RESUMEN
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.
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Microbioma Gastrointestinal , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Ratones , Animales , Vancomicina , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Trasplante Homólogo/efectos adversosRESUMEN
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.
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Homosexualidad Masculina , Microbiota , Conducta Sexual , Uretra , Uretritis , Humanos , Masculino , Adulto , Uretra/microbiología , Uretritis/microbiología , ARN Ribosómico 16S/genética , Adulto Joven , Estudios Longitudinales , Persona de Mediana Edad , Minorías Sexuales y de GéneroRESUMEN
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.
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Biología Computacional , Programas Informáticos , Humanos , Biología Computacional/métodos , Secuenciación Completa del Genoma , Genoma , Polimorfismo de Nucleótido Simple , Bacterias , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
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.
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Microbiota , Vagina , Vaginosis Bacteriana , Bacterias/clasificación , Femenino , Humanos , Posmenopausia , Premenopausia , ARN Ribosómico 16S/genética , Vagina/microbiología , Vaginosis Bacteriana/diagnósticoRESUMEN
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; Pâ =â .02) while alpha diversity remained stable (0.03; -.24 to .30; Pâ =â .83). Conversely, Nugent score remained relatively stable among non-HC users (change, -0.73; 95% CI, -2.18 to .73; Pâ =â .33) while alpha diversity decreased (-0.34; -.67 to -.001; Pâ =â .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; Pâ =â .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.
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Anticonceptivos Femeninos , Microbiota , Femenino , Humanos , Kenia , Acetato de Medroxiprogesterona , Periodo Posparto , VaginaRESUMEN
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.
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Microbiota , Infecciones por Mycoplasma , Mycoplasma penetrans , Minorías Sexuales y de Género , Uretritis , Chlamydia trachomatis , Femenino , Haemophilus influenzae , Homosexualidad Masculina , Humanos , Masculino , ARN Ribosómico 16S/genética , Conducta SexualRESUMEN
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.
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COVID-19 , Prueba de COVID-19 , Personal de Salud , Humanos , Prevalencia , SARS-CoV-2 , Washingtón/epidemiologíaRESUMEN
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.
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Metagenómica , ADN Bacteriano/genética , Genes de ARNr , Humanos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
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.
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Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/clasificación , Proteínas del Choque Térmico HSP40/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Cartilla de ADN/genética , ADN Bacteriano/análisis , Infecciones por Enterobacteriaceae/diagnóstico , Proteínas de Escherichia coli/genética , Genotipo , Humanos , Límite de Detección , Oligonucleótidos/genética , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
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.
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Microbiota , Personas Transgénero , Vagina/microbiología , Adolescente , Adulto , Estudios de Cohortes , Estrógenos/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Testosterona/administración & dosificación , Testosterona/sangre , Adulto JovenRESUMEN
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.
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Bacterias/aislamiento & purificación , Intestinos/microbiología , Microbiota , Algoritmos , Bacterias/genética , Humanos , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/clasificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismoRESUMEN
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.
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Bacterias Anaerobias/aislamiento & purificación , Gardnerella vaginalis/aislamiento & purificación , Microbiota , Vaginosis Bacteriana/microbiología , Bacterias Anaerobias/clasificación , Bacterias Anaerobias/citología , Bacterias Anaerobias/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Gardnerella vaginalis/clasificación , Gardnerella vaginalis/citología , Gardnerella vaginalis/genética , Humanos , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vagina/microbiologíaRESUMEN
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 (107 total genomes), we observed linear detection of a target organism over a 4-log concentration range (500-3 × 106 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.
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Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , ADN Bacteriano/genética , Técnicas Microbiológicas/normas , Análisis de Secuencia de ADN/tendencias , Humanos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/normasRESUMEN
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.
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Actinomyces/aislamiento & purificación , Coinfección/diagnóstico , Coinfección/microbiología , Fusobacterium nucleatum/aislamiento & purificación , Mastoiditis/diagnóstico , Mastoiditis/microbiología , Actinomicosis/diagnóstico , Actinomicosis/microbiología , Infecciones por Fusobacterium/diagnóstico , Infecciones por Fusobacterium/microbiología , Fusobacterium nucleatum/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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).
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Programas Informáticos , Algoritmos , Biología Computacional , HumanosRESUMEN
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.
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Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , ADN Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Ribosómico 16S/genética , Bacterias/clasificación , Bacterias/genética , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , HumanosRESUMEN
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.
RESUMEN
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.
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Sistemas de Información en Laboratorio Clínico , Infecciones por VIH , Humanos , Laboratorios Clínicos , Laboratorios , Côte d'Ivoire , ElectrónicaRESUMEN
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.