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Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2 IL4 ). Glucocorticoids (GC), widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2 GC ), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2 IL4 and M2 GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the glucocorticoid receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2 IL4 :M2 GC -shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design., (© 2024. The Author(s).)

Combination antiretroviral therapy (cART) suppresses viral replication but does not cure HIV infection because a reservoir of infectious (intact) HIV proviruses persists in long-lived CD4+T cells. However, a large majority (>95%) of HIV-infected cells that persist on effective cART carry defective (non-infectious) proviruses. Defective proviruses consisting of only a single LTR (solo long terminal repeat) are commonly found as endogenous retroviruses in many animal species, but the frequency of solo-LTR HIV proviruses has not been well defined. Here we show that, in five pediatric donors whose viremia was suppressed on cART for at least 5 years, the proviruses in the nine largest clones of HIV-infected cells were solo LTRs. The sizes of five of these clones were assayed longitudinally by integration site-specific quantitative PCR. Minor waxing and waning of the clones was observed, suggesting that these clones are generally stable over time. Our findings show that solo LTRs comprise a large fraction of the proviruses in infected cell clones that persist in children on long-term cART. IMPORTANCE This work highlights that severely deleted HIV-1 proviruses comprise a significant proportion of the proviral landscape and are often overlooked., Competing Interests: John W. Mellors reports additional research grant funding from USAID and Gilead Sciences, Inc., to the University of Pittsburgh; scientific advisory board member to Gilead Sciences, Inc.; shareholder in Abound Bio, Inc., and MingMed; and share option holder in Infectious Disease Connect.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors., Competing Interests: Declaration of interests J.D.B. holds patents related to ATAC-seq and scATAC-seq and serves on the Scientific Advisory Board of CAMP4 Therapeutics, seqWell, and CelSee. S.Z.J. and F.J.B. declare a related patent application: 10203-02-PC; EFS ID: 44924864 Enrichment and Characterization of Rare Circulating Cells, including Progenitor Cells from Peripheral Blood and Uses Thereof. F.J.B. is a co-founder and scientific advisor of IpiNovyx Bio. E.J.S. reports personal fees from NIAID through Axle Informatics for the subject matter expert program for the COVID-19 vaccine clinical trials. R.E.S. is on the scientific advisory board of Miromatrix Inc. and Lime Therapeutics and is a paid consultant and speaker for Alnylam Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The advent of combined antiretroviral therapy (cART) has been instrumental in controlling HIV-1 replication and transmission and decreasing associated morbidity and mortality. However, cART alone is not able to cure HIV-1 due to the presence of long-lived, latently infected immune cells, which re-seed plasma viremia when cART is interrupted. Assessment of HIV-cure strategies using ex vivo culture methods for further understanding of the diversity of reactivated HIV, viral outgrowth, and replication dynamics are enhanced using ultrasensitive digital ELISA based on single-molecule array (Simoa) technology to increase the sensitivity of endpoint detection. In viral outgrowth assays (VOA), exponential HIV-1 outgrowth has been shown to be dependent upon initial virus burst size surpassing a critical growth threshold of 5100 HIV-1 RNA copies. Here, we show an association between ultrasensitive HIV-1 Gag p24 concentrations and HIV-1 RNA copy number that characterize viral dynamics below the exponential replication threshold. Single-genome sequencing (SGS) revealed the presence of multiple identical HIV-1 sequences, indicative of low-level replication occurring below the threshold of exponential outgrowth early during a VOA. However, SGS further revealed diverse related HIV variants detectable by ultrasensitive methods that failed to establish exponential outgrowth. Overall, our data suggest that viral outgrowth occurring below the threshold necessary for establishing exponential growth in culture does not preclude replication competence of reactivated HIV, and ultrasensitive detection of HIV-1 p24 may provide a method to detect previously unquantifiable variants. These data strongly support the use of the Simoa platform in a multi-prong approach to measuring latent viral burden and efficacy of therapeutic interventions aimed at an HIV-1 cure., (© 2023. The Author(s).)

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5' LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5' LTR promoter.

Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs infected ex vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B Our analyses indicate that, despite marked differences in T-cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes. IMPORTANCE HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized., (Copyright © 2021 Bale et al.)

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JWM is a consultant to Gilead Sciences and Merck Co., Inc., and owns share options in Co-Crystal Pharmaceuticals, Inc. JMC is a member of the Scientific Advisory Board of ROME Therapeutics, Inc. All other authors declare no competing interests.

BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred by clinicians for nonsuppressible viremia (plasma HIV-1 RNA above 40 copies/mL) despite reported adherence to ART and the absence of drug resistance to the current ART regimen.METHODSSamples were collected from at least 2 time points from 8 donors who had nonsuppressible viremia for more than 6 months. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were amplified by PCR and sequenced for evidence of clones of cells that produced infectious viruses. Clones were confirmed by host-proviral integration site analysis.RESULTSHIV-1 genomic RNA with identical sequences were identified in plasma samples from all 8 donors. The identical viral RNA sequences did not change over time and did not evolve resistance to the ART regimen. In 4 of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication competent. Integration sites for infectious proviruses from those 4 donors were mapped to the introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were estimated to be from 50 million to 350 million cells.CONCLUSIONThese findings show that clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia. The mechanisms involved in clonal expansion and persistence need to be defined to effectively target viremia and the HIV-1 reservoir.FUNDINGNational Cancer Institute, NIH; Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute; Bill and Melinda Gates Foundation; Office of AIDS Research; American Cancer Society; National Cancer Institute through a Leidos subcontract; National Institute for Allergy and Infectious Diseases, NIH, to the I4C Martin Delaney Collaboratory; University of Rochester Center for AIDS Research and University of Rochester HIV/AIDS Clinical Trials Unit.

An amendment to this paper has been published and can be accessed via the original article.

Male genitals are highly divergent in animals with internal fertilization. Most studies attempting to explain this diversity have focused on testing the major hypotheses of genital evolution (the lock-and-key, pleiotropy, and sexual selection hypotheses), and quantifying the form of selection targeting male genitals has played an important role in this endeavor. However, we currently know far less about selection targeting female genitals or how male and female genitals interact during mating. Here, we use formal selection analysis to show that genital size and shape is subject to strong multivariate stabilizing sexual selection in both sexes of the red flour beetle, Tribolium castaneum. Moreover, we show significant sexual selection on the covariance between the sexes for specific aspects of genital shape suggesting that male and female genitalia also interact to determine the successful transfer of a spermatophore during mating. Our work therefore highlights the important role that both male and female genital morphologies play in determining mating success and that these effects can occur independently, as well as through their interaction. Moreover, it cautions against the overly simplistic view that the sexual selection targeting genital morphology will always be directional in form and restricted primarily to males., (© 2019 The Authors. Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.)

Background: All retroviruses, including human immunodeficiency virus (HIV), must integrate a DNA copy of their genomes into the genome of the infected host cell to replicate. Although integrated retroviral DNA, known as a provirus, can be found at many sites in the host genome, integration is not random. The adaption of linker-mediated PCR (LM-PCR) protocols for high-throughput integration site mapping, using randomly-sheared genomic DNA and Illumina paired-end sequencing, has dramatically increased the number of mapped integration sites. Analysis of samples from human donors has shown that there is clonal expansion of HIV infected cells and that clonal expansion makes an important contribution to HIV persistence. However, analysis of HIV integration sites in samples taken from patients requires extensive PCR amplification and high-throughput sequencing, which makes the methodology prone to certain specific artifacts., Results: To address the problems with artifacts, we use a comprehensive approach involving experimental procedures linked to a bioinformatics analysis pipeline. Using this combined approach, we are able to reduce the number of PCR/sequencing artifacts that arise and identify the ones that remain. Our streamlined workflow combines random cleavage of the DNA in the samples, end repair, and linker ligation in a single step. We provide guidance on primer and linker design that reduces some of the common artifacts. We also discuss how to identify and remove some of the common artifacts, including the products of PCR mispriming and PCR recombination, that have appeared in some published studies. Our improved bioinformatics pipeline rapidly parses the sequencing data and identifies bona fide integration sites in clonally expanded cells, producing an Excel-formatted report that can be used for additional data processing., Conclusions: We provide a detailed protocol that reduces the prevalence of artifacts that arise in the analysis of retroviral integration site data generated from in vivo samples and a bioinformatics pipeline that is able to remove the artifacts that remain.

In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age, one of whom had two identical intact sequences, suggestive of a cell clone harboring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably, as in adults, maintained by clonal expansion of cells infected before ART initiation. IMPORTANCE There are limited data about the proviral landscape in children exhibiting long-term suppression after early treatment, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through nearly full-length proviral amplification and sequencing (NFL-PAS), we generated 733 NFL-PAS amplicons from eight children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower ( P  < 0.05) than that reported for HIV subtype B-infected adults treated during early HIV infection. We show that cells harboring genetically intact HIV proviruses are rare in children exhibiting long-term suppression after early treatment and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs, particularly in pediatric studies where sample availability is limited., (Copyright © 2020 American Society for Microbiology.)

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for "viral reconstruction" to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality., Competing Interests: Competing interest statement: J.W.M. is a consultant to Gilead Sciences, Merck Research Laboratories, Janssen Pharmaceuticals, and AccelevirDx, and a share option holder of Co-Crystal, Inc. B.F.K. and J.A.H. are co-authors on an October 2015 article. The remaining authors have no potential conflicts.

Studies have demonstrated that intensive ART alone is not capable of eradicating HIV-1, as the virus rebounds within a few weeks upon treatment interruption. Viral rebound may be induced from several cellular subsets; however, the majority of proviral DNA has been found in antigen experienced resting CD4+ T cells. To achieve a cure for HIV-1, eradication strategies depend upon both understanding mechanisms that drive HIV-1 persistence as well as sensitive assays to measure the frequency of infected cells after therapeutic interventions. Assays such as the quantitative viral outgrowth assay (QVOA) measure HIV-1 persistence during ART by ex vivo activation of resting CD4+ T cells to induce latency reversal; however, recent studies have shown that only a fraction of replication-competent viruses are inducible by primary mitogen stimulation. Previous studies have shown a correlation between the acquisition of effector memory phenotype and HIV-1 latency reversal in quiescent CD4+ T cell subsets that harbor the reservoir. Here, we apply our mechanistic understanding that differentiation into effector memory CD4+ T cells more effectively promotes HIV-1 latency reversal to significantly improve proviral measurements in the QVOA, termed differentiation QVOA (dQVOA), which reveals a significantly higher frequency of the inducible HIV-1 replication-competent reservoir in resting CD4+ T cells., Competing Interests: The authors have declared that no competing interests exist.

We hypothesized that HIV-1 with dual-class but not single-class drug resistance mutations linked on the same viral genome, present in the virus population before initiation of antiretroviral therapy (ART), would be associated with failure of ART to suppress viremia. To test this hypothesis, we utilized an ultrasensitive single-genome sequencing assay that detects rare HIV-1 variants with linked drug resistance mutations (DRMs). A case (ART failure) control (nonfailure) study was designed to assess whether linkage of DRMs in pre-ART plasma samples was associated with treatment outcome in the nevirapine/tenofovir/emtricitabine arm of the AIDS Clinical Trials Group A5208/Optimal Combined Therapy After Nevirapine Exposure (OCTANE) Trial 1 among women who had received prior single-dose nevirapine. Ultrasensitive single-genome sequencing revealed a significant association between pre-ART HIV variants with DRMs to 2 drug classes linked on the same genome (dual class) and failure of combination ART with 3 drugs to suppress viremia. In contrast, linked, single-class DRMs were not associated with ART failure. We conclude that linked dual-class DRMs present before the initiation of ART are associated with ART failure, whereas linked single-class DRMs are not.

Background: HIV-1 proviruses can persist during ART in clonally-expanded populations of CD4+ T cells. To date, few examples of an expanded clones containing replication-competent proviruses exist, although it is suspected to be common. One such clone, denoted AMBI-1 (Maldarelli et al., 2014), was also a source of persistent viremia on ART, begging the question of how the AMBI-1 clone can survive despite infection with a replication-competent, actively-expressing provirus. We hypothesized that only a small fraction of cells within the AMBI-1 clone are activated to produce virus particles during cell division while the majority remain latent despite division, ensuring their survival. To address this question, we determined the fraction of HIV-1 proviruses within the AMBI-1 clone that expresses unspliced cell-associated RNA during ART and compared this fraction to 33 other infected T cell clones within the same individual., Results: In total, 34 different clones carrying either intact or defective proviruses in "Patient 1" from Maldarelli et al. (2014) were assessed. We found that 2.3% of cells within the AMBI-1 clone contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory (EM) T cell subset. The fraction of cells within clones that contained HIV-1 RNA was not different in clones with intact (median 2.3%) versus defective (median 3.5%) proviruses ( p = 0.2). However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia., Conclusion: These findings show that the vast majority of HIV-1 proviruses within expanded T cell clones, including intact proviruses, may be transcriptionally silent at any given time, implying that infected T cells may be able to be activated to proliferate without inducing the expression of the integrated provirus or, alternatelively, may be able to proliferate without cellular activation. The results of this study suggest that the long, presumed correlation between the level of cellular and proviral activation may not be accurate and, therefore, requires further investigation., (Copyright © 2019 Musick, Spindler, Boritz, Pérez, Crespo-Vélez, Patro, Sobolewski, Bale, Reid, Keele, Capoferri, Shao, Wiegand, Simonetti, Mellors, Hughes, Coffin, Maldarelli and Kearney.)

HIV persistence during combination antiretroviral therapy (cART) is the principal obstacle to cure. Mechanisms responsible for persistence remain uncertain; infections may be maintained by persistence and clonal expansion of infected cells or by ongoing replication in anatomic locations with poor antiretroviral penetration. These mechanisms require different strategies for eradication, and determining their contributions to HIV persistence is essential. We used phylogenetic approaches to investigate, at the DNA level, HIV populations in blood, lymphoid, and other infected tissues obtained at colonoscopy or autopsy in individuals who were on cART for 8 to 16 years. We found no evidence of ongoing replication or compartmentalization of HIV; we did detect clonal expansion of infected cells that were present before cART. Long-term persistence, and not ongoing replication, is primarily responsible for maintaining HIV. HIV-infected cells present when cART is initiated represent the only identifiable source of persistence and is the appropriate focus for eradication., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.

Characterizing HIV genetic diversity and evolution during antiretroviral therapy (ART) provides insights into the mechanisms that maintain the viral reservoir during ART. This review describes common methods used to obtain and analyze intra-patient HIV sequence data, the accumulation of diversity prior to ART and how it is affected by suppressive ART, the debate on viral replication and evolution in the presence of ART, HIV compartmentalization across various tissues, and mechanisms for the emergence of drug resistance. It also describes how CD4+ T cells that were likely infected with latent proviruses prior to initiating treatment can proliferate before and during ART, providing a renewable source of infected cells despite therapy. Some expanded cell clones carry intact and replication-competent proviruses with a small fraction of the clonal siblings being transcriptionally active and a source for residual viremia on ART. Such cells may also be the source for viral rebound after interrupting ART. The identical viral sequences observed for many years in both the plasma and infected cells of patients on long-term ART are likely due to the proliferation of infected cells both prior to and during treatment. Studies on HIV diversity may reveal targets that can be exploited in efforts to eradicate or control the infection without ART.

It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low. Eight children started ART at less than ten months of age and showed suppression of plasma viremia for seven to nine years. Two children had uncontrolled viremia for fifteen and thirty months, respectively, before viremia suppression, and served as positive controls for HIV replication and evolution. These latter 2 children showed clear evidence of virus evolution, whereas multiple methods of analysis bore no evidence of virus evolution in any of the 8 children with viremia suppression on ART. Phylogenetic trees simulated with the recently reported evolutionary rate of HIV-1 on ART of 6 × 10-4 substitutions/site/month bore no resemblance to the observed data. Taken together, these data refute the concept that ongoing HIV replication is common with ART and is the major barrier to curing HIV-1 infection.

Lorenzo-Redondo et al. recently analyzed HIV RNA sequences in plasma virus and proviral DNA sequences in lymph nodes (LN) and peripheral blood mononuclear cells (PBMC) from samples collected over a 6-month period from 3 individuals following initiation of antiretroviral therapy (ART) and concluded that ongoing HIV replication occurred in LN despite ART and that this replication maintained the HIV reservoir. We analyzed the same sequences and found that the dataset was very limited (median of 5 unique RNA or DNA sequences per sample) after accounting for polymerase chain reaction resampling and hypermutation and that the few remaining DNA sequences after 3 and 6 months on ART were not more diverse or divergent from those in pre-ART in any of the individuals studied. These findings, and others, lead us to conclude that the claims of ongoing replication on ART made by Lorenzo-Redondo et al. are not justified from the dataset analyzed in their publication.

Background: Oncomelania hupensis robertsoni is the sole intermediate host for Schistosoma japonicum in western China. Given the close co-evolutionary relationships between snail host and parasite, there is interest in understanding the distribution of distinct snail phylogroups as well as regional population structures. Therefore, this study focuses on these aspects in a re-emergent schistosomiasis area known to harbour representatives of two phylogroups - the Deyang-Mianyang area in Sichuan Province, China. Based on a combination of mitochondrial and nuclear DNA, the following questions were addressed: 1) the phylogeography of the two O. h. robertsoni phylogroups, 2) regional and local population structure in space and time, and 3) patterns of local dispersal under different isolation-by-distance scenarios., Results: The phylogenetic analyses confirmed the existence of two distinct phylogroups within O. h. robertsoni. In the study area, phylogroups appear to be separated by a mountain range. Local specimens belonging to the respective phylogroups form monophyletic clades, indicating a high degree of lineage endemicity. Molecular clock estimations reveal that local lineages are at least 0.69-1.58 million years (My) old and phylogeographical analyses demonstrate that local, watershed and regional effects contribute to population structure. For example, Analyses of Molecular Variances (AMOVAs) show that medium-scale watersheds are well reflected in population structures and Mantel tests indicate isolation-by-distance effects along waterways., Conclusions: The analyses revealed a deep, complex and hierarchical structure in O. h. robertsoni, likely reflecting a long and diverse evolutionary history. The findings have implications for understanding disease transmission. From a co-evolutionary standpoint, the divergence of the two phylogroups raises species level questions in O. h. robertsoni and also argues for future studies relative to the distinctness of the respective parasites. The endemicity of snail lineages at the regional level supports the concept of endemic schistosomiasis areas and calls for future geospatial analyses for a better understanding of respective boundaries. Finally, local snail dispersal mainly occurs along waterways and can be best described by using cost distance, thus potentially enabling a more precise modelling of snail, and therefore, parasite dispersal.

The natural history of the carriage of methicillin-resistant Staphylococcus aureus (MRSA) was examined in a 9-year retrospective cohort study of 102 known carriers. The populations studied consisted of patients admitted to a university hospital from 1989 through 1991; a review extending back to January 1983 was conducted. The focuses of the study included the duration of carriage among patients who were known to have carried MRSA previously and who were readmitted to the hospital (36 patients) and the optimal anatomic site for screening (66 patients). Cultures of the nares (sensitivity, 93%; negative predictive value, 95%) were considerably more valuable for the detection of MRSA colonization than were cultures of cutaneous sites of the axilla, groin, and perineum (sensitivity, < or = 39%; negative predictive value, < or = 69%). The estimated half-life of MRSA colonization in this special population of patients was approximately 40 months. Restriction enzyme analysis of plasmid types of paired isolates from the 12 patients with MRSA carriage persisting for > 12 months revealed five instances (42%) in which both isolates were of the same type. In summary, our results indicate that the majority of readmitted carriers harbor MRSA for > 3 years and that, in this population, culture of the anterior nares alone (with culture of wound or sputum, when present) is a valid and efficient method for the detection of persistent MRSA carriage.

This comparative study determined the effect of blood on the antienterococcal activities of the newer cephalosporins. Standardized disk diffusion susceptibility tests were performed with 57 strains of enterococci (30 Enterococcus faecalis strains) on Mueller-Hinton agar with and without 5% sheep blood supplementation. Twelve cephalosporins representing five different structural groups (based on the 7-alpha position substitution) were tested. The greatest frequency of activity enhancement by blood was observed with cefdaloxime and cefdinir (7-alpha hydroxyimino group) against E. faecalis. Cephalosporins with a 7-alpha methoxyimino group (cefpodoxime, cefepime, and cefpirome) had marked increases in zone diameters (3 to > 9 mm) when tested with the blood supplement. Cephems with 7-alpha amino or carboxy substitutions did not demonstrate any enhanced activity. Awareness of this phenomenon is important for the interpretation and accuracy of cephalosporin susceptibility testing.

Several multilaboratory studies to determine quality control (QC) ranges for a variety of National Committee for Clinical Laboratory Standards (NCCLS) susceptibility tests are summarized. Replicate testing used multiple lots of media and antimicrobial disks in accordance with NCCLS recommendations, including the appropriate medium modifications for tests with Haemophilus spp. and Neisseria gonorrhoeae. QC ranges for MIC and disk diffusion testing of N. gonorrhoeae ATCC 49226 were proposed for cefepime, cefetamet, cefmetazole, and cefpodoxime. Disk diffusion QC ranges for Haemophilus influenzae ATCC 49247 or ATCC 49766 were recommended with cefepime, cefetamet (10- and 30-microgram disks), cefmetazole, cefpodoxime, and cefprozil. Disk diffusion QC ranges for Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 with cefdinir and clinafloxacin and those for Pseudomonas aeruginosa ATCC 27853 with clinafloxacin were also proposed.

Pseudomonas aeruginosa was isolated from nine patients (16.2 isolations/1,000 patient-days) in a surgical intensive care unit during an outbreak in November 1990; this rate of isolation was three times higher than that noted previously on this unit. Three patients were infected with the same strain, as defined by identical serotypes, pyocin types, and contour-clamped homogeneous electric field (CHEF) electrophoresis patterns of digested genomic DNA. The hands of 80 health care workers were cultured, and a strain of P. aeruginosa identical to that infecting the three patients was isolated from the hands of a nurse providing care to all three. Environmental surfaces, medical devices, and ward stock supplies were cultured; none of these cultures yielded this strain. No clusters of infection with this strain or other strains of P. aeruginosa were observed after compliance with hand-washing and universal precautions was reemphasized. Thus this outbreak was linked to the carriage of P. aeruginosa on the hands of a health care worker. It could not be determined definitively whether this carriage was the source of the cluster or a consequence of it. However, the geographic and temporal clustering of carriage with an outbreak due to a strain of an apparently identical molecular type underlines the importance of routine hand washing between contacts with different patients.

A multilaboratory study to determine disk diffusion quality control ranges for Haemophilus influenzae ATCC 49247 and five investigational drugs was performed. Multiple lots of Haemophilus Test Medium and antibiotic disks were used for replicate testing in conformance with the recommendations of the National Committee for Clinical Laboratory Standards. Quality control disk zone diameter ranges were proposed for cefdinir, CI-960, fleroxacin, temafloxacin, and trospectomycin.

A multilaboratory study was performed to establish broth microdilution MIC quality control (QC) guidelines for 10 investigational drugs which previously demonstrated significant activity against Haemophilus influenzae. MIC QC ranges for H. influenzae ATCC 49247 with Haemophilus test medium were determined by using multiple contemporary lots of Haemophilus test medium and the National Committee for Clinical Laboratory Standards' recommended numbers of replicate tests. On the basis of these results, QC ranges (generally modal MIC +/- one log2 dilution) are proposed for cefdinir, cefepime, cefetamet, cefpirome, ceftibuten, fleroxacin, temafloxacin, clarithromycin, RP59500, and trospectomycin. The proposed QC guidelines for clarithromycin and temafloxacin were recently accepted by the National Committee for Clinical Laboratory Standards.

A total of 899 urine cultures were evaluated to assess the need for and cost-effectiveness of using a gram-positive coccal selective plate in the initial plating of urine cultures. Of these cultures, 437 were examined retrospectively and 462 were examined prospectively. Urines were quantitatively plated to three media: sheep blood sugar, MacConkey agar, and phenyl ethyl alcohol agar. Of all urine samples in both studies, 52% yielded no growth on any of the three media. Of all 899 urine cultures, there were only 5 cultures (less than 1%) in which a significant count of a gram-positive organism was detected only on the phenyl ethyl alcohol agar plate and not recoverable on the sheep blood agar plate. In each of these five instances, the need for the use of the selective plate occurred when a Proteus mirabilis strain swarmed over an enterococcus. The inclusion of a selective gram-positive coccal medium for initial plating of urine cultures is unnecessary and not cost-effective. When Proteus swarms on sheep blood agar, a sweep should be made with an inoculating loop from the sheep blood agar and streaked to phenyl ethyl alcohol agar or a similar gram-positive coccal selective medium.

In situ mating experiments were done in the River Taff, South Wales, United Kingdom, by using a natural mercury resistance plasmid (pQM1) isolated from a mixture of epilithic bacteria in vitro. The river temperature from March to November was found to influence transfer frequencies strongly (6.8 x 10 to 1.5 x 10 per recipient). A linear relationship existed between log(10) transfer frequency and river temperature (6 to 21 degrees C), a 2.6 degrees C change in temperature giving a 10-fold change in transfer frequency. In vitro experiments showed that pQM1 transferred most efficiently between fluorescent pseudomonads and that one epilithic isolate (Pseudomonas fluorescens) was an efficient donor in situ. Experiments with a P. putida recipient showed that intact epilithic bacterial communities could transfer mercury resistance plasmids in situ at frequencies of up to 3.75 x 10 per recipient. Nineteen of the large (>250-kilobase) plasmids isolated by transfer into P. putida were studied in detail and grouped into seven types by restriction digests. Mercury resistance and UV resistance were found to be common linked phenotypes in 19 of the 23 plasmids tested.

Pseudomonas aeruginosa is the only gram-negative bacillus capable of producing the very distinctive water-soluble pigment pyocyanin. We evaluated the reliability of this characteristic as a unique test for the identification of this organism by using Tech agar (BBL Microbiology Systems, Cockeysville, Md.) medium. A retrospective and prospective analysis was performed with a total of 835 strains of P. aeruginosa; 818 (98%) produced pigment within 48 h of incubation, and 96% of those which produced pigment were positive after overnight incubation. Seventeen strains (2.0%) failed to produce pigment; 15 were mucoid strains from patients with cystic fibrosis. Tech agar is an effective, simple, and inexpensive medium for P. aeruginosa identification and may be used as a unique test for all potential P. aeruginosa isolates (beta hemolytic on blood agar; lactose-negative, oxidase-positive colonies). Nonpigmented mucoid strains, as well as other nonpigmented organisms, will require additional testing to ensure proper identification.

A total of 730 Enterobacteriaceae strains isolated from 567 cultures were evaluated by a rapid kit method (Micro-ID; General Diagnostics, Morris Plains, N.J.; 4 h), an overnight incubation kit method (API 20E; Analytab Products, Plainview, N.Y.), and conventional biochemical test methodology (mostly overnight incubation and some rapid methods) to compare the amount of laboratory effort required, timing, and cost parameters. We assessed the amount of technologist time expended, the time sequence of culture reporting to physicians, the number of isolates requiring repeat testing or additional biochemical testing, the number of cultures held due to the need for identification of other organisms, the cost of total work-up, etc. Cultures evaluated included urines, respiratory cultures, wound cultures, body fluids, genital cultures, and cultures from miscellaneous categories. A total of 64% of the Enterobacteriaceae strains processed by the Micro-ID method could be identified within 24 h of receipt of the specimens in the clinical laboratories, in contrast to the need for an additional day required by the API or conventional biochemical methods. The Micro-ID method also required less technologist time (4.5 min) for set-up and interpretation than did either the API method (6 min) or conventional methods (7 min). Total direct costs (June 1981) per organism identified were: Micro-ID, $4.30; API 20E, $4.96; conventional biochemicals with commercially prepared media, $5.66. An estimate of 80% technologist time efficiency was made in all procedures.

A method for in situ mating experiments is described which involved overnight incorporation of donors containing the mercury resistance plasmid pQM1 and recipients into the epilithon on separate river stones. The stones were then joined to begin the mating. Transfer frequencies obtained were between 2.2 x 10(-1) and 2.5 x 10(-6) per recipient and appeared to depend on the donor-to-recipient ratio (489/1 to 0.0047/1) and not on the river temperature (12 to 19 degrees C). Controls showed that the low density of donors and recipients at the end of the experiment (3.4 x 10(2) to 7.0 x 10(5) cm-2) did not significantly affect the heterotrophic bacterial count (1.43 x 10(6) to 6.39 x 10(6) cm-2) nor the fluorescent-pseudomonad count (2.3 x 10(4) to 9.33 x 10(4) cm-2).

The Quantum II Bacterial Identification System (BID; Abbott Laboratories) is a microprocessor-based spectrophotometric system for identification within 4 to 5 h of both enteric and nonenteric gram-negative bacilli. We compared the BID with the AutoMicrobic System (AMS; Vitek Systems, Inc.), using the most recent gram-negative identification card and software (AMS-GNI), for the identification of 501 clinical isolates of gram-negative bacilli, including 382 belonging to the Enterobacteriaceae and 119 nonenteric organisms. The API 20E (Analytab Products) was used as the reference system. The BID correctly identified 375 (98.2%) of the Enterobacteriaceae isolates and 111 (93.2%) of the nonenteric isolates; the AMS-GNI correctly identified 374 (97.9%) and 115 (96.6%) isolates, respectively. The BID identified all isolates within 5 h, whereas the AMS-GNI identified only 35% within this time period. The BID performed comparably to the AMS-GNI for the identification of most gram-negative bacilli. Simplicity, speed, and relatively low reagent cost make the BID a competitive system for many clinical laboratories.

Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log10 molecular size against log10 relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates (less than or equal to 3.0 +/- 1.5%). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.

The usefulness of a test for slime production as a marker for clinically significant infections with coagulase-negative staphylococci and its implications for therapy were examined. Hospital records were reviewed for 59 patients from each of whom more than one isolate of coagulase-negative staphylococci was obtained. In patients with a prosthetic device, 81% of 59 infectious episodes were due to a slime-positive coagulase-negative staphylococci. In contrast, 22 noninfectious episodes (in which the organisms were contaminants) were equally distributed between episodes due to slime-positive or slime-negative isolates (P = .005). Only 32% of infections caused by slime-positive organisms, in contrast to 100% of infections caused by slime-negative organisms, were improved by treatment with antibiotics alone (P = .02). Prosthetic device removal in addition to antibiotic treatment significantly improved the outcome in patients with infections due to slime-positive organisms when compared with treatment with antibiotics alone (93% vs. 32% improvement; P = .00025).

A naturally occurring mercury-resistance, conjugative plasmid, designated pQM1, was isolated from a bacterial population on the surface of stones from a river using Pseudomonas aeruginosa as a recipient. This was a narrow-host-range plasmid [IncP-13; 165 MDa; Tra+, Hgr, fluorescein mercuric acetater, merbrominr, Phi(E79), UVr] confined to some Pseudomonas spp. It was used to demonstrate transfer between bacteria on stones in laboratory microcosm experiments and in situ. Transfer occurred (3.3 X 10(-1) to 6.8 X 10(-9) per recipient) at all the temperatures used (6-20 degrees C), although frequencies were lower in the cold. Nutrient status also affected transfer frequency, rich conditions promoting transfer. The presence of competing bacteria in the natural epilithon lowered transfer frequencies, but when unscrubbed stones were heat treated, transfer was enhanced, perhaps because of nutrient release from the heated epilithon.