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Human immunodeficiency virus (HIV)-infected macrophages are long-lived cells that sustain persistent virus expression, which is both a barrier to viral eradication and contributor to neurological complications in patients despite antiretroviral therapy (ART). To better understand the regulation of HIV-1 in macrophages, we compared HIV-infected primary human monocyte-derived macrophages (MDM) to acutely infected primary CD4 T cells and Jurkat cells latently infected with HIV (JLAT 8.4). HIV genomes in MDM were actively transcribed despite enrichment with heterochromatin-associated H3K9me3 across the complete HIV genome in combination with elevated activation marks of H3K9ac and H3K27ac at the long terminal repeat (LTR). Macrophage patterns contrasted with JLAT cells, which showed conventional bivalent H3K4me3/H3K27me3, and acutely infected CD4 T cells, which showed an intermediate epigenotype. 5'-Methylcytosine (5mC) was enriched across the HIV genome in latently infected JLAT cells, while 5'-hydroxymethylcytosine (5hmC) was enriched in CD4 cells and MDMs. HIV infection induced multinucleation of MDMs along with DNA damage-associated p53 phosphorylation, as well as loss of TET2 and the nuclear redistribution of 5-hydoxymethylation. Taken together, our findings suggest that HIV induces a unique macrophage nuclear and transcriptional profile, and viral genomes are maintained in a noncanonical bivalent epigenetic state. IMPORTANCE Macrophages serve as a reservoir for long-term persistence and chronic production of HIV. We found an atypical epigenetic control of HIV in macrophages marked by heterochromatic H3K9me3 despite active viral transcription. HIV infection induced changes in macrophage nuclear morphology and epigenetic regulatory factors. These findings may identify new mechanisms to control chronic HIV expression in infected macrophages.

In October of 2020, researchers from around the world met online for the sixth annual International Workshop on Microbiome in HIV Pathogenesis, Prevention, and Treatment. New research was presented on the roles of the microbiome on immune response and HIV transmission and pathogenesis and the potential for alterations in the microbiome to decrease transmission and affect comorbidities. This article presents a summary of the findings reported.

Background: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations., Methods: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients., Results: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden., Conclusions: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Sarcoidosis is a multisystem granulomatous disease of unknown origin with a variable and often unpredictable course and pattern of organ involvement. In this study we sought to identify specific bronchoalveolar lavage (BAL) cell gene expression patterns indicative of distinct disease phenotypic traits., Methods: RNA sequencing by Ion Torrent Proton was performed on BAL cells obtained from 215 well-characterised patients with pulmonary sarcoidosis enrolled in the multicentre Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Weighted gene co-expression network analysis and nonparametric statistics were used to analyse genome-wide BAL transcriptome. Validation of results was performed using a microarray expression dataset of an independent sarcoidosis cohort (Freiburg, Germany; n=50)., Results: Our supervised analysis found associations between distinct transcriptional programmes and major pulmonary phenotypic manifestations of sarcoidosis including T-helper type 1 (Th1) and Th17 pathways associated with hilar lymphadenopathy, transforming growth factor-β1 (TGFB1) and mechanistic target of rapamycin (MTOR) signalling with parenchymal involvement, and interleukin (IL)-7 and IL-2 with airway involvement. Our unsupervised analysis revealed gene modules that uncovered four potential sarcoidosis endotypes including hilar lymphadenopathy with increased acute T-cell immune response; extraocular organ involvement with PI3K activation pathways; chronic and multiorgan disease with increased immune response pathways; and multiorgan involvement, with increased IL-1 and IL-18 immune and inflammatory responses. We validated the occurrence of these endotypes using gene expression, pulmonary function tests and cell differentials from Freiburg., Conclusion: Taken together, our results identify BAL gene expression programmes that characterise major pulmonary sarcoidosis phenotypes and suggest the presence of distinct disease molecular endotypes., Competing Interests: Conflict of interest: M. Vukmirovic has nothing to disclose. Conflict of interest: X. Yan has nothing to disclose. Conflict of interest: K.F. Gibson has nothing to disclose. Conflict of interest: M. Gulati reports grants from NIH, during the conduct of the study; personal fees for advisory board work and other (PI/publication committee) from Boehringer Ingelheim, other (lectures) from France Foundation, other (PI/centre director) from Pulmonary Fibrosis Foundation, grants from NIH and Sarcoidosis Research Foundation, outside the submitted work. Conflict of interest: J.C. Schupp has nothing to disclose. Conflict of interest: G. DeIuliis has nothing to disclose. Conflict of interest: T.S. Adams has nothing to disclose. Conflict of interest: B. Hu has nothing to disclose. Conflict of interest: A. Mihaljinec has nothing to disclose. Conflict of interest: T.N. Woolard has nothing to disclose. Conflict of interest: H. Lynn has nothing to disclose. Conflict of interest: N. Emeagwali has nothing to disclose. Conflict of interest: E.L Herzog reports grants from NIH, Sanofi, Bristol Myers and Promedior, personal fees for consultancy from Boehringer Ingelheim and Pfizer, outside the submitted work. Conflict of interest: E.S. Chen has nothing to disclose. Conflict of interest: A. Morris reports grants from NIH, during the conduct of the study. Conflict of interest: J.K. Leader has nothing to disclose. Conflict of interest: Y. Zhang has nothing to disclose. Conflict of interest: J.G.N. Garcia has nothing to disclose. Conflict of interest: L.A. Maier grants from NIH (1U01 HL112695-01, U01 HL112707-03) and NIH/NCRR (UL1TRR002535), during the conduct of the study; grants from National Institutes of Health (1R01 HL127461-01A1, R01HL136681-01A1, 1R01 HL140357-01A1, R01HL136681-01A1), FSR, University of Cinncinati under a Mallinckrodt foundation, MNK14344100, ATYR1923-C-002, outside the submitted work; and is a member of the FSR scientific advisory board, for which no compensation is received. Conflict of interest: R.G. Collman reports grants from National Institutes of Health, during the conduct of the study. Conflict of interest: W.P. Drake has nothing to disclose. Conflict of interest: M.J. Becich reports grants from NCATS, NCI, PCORI, NHLBI and CDC NIOSH, other (startup) from SpIntellx, during the conduct of the study; other (startup) from SpIntellx, outside the submitted work; and has patents SpIntellx (multiple) pending. Conflict of interest: H. Hochheiser has nothing to disclose. Conflict of interest: S.R. Wisniewski has nothing to disclose. Conflict of interest: P.V. Benos has nothing to disclose. Conflict of interest: D.R. Moller reports grants from NHLBI (1U01HL112708), during the conduct of the study; personal fees for consultancy from Merck, aTYR and Roivant, personal fees for advisory board work from SarcoMed, personal fees for consultancy/witness from Legal Expert, other (royalties) from Hodder Education and Taylor & Francis Group, outside the submitted work; has patents number 9,683,999 B2 issued, and number 9,977,029 B2 issued; is Chairman and Chief Technical Officer of Sarcoidosis Diagnostic Testing, LLC (a company whose goal is to develop a diagnostic blood test for sarcoidosis) and has received funding including past salary support under the NHLBI STTR programme, grant R41 HL129728 more than 3 years ago; and is a former member of the Scientific Advisory Board of the Foundation for Sarcoidosis Research. Conflict of interest: A. Prasse reports personal fees for lectures and consultancy and non-financial support for meeting attendance from Boehringer Ingelheim and Roche, personal fees for lectures from Novartis and AstraZeneca, personal fees for consultancy from Amgen, Pliant and Nitto Denko, outside the submitted work. Conflict of interest: L.L. Koth has nothing to disclose. Conflict of interest: N. Kaminski reports personal fees for consultancy and/or advisory board work from Biogen Idec, Boehringer Ingelheim, Third Rock, Samumed, NuMedii, Indaloo, Theravance, LifeMax, Three Lake Partners, RohBar and Pliant, non-financial support from Miragen, equity with Pliant, a grant from Veracyte; all outside the submitted work; and has a patent New Therapies in Pulmonary Fibrosis and on Peripheral Blood Gene Expression that have been licensed to Biotech., (Copyright ©The authors 2021. For reproduction rights and permissions contact permissions@ersnet.org.)

Redondoviridae is a newly established family of circular Rep-encoding single-stranded (CRESS) DNA viruses found in the human ororespiratory tract. Redondoviruses were previously found in ∼15% of respiratory specimens from U.S. urban subjects; levels were elevated in individuals with periodontitis or critical illness. Here, we report higher redondovirus prevalence in saliva samples: four rural African populations showed 61 to 82% prevalence, and an urban U.S. population showed 32% prevalence. Longitudinal, limiting-dilution single-genome sequencing revealed diverse strains of both redondovirus species ( Brisavirus and Vientovirus ) in single individuals, persistence over time, and evidence of intergenomic recombination. Computational analysis of viral genomes identified a recombination hot spot associated with a conserved potential DNA stem-loop structure. To assess the possible role of this site in recombination, we carried out in vitro studies which showed that this potential stem-loop was cleaved by the virus-encoded Rep protein. In addition, in reconstructed reactions, a Rep-DNA covalent intermediate was shown to mediate DNA strand transfer at this site. Thus, redondoviruses are highly prevalent in humans, found in individuals on multiple continents, heterogeneous even within individuals and encode a Rep protein implicated in facilitating recombination. IMPORTANCE Redondoviridae is a recently established family of DNA viruses predominantly found in the human respiratory tract and associated with multiple clinical conditions. In this study, we found high redondovirus prevalence in saliva from urban North American individuals and nonindustrialized African populations in Botswana, Cameroon, Ethiopia, and Tanzania. Individuals on both continents harbored both known redondovirus species. Global prevalence of both species suggests that redondoviruses have long been associated with humans but have remained undetected until recently due to their divergent genomes. By sequencing single redondovirus genomes in longitudinally sampled humans, we found that redondoviruses persisted over time within subjects and likely evolve by recombination. The Rep protein encoded by redondoviruses catalyzes multiple reactions in vitro , consistent with a role in mediating DNA replication and recombination. In summary, we identify high redondovirus prevalence in humans across multiple continents, longitudinal heterogeneity and persistence, and potential mechanisms of redondovirus evolution by recombination.

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.

Background: Many people living with HIV have persistent monocyte activation despite viral suppression by antiretroviral therapy (ART), which contributes to non-AIDS complications including neurocognitive and other disorders. Statins have immunomodulatory properties that might be beneficial by reducing monocyte activation., Methods: We previously characterized monocyte gene expression and inflammatory markers in 11 HIV-positive individuals on long-term ART (HIV/ART) at risk for non-AIDS complications because of low nadir CD4+ counts (median 129 cells/uL) and elevated hsCRP. Here, these individuals participated in a double-blind, randomized, placebo-controlled crossover study of 12 weeks of atorvastatin treatment. Monocyte surface markers were assessed by flow cytometry, plasma mediators by ELISA and Luminex, and monocyte gene expression by microarray analysis., Results: Among primary outcome measures, 12 weeks of atorvastatin treatment led to an unexpected increase in CCR2+ monocytes ( P =0.04), but did not affect CD16+ or CD163+ monocytes, nor levels in plasma of CCL2/MCP-1 or sCD14. Among secondary outcomes, atorvastatin treatment was associated with decreased plasma hsCRP ( P =0.035) and IL-2R ( P =0.012). Treatment was also associated with increased total CD14+ monocytes ( P =0.015), and increased plasma CXCL9 ( P =0.003) and IL-12 ( P <0.001). Comparable results were seen in a subgroup that had inflammatory marker elevations at baseline. Atorvastatin treatment did not significantly alter monocyte gene expression or normalize aberrant baseline transcriptional patterns., Conclusions: In this study of aviremic HIV+ individuals at high risk of non-AIDS events, 12 weeks of atorvastatin did not normalize monocyte gene expression patterns nor lead to significant changes in monocyte surface markers or plasma mediators linked to non-AIDS comorbidities., Competing Interests: The authors declare no competing financial or other conflicts of interest., (Copyright © Pathogens and Immunity 2021.)

Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition that causes early onset pulmonary emphysema and airways obstruction. The complete mechanisms via which AATD causes lung disease are not fully understood. To improve our understanding of the pathogenesis of AATD, we investigated gene expression profiles of bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMCs) in AATD individuals., Methods: We performed RNA-Seq on RNA extracted from matched BAL and PBMC samples isolated from 89 subjects enrolled in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Subjects were stratified by genotype and augmentation therapy. Supervised and unsupervised differential gene expression analyses were performed using Weighted Gene Co-expression Network Analysis (WGCNA) to identify gene profiles associated with subjects' clinical variables. The genes in the most significant WGCNA module were used to cluster AATD individuals. Gene validation was performed by NanoString nCounter Gene Expression Assay., Result: We observed modest effects of AATD genotype and augmentation therapy on gene expression. When WGCNA was applied to BAL transcriptome, one gene module, ME31 (2312 genes), correlated with the highest number of clinical variables and was functionally enriched with numerous immune T-lymphocyte related pathways. This gene module identified two distinct clusters of AATD individuals with different disease severity and distinct PBMC gene expression patterns., Conclusions: We successfully identified novel clusters of AATD individuals where severity correlated with increased immune response independent of individuals' genotype and augmentation therapy. These findings may suggest the presence of previously unrecognised disease endotypes in AATD that associate with T-lymphocyte immunity and disease severity., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Viruses in the family Redondoviridae have a circular genome of 3.0 kb with three open reading frames. The packaged genome is inferred to be single-stranded DNA by analogy to related viruses. Redondoviruses were discovered through metagenomic sequencing methods in samples from human subjects and are inferred to replicate in humans. Evidence of redondovirus infection is associated with periodontitis and critical illness, but redondoviruses have not been shown to be the causative agent of any diseases. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Redondoviridae , which is available at ictv.global/report/redondoviridae.

People living with HIV (PLWH) in the antiretroviral therapy (ART) era may lose more life-years to tobacco use than to HIV. Yet, smoking rates are more than twice as high among PLWH than the general population, contributing not just to mortality but to other adverse health outcomes, including neurocognitive deficits (neuroHIV). There is growing evidence that synergy with chronic inflammation and immune dysregulation that persists despite ART may be one mechanism by which tobacco smoking contributes to neuroHIV. This review will summarize the differential effects of nicotine vs tobacco smoking on inflammation in addition to the effects of tobacco smoke components on HIV disease progression. We will also discuss biomarkers of inflammation via neuroimaging as well as biomarkers of nicotine dependence (e.g., nicotine metabolite ratio). Tobacco smoking and nicotine may impact ART drug metabolism and conversely, certain ARTs may impact nicotine metabolism. Thus, we will review these bidirectional relationships and how they may contribute to neuroHIV and other adverse outcomes. We will also discuss the effects of tobacco use on the interaction between peripheral organs (lungs, heart, kidney) and subsequent CNS function in the context of HIV. Lastly, given the dramatic rise in the use of electronic nicotine delivery systems, we will discuss the implications of vaping on these processes. Despite the growing recognition of the importance of addressing tobacco use among PLWH, more research is necessary at both the preclinical and clinical level to disentangle the potentially synergistic effects of tobacco use, nicotine, HIV, cognition and immune dysregulation, as well as identify optimal approaches to reduce tobacco use. Graphical Abstract Proposed model of the relationships among HIV, ART, smoking, inflammation, and neurocognition. Solid lines represent relationships supported by evidence. Dashed lines represent relationships for which there is not enough evidence to make a conclusion. (a) HIV infection produces elevated levels of inflammation even among virally suppressed individuals. (b) HIV is associated with deficits in cognition function. (c) Smoking rates are higher among PLWH, compared to the general population. (d) The nicotine metabolite ratio (NMR) is associated with smoking behavior. (e) HIV and tobacco use are both associated with higher rates of psychiatric comorbidities, such as depression, and elevated levels of chronic stress. These factors may represent other mechanisms linking HIV and tobacco use. (f) The relationship between nicotine, tobacco smoking, and inflammation is complex, but it is well-established that smoking induces inflammation; the evidence for nicotine as anti-inflammatory is supported in some studies, but not others. (g) The relationship between tobacco use and neurocognition may differ for the effects of nicotine (acute nicotine use may have beneficial effects) vs. tobacco smoking (chronic use may impair cognition). (h) Elevated levels of inflammation may be associated with deficits in cognition. (i) PLWH may metabolize nicotine faster than those without HIV; the mechanism is not yet known and the finding needs validation in larger samples. We also hypothesize that if HIV-infection increases nicotine metabolism, then we should observe an attenuation effect once ART is initiated. (j) It is possible that the increase in NMR is due to ART effects on CYP2A6. (k) We hypothesize that faster nicotine metabolism may result in higher levels of inflammation since nicotine has anti-inflammatory properties.

In October of 2019, researchers and community members from around the world met at the NIH for the fifth annual International Workshop on Microbiome in HIV. New research was presented on the role of the microbiome on chronic inflammation and vaccine design, interactions of genetics, environment, sexual practice and HIV infection with the microbiome and the development and clinical trials of microbiome-based therapeutic approaches intended to decrease the probability of HIV acquisition/transmission or ameliorate sequelae of HIV. The keynote address by Dr. Jacques Ravel focused on his work on the vaginal microbiome and efforts to improve the analysis and resolution of microbiome data.

People with HIV (PWH) experience accentuated biological aging, as defined by markers of inflammation, immune dysfunction, and the epigenetic clock. They also have an elevated risk of multiple age-associated comorbidities. To discuss current knowledge, research gaps, and priorities in aging and age-related comorbidities in treated HIV infection, the NIH program staff organized a workshop held in Bethesda, Maryland in September 2019. This review article describes highlights of discussions led by the Pathogenesis/Basic Science Research working group that focused on three high priority topics: immunopathogenesis; the microbiome/virome; and aging and senescence. We summarize knowledge in these fields and describe key questions for research on the pathogenesis of aging and age-related comorbidities in PWH. Understanding the drivers and mechanisms underlying accentuated biological aging is a high priority that will help identify potential therapeutic targets to improve healthspan in older PWH., Competing Interests: Nicholas Funderberg serves as a consultant for Gilead. Michael M. Lederman is the senior editor for Pathogens and Immunity. Steven G. Deeks, Dirk P. Dittmer, Nicholas T. Funderburg, and Peter W. Hunt serve as associate editors for Pathogens and Immunity., (© Pathogens and Immunity 2020.)

Each year, a growing international collection of researchers meets at the NIH to share and discuss developments in the microbiome HIV story. This past year has seen continued progress toward a detailed understanding of host-microbe interactions both within and outside the field of HIV. Commensal microbes are being linked to an ever-growing list of maladies and physiologic states, including major depressive disorder, chronic kidney disease, and Parkinson disease. PubMed citations for "microbiome" are growing at an exponential rate with over 11,000 in 2018. Various microbial taxa have been associated with HIV infection, and some of these taxa associated with HIV infection have also been associated with systemic markers of inflammation in HIV infected individuals. Causality remains unclear however as environmental and behavioral factors may drive HIV risk, inflammation, and gut enterotype. Much of the work currently being done addresses potential mechanisms by which gut microbes influence immune and inflammatory pathways. No portion of the microbiome landscape has grown as rapidly as study of the interplay between gut microbes and response to cancer immunotherapy. As Dr. Wargo discussed in her keynote address, this area has opened the door to better understanding on how commensal microbes interact with the human immune system.

Background: People living with HIV on antiretroviral therapy (HIV/ART) experience excess non-AIDS comorbidities, and also remain at increased risk for certain infections and viral malignancies. Monocytes/macrophages are central to many of these comorbidities, and elevated plasma cytokines and immune activation during untreated infection are often incompletely reversed by ART and are also associated with comorbidities., Methods: We investigated monocyte surface markers, gene expression, and plasma cytokines in 11 HIV-infected older individuals (median 53 years) who started therapy with low CD4 counts (median 129 cells/µl), with elevated hsCRP (≥ 2mg/L) despite long-term ART (median 7.4 years), along with matched controls., Results: Frequency of monocyte subsets (based on CD14/CD16/CD163), were not different from controls, but surface expression of CD163 was increased ( P = 0.021) while PD1 was decreased ( P = 0.013) along with a trend for higher tissue factor ( P = 0.096). As a group, HIV/ART participants had elevated plasma CCL2 (MCP-1; P = 0.0001), CXCL9 (MIG; P = 0.04), and sIL2R ( P = 0.015), which were correlated, while sCD14 was not elevated. Principal component analysis of soluble markers revealed that 6/11 HIV/ART participants clustered with controls, while 5 formed a distinct group, driven by IL-10, CCL11, CXCL10, CCL2, CXCL9, and sIL2R. These individuals were significantly older than those clustering with controls. Transcriptomic analysis revealed multiple genes linked to immune functions including inflammation, immune cell development, and cell-cell signaling that were downregulated in HIV/ART monocytes and distinct from patterns in untreated subjects., Conclusions: Long-term ART-treated individuals normalize monocyte subsets but exhibit immune dysregulation involving both aberrant inflammation and monocyte dysfunction, as well as inter-individual heterogeneity, suggesting complex mechanisms linking monocytes and HIV/ART comorbidities., Competing Interests: The authors declare no competing financial or other conflicts of interest., (© Pathogens and Immunity 2019.)

Objective: HIV-infected smokers lose more life years to tobacco use than to HIV infection. The nicotine metabolite ratio (NMR), a biomarker of CYP2A6, represents individual variation in the rate at which nicotine is metabolized and is associated with response to smoking cessation treatments. We evaluated whether HIV-infected smokers metabolize nicotine faster than HIV-uninfected smokers, which may contribute to the disproportionate smoking burden and may have important treatment implications., Design: We analysed baseline data from two clinical trials (NCT01710137; NCT01314001) to compare the NMR in HIV-infected smokers (N = 131) to HIV-uninfected smokers (N = 199)., Methods: Propensity scores were used to match the groups 2 : 1 on characteristics that influence NMR: sex, race, BMI and smoking rate. Nicotine metabolites were assessed via liquid chromatography-tandem mass spectrometry methods and the ratio of 3-hydroxycotinine:cotinine was used to compute the NMR., Results: HIV-infected smokers had significantly higher NMR (mean = 0.47, SEM = 0.02) and were more likely to be in the highest NMR quartile compared with HIV-uninfected smokers (mean = 0.34, SEM = 0.02; Ps < 0.001)., Conclusion: The higher NMR observed among HIV-infected smokers may partially explain higher smoking rates and lower response to transdermal nicotine therapy. Understanding the mechanisms by which HIV and/or ART contribute to faster nicotine metabolism may guide the use of the NMR to personalize tobacco cessation strategies in this underserved population.

Solid organ transplantation disrupts virus-host relationships, potentially resulting in viral transfer from donor to recipient, reactivation of latent viruses, and new viral infections. Viral transfer, colonization, and reactivation are typically monitored using assays for specific viruses, leaving the behavior of full viral populations (the "virome") understudied. Here we sought to investigate the temporal behavior of viruses from donor lungs and transplant recipients comprehensively. We interrogated the bronchoalveolar lavage and blood viromes during the peritransplant period and 6-16 months posttransplant in 13 donor-recipient pairs using shotgun metagenomic sequencing. Anelloviridae, ubiquitous human commensal viruses, were the most abundant human viruses identified. Herpesviruses, parvoviruses, polyomaviruses, and bacteriophages were also detected. Anelloviridae populations were complex, with some donor organs and hosts harboring multiple contemporaneous lineages. We identified transfer of Anelloviridae lineages from donor organ to recipient serum in 4 of 7 cases that could be queried, and immigration of lineages from recipient serum into the allograft in 6 of 10 such cases. Thus, metagenomic analyses revealed that viral populations move between graft and host in both directions, showing that organ transplantation involves implantation of both the allograft and commensal viral communities., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Rationale: The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers., Objectives: We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination., Methods: We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing., Measurements and Main Results: In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons., Conclusions: Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives.

Sarcoidosis is a complex, polygenic disease of unknown cause with diverse clinical phenotypes, ranging from self-limited, asymptomatic disease to life-altering symptoms and early disease-related mortality. It is unlikely that a single common environmental exposure (e.g., infection, antigen) entirely explains the disease, and numerous genetic mutations are associated with the disease. As such, it is reasonable to assume, as with other phenotypically diverse diseases, that distinct genetic mechanisms and related biological biomarkers will serve to further define sarcoidosis subphenotypes, mechanisms, and possibly etiology, thus guiding personalized care. The fields of "omics" and systems biology research are widely applied to understand polygenic and phenotypically diverse diseases, such as sarcoidosis. "Omics" refers to technologies that allow comprehensive profiling of sets of molecules in an organism. Systems biology applies advanced computational approaches to make sense of the enormous data sets that are typically generated from "omics" platforms. The primary objectives of this article are to review the available "omics" tools, assess the current status of "omics" and systems biology research in the field of sarcoidosis, and consider how this technology could be applied to advance our understanding of the mechanistic underpinnings of disease and to develop novel treatments.

HIV-1-associated neurocognitive disorder (HAND) remains a persistent problem despite antiretroviral therapy (ART), largely a result of continued inflammation in the periphery and the brain and neurotoxin release from activated myeloid cells in the CNS. CD14+CD16+ inflammatory monocytes, expanded in HIV infection, play a central role in the pathogenesis of HAND and have parallels with monocyte-dependent inflammatory mechanisms in atherosclerosis. Statins, through their HMG-CoA reductase inhibitor activity, have pleiotropic immunomodulatory properties that contribute to their benefit in atherosclerosis beyond lipid lowering. Here, we investigated whether statins would modulate the monocyte phenotype and function associated with HIV-1 neuropathogenesis. Treatment ex vivo with simvastatin and atorvastatin reduced the proportion of CD16+ monocytes in peripheral blood mononuclear cells, as well as in purified monocytes, especially CD14++CD16+ "intermediate" monocytes most closely associated with neurocognitive disease. Statin treatment also markedly reduced expression of CD163, which is also linked to HAND pathogenesis. Finally, simvastatin inhibited production of monocyte chemoattractant protein-1 (MCP-1) and other inflammatory cytokines following LPS stimulation and reduced monocyte chemotaxis in response to MCP-1, a major driver of myeloid cell accumulation in the CNS in HAND. Together, these findings suggest that statin drugs may be useful to prevent or reduce HAND in HIV-1-infected subjects on ART with persistent monocyte activation and inflammation., Competing Interests: The authors A. Yadav, M.R. Betts and R.G. Collman declare that they have no conflict of interest.

The role of microbiota in the pathogenesis of HIV infection has become the subject of intense research in recent years. A rapidly growing amount of data suggest that microbial dysbiosis-in the gut or the genital tract-can influence HIV transmission and/or disease progression; however, a deeper understanding of the mechanisms involved is lacking. To better understand the relationship between the microbiome and HIV infection, investigators from a wide variety of disciplines, including those working in basic and clinical HIV studies, cardiovascular disease, reproductive health, and bioinformatics, gathered at the first International Workshop on Microbiome in HIV Pathogenesis, Prevention and Treatment, at NIH on 7 and 8 April, 2015.

Microaspiration is a common phenomenon in healthy subjects, but its frequency is increased in chronic inflammatory airway diseases, and its role in inflammatory and immune phenotypes is unclear. We have previously demonstrated that acellular bronchoalveolar lavage samples from half of the healthy people examined are enriched with oral taxa (here called pneumotypeSPT) and this finding is associated with increased numbers of lymphocytes and neutrophils in bronchoalveolar lavage. Here, we have characterized the inflammatory phenotype using a multi-omic approach. By evaluating both upper airway and acellular bronchoalveolar lavage samples from 49 subjects from three cohorts without known pulmonary disease, we observed that pneumotypeSPT was associated with a distinct metabolic profile, enhanced expression of inflammatory cytokines, a pro-inflammatory phenotype characterized by elevated Th-17 lymphocytes and, conversely, a blunted alveolar macrophage TLR4 response. The cellular immune responses observed in the lower airways of humans with pneumotypeSPT indicate a role for the aspiration-derived microbiota in regulating the basal inflammatory status at the pulmonary mucosal surface.

Rationale: Improved understanding of the lung microbiome in HIV-infected individuals could lead to better strategies for diagnosis, therapy, and prophylaxis of HIV-associated pneumonias. Differences in the oral and lung microbiomes in HIV-infected and HIV-uninfected individuals are not well defined. Whether highly active antiretroviral therapy influences these microbiomes is unclear., Objectives: We determined whether oral and lung microbiomes differed in clinically healthy groups of HIV-infected and HIV-uninfected subjects., Methods: Participating sites in the Lung HIV Microbiome Project contributed bacterial 16S rRNA sequencing data from oral washes and bronchoalveolar lavages (BALs) obtained from HIV-uninfected individuals (n = 86), HIV-infected individuals who were treatment naive (n = 18), and HIV-infected individuals receiving antiretroviral therapy (n = 38)., Measurements and Main Results: Microbial populations differed in the oral washes among the subject groups (Streptococcus, Actinomyces, Rothia, and Atopobium), but there were no individual taxa that differed among the BALs. Comparison of oral washes and BALs demonstrated similar patterns from HIV-uninfected individuals and HIV-infected individuals receiving antiretroviral therapy, with multiple taxa differing in abundance. The pattern observed from HIV-infected individuals who were treatment naive differed from the other two groups, with differences limited to Veillonella, Rothia, and Granulicatella. CD4 cell counts did not influence the oral or BAL microbiome in these relatively healthy, HIV-infected subjects., Conclusions: The overall similarity of the microbiomes in participants with and without HIV infection was unexpected, because HIV-infected individuals with relatively preserved CD4 cell counts are at higher risk for lower respiratory tract infections, indicating impaired local immune function.

Motivation: The variation in community composition between microbiome samples, termed beta diversity, can be measured by pairwise distance based on either presence-absence or quantitative species abundance data. PERMANOVA, a permutation-based extension of multivariate analysis of variance to a matrix of pairwise distances, partitions within-group and between-group distances to permit assessment of the effect of an exposure or intervention (grouping factor) upon the sampled microbiome. Within-group distance and exposure/intervention effect size must be accurately modeled to estimate statistical power for a microbiome study that will be analyzed with pairwise distances and PERMANOVA., Results: We present a framework for PERMANOVA power estimation tailored to marker-gene microbiome studies that will be analyzed by pairwise distances, which includes: (i) a novel method for distance matrix simulation that permits modeling of within-group pairwise distances according to pre-specified population parameters; (ii) a method to incorporate effects of different sizes within the simulated distance matrix; (iii) a simulation-based method for estimating PERMANOVA power from simulated distance matrices; and (iv) an R statistical software package that implements the above. Matrices of pairwise distances can be efficiently simulated to satisfy the triangle inequality and incorporate group-level effects, which are quantified by the adjusted coefficient of determination, omega-squared (ω2). From simulated distance matrices, available PERMANOVA power or necessary sample size can be estimated for a planned microbiome study., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen., Importance: Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1-independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.

Approximately 1.1 million, or 13 %, of all TB cases in 2013 were coinfected with HIV, and in some African countries, such as Botswana and Swaziland, 60-80 % of TB cases are coinfected with HIV. Effective therapies for both HIV and TB exist, yet patients presenting with TB and advanced HIV still experience high rates of morbidity and mortality despite initiation of both antitubercular and antiretroviral therapy (ART). Previous reviews and research have focused largely on TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) as a type of complicated outcome on ART in advanced HIV/TB, but recent data indicate that immunologic failure despite suppressive ART is associated with early mortality. In this review, we examine recent findings regarding early mortality in HIV/TB and emerging concepts in the pathophysiology of TB-IRIS, in order to provide an integrated view of factors determining outcomes in coinfected people as well as highlight key needs for future research and therapeutic development., Competing Interests: Gregory Bisson declares a grant from NIAID, is a consultant to Pfizer and Celgene, and has provided expert testimony for Levaquin Litigation Fund, LLC. Nicola Zetola declares that she has no conflict of interest. Ronald Collman declares multiple grants from NIH.

In rhesus macaques (RMs), experimental depletion of CD4+ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4+ T-cells prior to SIVmac251 infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA+ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4+ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.

Background: Nosocomial transmission of pulmonary tuberculosis (PTB) is a problem in resource-limited settings. However, the degree of TB exposure and the intermediate- and long-term morbidity and mortality of hospital-associated TB is unclear. In this study we determined: 1) the nature, patterns and intensity of TB exposure occurring in the context of current TB cohorting practices in medical centre with a high prevalence of TB and HIV; 2) the one-year TB incidence after discharge; and 3) one-year TB-related mortality after hospital discharge., Methods: Factors leading to nosocomial TB exposure were collected daily over a 3-month period. Patients were followed for 1-year after discharge. TB incidence and mortality were calculated and logistic regression was used to determine the factors associated with TB incidence and mortality during follow up., Results: 1,094 patients were admitted to the medical wards between May 01 and July 31, 2010. HIV was confirmed in 690/1,094 (63.1%) of them. A total of 215/1,094 (19.7%) patients were diagnosed with PTB and 178/1,094 (16.3%) patients died during the course of their hospitalization; 12/178 (6.7%) patients died from TB-related complications. Of the 896 (83.7%) discharged patients, 41 (4.6%) [corrected] were diagnosed with TB during the year of follow up. Overall, 123/896 (14%) patients died during the follow up period, of whom 26/123 (21%) died from TB. [corrected] One-year TB incidence rate and TB-associated mortality were associated with the number of days that the patient remained hospitalized, the number of days spent in the cohorting bay (regardless of whether the patient was eventually diagnosed with TB or not), and the number and proximity to TB index cases. There was no difference in the performance of each of these 3 measurements of nosocomial TB exposure for the prediction of one-year TB incidence., Conclusion: Substantial TB exposure, particularly among HIV-infected patients, occurs in nosocomial settings despite implementation of cohorting measures. Nosocomial TB exposure is strongly associated with one-year TB incidence and TB-related mortality. Further studies are needed to identify strategies to reduce such exposure among susceptible patients.

The Xpert MTB/RIF (Xpert) assay is becoming a principal screening tool for diagnosing rifampin-resistant Mycobacterium tuberculosis complex (MTBC) infection. However, little is known about the performance of the Xpert assay in infections with both drug-sensitive and drug-resistant strains (mixed MTBC infections). We assessed the performance of the Xpert assay for detecting rifampin resistance using phenotypic drug sensitivity testing (DST) as the reference standard in 370 patients with microbiologically proven pulmonary tuberculosis. Mixed MTBC infections were identified genetically through 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Logistic regression was used to identify the factors associated with poor (defined as treatment failure, default, and death from any cause) or good (defined as cure or successful treatment completion) clinical outcomes. The analytic sensitivity of the Xpert assay for detecting rifampin resistance was assessed in vitro by testing cultures containing different ratios of drug-sensitive and drug-resistant organisms. Rifampin resistance was detected by the Xpert assay in 52 (14.1%) and by phenotypic DST in 55 (14.9%) patients. Mixed MTBC infections were identified in 37 (10.0%) patients. The Xpert assay was 92.7% (95% confidence interval [CI], 82.4% to 97.9%) sensitive for detecting rifampin resistance and 99.7% (95% CI, 98.3% to 99.9%) specific. When restricted to patients with mixed MTBC infections, Xpert sensitivity was 80.0% (95% CI, 56.3 to 94.3%). False-negative Xpert results (adjusted odds ratio [aOR], 6.6; 95% CI,1.2 to 48.2) and mixed MTBC infections (aOR, 6.5; 95% CI, 2.1 to 20.5) were strongly associated with poor clinical outcome. The Xpert assay failed to detect rifampin resistance in vitro when <90% of the organisms in the sample were rifampin resistant. Our study indicates that the Xpert assay has an increased false-negative rate for detecting rifampin resistance with mixed MTBC infections. In hyperendemic settings where mixed infections are common, the Xpert results might need further confirmation., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Patients with multidrug-resistant (MDR) tuberculosis may have phenotypic heterogeneity in results of drug-susceptibility tests (DSTs). However, the impact of this on clinical outcomes among patients treated for MDR tuberculosis is unknown., Methods: Phenotypic DST heterogeneity was defined as presence of at least 1 Mycobacterium tuberculosis isolate susceptible to rifampicin and isoniazid recovered <3 months after MDR tuberculosis treatment initiation from a patient with previous documented tuberculosis due to M. tuberculosis resistant to at least rifampicin and isoniazid. The primary outcome was defined as good (ie, cure or treatment completion) or poor (ie, treatment failure, treatment default, or death). A secondary outcome was time to culture conversion. Cox proportional hazard models were used to determine the association between phenotypic DST heterogeneity and outcomes., Results: Phenotypic DST heterogeneity was identified in 33 of 475 patients (7%) with MDR tuberculosis. Poor outcome occurred in 126 patients (28%). Overall, patients with MDR tuberculosis who had phenotypic DST heterogeneity were at greater risk of poor outcome than those with MDR tuberculosis but no phenotypic DST heterogeneity (adjusted hazard ratio [aHR], 2.1; 95% confidence interval [CI], 1.2-3.6). Among HIV-infected patients with MDR tuberculosis, the adjusted hazard for a poor outcome for those with phenotypic DST heterogeneity was 2.4 (95% CI, 1.3-4.2) times that for those without phenotypic DST heterogeneity, whereas among HIV-negative patients with MDR tuberculosis, the adjusted hazard for those with phenotypic DST heterogeneity was 1.5 (95% CI, .5-4.3) times that for those without phenotypic DST heterogeneity. HIV-infected patients with MDR tuberculosis with phenotypic DST heterogeneity also had a longer time to culture conversion than with HIV-infected patients with MDR tuberculosis without phenotypic DST heterogeneity (aHR, 2.9; 95% CI, 1.4-6.0)., Conclusions: Phenotypic DST heterogeneity among persons with HIV infection who are being treated for MDR tuberculosis is associated with poor outcomes and longer times to culture conversion.

Recent studies have identified a subset of memory T cells with stem cell-like properties (T(SCM)) that include increased longevity and proliferative potential. In this study, we examined the dynamics of CD4(+) T(SCM) during pathogenic SIV infection of rhesus macaques (RM) and nonpathogenic SIV infection of sooty mangabeys (SM). Whereas SIV-infected RM show selective numeric preservation of CD4(+) T(SCM), SIV infection induced a complex perturbation of these cells defined by depletion of CD4(+)CCR5(+) T(SCM), increased rates of CD4(+) T(SCM) proliferation, and high levels of direct virus infection. The increased rates of CD4(+) T(SCM) proliferation in SIV-infected RM correlated inversely with the levels of central memory CD4(+) T cells. In contrast, nonpathogenic SIV infection of SM evidenced preservation of both CD4(+) T(SCM) and CD4(+) central memory T cells, with normal levels of CD4(+) T(SCM) proliferation, and lack of selective depletion of CD4(+)CCR5(+) T(SCM). Importantly, SIV DNA was below the detectable limit in CD4(+) T(SCM) from 8 of 10 SIV-infected SM. We propose that increased proliferation and infection of CD4(+) T(SCM) may contribute to the pathogenesis of SIV infection in RM.

Objectives: Alcohol use has been linked to risky sexual behaviour and it has been identified as an important modifiable factor to prevent HIV infection. However, the evidence of a link between alcohol use and risky sexual behaviour is mixed. In this paper, we examine the role of alcohol use in sexual risk taking among women in Botswana., Methods: Participants were recruited by stratified proportional random sampling and were administered a survey interview that collected information on HIV/AIDS knowledge, risky sexual behaviour and alcohol use. Logistic regression and bivariate probit analyses were used to examine the association between alcohol use and high-risk sexual behaviour., Results: 239 women were interviewed. 168 (70%) had high levels of HIV/AIDS knowledge. We found no significant protective effect of good HIV/AIDS knowledge over high-risk sex behaviour (adjusted OR 0.74, 95% CI 0.38 to 1.42). However, alcohol use before sex was associated with high-risk sex behaviour (adjusted OR 3.04, 95% CI 1.11 to 6.45). However, bivariate probit analysis that simultaneously estimates risky sexual behaviour and alcohol use revealed an insignificant association between alcohol use and risky sex, highlighting the potential presence of other unobserved individual factors that are associated with alcohol use and risky sex., Conclusions: Knowledge about HIV may not be sufficient to decrease risky sexual behaviour. Alcohol consumption was associated with an increased probability of high-risk sexual intercourse. However, the relationship between alcohol use and risky sex may also be a marker of a third omitted variable (such as overall risk-taking propensity). Further research is needed to identify factors associated with alcohol use and high-risk sex.

Mother-to-infant transmission (MTIT) of HIV is a serious global health concern, with over 300,000 children newly infected in 2011. SIV infection of rhesus macaques (RMs) results in similar rates of MTIT to that of HIV in humans. In contrast, SIV infection of sooty mangabeys (SMs) rarely results in MTIT. The mechanisms underlying protection from MTIT in SMs are unknown. In this study we tested the hypotheses that breast milk factors and/or target cell availability dictate the rate of MTIT in RMs (transmitters) and SMs (non-transmitters). We measured viral loads (cell-free and cell-associated), levels of immune mediators, and the ability to inhibit SIV infection in vitro in milk obtained from lactating RMs and SMs. In addition, we assessed the levels of target cells (CD4+CCR5+ T cells) in gastrointestinal and lymphoid tissues, including those relevant to breastfeeding transmission, as well as peripheral blood from uninfected RM and SM infants. We found that frequently-transmitting RMs did not have higher levels of cell-free or cell-associated viral loads in milk compared to rarely-transmitting SMs. Milk from both RMs and SMs moderately inhibited in vitro SIV infection, and presence of the examined immune mediators in these two species did not readily explain the differential rates of transmission. Importantly, we found that the percentage of CD4+CCR5+ T cells was significantly lower in all tissues in infant SMs as compared to infant RMs despite robust levels of CD4+ T cell proliferation in both species. The difference between the frequently-transmitting RMs and rarely-transmitting SMs was most pronounced in CD4+ memory T cells in the spleen, jejunum, and colon as well as in central and effector memory CD4+ T cells in the peripheral blood. We propose that limited availability of SIV target cells in infant SMs represents a key evolutionary adaptation to reduce the risk of MTIT in SIV-infected SMs.

Background: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe., Methods: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance., Results: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients., Conclusions: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).

Background: Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources., Results: Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure., Conclusions: We find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci,a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

Myeloid cells residing in the CNS and lymphoid tissues are targets for productive HIV-1 replication, and their infection contributes to the pathological manifestations of HIV-1 infection. The Envs can adopt altered configurations to overcome entry restrictions in macrophages via a more efficient and/or altered mechanism of engagement with cellular receptors. This review highlights evidence supporting an important role for macrophages in HIV-1 pathogenesis and persistence, which need to be considered for strategies aimed at achieving a functional or sterilizing cure. We also highlight that the molecular mechanisms underlying HIV-1 tropism for macrophages are complex, involving enhanced and/or altered interactions with CD4, CCR5, and/or CXCR4, and that the nature of these interactions may depend on the anatomical location of the virus.

Background: HIV and SIV generally require CD4 binding prior to coreceptor engagement, but Env can acquire the ability to use CCR5 independently of CD4 under various circumstances. The ability to use CCR5 coupled with low-to-absent CD4 levels is associated with enhanced macrophage infection and increased neutralization sensitivity, but the additional features of these Envs that may affect cell targeting is not known., Results: Here we report that CD4-independent SIV variants that emerged in vivo in a CD4+ T cell-depleted rhesus macaque model display markedly decreased plasticity of co-receptor use. While CD4-dependent Envs can use low levels of macaque CCR5 for efficient entry, CD4-independent variants required high levels of CCR5 even in the presence of CD4. CD4-independent Envs were also more sensitive to the CCR5 antagonist Maraviroc. CD4-dependent variants mediated efficient entry using human CCR5, whereas CD4-independent variants had impaired use of human CCR5. Similarly, CD4-independent Envs used the alternative coreceptors GPR15 and CXCR6 less efficiently than CD4-dependent variants. Env amino acids D470N and E84K that confer the CD4-independent phenotype also regulated entry through low CCR5 levels and GPR15, indicating a common structural basis. Treatment of CD4-dependent Envs with soluble CD4 enhanced entry through CCR5 but reduced entry through GPR15, suggesting that induction of CD4-induced conformational changes by non-cell surface-associated CD4 impairs use of this alternative co-receptor., Conclusions: CD4 independence is associated with more restricted coreceptor interactions. While the ability to enter target cells through CCR5 independently of CD4 may enable infection of CD4 low-to-negative cells such as macrophages, this phenotype may conversely reduce the potential range of targets such as cells expressing low levels of CCR5, conformational variants of CCR5, or possibly even alternative coreceptors.

Often, high-sensitivity, point-of-care (POC) clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low-abundance target molecules. We report on a simple-to-use, low-cost, pump-free, membrane-based, sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. This plasma separator consists of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. The plasma separator demonstrated a "blood in-plasma out" capability, consistently extracting 275 ± 33.5 μL of plasma from 1.8 mL of undiluted whole blood within less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ± 3.5%, 88.0% ± 9.5%, and 81.5% ± 12.1% for viral loads of 35,000, 3500, and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into our custom-made microfluidic chip for nucleic acid testing and was successfully subjected to reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP), demonstrating that the plasma is sufficiently pure to support high-efficiency nucleic acid amplification.

CD4(+) T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4(+) T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4(+) T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV(+)) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV(+) plasma neutralization. However, plasma from SIV-infected CD4(+) T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4(+) T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4(+) target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.

Study of the human lung microbiome in the context of pulmonary health and disease is an area of emerging research interest that is being driven by several contributing factors. These factors include increased recognition of the diversity of human-associated microbiota, their roles in health and in diseases associated with chronic inflammation, and advancements in technologies and tools that have facilitated such discoveries about the microbiota in organ systems outside of the lung. Therefore, the overarching goals of lung microbiome research are: to identify and characterize microbial populations associated with the respiratory tract and lungs; to understand their roles in lung health and disease; and, we hope, to allow the development of improved approaches for diagnosing and treating chronic respiratory diseases in which the microbiome has a role. Recent studies of the lung microbiome have yielded a number of interesting findings but also highlighted questions and challenges for researchers and clinicians. In December 2011, the National Heart, Lung, and Blood Institute convened a workshop to identify key issues and areas for further attention or development to advance research on the lung microbiome. Current knowledge and the state of research on the lung and related areas of human microbiome investigation were reviewed and discussed.

Rationale: Lung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV., Objectives: To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota., Methods: We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects., Measurements and Main Results: Sequences representing Tropheryma whipplei, the etiologic agent of Whipple's disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple's disease., Conclusions: Asymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.

We report the safety and tolerability of 87 infusions of lentiviral vector–modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector–transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.

Rationale: Long-term survival after lung transplantation is limited by infectious complications and by bronchiolitis obliterans syndrome (BOS), a form of chronic rejection linked in part to microbial triggers., Objectives: To define microbial populations in the respiratory tract of transplant patients comprehensively using unbiased high-density sequencing., Methods: Lung was sampled by bronchoalveolar lavage (BAL) and upper respiratory tract by oropharyngeal wash (OW). Bacterial 16S rDNA and fungal internal transcribed spacer sequencing was used to profile organisms present. Outlier analysis plots defining taxa enriched in lung relative to OW were used to identify bacteria enriched in lung against a background of oropharyngeal carryover., Measurements and Main Results: Lung transplant recipients had higher bacterial burden in BAL than control subjects, frequent appearance of dominant organisms, greater distance between communities in BAL and OW indicating more distinct populations, and decreased respiratory tract microbial richness and diversity. Fungal populations were typically dominated by Candida in both sites or by Aspergillus in BAL but not OW. 16S outlier analysis identified lung-enriched taxa indicating bacteria replicating in the lower respiratory tract. In some cases this confirmed respiratory cultures but in others revealed enrichment by anaerobic organisms or mixed outgrowth of upper respiratory flora and provided quantitative data on relative abundances of bacteria found by culture., Conclusions: Respiratory tract microbial communities in lung transplant recipients differ in structure and composition from healthy subjects. Outlier analysis can identify specific bacteria replicating in lung. These findings provide novel approaches to address the relationship between microbial communities and transplant outcome and aid in assessing lung infections.

Motivation: The human microbiome plays an important role in human disease and health. Identification of factors that affect the microbiome composition can provide insights into disease mechanism as well as suggest ways to modulate the microbiome composition for therapeutical purposes. Distance-based statistical tests have been applied to test the association of microbiome composition with environmental or biological covariates. The unweighted and weighted UniFrac distances are the most widely used distance measures. However, these two measures assign too much weight either to rare lineages or to most abundant lineages, which can lead to loss of power when the important composition change occurs in moderately abundant lineages., Results: We develop generalized UniFrac distances that extend the weighted and unweighted UniFrac distances for detecting a much wider range of biologically relevant changes. We evaluate the use of generalized UniFrac distances in associating microbiome composition with environmental covariates using extensive Monte Carlo simulations. Our results show that tests using the unweighted and weighted UniFrac distances are less powerful in detecting abundance change in moderately abundant lineages. In contrast, the generalized UniFrac distance is most powerful in detecting such changes, yet it retains nearly all its power for detecting rare and highly abundant lineages. The generalized UniFrac distance also has an overall better power than the joint use of unweighted/weighted UniFrac distances. Application to two real microbiome datasets has demonstrated gains in power in testing the associations between human microbiome and diet intakes and habitual smoking., Availability: http://cran.r-project.org/web/packages/GUniFrac