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Background: Few investigations have assessed contributions of both vaginal bacteria and proinflammatory immune mediators to human immunodeficiency virus (HIV) acquisition risk in a prospective cohort., Methods: We conducted a nested case-control study of African women who participated in a randomized placebo-controlled trial of daily oral versus vaginal tenofovir-based preexposure prophylaxis for HIV infection. Vaginal concentrations of 23 bacterial taxa and 16 immune mediators were measured. Relationships between individual bacterial concentrations or immune mediators and HIV risk were analyzed using generalized estimating equations in a multivariable model. Factor analysis assessed relationships between combinations of bacterial taxa, immune mediators, and HIV acquisition risk., Results: We identified 177 HIV pre-seroconversion visits from 150 women who acquired HIV and 531 visits from 436 women who remained HIV uninfected. Fourteen bacterial taxa and 6 proinflammatory cytokines and chemokines were individually associated with greater HIV risk after adjusting for confounders. Women with all 14 taxa versus <14 taxa (adjusted odds ratio [aOR], 4.45 [95% confidence interval {CI}, 2.20-8.98]; P < .001) or all 6 immune mediators versus <6 mediators (aOR, 1.77 [95% CI, 1.24-2.52]; P < .001) had greater risk for HIV acquisition. Factor analysis demonstrated that a bacterial factor comprised of 14 high-risk bacterial taxa (aOR, 1.57 [95% CI, 1.27-1.93]; P < 0.001) and the interferon gamma-induced protein 10 (highest quartile: aOR, 3.19 [95% CI, 1.32-7.72]; P = 0.002) contributed to the highest HIV risk., Conclusions: Bacterial and host biomarkers for predicting HIV acquisition risk identify women at greatest risk for HIV infection and can focus prevention efforts., Competing Interests: Potential conflicts of interest. D. N. F. and T. L. F. receive royalties from Becton Dickinson. S. S. has received speaking honoraria from Lupin Inc. S. S. and D. N. F. have developed intellectual property around the molecular detection of bacteria associated with HIV risk. During the course of this study, S. L. H. received consulting fees from Dare Biosciences and Lupin Inc, and her institution received support from Dickinson, Lupin, Dare, and Curetek. B. A. R. has received payment from Gilead Sciences Inc for service as a data and safety monitoring board member for previous and ongoing clinical trials. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purpose: The goal of this preliminary study is to describe the vaginal microbiome of transgender and gender nonbinary (TGNB) individuals using nonculture-based techniques. TGNB individuals may undergo gender-affirming surgical procedures, which can include the creation of a neovagina. Little is known about microbial species that comprise this environment in states of health or disease., Methods: In this pilot study, vaginal swabs were self-collected from 15 healthy self-identified TGNB participants (age 26-69 years) and 8 cisgender comparator participants (age 27-50 years) between 2017 and 2018. Next-generation 16S ribosomal RNA sequencing was used to profile individual bacterial communities from all study samples., Results: The TGNB cohort demonstrated significantly higher intraindividual (alpha) diversity than the cisgender group ( p =0.0003). Microbial species commensal to the gut and skin were identified only in specimens from TGNB participants. Although Lactobacillus species were dominant in all cisgender comparator samples, they were found at low relative abundance (≤3%) in TGNB samples., Conclusion: In this study, specimens collected from neovaginas showed increased alpha diversity and substantially different composition compared with natal vaginas. In contrast to natal vaginas, neovaginas were not dominated by Lactobacillus , but were hosts to many microbial species. Studies that help to improve our understanding of the neovaginal microbiome may enable clinicians to differentiate between healthy and diseased neovaginal states., (Copyright 2023, Mary Ann Liebert, Inc., publishers.)

Background: Sexual behavior may influence the composition of the male urethral microbiota, but this hypothesis has not been tested in longitudinal studies of men who have sex with men (MSM)., Methods: From December 2014 to July 2018, we enrolled MSM with nongonococcal urethritis (NGU) attending a sexual health clinic. Men attended 5 in-clinic visits at 3-week intervals, collected weekly urine specimens at home, and reported daily antibiotics and sexual activity on weekly diaries. We applied broad-range 16S rRNA gene sequencing to urine. We used generalized estimating equations to estimate the association between urethral sexual exposures in the prior 7 days (insertive oral sex [IOS] only, condomless insertive anal intercourse [CIAI] only, IOS with CIAI [IOS + CIAI], or none) and Shannon index, number of species (observed, oral indicator, and rectal indicator), and specific taxa, adjusting for recent antibiotics, age, race/ethnicity, HIV, and preexposure prophylaxis., Results: Ninety-six of 108 MSM with NGU attended ≥1 follow-up visit. They contributed 1140 person-weeks of behavioral data and 1006 urine specimens. Compared with those with no urethral sexual exposures, those with IOS only had higher Shannon index ( P = 0.03 ) but similar number of species and presence of specific taxa considered, adjusting for confounders; the exception was an association with Haemophilus parainfluenzae . CIAI only was not associated with measured aspects of the urethral microbiota. IOS + CIAI was only associated with presence of H. parainfluenzae and Haemophilus ., Conclusions: Among MSM after NGU, IOS and CIAI did not seem to have a substantial influence on measured aspects of the composition of the urethral microbiota., Competing Interests: Conflict of Interest of Sources of Funding: This work was supported by the National Institutes of Health (grant number U19 AI113173). L.C.C. was supported by the National Institutes of Health (grant number TL1 TR002318 trainee support). K.A.T. was supported by the University of Washington/Fred Hutch Center for AIDS Research, a program funded by the National Institutes of Health (grant number P30 AI027757). Study data were collected and managed using Research Electronic Data Capture (REDCap) tools hosted at the University of Washington Institute of Translational Health Sciences and supported by the National Institutes of Health (grant number UL1 TR002319). C.M.K. has received donations of test kits and reagents from Hologic, Inc. M.R.G. has conducted studies unrelated to this work supported by grants from Hologic, Inc. L.E.M. has received research support and honoraria from Hologic, Inc., and Nabriva Therapeutics. All other authors declare that they have no conflict of interest., (Copyright © 2024 American Sexually Transmitted Diseases Association. All rights reserved.)

Background: The Ministry of Health in Côte d'Ivoire and the International Training and Education Center for Health at the University of Washington, funded by the United States President's Emergency Plan for AIDS Relief, have been collaborating to develop and implement the Open-Source Enterprise-Level Laboratory Information System (OpenELIS). The system is designed to improve HIV-related laboratory data management and strengthen quality management and capacity at clinical laboratories across the nation., Objective: This evaluation aimed to quantify the effects of implementing OpenELIS on data quality for laboratory tests related to HIV care and treatment., Methods: This evaluation used a quasi-experimental design to perform an interrupted time-series analysis to estimate the changes in the level and slope of 3 data quality indicators (timeliness, completeness, and validity) after OpenELIS implementation. We collected paper and electronic records on clusters of differentiation 4 (CD4) testing for 48 weeks before OpenELIS adoption until 72 weeks after. Data collection took place at 21 laboratories in 13 health regions that started using OpenELIS between 2014 and 2020. We analyzed the data at the laboratory level. We estimated odds ratios (ORs) by comparing the observed outcomes with modeled counterfactual ones when the laboratories did not adopt OpenELIS., Results: There was an immediate 5-fold increase in timeliness (OR 5.27, 95% CI 4.33-6.41; P<.001) and an immediate 3.6-fold increase in completeness (OR 3.59, 95% CI 2.40-5.37; P<.001). These immediate improvements were observed starting after OpenELIS installation and then maintained until 72 weeks after OpenELIS adoption. The weekly improvement in the postimplementation trend of completeness was significant (OR 1.03, 95% CI 1.02-1.05; P<.001). The improvement in validity was not statistically significant (OR 1.34, 95% CI 0.69-2.60; P=.38), but validity did not fall below pre-OpenELIS levels., Conclusions: These results demonstrate the value of electronic laboratory information systems in improving laboratory data quality and supporting evidence-based decision-making in health care. These findings highlight the importance of OpenELIS in Côte d'Ivoire and the potential for adoption in other low- and middle-income countries with similar health systems., (©Yao He, Yves-Rolland Kouabenan, Paul Henri Assoa, Nancy Puttkammer, Bradley H Wagenaar, Hong Xiao, Stephen Gloyd, Noah G Hoffman, Pascal Komena, N'zi Pierre Fourier Kamelan, Casey Iiams-Hauser, Adama Sanogo Pongathie, Alain Kouakou, Jan Flowers, Nadine Abiola, Natacha Kohemun, Jean-Bernard Amani, Christiane Adje-Toure, Lucy A Perrone. Originally published in JMIR Public Health and Surveillance (https://publichealth.jmir.org), 20.03.2024.)

For studies using microbiome data, the ability to robustly combine data from technically and biologically distinct microbiome studies is a crucial means of supporting more robust and clinically relevant inferences. Formidable technical challenges arise when attempting to combine data from technically diverse 16S rRNA gene variable region amplicon sequencing (16S) studies. Closed operational taxonomic units and taxonomy are criticized as being heavily dependent upon reference sets and with limited precision relative to the underlying biology. Phylogenetic placement has been demonstrated to be a promising taxonomy-free manner of harmonizing microbiome data, but it has lacked a validated count-based feature suitable for use in machine learning and association studies. Here we introduce a phylogenetic-placement-based, taxonomy-independent, compositional feature of microbiota: phylotypes. Phylotypes were predictive of clinical outcomes such as obesity or pre-term birth on technically diverse independent validation sets harmonized post hoc. Thus, phylotypes enable the rigorous cross-validation of 16S-based clinical prognostic models and associative microbiome studies., Competing Interests: Declaration of interests A patent has been filed for portions of the phylotype generation process., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In this paper, we consider the current and potential role of the latest generation of Large Language Models (LLMs) in medical informatics, particularly within the realms of clinical and anatomic pathology. We aim to provide a thorough understanding of the considerations that arise when employing LLMs in healthcare settings, such as determining appropriate use cases and evaluating the advantages and limitations of these models. Furthermore, this paper will consider the infrastructural and organizational requirements necessary for the successful implementation and utilization of LLMs in healthcare environments. We will discuss the importance of addressing education, security, bias, and privacy concerns associated with LLMs in clinical informatics, as well as the need for a robust framework to overcome regulatory, compliance, and legal challenges., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Identification and analysis of clinically relevant strains of bacteria increasingly relies on whole-genome sequencing. The downstream bioinformatics steps necessary for calling variants from short-read sequences are well-established but seldom validated against haploid genomes. We devised an in silico workflow to introduce single nucleotide polymorphisms (SNP) and indels into bacterial reference genomes, and computationally generate sequencing reads based on the mutated genomes. We then applied the method to Mycobacterium tuberculosis H37Rv, Staphylococcus aureus NCTC 8325, and Klebsiella pneumoniae HS11286, and used the synthetic reads as truth sets for evaluating several popular variant callers. Insertions proved especially challenging for most variant callers to correctly identify, relative to deletions and single nucleotide polymorphisms. With adequate read depth, however, variant callers that use high quality soft-clipped reads and base mismatches to perform local realignment consistently had the highest precision and recall in identifying insertions and deletions ranging from1 to 50 bp. The remaining variant callers had lower recall values associated with identification of insertions greater than 20 bp., Competing Interests: The authors declare no conflict of interest.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4 + T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity., Competing Interests: Declaration of interests W.M.G. is a scientific advisory board member for Freenome, Guardant Health, and SEngine and consultant for DiaCarta, Natera, Guidepoint, and GLG. He is an investigator in a clinical trial sponsored by Janssen Pharmaceuticals and receives research support from Tempus and LucidDx. M.R.M.v.d.B. has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; he has received royalties from Wolters Kluwer; has consulted, received honorarium from or participated in advisory boards for Seres Therapeutics, Vor Biopharma, Rheos Medicines, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Ceramedix, Lygenesis, Pluto Therapeutics, GlaskoSmithKline, Da Volterra, Thymofox, Garuda, Novartis (Spouse), Synthekine (Spouse), Beigene (Spouse), and Kite (Spouse); has IP Licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a nonprofit organization). Memorial Sloan Kettering has institutional financial interests relative to Seres Therapeutics. K.A.M. serves in an advisory role and holds stock in PostBiotics Plus Research and has consulted for Incyte. D.N.F. and T.L.F. have financial relationships with BD for licensure of molecular diagnosis of bacterial vaginosis, unrelated to the research presented in this article. G.R.H. has consulted for Generon Corporation, NapaJen Pharma, iTeos Therapeutics, and Neoleukin Therapeutics and receives research funding from Compass Therapeutics, Syndax Pharmaceuticals, Applied Molecular Transport, Serplus Technology, Heat Biologics, Laevoroc Oncology,iTeos Pharmaceuticals, and Genentech., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Reflexive laboratory testing workflows can improve the assessment of patients receiving pain medications chronically, but complex workflows requiring pathologist input and interpretation may not be well-supported by traditional laboratory information systems. In this work, we describe the development of a web application that improves the efficiency of pathologists and laboratory staff in delivering actionable toxicology results., Method: Before designing the application, we set out to understand the entire workflow including the laboratory workflow and pathologist review. Additionally, we gathered requirements and specifications from stakeholders. Finally, to assess the performance of the implementation of the application, we surveyed stakeholders and documented the approximate amount of time that is required in each step of the workflow., Results: A web-based application was chosen for the ease of access for users. Relevant clinical data was routinely received and displayed in the application. The workflows in the laboratory and during the interpretation process served as the basis of the user interface. With the addition of auto-filing software, the return on investment was significant. The laboratory saved the equivalent of one full-time employee in time by automating file management and result entry., Discussion: Implementation of a purpose-built application to support reflex and interpretation workflows in a clinical pathology practice has led to a significant improvement in laboratory efficiency. Custom- and purpose-built applications can help reduce staff burnout, reduce transcription errors, and allow staff to focus on more critical issues around quality., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background: Bacterial colonization and associations with bacterial vaginosis (BV) signs and symptoms (Amsel criteria) may vary between populations. We assessed relationships between vaginal bacteria and Amsel criteria among two populations., Methods: Kenyan participants from the placebo arm of the Preventing Vaginal Infections (PVI) trial and participants from a Seattle-based cross-sectional BV study were included. Amsel criteria were recorded at study visits, and the vaginal microbiota was characterized using 16S rRNA gene sequencing. Logistic regression models, accounting for repeat visits as appropriate, were fit to evaluate associations between bacterial relative abundance and each Amsel criterion., Results: Among 84 PVI participants (496 observations) and 220 Seattle participants, the prevalence of amine odor was 25% and 40%, clue cells 16% and 37%, vaginal discharge 10% and 52%, elevated vaginal pH 69% and 67%, and BV 13% and 44%, respectively. BV-associated bacterium 1 (BVAB1) was positively associated with all Amsel criteria in both populations. Eggerthella type 1, Fannyhessea ( Atopobium) vaginae , Gardnerella spp., Sneathia amnii , and Sneathia sanguinegens were positively associated with all Amsel criteria in the Seattle study, and all but discharge in the PVI trial., Conclusions: Core vaginal bacteria are consistently associated with BV signs and symptoms across two distinct populations of women., Competing Interests: JB has received honoraria from BD for consulting. RM has received research funding, paid to the University of Washington, from Hologic Corporation and has received honoraria for consulting from Lupin Pharmaceuticals. SS has received consulting honoraria from Lupin Pharmaceuticals. DF and TF received a royalty from BD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Preventing Vaginal Infections trial received funding (donated study product) from Embil Pharmaceutical Company. Embil Pharmaceutical Company was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2022 Carter, Balkus, Anzala, Kimani, Hoffman, Fiedler, Mochache, Fredricks, McClelland and Srinivasan.)

Background: Nongonococcal urethritis (NGU) is a common syndrome with no known etiology in ≤50% of cases. We estimated associations between urethral bacteria and NGU in men who have sex with men (MSM) and men who have sex with women (MSW)., Methods: Urine was collected from NGU cases (129 MSM, 121 MSW) and controls (70 MSM, 114 MSW) attending a Seattle STD clinic. Cases had ≥5 polymorphonuclear leukocytes on Gram stain plus symptoms or discharge; controls had <5 PMNs, no symptoms, no discharge. NGU was considered idiopathic when Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, adenovirus, and herpes simplex virus were absent. The urethral microbiota was characterized using 16S rRNA gene sequencing. Compositional lasso analysis was conducted to identify associations between bacterial taxa and NGU and to select bacteria for targeted qPCR., Results: Among NGU cases, 45.2% were idiopathic. Based on compositional lasso analysis, we selected Haemophilus influenzae (HI) and Mycoplasma penetrans (MP) for targeted qPCR. Compared with 182 men without NGU, the 249 men with NGU were more likely to have HI (14% vs 2%) and MP (21% vs 1%) (both P ≤ .001). In stratified analyses, detection of HI was associated with NGU among MSM (12% vs 3%, P = .036) and MSW (17% vs 1%, P < .001), but MP was associated with NGU only among MSM (13% vs 1%, P = .004). Associations were stronger in men with idiopathic NGU., Conclusions: HI and MP are potential causes of male urethritis. MP was more often detected among MSM than MSW with urethritis., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: The vaginal microbiome plays a key role in women's reproductive health. Use of exogenous hormones, such as intramuscular depot medroxyprogesterone acetate (DMPA-IM), may alter the composition of vaginal bacterial community., Methods: Vaginal swab samples were collected from postpartum Kenyan women initiating DMPA-IM or nonhormonal contraception (non-HC). Bacterial vaginosis was assessed by Nugent score (Nugent-BV) and bacterial community composition was evaluated using broad-range 16S ribosomal RNA gene polymerase chain reaction with high-throughput sequencing. Changes in Nugent score, alpha diversity (Shannon diversity index), and total bacterial load between contraceptive groups from enrollment to 3 months after initiation were estimated using multivariable linear mixed effects regression., Results: Among 54 human immunodeficiency virus-negative women, 33 choosing DMPA-IM and 21 choosing non-HC, Nugent-BV was more common among DMPA-IM users at enrollment. At follow-up, Nugent score had decreased significantly among DMPA-IM users (change, -1.89; 95% confidence interval [CI], -3.53 to -.25; P = .02) while alpha diversity remained stable (0.03; -.24 to .30; P = .83). Conversely, Nugent score remained relatively stable among non-HC users (change, -0.73; 95% CI, -2.18 to .73; P = .33) while alpha diversity decreased (-0.34; -.67 to -.001; P = .05). The total bacterial load decreased slightly in DMPA-IM users and increased slightly among non-HC users, resulting in a significant difference in change between the contraceptive groups (difference, -0.64 log10 gene copies per swab sample; 95% CI, -1.19 to -.08; P = .02). While significant changes in Nugent score and alpha diversity were observed within contraceptive groups, changes between groups were not significantly different., Conclusions: Postpartum vaginal bacterial diversity did not change in DMPA-IM users despite a reduction in Nugent-BV, but it decreased significantly among women using non-HC. Choice of contraception may influence Lactobacillus recovery in postpartum women., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The Nugent score is the reference standard for bacterial vaginosis (BV) diagnosis but has not been validated in postmenopausal women. We compared relative abundances from 16S ribosomal RNA gene sequencing of vaginal microbiota with Nugent score in cohorts of premenopausal (n = 220) and postmenopausal (n = 144) women. In premenopausal women, 33 taxa were significantly correlated with Nugent score, including the classic BV-associated taxa Gardnerella, Atopobium, Sneathia, Megasphaera, and Prevotella. In postmenopausal women, 11 taxa were significantly associated with Nugent score, including Prevotella but no other BV-associated genera. High Nugent scores should not be used to infer BV in postmenopausal women., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Healthcare workers (HCWs) who serve on the front lines of the coronavirus disease 2019 (COVID-19) pandemic have been at increased risk for infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in some settings. Healthcare-acquired infection has been reported in similar epidemics, but there are limited data on the prevalence of COVID-19 among HCWs and their associated clinical outcomes in the United States., Methods: We established 2 high-throughput employee testing centers in Seattle, Washington, with drive-through and walk-through options for symptomatic employees in the University of Washington Medicine system and its affiliated organizations. Using data from these testing centers, we report the prevalence of SARS-CoV-2 infection among symptomatic employees and describe the clinical characteristics and outcomes among employees with COVID-19., Results: Between 12 March 2020 and 23 April 2020, 3477 symptomatic employees were tested for COVID-19 at 2 employee testing centers; 185 (5.3%) employees tested positive for COVID-19. The prevalence of SARS-CoV-2 was similar when comparing frontline HCWs (5.2%) with nonfrontline staff (5.5%). Among 174 positive employees reached for follow-up at least 14 days after diagnosis, 6 reported COVID-related hospitalization; all recovered., Conclusions: During the study period, we observed that the prevalence of positive SARS-CoV-2 tests among symptomatic HCWs was comparable to that of symptomatic nonfrontline staff. Reliable and rapid access to testing for employees is essential to preserve the health, safety, and availability of the healthcare workforce during this pandemic and to facilitate the rapid return of SARS-CoV-2-negative employees to work., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture ("16S Capture"). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA., (Copyright © 2020 American Society for Microbiology.)

Whereas 16S rRNA gene amplicon sequencing quantifies relative abundances of bacterial taxa, variation in total bacterial load between samples restricts its ability to reflect absolute concentrations of individual bacterial species. Quantitative PCR (qPCR) can quantify individual species, but it is not practical to develop a suite of qPCR assays for every bacterium present in a diverse sample. We sought to determine the accuracy of an inferred measure of bacterial concentration using total bacterial load and relative abundance. We analyzed 1,320 samples from 20 women with a history of frequent bacterial vaginosis who self-collected vaginal swabs daily over 60 days. We inferred bacterial concentrations by taking the product of species relative abundance (assessed by 16S rRNA gene amplicon sequencing) and bacterial load (measured by broad-range 16S rRNA gene qPCR). Log 10 -converted inferred concentrations correlated with targeted qPCR ( r  = 0. 935, P < 2.2e-16) for seven key bacterial species. The mean inferred concentration error varied across bacteria, with rarer bacteria associated with larger errors. A total of 92% of the >0.5-log 10 errors occurred when the relative abundance was <10%. Many errors occurred during early bacterial expansion from or late contraction to low abundance. When the relative abundance of a species is >10%, inferred concentrations are reliable proxies for targeted qPCR in the vaginal microbiome. However, targeted qPCR is required to capture bacteria at low relative abundance and is preferable for characterizing growth and decay kinetics of single species. IMPORTANCE Microbiome studies primarily use 16S rRNA gene amplicon sequencing to assess the relative abundance of bacterial taxa in a community. However, these measurements do not accurately reflect absolute taxon concentrations. We sought to determine whether the product of species' relative abundance and total bacterial load measured by broad-range qPCR is an accurate proxy for individual species' concentrations, as measured by taxon-specific qPCR assays. Overall, the inferred bacterial concentrations were a reasonable proxy of species-specific qPCR values, particularly when bacteria are present at a higher relative abundance. This approach offers an opportunity to assess the concentrations of bacterial species and how they change in a community over time without developing individual qPCR assays for each taxon., (Copyright © 2020 Tettamanti Boshier et al.)

Optimal clinical decision-making depends on identification of clinically relevant organisms present in a sample. Standard microbiological culture may fail to identify unusual or fastidious organisms and can misrepresent relative abundance of sample constituents. Culture-independent methods have improved our ability to deconvolute polymicrobial patient samples. We used next-generation 16S rRNA gene sequencing (NGS16S) to determine how often cultivatable organisms in complex polymicrobial samples are not reported by standard culture. Twenty consecutive bronchoalveolar lavage (BAL) samples were plated to standard and additional media; bacteria were identified by NGS16S analysis of DNA extracted directly from samples or from washed culture plates. 96% of organisms identified were cultivable, but only 21% were reported by standard culture, indicating that standard work-up provides an incomplete assessment of microbial constituents. Direct NGS16S correlated well with standard culture, identifying the same predominant organism in 50% of samples. When predominant organisms differed, NGS16S most often detected anaerobes, whose growth is unsupported by standard culture conditions for this specimen. NGS16S identified more organisms per sample and allowed identification of fastidious organisms, while culture was better at capturing organisms when bacterial load was low, and allowed incidental recovery of non-bacterial pathogens. Molecular and culture-based methods together detect more organisms than either method alone.

Enterobacteriaceae represent a diverse and medically important family of bacteria that are difficult to identify to the species level using the standard molecular method of 16S rRNA gene sequencing. Prior work has demonstrated the value of dnaJ gene sequence analysis in resolving different members of the family. However, existing protocols are not optimized for clinical use and exhibit several limitations in practice. Here, we describe an improved assay for dnaJ -based identification of Enterobacteriaceae which boasts increased broad-range specificity across genera, shorter amplicon sizes that are suitable for use with formalin-fixed or direct patient specimens, and enhanced amplification efficiency and assay sensitivity through the incorporation of locked nucleic acid chemistries. Sequence analysis of public databases indicates that the partial dnaJ sequence interrogated by this design retains high discriminatory power among Enterobacteriaceae genera and species, with only particular lineages of Shigella sp. and Escherichia coli proving unresolvable. Limits of detection studies using 8 disparate species indicated that amplification was consistently achievable across organisms and allowed robust dideoxynucleotide chain terminator sequencing from as little as 10 genome equivalents of template, depending on the species interrogated. Retrospective application of the dnaJ assay to patient specimens enabled unambiguous classification of Enterobacteriaceae to the species level in 22 of 27 (81.5%) positive specimens examined, with most remaining cases representing unresolvable calls between closely related Escherichia coli and Shigella species. We expect that this assay will facilitate the accurate molecular identification of species from the Enterobacteriaceae family in a variety of clinical specimens and diagnostic contexts., (Copyright © 2019 American Society for Microbiology.)

Background: Hormonal changes influence the composition of vaginal flora, which is directly related to the health of an individual. Transgender men prescribed testosterone experience a vaginal hormone composition that differs from cisgender women. To the author's knowledge, there are no clinical studies evaluating the influence that testosterone administration has on the vaginal microbiome., Methods: Vaginal swabs were self-collected by a cohort of self-identified healthy transgender men prescribed testosterone for at least 1 year (n = 28) and from cisgender women who were used as the comparator (n = 8). Participants completed a questionnaire to indicate the mode and dose of testosterone administration, sexual history, and vaginal health. Serum was collected for hormone analysis. Bacterial community profiles were assessed with broad-range PCR primers targeting the V3-V4 hypervariable region of the 16S bacterial rRNA, next-generation sequencing, and analysis by phylogenetic placement., Results: Compared to cisgender women, the vaginal floras of transgender men were less likely to have Lactobacillus as their primary genus. Intravaginal estrogen administration was positively associated with the presence of Lactobacillus in transgender men ( P = 0.045). Transgender men had a significantly increased relative abundance of >30 species and a significantly higher α diversity ( P = 0.0003). The presence of Lactobacillus was significantly associated with a lower α diversity index ( P = 0.017)., Conclusions: The vaginal microbiome of transgender men who were assigned a female sex at birth and use testosterone may differ from that of cisgender women. Intravaginal estrogen administration may reduce these differences by promoting colonization with Lactobacillus species and decreasing α diversity., (© 2018 American Association for Clinical Chemistry.)

Objective: The aim of the study was to identify associations between improvement in genitourinary symptoms of menopause (GSM) and vaginal microbiota, vaginal glycogen, and serum estrogen., Methods: Thirty postmenopausal women enrolled in a hot flash treatment trial (oral estradiol vs venlafaxine vs placebo) who reported GSM and provided vaginal swabs at 0, 4, and 8 weeks were studied. Bacterial communities were characterized using deep sequencing targeting the 16S rRNA gene V3-V4 region. Participants selected a most bothersome genitourinary symptom (dryness, discharge, pain, itch/burn, or inability to have sex) and rated severity on a 10-point scale at baseline and 8 weeks. Vaginal glycogen and serum estradiol and estrone were measured at enrollment and 8 weeks. Comparisons according to improvement in most bothersome symptom (MBS) were made using χ, Wilcoxon signed-rank test, or Hotelling's t test., Results: Of 30 participants, 21 (70%) had improvement in MBS over the 8-week study and 9 (30%) had no improvement or worsening of MBS. A higher proportion of women receiving estradiol or venlafaxine reported improvement in MBS (88%, 78%) compared with placebo (54%; P = 0.28). MBS improvement was associated with Lactobacillus-dominant vaginal microbiota at enrollment (57% vs 22%, P = 0.08). Vaginal glycogen, serum estradiol, and estrone significantly increased in women whose MBS improved., Conclusions: A larger proportion of women whose MBS improved had a Lactobacillus dominant microbiota at enrollment than those who had no improvement during the trial, though this difference was not statistically significant. Larger trials are needed to determine whether vaginal microbiota modify or mediate treatment responses in women with GSM.

Objective: To examine associations between the composition of the vaginal microbiota and genitourinary menopausal symptoms, serum estrogen, and vaginal glycogen., Methods: For this cross-sectional study, 88 women aged 40 to 62 years, enrolled in a hot flash treatment trial, provided vaginal swabs and a blood sample at enrollment. Bacterial communities were characterized using 16S rRNA PCR and deep sequencing targeting the V3-V4 region. Quantities of Lactobacillus crispatus and Lactobacillus iners were measured using qPCR. Self-reported genitourinary symptoms included: presence and severity of individual symptoms and identification of most bothersome symptom. Glycogen was measured fluorometrically in swab eluate. Serum estradiol (E2) and estrone (E1) were measured by liquid chromatography/mass spectrometry. Associations between bacteria, symptoms, glycogen, and serum estrogens were tested by linear regression or Wilcoxon signed-rank test, adjusted for multiple comparisons. Comparisons between groups used Kruskall-Wallis or Fisher's exact test., Results: Of the 88 women, 33 (38%) had a majority of Lactobacillus species, whereas 58 (66%) had any Lactobacillus detected. Over half (53%) reported at least one vulvovaginal symptom (most commonly dryness), but symptoms were not associated with the presence of Lactobacillus species. Women with Lactobacillus-dominant communities had higher unconjugated serum estrone, but no difference in vaginal glycogen levels, compared with those with non-Lactobacillus-dominant communities. Higher serum E2 and E1 were not associated with either higher vaginal glycogen or detection of individual genera., Conclusions: Presence of Lactobacillus-dominant vaginal microbiota was not associated with fewer vulvovaginal symptoms. Serum estrone was higher in women with Lactobacillus dominance, but vaginal-free glycogen was not associated with composition of the vaginal microbiota.

Background: Microbiome studies commonly use 16S rRNA gene amplicon sequencing to characterize microbial communities. Errors introduced at multiple steps in this process can affect the interpretation of the data. Here we evaluate the accuracy of operational taxonomic unit (OTU) generation, taxonomic classification, alpha- and beta-diversity measures for different settings in QIIME, MOTHUR and a pplacer-based classification pipeline, using a novel software package: DECARD., Results: In-silico we generated 100 synthetic bacterial communities approximating human stool microbiomes to be used as a gold-standard for evaluating the colligative performance of microbiome analysis software. Our synthetic data closely matched the composition and complexity of actual healthy human stool microbiomes. Genus-level taxonomic classification was correctly done for only 50.4-74.8% of the source organisms. Miscall rates varied from 11.9 to 23.5%. Species-level classification was less successful, (6.9-18.9% correct); miscall rates were comparable to those of genus-level targets (12.5-26.2%). The degree of miscall varied by clade of organism, pipeline and specific settings used. OTU generation accuracy varied by strategy (closed, de novo or subsampling), reference database, algorithm and software implementation. Shannon diversity estimation accuracy correlated generally with OTU-generation accuracy. Beta-diversity estimates with Double Principle Coordinate Analysis (DPCoA) were more robust against errors introduced in processing than Weighted UniFrac. The settings suggested in the tutorials were among the worst performing in all outcomes tested., Conclusions: Even when using the same classification pipeline, the specific OTU-generation strategy, reference database and downstream analysis methods selection can have a dramatic effect on the accuracy of taxonomic classification, and alpha- and beta-diversity estimation. Even minor changes in settings adversely affected the accuracy of the results, bringing them far from the best-observed result. Thus, specific details of how a pipeline is used (including OTU generation strategy, reference sets, clustering algorithm and specific software implementation) should be specified in the methods section of all microbiome studies. Researchers should evaluate their chosen pipeline and settings to confirm it can adequately answer the research question rather than assuming the tutorial or standard-operating-procedure settings will be adequate or optimal.

Objective: To reduce wasteful ordering of rare 1,25-OH vitamin D lab tests through use of a noninterruptive decision support tool., Materials and Methods: We conducted a time series quality improvement study at 2 academic hospitals. The titles of vitamin D tests and the order in which they appeared in search results were changed to reflect the purpose and rarity of the tests. We used interruptive time series analyses to evaluate the changes we made., Results: The estimated number of monthly tests ordered at the 2 hospitals increased, by 24.8 and 14.2, following the introduction of computerized provider order entry (CPOE) (both P < .001). When we changed the titles of the tests, the estimated number of monthly tests decreased at the 2 hospitals, by 22.1 and 11.3 (both P < .001). The search order did not affect test utilization., Discussion: Changing catalog names in CPOE systems for infrequently used tests can reduce unintentional overuse. Users may prefer this to interruptive or restrictive interventions., Conclusion: CPOE vendors and users should refine interfaces by incorporating human factors engineering. Health care institutions should monitor test utilization for unintended changes after CPOE implementation., (© The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Humans suffer from infections caused by single species or more complex polymicrobial communities. Identification of infectious bacteria commonly employs microbiological culture, which depends upon the in vitro propagation and isolation of viable organisms. In contrast, detection of bacterial DNA using next generation sequencing (NGS) allows culture-independent microbial profiling, potentially providing important new insights into the microbiota in clinical specimens., Methods: NGS 16S rRNA gene sequencing (NGS16S) was compared with culture using (a) synthetic polymicrobial samples for which the identity and abundance of organisms present were precisely defined and (b) primary clinical specimens., Results: Complex mixtures of at least 20 organisms were well resolved by NGS16S with excellent reproducibility. In mixed bacterial suspensions (10 7 total genomes), we observed linear detection of a target organism over a 4-log concentration range (500-3 × 10 6 genomes). NGS16S analysis more accurately recapitulated the known composition of synthetic samples than standard microbiological culture using nonselective media, which distorted the relative abundance of organisms and frequently failed to identify low-abundance pathogens. However, extended quantitative culture using selective media for each of the component species recovered the expected organisms at the proper abundance, validating NGS16S results. In an analysis of sputa from cystic fibrosis patients, NGS16S identified more clinically relevant pathogens than standard culture., Conclusions: Biases in standard, nonselective microbiological culture lead to a distorted characterization of polymicrobial mixtures. NGS16S demonstrates enhanced reproducibility, quantification, and classification accuracy compared with standard culture, providing a more comprehensive, accurate, and culture-free analysis of clinical specimens., (© 2016 American Association for Clinical Chemistry.)

Background: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture., Methods: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing., Results: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV., Conclusions: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Objectives: To apply techniques for ancestry and sex computation from next-generation sequencing (NGS) data as an approach to confirm sample identity and detect sample processing errors., Methods: We combined a principal component analysis method with k-nearest neighbors classification to compute the ancestry of patients undergoing NGS testing. By combining this calculation with X chromosome copy number data, we determined the sex and ancestry of patients for comparison with self-report. We also modeled the sensitivity of this technique in detecting sample processing errors., Results: We applied this technique to 859 patient samples with reliable self-report data. Our k-nearest neighbors ancestry screen had an accuracy of 98.7% for patients reporting a single ancestry. Visual inspection of principal component plots was consistent with self-report in 99.6% of single-ancestry and mixed-ancestry patients. Our model demonstrates that approximately two-thirds of potential sample swaps could be detected in our patient population using this technique., Conclusions: Patient ancestry can be estimated from NGS data incidentally sequenced in targeted panels, enabling an inexpensive quality control method when coupled with patient self-report., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Bacterial vaginosis (BV) is characterized by shifts in the vaginal microbiota from Lactobacillus dominant to a microbiota with diverse anaerobic bacteria. Few studies have linked specific metabolites with bacteria found in the human vagina. Here, we report dramatic differences in metabolite compositions and concentrations associated with BV using a global metabolomics approach. We further validated important metabolites using samples from a second cohort of women and a different platform to measure metabolites. In the primary study, we compared metabolite profiles in cervicovaginal lavage fluid from 40 women with BV and 20 women without BV. Vaginal bacterial representation was determined using broad-range PCR with pyrosequencing and concentrations of bacteria by quantitative PCR. We detected 279 named biochemicals; levels of 62% of metabolites were significantly different in women with BV. Unsupervised clustering of metabolites separated women with and without BV. Women with BV have metabolite profiles marked by lower concentrations of amino acids and dipeptides, concomitant with higher levels of amino acid catabolites and polyamines. Higher levels of the signaling eicosanoid 12-hydroxyeicosatetraenoic acid (12-HETE), a biomarker for inflammation, were noted in BV. Lactobacillus crispatus and Lactobacillus jensenii exhibited similar metabolite correlation patterns, which were distinct from correlation patterns exhibited by BV-associated bacteria. Several metabolites were significantly associated with clinical signs and symptoms (Amsel criteria) used to diagnose BV, and no metabolite was associated with all four clinical criteria. BV has strong metabolic signatures across multiple metabolic pathways, and these signatures are associated with the presence and concentrations of particular bacteria., Importance: Bacterial vaginosis (BV) is a common but highly enigmatic condition that is associated with adverse outcomes for women and their neonates. Small molecule metabolites in the vagina may influence host physiology, affect microbial community composition, and impact risk of adverse health outcomes, but few studies have comprehensively studied the metabolomics profile of BV. Here, we used mass spectrometry to link specific metabolites with particular bacteria detected in the human vagina by PCR. BV was associated with strong metabolic signatures across multiple pathways affecting amino acid, carbohydrate, and lipid metabolism, highlighting the profound metabolic changes in BV. These signatures were associated with the presence and concentrations of particular vaginal bacteria, including some bacteria yet to be cultivated, thereby providing clues as to the microbial origin of many metabolites. Insights from this study provide opportunities for developing new diagnostic markers of BV and novel approaches for treatment or prevention of BV., (Copyright © 2015 Srinivasan et al.)

Driving while impaired (DWI) is a frequently committed crime with enormous individual and social costs. The type of disposition and/or treatment appropriate for an individual offender is often determined, in part, by diagnostic criteria based on the American Psychiatric Association's Diagnostic and Statistics Manual. The DSM-5 significantly modified these criteria by eliminating legal problems as a criterion and dropping the categories of abuse and dependence. A brief substance abuse focused interview was conducted with 658 consecutive first-time DUI offenders who were arrested for driving under the influence of alcohol. Most were white, well-educated males. Contingency analyses were utilized to compare the current with the new diagnostic criteria based on algorithms for both diagnostic formulations. The major change observed when moving from DSM-IV-TR to DSM-5 criteria was that, approximately 54% of first-time DUI/DWI offenders would no longer meet diagnostic criteria based on the DSM-5. Of the nearly 17% who met dependence criteria, the majority were in the severe designation of the DSM-5.

High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common "benchtop" sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Some bacterial infections involve potentially complex mixtures of species that can now be distinguished using next-generation DNA sequencing. We present a case of mastoiditis where Gram stain, culture, and molecular diagnosis were nondiagnostic or discrepant. Next-generation sequencing implicated coinfection of Fusobacterium nucleatum and Actinomyces israelii, resolving these diagnostic discrepancies.

Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms.

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the "Bacteroides morphotype," whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.

Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.

Purpose: Current Brain Trauma Foundation guidelines recommend avoiding hypoxemia after severe pediatric traumatic brain injury (TBI). Yet, recent studies on optimum admission oxygenation and ventilation parameters associated with discharge survival in pediatric TBI are lacking., Materials and Methods: After IRB approval, a retrospective study involving pediatric patients ages ≤14 years with severe TBI (head Abbreviated Injury Scale (AIS) score of ≥3, Glasgow Coma Scale score of ≤8 on admission) admitted to Harborview Medical Center (level 1 pediatric trauma center), Seattle, WA, during 2003 to 2007 was performed. Admission demographics, clinical data, and laboratory characteristics were abstracted. Hypoxemia was defined as PaO2 < 60 mmHg, hypocarbia was defined as PaCO2 ≤ 35 mmHg, and hypercarbia was defined as PaCO2 ≥ 46 mmHg., Results: One hundred ninety-four patients met inclusion criteria of which 162 (83.5 %) patients survived. Admission hypoxemia occurred in nine (5.6 %) patients who survived and eight (25 %) patients who died (p < 0.001). Children with admission PaCO2 between 36 and 45 mmHg had greater discharge survival compared with those with both admission hypocarbia (PaCO2 ≤ 35 mmHg) and hypercarbia (PaCO2 ≥ 46 mmHg). Admission PaO2 301-500 mmHg (adjusted odds ratio (AOR), 8.02 (95 % confidence interval (CI), 1.73-37.10); p = 0.008) and admission PaCO2 = 36-45 mmHg (AOR, 5.47 (95 % CI, 1.30-23.07); p = 0.02) were independently associated with discharge survival., Conclusions: Discharge survival after severe pediatric TBI was associated with admission PaO2 301-500 mmHg and PaCO2 = 36-45 mmHg. Admission hypocarbia and hypercarbia were each associated with increased discharge mortality.

Unlabelled: The execution of a software application or pipeline using various combinations of parameters and inputs is a common task in bioinformatics. In the absence of a specialized tool to organize, streamline and formalize this process, scientists must write frequently complex scripts to perform these tasks. We present nestly, a Python package to facilitate running tools with nested combinations of parameters and inputs. nestly provides three components. First, a module to build nested directory structures corresponding to choices of parameters. Second, the nestrun script to run a given command using each set of parameter choices. Third, the nestagg script to aggregate results of the individual runs into a CSV file, as well as support for more complex aggregation. We also include a module for easily specifying nested dependencies for the SCons build tool, enabling incremental builds., Availability: Source, documentation and tutorial examples are available at http://github.com/fhcrc/nestly. nestly can be installed from the Python Package Index via pip; it is open source (MIT license).

Background: Mass spectrometry provides a powerful platform for performing quantitative, multiplexed assays in the clinical laboratory, but at the cost of increased complexity of analysis and quality assurance calculations compared to other methodologies., Methods: Here we describe the design and implementation of a software application that performs quality control calculations for a complex, multiplexed, mass spectrometric analysis of opioids and opioid metabolites., Results: The development and implementation of this application improved our data analysis and quality assurance processes in several ways. First, use of the software significantly improved the procedural consistency for performing quality control calculations. Second, it reduced the amount of time technologists spent preparing and reviewing the data, saving on average over four hours per run, and in some cases improving turnaround time by a day. Third, it provides a mechanism for coupling procedural and software changes with the results of each analysis. We describe several key details of the implementation including the use of version control software and automated unit tests., Conclusions: These generally useful software engineering principles should be considered for any software development project in the clinical lab., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have developed a unified format for phylogenetic placements, that is, mappings of environmental sequence data (e.g., short reads) into a phylogenetic tree. We are motivated to do so by the growing number of tools for computing and post-processing phylogenetic placements, and the lack of an established standard for storing them. The format is lightweight, versatile, extensible, and is based on the JSON format, which can be parsed by most modern programming languages. Our format is already implemented in several tools for computing and post-processing parsimony- and likelihood-based phylogenetic placements and has worked well in practice. We believe that establishing a standard format for analyzing read placements at this early stage will lead to a more efficient development of powerful and portable post-analysis tools for the growing applications of phylogenetic placement.

Background: Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV., Methodology/principal Findings: Broad-range 16S rRNA gene PCR and pyrosequencing were performed on vaginal swabs from 220 women with and without BV. BV was assessed by Amsel's clinical criteria and confirmed by Gram stain. Taxonomic classification was performed using phylogenetic placement tools that assigned 99% of query sequence reads to the species level. Women with BV had heterogeneous vaginal bacterial communities that were usually not dominated by a single taxon. In the absence of BV, vaginal bacterial communities were dominated by either Lactobacillus crispatus or Lactobacillus iners. Leptotrichia amnionii and Eggerthella sp. were the only two BV-associated bacteria (BVABs) significantly associated with each of the four Amsel's criteria. Co-occurrence analysis revealed the presence of several sub-groups of BVABs suggesting metabolic co-dependencies. Greater abundance of several BVABs was observed in Black women without BV., Conclusions/significance: The human vaginal bacterial biota is heterogeneous and marked by greater species richness and diversity in women with BV; no species is universally present. Different bacterial species have different associations with the four clinical criteria, which may account for discrepancies often observed between Amsel and Nugent (Gram stain) diagnostic criteria. Several BVABs exhibited race-dependent prevalence when analyzed in separate groups by BV status which may contribute to increased incidence of BV in Black women. Tools developed in this project can be used to study microbial ecology in diverse settings at high resolution.

The diagnosis and classification of many cancers depends in part on the identification of large-scale genomic aberrations such as chromosomal deletions, duplications, and balanced translocations. Array-based comparative genomic hybridization (array CGH) can detect chromosomal imbalances on a genome-wide scale but cannot reliably identify balanced chromosomal rearrangements. We describe a simple modification of array CGH that enables simultaneous identification of recurrent balanced rearrangements and genomic imbalances on the same microarray. Using custom tiling oligonucleotide arrays and gene-specific linear amplification primers, translocation CGH (tCGH) maps balanced rearrangements to ∼100-base resolution and facilitates the rapid cloning and sequencing of novel rearrangement breakpoints. As proof of principle, we used tCGH to characterize nine of the most common gene fusions in mature B-cell neoplasms and myeloid leukemias. Because tCGH can be performed in any CGH-capable laboratory and can screen for multiple recurrent translocations and genome-wide imbalances, it should be of broad utility in the diagnosis and classification of various types of lymphomas, leukemias, and solid tumors., (Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Despite their widespread use, the performance of delta check rules is rarely evaluated because errors are rare and lack a gold standard for detection. In this study we used a simulation-based approach to compare strategies for empirically defining criteria for univariate delta checks, and assessed the performance of these rules for detecting mislabeled specimens in 2 inpatient populations., Methods: We performed simulations using historical laboratory test results by randomly sampling pairs of specimens successively drawn from the same patient or two different patients. We evaluated the performance of delta check rules using a variety of thresholds, including those currently in use in our laboratory., Result: Mean corpuscular volume had the highest positive predictive value for specimen mislabeling, and produced the fewest false positives. Conversely, rules using other laboratory tests had considerably poorer performance. Several of the "best guess" thresholds historically used in our laboratory, notably those for potassium and anion gap, were predicted to have extremely low yields. In addition, rule performance was not consistent between the two patient populations., Conclusions: The low yield of delta checks based on any single analyte should prompt careful evaluation of their practical utility. Furthermore, our results indicate that it may not be possible to generalize delta rules across institutions., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Hyperglycemia after traumatic brain injury (TBI) is associated with poor outcome, but previous studies have not addressed intraoperative hyperglycemia in adult TBI. In this study, we examined glucose value variability and risk factors for hyperglycemia during craniotomy in adults with TBI., Methods: A retrospective cohort study of patients ≥18 years who underwent urgent or emergent craniotomy for TBI at Harborview Medical Center (level 1 adult and pediatric trauma center) between October 2007 and May 2010 was performed. Preoperative (within 24 hours of anesthesia start) and intraoperative (during anesthesia) glucose values for each patient were retrieved. The prevalence of intraoperative hyperglycemia (glucose ≥200 mg/dL), hypoglycemia (glucose <60 mg/dL), and glycemic trends was determined. Generalized Estimating Equations was used to determine the independent predictors of intraoperative hyperglycemia. Data are presented as adjusted odds ratio (AOR) (95% confidence interval [CI]), and P < 0.05 reflects significance., Results: Intraoperative hyperglycemia was common (26 [15%]) and intraoperative hypoglycemia was not observed. Independent risk factors of intraoperative hyperglycemia were age ≥65 years (AOR 3.9 [95% CI: 1.4-10.3]; P = 0.007), Glasgow Coma Scale score <9 (AOR 4.9 [95% CI: 1.6-15.1]; P = 0.006), preoperative hyperglycemia (AOR 4.4 [95% CI: 1.7-11.6]; P = 0.003), and subdural hematoma (AOR 5.6 [95% CI: 1.4-22.2]; P = 0.02). Mean intraoperative glucose was highest in severe TBI patients (P = 0.02). There was both between-patient (79.5% variance; P < 0.001) and within-patient (20.5% variance; P < 0.001) intraoperative glucose value variability. Patients with intraoperative hyperglycemia had higher in-hospital mortality (8 [31%] vs 20 [13%]; P < 0.02)., Conclusion: Intraoperative hyperglycemia was common in adults undergoing urgent/emergent craniotomy for TBI and was predicted by severe TBI, the presence of subdural hematoma, preoperative hyperglycemia, and age ≥65 years. However, there was significant variability in intraoperative glucose values.

BK virus (BKV) is the infectious cause of polyomavirus-associated nephropathy. Screening guidelines for renal-transplant recipients define levels of viremia and viruria that are actionable for additional testing or intervention. However, standardized real-time PCR primers, probes, and standards are unavailable, and the extent of agreement among published assays is unknown. We compared seven TaqMan real-time PCR primer/probe sets (three designed at this institution, three described in the literature, and one purchased) in conjunction with two different standards to prospectively measure BKV titers in 251 urine specimens submitted to our clinical laboratory. We observed substantial disagreement among assays attributable both to features of primer and probe design and to choice of reference material. The most significant source of error among individual specimens was primer or probe mismatch due to subtype-associated polymorphisms, primarily among subtype III and IV isolates. In contrast, measurement of the most abundant subtypes (Ia, V, and VI) were typically uniform among all seven assays. Finally, we describe and validate a new clinical assay designed to reliably measure all subtypes encountered in our study population (Ia, Ic, III, IV, and VI). Consideration of available BKV sequence information in conjunction with details of subtype distribution allowed us to develop a redesigned assay with markedly improved performance. These results suggest that both accurate BKV measurement and the uniform application of BKV screening guidelines could be significantly improved by the use of standardized reference materials and PCR primers and probes.

There is growing evidence that interactions between biological molecules (e.g., RNA-RNA, protein-protein, RNA-protein) place limits on the rate and trajectory of molecular evolution. Here, by extending Kimura's model of compensatory evolution at interacting sites, we show that the ratio of transition to transversion substitutions (kappa) at interacting sites should be equal to the square of the ratio at independent sites. Because transition mutations generally occur at a higher rate than transversions, the model predicts that kappa should be higher at interacting sites than at independent sites. We tested this prediction in 10 RNA secondary structures by comparing phylogenetically derived estimates of kappa in paired sites within stems (kappa(p)) and unpaired sites within loops (kappa(u)). Eight of the 10 structures showed an excellent match to the quantitative predictions of the model, and 9 of the 10 structures matched the qualitative prediction kappa(p) > kappa(u). Only the Rev response element from the human immunovirus (HIV) genome showed the reverse pattern, with kappa(p) < kappa(u). Although a variety of evolutionary forces could produce quantitative deviations from the model predictions, the reversal in magnitude of kappa(p) and kappa(u) could be achieved only by violating the model assumption that the underlying transition (or transversion) mutation rates were identical in paired and unpaired regions of the molecule. We explore the ability of the APOBEC3 enzymes, host defense mechanisms against retroviruses, which induce transition mutations preferentially in single-stranded regions of the HIV genome, to explain this exception to the rule. Taken as a whole, our findings suggest that kappa may have utility as a simple diagnostic to evaluate proposed secondary structures.

The V3 region of the human immunodeficiency virus type 1 gp120 Env protein is a key domain in Env due to its role in interacting with the coreceptors CCR5 and CXCR4. We examined potential subtype-specific V3 region differences by comparing patterns of amino acid variability and probing for subtype-specific structures using 11 anti-V3 monoclonal antibodies (V3 MAbs). Differences between the subtypes in patterns of variability were most evident in the stem and turn regions of V3 (positions 9 to 24), with the two subtypes being very similar in the base region. The characteristics of the binding of V3 MAbs to Env proteins of the subtype B virus JR-FL and the subtype C virus BR025 suggested three patterns, as each group of MAbs recognized a specific conformation- or sequence-based epitope. Viruses pseudotyped with Env from JR-FL and BR025 were resistant to neutralization by the V3 MAbs, although the replacement of the Env V3 region of the SF162 virus with the JR-FL V3 created a pseudotyped virus that was hypersensitive to neutralization. A single mutation in V3 (H13R) made this chimeric Env selectively resistant to one group of V3 MAbs, consistent with the mAb binding properties. We hypothesize that there are intrinsic differences in V3 conformation between subtype B and subtype C that are localized to the stem and turn regions and that these differences have two important biological consequences: first, subtype B and subtype C V3 regions can have subtype-specific epitopes that will inherently limit antibody cross-reactivity, and second, V3 conformational differences may potentiate the frequent evolution of R5- into X4-tropic variants of subtype B but limit subtype C virus from using the same mechanism to evolve X4-tropic variants as efficiently.

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) through breast milk is a significant mechanism of infection in many regions of the world. We compared the HIV-1 populations in paired blood and breast milk samples using a heteroduplex tracking assay (HTA) for the V1/V2 regions of env (V1/V2-HTA). V1/V2-HTA patterns were similar in the eight pairs of samples for which adequate template sampling could be demonstrated. No unique variants existed in either compartment, and differences detected in the relative abundance of variants between compartments were small, occurred among low abundance variants, and were not statistically significant. We also documented the impact of template sampling as a limiting feature in comparing two viral populations. The absence of unique variants and the lack of significant differences in the relative abundance of variants between these compartments support the conclusion that viruses in the blood plasma and breast milk are well equilibrated.

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.

Background: The HIV-1 envelope third variable loop (V3 loop) is an important determinant of viral phenotype and co-receptor usage. We wished to determine the impact of specific V3 genotypes associated with viral phenotype and co-receptor usage on response to initial triple antiretroviral therapy., Methods: Pre-therapy plasma samples from the HOMER cohort of 1191 antiretroviral-naive, HIV-infected adults who initiated triple therapy in British Columbia, Canada between August 1996 and September 1999 were genotyped for V3 loop sequence. V3 sequences were dichotomized by the presence or absence of positively charged residues at codons 11 and/or 25 (an '11/25' genotype). Neural network (NN) and Position Specific Scoring Matrix (PSSM) approaches were used as alternative V3 sequence interpretation methods. The association of V3 genotypes with clinical endpoints was assessed over a median of 43 months of follow up., Results: One-hundred and eighteen (10.9%) of the 1085 isolates successfully genotyped for V3 displayed the 11/25 genotype. In multivariate analyses, this genotype was associated with a more rapid CD4 decline [risk ratio, (RR), 1.38; P = 0.012] and earlier mortality (RR, 1.70; P = 0.027), despite comparable viral load suppression below 500 HIV RNA copies/ml. We observed no influence of the 11/25 genotype on time to viral rebound or the development of drug resistance. PSSM-based sequence categories were similarly predictive of outcomes. NN sequence categories were not associated with any endpoints., Conclusion: The 11/25 genotype of the HIV V3 loop is an independent predictor of poor immunological response and more rapid mortality even after starting triple antiretroviral therapy. These results may prove to be useful for the clinical management of HIV-infected individuals.