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BACKGROUND: Adenoviruses (Ads) cause continuous outbreaks of acute respiratory disease (ARD) in US military training facilities. In 1996, the loss of vaccines targeting the dominant recruit-associated serotypes precipitated the reemergence of Ads in these populations. Between 1999 and 2002, serotype 4 accounted for >95% of Ads isolated from recruits and for >50% of ARD cases in training facilities (15,000 cases/year). METHODS: Ads (n=1867) collected between 2002 and 2006 from recruits with ARD at 8 military training facilities in the United States were serotyped by serum neutralization and polymerase chain reaction. RESULTS: The dominance of Ad4 continued through 2005, followed by a simultaneous emergence of diverse species B serotypes at the majority of sites. This included the subspecies B1 serotypes 3, 7, and 21 and the subspecies B2 serotype 14. Ad14 was the most prevalent species B serotype, appearing in high numbers at 3 sites and becoming dominant at 1. CONCLUSIONS: Subspecies B2 Ads have rarely been associated with ARD, and only in Eurasia. This survey represents the first report of AdB2-associated ARD in the Western Hemisphere. The simultaneous emergence of several species B Ads suggests a common external source (the civilian population) and a decrease in preexisting immunity to species B Ads. [ABSTRACT FROM AUTHOR]

The incidence of invasive fungal infections is on the rise worldwide due to the growth of the immunocompromised population. We report here the use of a diagnostic assay that utilizes a universal extraction method, broad spectrum PCR amplification and analysis via electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify more than 200 pathogenic fungi directly from bronchoalveolar lavage (BAL) specimens in less than 8 hours. In this study, we describe both analytical and clinical performance of the assay, when run with prospectively collected clinical BAL specimens. In 146 patients with probable and possible fungal infections defined by EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria, the PCR/ESI-MS assay demonstrated a sensitivity of 90.9% (95% CI: 76.4-96.9%) and a specificity of 82.3% (95% CI: 74.2-88.2%). This data demonstrates the utility of a non-culture based broad fungal targets molecular diagnostic tool for rapid and accurate diagnosis of invasive fungal infections in patients at risk of developing fungal diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Clinical samples were evaluated with the Mobile Analysis Platform (MAP) to determine platform performance for detecting respiratory viruses in samples previously characterized using clinical reverse transcriptase polymerase chain reaction assays. The percent agreement between MAP and clinical results was 97% for influenza A (73/75), 100% (21/21) for influenza B, 100% (6/6) for respiratory syncytial virus (RSV), and 80% (4/5) for negative specimens. The approximate limit of detection of the MAP was 30 copies/assay for RSV and 1500 copies/assay for Middle East respiratory syndrome coronavirus., (Copyright © 2018. Published by Elsevier Inc.)

Unlabelled: Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours., Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States.

Background: The circulation of human adenovirus type 21 (HAdV21) in the United States has been documented since the 1960s in association with outbreaks of febrile respiratory illness (FRI) in military boot camps and civilian cases of respiratory disease., Methods: To describe the molecular epidemiology of HAdV21 respiratory infections across the country, 150 clinical respiratory isolates obtained from continuous surveillance of military recruit FRI, and 23 respiratory isolates recovered from pediatric and adult civilian cases of acute respiratory infection were characterized to compile molecular typing data spanning 37 years (1978-2014)., Results: Restriction enzyme analysis and genomic sequencing identified 2 clusters of closely related genomic variants readily distinguishable from the prototype and designated 21a-like and 21b-like. A-like variants predominated until 1999. A shift to b-like variants was noticeable by 2007 after a 7-year period (2000-2006) of cocirculation of the 2 genome types. US strains are phylogenetically more closely related to European and Asian strains isolated over the last 4 decades than to the Saudi Arabian prototype strain AV-1645 isolated in 1956., Conclusions: Knowledge of circulating HAdV21 variants and their epidemic behavior will be of significant value to local and global FRI surveillance efforts., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Diverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes., Objectives: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens., Study Design: We developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to simultaneously detect and identify adenovirus, enterovirus, polyomaviruses JC and BK, parvovirus B19, HSV-1, HSV-2, VZV, EBV, CMV, and herpesviruses 6-8 in plasma specimens. The assay performance was characterized analytically, and the results from clinical plasma samples were compared to the results obtained from single-analyte real time PCR tests currently used in clinical practice., Results: The assay demonstrated sensitivity and specificity to diverse strains of the targeted viral families and robustness to interfering substances and potentially cross reacting organisms. The assay yielded 94% sensitivity when testing clinical plasma samples previously identified as positive using standard-of-care real-time PCR tests for a single target virus (available samples included positive samples for 11 viruses targeted by the assay)., Conclusions: The assay functioned as designed, providing simultaneous broad-spectrum detection and identification of diverse agents of disseminated viral infection. Among 156 clinical samples tested, 37 detections were made in addition to the detections matching the initial clinical positive results., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Simple sequence repeats (SSRs), or microsatellites are hyper-mutable and can lead to disorders. Here we explore SSR distribution in cell cycle-associated genes [grouped into: checkpoint; regulation; replication, repair, and recombination (RRR); and transition] in humans and orthologues of eight mammals. Among the gene groups studied, transition genes have the highest SSR density. Trinucleotide repeats are not abundant and introns have higher repeat density than exons. Many repeats in human genes are conserved; however, CG motifs are conserved only in regulation genes. SSR variability in cell cycle genes represents a genetic Achilles' heel, yet SSRs are common in all groups of genes. This tolerance many be due to i) positions in introns where they do not disrupt gene function, ii) essential roles in regulation, iii) specific value of adaptability, and/or iv) lack of negative selection pressure. Present study may be useful for further exploration of their medical relevance and potential functionality.

Broad spectrum biosensors capable of identifying diverse organisms are transitioning from the realm of research into the clinic. These technologies simultaneously capture signals from a wide variety of biological entities using universal processes. Specific organisms are then identified through bioinformatic signature-matching processes. This is in contrast to currently accepted molecular diagnostic technologies, which utilize unique reagents and processes to detect each organism of interest. This paradigm shift greatly increases the breadth of molecular diagnostic tools with little increase in biochemical complexity, enabling simultaneous diagnostic, epidemiologic, and biothreat surveillance capabilities at the point of care. This, in turn, offers the promise of increased biosecurity and better antimicrobial stewardship. Efficient realization of these potential gains will require novel regulatory paradigms reflective of the generalized, information-based nature of these assays, allowing extension of empirical data obtained from readily available organisms to support broader reporting of rare, difficult to culture, or extremely hazardous organisms.

We describe an assay which uses broad-spectrum, conserved-site PCR paired with mass spectrometry analysis of amplicons (PCR/electrospray ionization-mass spectrometry [ESI-MS]) to detect and identify diverse bacterial and Candida species in uncultured specimens. The performance of the assay was characterized using whole-blood samples spiked with low titers of 64 bacterial species and 6 Candida species representing the breadth of coverage of the assay. The assay had an average limit of detection of 100 CFU of bacteria or Candida per milliliter of blood, and all species tested yielded limits of detection between 20 and 500 CFU per milliliter. Over 99% of all detections yielded correct identifications, whether they were obtained at concentrations well above the limit of detection or at the lowest detectable concentrations. This study demonstrates the ability of broad-spectrum PCR/ESI-MS assays to detect and identify diverse organisms in complex natural matrices that contain high levels of background DNA.

Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 molds and 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification, and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of molds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species-level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses.

As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.

Background: The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts., Objectives: To determine the analytical sensitivity of RT-PCR/ESI-MS influenza typing assay for identifying the pandemic H1N1 strain and describe its ability to assess viral genetic diversity., Study Design: Two sets of pandemic H1N1 samples, 190 collected between April and June of 2009, and 69 collected between October 2009 and January 2010, were processed by the RT-PCR/ESI-MS influenza typing assay, and the spectral results were compared to reference laboratory results and historical sequencing data from the Nucleotide Database of the National Center for Biotechnology Information (NCBI)., Results: Strain typing concordance with reference standard testing was 100% in both sample sets, and the assay demonstrated a significant increase in influenza genetic diversity, from 10.5% non-wildtype genotypes in early samples to 69.9% in late samples (P<0.001). An NCBI search demonstrated a similar increase, from 13.4% to 45.2% (P<0.001)., Conclusions: This comparison of early versus late influenza samples analyzed by RT-PCR/ESI-MS demonstrates the influenza typing assay's ability as a universal influenza detection platform to provide high-fidelity pH1N1 strain identification over time, despite increasing genetic diversity in the circulating virus. The genotyping data can also be leveraged for high-throughput influenza surveillance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Five genomes of human subspecies B1 adenoviruses isolated from cases of acute respiratory disease have been sequenced and archived for reference. These include representatives of two prevalent genomic variants of HAdV-7, i.e., HAdV-7h and HAdV-7d2. The other three are HAdV-3/16, HAdV-16 strain E26, and HAdV-3+7 strain Takeuchi. All are recombinant genomes. Genomics and bioinformatics provide detailed views into the genetic makeup of these pathogens and insight into their molecular evolution. Retrospective characterization of particularly problematic older pathogens such as HAdV-7h (1987) and intriguing isolates such as HAdV-3+7 strain Takeuchi (1958) may provide clues to their phenotypes and serology and may suggest protocols for prevention and treatment.

The M protein coats group A streptococci (GAS) and acts as the primary antigen and determinant of type-specific immunity. M is essential for GAS virulence, providing antiphagocytic functions critical to survival in human tissues and fluids. Specific regions of M protein also serve as shared antigens, and cross-reactivity between these epitopes and human proteins may be the source of autoimmune sequelae such as rheumatic heart disease. The M protein is hypervariable, and has long served as the primary target for epidemiological typing of GAS. Though other markers or genotyping methods may be necessary to increase strain resolution when clones of a given M type differ in clinically critical ways, M typing remains the most directly informative and well-documented method for tracking outbreaks of GAS, predicting clinical outcomes during those outbreaks, and measuring the general threat presented by GAS at any given time and place.