and those with compromised immunity (7,8). Therefore, there is increasing interest in the application of novel technologies, such as next-generation sequencing (NGS), for unbiased detection of pathogens in clinical samples.Among the various challenges with the implementation of NGS for routine pathogen detection using metagenomics, the presence of an overwhelming amount of host DNA is one of the most important problems to be addressed. A previous metagenomic study on nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections revealed that up to ϳ95% of raw NGS reads were of human DNA (9). The su...

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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Smoking during pregnancy, place of residence, Inuit race, lack of breast-feeding, and overcrowding were all independently associated with increased risk of hospital admission for LRTI among Inuit children less than 2 years of age. Future research on the role of adoption and genetics on the health of Inuit children are required.

in symptomatic children presenting to the emergency room, total viral load is related to clinical diagnosis; specific viruses are associated with particular clinical diagnoses, and qPCR has a higher yield than other viral diagnostic methods.