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Context: While interleukin 2 (IL-2) is capable of inducing a marked expansion of the CD4 T-lymphocyte pool, limited data exist on whether IL-2 treatment can add significantly to the immunologic and virologic effects of potent antiretroviral therapy (ART).Objective: To determine the rate and magnitude of CD4 cell recovery and viral suppression when using a combination therapy of IL-2 and ART compared with ART alone.Design and Setting: Randomized, controlled multicenter trial conducted from April 1996 through April 1998 at 8 clinical sites in the United States.Patients: Eighty-two adult outpatients who were infected with human immunodeficiency virus (HIV) and had baseline CD4 cell counts of 200 x 10(6)/L to 500 x 10(6)/L and baseline RNA levels of fewer than 10,000 copies/mL were randomized; 78 completed the study.Interventions: Thirty-nine patients were randomly assigned to receive a combination therapy of subcutaneous IL-2 (administered in 5-day courses every 8 weeks at a starting dosage of 7.5 mIU twice per day) and ART; 43 were to receive ART therapy alone.Main Outcome Measures: Interleukin 2 safety and differential effects on CD4 cell counts, CD4 cell percentages, and plasma HIV RNA levels.Results: The mean (SD) percentage increase in CD4 cell counts at 1 year for patients who received IL-2 was 112% (113%) compared with 18% (35%) in recipients of ART alone (P<.001). Both groups had mean (SD) increases in CD4 cell percentage: from 20.4% (6.3%) to 32.3% (12.4%) for the combination therapy group compared with 20.4% (5.1%) to 23.0% (7.2%) for recipients of ART alone (P<.001). Using a sensitive viral RNA assay, mean viral load changes were -0.28 and 0.09 log(10) copies for IL-2 recipients and control patients, respectively (P=.03). Twenty (67%) of 30 evaluable patients receiving IL-2 achieved final viral loads of fewer than 50 copies/mL compared with 13 (36%) of 36 control patients (P=.02). Toxic effects were common among patients who received IL-2 and were managed with antipyretics, hydration, rest, and dosage reduction as needed.Conclusions: Intermittent therapy with IL-2 and ART produced a substantially greater increase in CD4 cells and was associated with a larger decrease in viral load than ART alone. Clinical end-point trials will be necessary to determine whether the enhanced viral suppression and CD4 cell increases associated with IL-2 therapy will translate into improved clinical outcomes. JAMA. 2000;284:183-189 [ABSTRACT FROM AUTHOR]

Background: PECOS is an ongoing study aimed to characterize long-term outcomes following pediatric SARS-CoV-2 infection., Methods: This is a cross-sectional analysis of infected and uninfected cohorts at baseline. Participants (0-21 years) with laboratory-confirmed SARS-CoV-2 infection were enrolled as infected. Uninfected were defined as individuals without history or laboratory evidence of SARS-CoV-2 infection. Outcome measures included demographics, medical history, review of symptoms, physical exam, cardiopulmonary evaluation and validated psychological and developmental surveys. Primary outcomes were cohort comparisons for abnormalities on all measures., Results: 654 participants (541 infected, 113 uninfected) completed baseline visits by June 30, 2023. Infected participants were more likely to report constitutional (OR: 2.24), HEENT (OR: 3.74); respiratory (OR: 2.41), or gastrointestinal (OR: 2.58) symptoms. Infected had worse scores in domains of Pain, Fatigue, Global Health, Physical and Cognitive functioning, Mobility and Sleep disturbances when compared to uninfected controls using Patient Reported Outcomes. Cardiopulmonary findings were similar among cohorts., Conclusions: The first report of this ongoing longitudinal study demonstrates that infected participants were more likely to report symptoms compared to uninfected controls, which may affect performance and quality of life of these individuals. Longitudinal data will increase understanding of long-term effects of SARS-CoV-2 infection in children., Clinicaltrials: gov Identifier: NCT04830852 IMPACT: This study establishes a large, diverse, prospective, longitudinal, multi-center cohort of children with history of SARS-CoV-2 infection compared to an uninfected cohort to be followed for 3 years. Cross-sectional cohort analysis at study entry showed infected participants were more likely to report constitutional, respiratory, and GI symptoms compared to uninfected controls. Infected participants were more likely to have significantly worse parent-reported performance in 6 of 10 Patient Reported Outcome Measures domains. Continued study of this cohort will help identify clinical sequelae of COVID-19, characterize the immune response to SARS-CoV-2 infection, and identify potential genetic/immunologic factors associated with long-term outcomes., Competing Interests: Competing interests: The authors declare no competing interests. Informed consent: Written Informed Consent was obtained from all participants ≥18 years of age and at least one parent or legal guardian per participant <18 years of age. Written Assent was obtained for all participants 12–17 years of age., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Extrapulmonary complications (EPCs) are common in patients hospitalized for coronavirus disease 2019 (COVID-19), but data on their clinical consequences and association with viral replication and systemic viral dissemination are lacking., Methods: Patients hospitalized for COVID-19 and enrolled in the Therapeutics for Inpatients with COVID-19 (TICO) platform trial at 114 international sites between August 2020 and November 2021 were included in a prospective cohort study. We categorized EPCs into 39 event types within 9 categories and estimated their frequency through day 28 and their association with clinical outcomes through day 90. We analyzed the association between baseline viral burden (plasma nucleocapsid antigen [N-Ag] level and upper airway viral load) and EPCs, adjusting for other baseline factors., Results: A total of 2625 trial participants were included in the study. Their median age was 57 years (interquartile range, 46-68 years), 57.7% were male, and 537 (20.5%) had ≥1 EPC. EPCs were associated with higher day-90 all-cause mortality rate (hazard ratio, 9.6 [95% confidence interval, 7.3-12.7]) after adjustment for other risk factors. The risk of EPCs increased with increasing baseline plasma N-Ag level (hazard ratio, 1.21 per log10 ng/L increase [95% confidence interval, 1.09-1.34]), and upper airway viral load (1.12 per log10 copies/mL increase [1.04-1.19), after adjustment for comorbid conditions, disease severity, inflammatory markers, and other baseline factors. Trial treatment allocation had no effect on EPC risk., Conclusions: Systemic viral dissemination as evidenced by high plasma N-Ag level and high respiratory viral burden are associated with development of EPCs in COVID-19, which in turn are associated with higher 90-day mortality rates., Competing Interests: Potential conflicts of interest. N. J. D. received institutional funding from Gilead Sciences. S. L. P. reports institutional grants from the National Institute for Health and Care Research, the European and Developing Countries Clinical Trials Partnership, the Medical Research Council, Gilead Sciences, Janssen-Cilag, and ViiV Healthcare and also serves as a member of the data and safety monitoring board for the TIPAL trial. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Serial measurement of virological and immunological biomarkers in patients admitted to hospital with COVID-19 can give valuable insight into the pathogenic roles of viral replication and immune dysregulation. We aimed to characterise biomarker trajectories and their associations with clinical outcomes., Methods: In this international, prospective cohort study, patients admitted to hospital with COVID-19 and enrolled in the Therapeutics for Inpatients with COVID-19 platform trial within the Accelerating COVID-19 Therapeutic Interventions and Vaccines programme between Aug 5, 2020 and Sept 30, 2021 were included. Participants were included from 108 sites in Denmark, Greece, Poland, Singapore, Spain, Switzerland, Uganda, the UK, and the USA, and randomised to placebo or one of four neutralising monoclonal antibodies: bamlanivimab (Aug 5 to Oct 13, 2020), sotrovimab (Dec 16, 2020, to March 1, 2021), amubarvimab-romlusevimab (Dec 16, 2020, to March 1, 2021), and tixagevimab-cilgavimab (Feb 10 to Sept 30, 2021). This trial included an analysis of 2149 participants with plasma nucleocapsid antigen, anti-nucleocapsid antibody, C-reactive protein (CRP), IL-6, and D-dimer measured at baseline and day 1, day 3, and day 5 of enrolment. Day-90 follow-up status was available for 1790 participants. Biomarker trajectories were evaluated for associations with baseline characteristics, a 7-day pulmonary ordinal outcome, 90-day mortality, and 90-day rate of sustained recovery., Findings: The study included 2149 participants. Participant median age was 57 years (IQR 46-68), 1246 (58·0%) of 2149 participants were male and 903 (42·0%) were female; 1792 (83·4%) had at least one comorbidity, and 1764 (82·1%) were unvaccinated. Mortality to day 90 was 172 (8·0%) of 2149 and 189 (8·8%) participants had sustained recovery. A pattern of less favourable trajectories of low anti-nucleocapsid antibody, high plasma nucleocapsid antigen, and high inflammatory markers over the first 5 days was observed for high-risk baseline clinical characteristics or factors related to SARS-CoV-2 infection. For example, participants with chronic kidney disease demonstrated plasma nucleocapsid antigen 424% higher (95% CI 319-559), CRP 174% higher (150-202), IL-6 173% higher (144-208), D-dimer 149% higher (134-165), and anti-nucleocapsid antibody 39% lower (60-18) to day 5 than those without chronic kidney disease. Participants in the highest quartile for plasma nucleocapsid antigen, CRP, and IL-6 at baseline and day 5 had worse clinical outcomes, including 90-day all-cause mortality (plasma nucleocapsid antigen hazard ratio (HR) 4·50 (95% CI 3·29-6·15), CRP HR 3·37 (2·30-4·94), and IL-6 HR 5·67 (4·12-7·80). This risk persisted for plasma nucleocapsid antigen and CRP after adjustment for baseline biomarker values and other baseline factors., Interpretation: Patients admitted to hospital with less favourable 5-day biomarker trajectories had worse prognosis, suggesting that persistent viral burden might drive inflammation in the pathogenesis of COVID-19, identifying patients that might benefit from escalation of antiviral or anti-inflammatory treatment., Funding: US National Institutes of Health., Competing Interests: Declaration of interests ALG reports receiving institutional funding from Novavax, institutional partnership with AstraZeneca, and being personally named as inventor on a patent used by AstraZeneca. DDM reports receiving grants from the Danish National Research Foundation. DL reports participation in a study funded by Gilead with no salary support. EM reports payments to his institution received from SciClone Pharmaceuticals, Regeneron Pharmaceuticals, Pfizer, Chemic Labs and KODA Therapeutics, and Cidara, and payment for participating in an advisory board for Basilea. KW reports honorarium for Acute Respiratory Distress Syndrome (ARDS) International Conference in 2023, and travel support from University of California San Francisco. MAM reports institutional funding by the US Department of Defence, Roche-Genetech, and Quantum Health, and personal consulting fees from Gilead, Novartis, and Johnson & Johnson. MKJ reports grants received from Regeneron, Laurent, and Gilead, honoraria from Gilead, and a leadership role with the HIV Medicine Association Board of Directors. PC reports consulting fees from Eli Lilly and Regeneron. All other authors declare no competing interests. All authors were not precluded from accessing data in the study., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Neutralizing monoclonal antibodies (nmAbs) failed to show clear benefit for hospitalized patients with coronavirus disease 2019 (COVID-19). Dynamics of virologic and immunologic biomarkers remain poorly understood., Methods: Participants enrolled in the Therapeutics for Inpatients with COVID-19 trials were randomized to nmAb versus placebo. Longitudinal differences between treatment and placebo groups in levels of plasma nucleocapsid antigen (N-Ag), anti-nucleocapsid antibody, C-reactive protein, interleukin-6, and D-dimer at enrollment, day 1, 3, and 5 were estimated using linear mixed models. A 7-point pulmonary ordinal scale assessed at day 5 was compared using proportional odds models., Results: Analysis included 2149 participants enrolled between August 2020 and September 2021. Treatment resulted in 20% lower levels of plasma N-Ag compared with placebo (95% confidence interval, 12%-27%; P < .001), and a steeper rate of decline through the first 5 days (P < .001). The treatment difference did not vary between subgroups, and no difference was observed in trajectories of other biomarkers or the day 5 pulmonary ordinal scale., Conclusions: Our study suggests that nmAb has an antiviral effect assessed by plasma N-Ag among hospitalized patients with COVID-19, with no blunting of the endogenous anti-nucleocapsid antibody response. No effect on systemic inflammation or day 5 clinical status was observed., Clinical Trials Registration: NCT04501978., Competing Interests: Potential conflicts of interest. A. L. G. reports receiving institutional funding from Novavax, insititutaional partnership with Astra Zeneca, and reports being personally named as inventor on a patent used by Astra Zeneca. D. D. M. reports receiving grant from the Danish National research Foundation. E. M. reports payments to his institution received from SciClone Pharmaceuticals, Regeneron Pharmaceuticals, Pfizer, Chemic Labs/KODA Therapeutics, Cidara, and Leidos Biomedical Research Inc; E. M. also reports being on an advisory board for Basilea. K. W. reports receiving a honorarium for international ARDS conference and travel support from UCSF. M. A. reports a grant from NINDS. M. A. M. reports institutional grants from NHLBI/NIAID, the department of defence, California Institute of Regeneration, Roche-Genentech, and Quantum Health. M. A. M. has also received personal consulting fees from Novartis, Pliant Therapeutics, Johnson and Johnson, Gilead, and Citius Pharmaceuticals. M. K. J. reports institutional grants from Gilead, Laurent, and Regeneron; personal consulting fees from Gilead; and personal honoraria from Cooper Clinic. S. T. is funded by grants from the Australian Medical Foundation and New South Wales Health. The remaining authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: There is a clinical need for therapeutics for COVID-19 patients with acute hypoxemic respiratory failure whose 60-day mortality remains at 30-50%. Aviptadil, a lung-protective neuropeptide, and remdesivir, a nucleotide prodrug of an adenosine analog, were compared with placebo among patients with COVID-19 acute hypoxaemic respiratory failure., Methods: TESICO was a randomised trial of aviptadil and remdesivir versus placebo at 28 sites in the USA. Hospitalised adult patients were eligible for the study if they had acute hypoxaemic respiratory failure due to confirmed SARS-CoV-2 infection and were within 4 days of the onset of respiratory failure. Participants could be randomly assigned to both study treatments in a 2 × 2 factorial design or to just one of the agents. Participants were randomly assigned with a web-based application. For each site, randomisation was stratified by disease severity (high-flow nasal oxygen or non-invasive ventilation vs invasive mechanical ventilation or extracorporeal membrane oxygenation [ECMO]), and four strata were defined by remdesivir and aviptadil eligibility, as follows: (1) eligible for randomisation to aviptadil and remdesivir in the 2 × 2 factorial design; participants were equally randomly assigned (1:1:1:1) to intravenous aviptadil plus remdesivir, aviptadil plus remdesivir matched placebo, aviptadil matched placebo plus remdesvir, or aviptadil placebo plus remdesivir placebo; (2) eligible for randomisation to aviptadil only because remdesivir was started before randomisation; (3) eligible for randomisation to aviptadil only because remdesivir was contraindicated; and (4) eligible for randomisation to remdesivir only because aviptadil was contraindicated. For participants in strata 2-4, randomisation was 1:1 to the active agent or matched placebo. Aviptadil was administered as a daily 12-h infusion for 3 days, targeting 600 pmol/kg on infusion day 1, 1200 pmol/kg on day 2, and 1800 pmol/kg on day 3. Remdesivir was administered as a 200 mg loading dose, followed by 100 mg daily maintenance doses for up to a 10-day total course. For participants assigned to placebo for either agent, matched saline placebo was administered in identical volumes. For both treatment comparisons, the primary outcome, assessed at day 90, was a six-category ordinal outcome: (1) at home (defined as the type of residence before hospitalisation) and off oxygen (recovered) for at least 77 days, (2) at home and off oxygen for 49-76 days, (3) at home and off oxygen for 1-48 days, (4) not hospitalised but either on supplemental oxygen or not at home, (5) hospitalised or in hospice care, or (6) dead. Mortality up to day 90 was a key secondary outcome. The independent data and safety monitoring board recommended stopping the aviptadil trial on May 25, 2022, for futility. On June 9, 2022, the sponsor stopped the trial of remdesivir due to slow enrolment. The trial is registered with ClinicalTrials.gov, NCT04843761., Findings: Between April 21, 2021, and May 24, 2022, we enrolled 473 participants in the study. For the aviptadil comparison, 471 participants were randomly assigned to aviptadil or matched placebo. The modified intention-to-treat population comprised 461 participants who received at least a partial infusion of aviptadil (231 participants) or aviptadil matched placebo (230 participants). For the remdesivir comparison, 87 participants were randomly assigned to remdesivir or matched placebo and all received some infusion of remdesivir (44 participants) or remdesivir matched placebo (43 participants). 85 participants were included in the modified intention-to-treat analyses for both agents (ie, those enrolled in the 2 x 2 factorial). For the aviptadil versus placebo comparison, the median age was 57 years (IQR 46-66), 178 (39%) of 461 participants were female, and 246 (53%) were Black, Hispanic, Asian or other (vs 215 [47%] White participants). 431 (94%) of 461 participants were in an intensive care unit at baseline, with 271 (59%) receiving high-flow nasal oxygen or non-invasive ventiliation, 185 (40%) receiving invasive mechanical ventilation, and five (1%) receiving ECMO. The odds ratio (OR) for being in a better category of the primary efficacy endpoint for aviptadil versus placebo at day 90, from a model stratified by baseline disease severity, was 1·11 (95% CI 0·80-1·55; p=0·54). Up to day 90, 86 participants in the aviptadil group and 83 in the placebo group died. The cumulative percentage who died up to day 90 was 38% in the aviptadil group and 36% in the placebo group (hazard ratio 1·04, 95% CI 0·77-1·41; p=0·78). The primary safety outcome of death, serious adverse events, organ failure, serious infection, or grade 3 or 4 adverse events up to day 5 occurred in 146 (63%) of 231 patients in the aviptadil group compared with 129 (56%) of 230 participants in the placebo group (OR 1·40, 95% CI 0·94-2·08; p=0·10)., Interpretation: Among patients with COVID-19-associated acute hypoxaemic respiratory failure, aviptadil did not significantly improve clinical outcomes up to day 90 when compared with placebo. The smaller than planned sample size for the remdesivir trial did not permit definitive conclusions regarding safety or efficacy., Funding: National Institutes of Health., Competing Interests: Declaration of interests SMB reports funding from the National Institutes of Health during the conduct of the study and chairing a Data and Safety Monitoring Board (DSMB) for Hamilton Ventilators, outside of the study. CEB reports funding from the National Institutes of Health (NIH) for the Aviptadil study, during the conduct of the study. BG reports grants from the NIH, during the conduct of the study. SS reports a grant from the NIH, during the conduct of the study. ANP reports grants from the Wellcome Trust, the National Institute for Health and Care Research, UK Research and Innovation (UKRI), and the Bill & Melinda Gates Foundation, and consulting fees from the Bill & Melinda Gates Foundation, outside of the submitted work. IDP reports funding from NIH and the National Institute of General Medical Sciences, during the conduct of the study, a grant from Janssen for a study of influenza patient reported outcomes, and a contract with Regeneron for a COVID-19 therapy trial, outside of the submitted work. JRB reports a grant from the NIH, during the conduct of the study, grants from the NIH, Quantum Leap Healthcare Collaborative, and Sedana Medical, consulting fees from Sedana Medical, Biomarck, and Global Blood Therapeutics, and compensation from Hamilton Medical for participation as a medical monitor, outside of the submitted work. ESHa reports study materials from NeuroRx and Gilead through a National Heart, Lung, and Blood Institute (NHLBI) subcontract, during the conduct of the study, subcontracts with Bristol Meyers Squibb (BMS), Allergan, Gilead, and Janssen for the Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV-1) clinical trial, subcontracts with AstraZeneca, Brii Biosciences, Vir Biotechnology, and Eli Lilly for the Therapeutics for Inpatients with COVID-19 (TICO) clinical trial, and additional subcontracts with Rigel, APEIRON Biologics, and Trevena for the Novel Experimental COVID-19 Therapies Affecting Host Response (NECTAR) clinical trial, outside of the submitted work. EAM reports study materials from NeuroRx and Gilead through an NHLBI subcontract, during the conduct of the study, subcontracts with BMS, Allergan, Gilead, and Janssen for the ACTIV-1 clinical trial, subcontracts with AstraZeneca, Brii Biosciences, Vir Biotechnology, and Eli Lilly for the TICO clinical trial, and additional subcontracts with Rigel, APEIRON Biologics, and Trevena for the NECTAR clinical trial, outside of the submitted work. MAGB reports study materials from NeuroRx and Gilead through an NHLBI subcontract, during the conduct of the study, subcontracts with BMS, Allergan, Gilead, and Janssen for the ACTIV-1 clinical trial, subcontracts with AstraZeneca, Brii Biosciences, Vir Biotechnology, and Eli Lilly for the TICO clinical trial, and subcontracts with Rigel, APEIRON Biologics, and Trevena for the NECTAR clinical trial, outside of the submitted work. KSM reports grants and contracts from NIH, NHLBI, and the Society for Critical Care Medicine, participation as a steering committee member for Roivant-Kinevant Sciences, and employment as a clinical research physician at Chiesi USA, outside of submitted work. JM reports receiving study materials and funding from the Albert Einstein College of Medicine for the study protocol, during the conduct of the study. CH reports funding from the National Institute of Allergy and Infectious Diseases (NIAID) in the form of per-patient payments for A Multicenter, Adaptive, Randomized, Blinded Controlled Trial of the Safety and Efficacy of Investigational Therapeutics for Hospitalized Patients with Acute Respiratory Distress Syndrome Associated with COVID-19, during the conduct of the study. AK reports grants from Eli Lilly, AstraZeneca, 4D Medical, United Therapeutics, Regeneron Pharmaceuticals, and Dompe Pharmaceuticals and consulting fees from Dompe Pharmaceuticals for clinical trial design, outside of the submitted work. AD reports grants from NHLBI Prevention and Early Treatment of Acute Lung Injury network and the US Centers for Disease Control and Prevention (CDC) and data safety monitoring board (DSMB) or advisory board participation for Alung Technologies, outside of the submitted work. SD reports a grant from Chest Sonosite Ultrasound to study the incidence of deep vein thrombosis in patients with COVID-19, during the conduct of the study. AJG reports payment from Sound Pharmaceuticals for participation as a medical monitor for a COVID therapeutic trial, outside of the submitted work. USS reports consulting fees from Shionogi, Paratek, and ViiV Healthcare for participation on advisory boards and speaking fees from Shionogi and Paratek, outside of the submitted work. NJJ reports grants from the CDC, the US Department of Defense (DOD), and NIH and participation on a DSMB for the Legacy Health System, outside of the submitted work. MAM reports grants from NIH and NIAID, during the conduct of the study. NRA reports funding from NIH, during the conduct of the study. JDC reports grants from NIH and DOD, outside of the submitted work. DDM reports funding from the Danish National Research Foundation (DNRF126), during the conduct of study. AAG reports funding from NIH, during the conduct of the study, grants or contracts from NIH, DOD, CDC, Faron Pharmaceuticals, and Abbvie, and participation on a DSMB or advisory board for NIH, outside of the submitted work. WHS reports funding from NIH and NIAID, during the conduct of the study. CFO reports contracts with NIH and NHLBI, outside of the submitted work. BTT reports a grant from NHLBI, consulting fees from Bayer, Genetec, and Novartis, and participation on a DSMB or advisory board for Aperion, outside of the submitted work. During a portion of this research, BTT had a financial interest in Direct Biologics, a developer and manufacturer of regenerative biologic products, including an investigational treatment of COVID-19-associated ARDS. BTT's interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies, and had no relationship to the agents studied. VK reports subcontracts with University of Minnesota, NIAID, and NIH for the TICO and Therapeutics for Severely Ill Inpatients with COVID-19 platform trials, outside of the submitted work. AGB reports a grant from University of Minnesota, during the conduct of the study, grants from the Medical Reserve Corps and UKRI, payment from NIAID for participation on a COVID-19 Vaccines DSMB, and participation on the WHO Trial Data and Safety Monitoring Committee, outside of the submitted work. JDN reports grants from NIAID, NIH, and Leidos Biomedical, outside of the submitted work. HCL reports employment from NIAID, during the conduct of the study. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The levels of immune response to SARS-CoV-2 infection or vaccination are poorly understood in African populations and is complicated by cross-reactivity to endemic pathogens as well as differences in host responsiveness. To begin to determine the best approach to minimize false positive antibody levels to SARS-CoV-2 in an African population, we evaluated three commercial assays, namely Bio-Rad Platelia SARS-CoV-2 Total Antibody (Platelia), Quanterix Simoa Semi-Quantitative SARS-CoV-2 IgG Antibody Test (anti-Spike), and the GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (cPass) using samples collected in Mali in West Africa prior to the emergence of SARS-CoV-2. A total of one hundred samples were assayed. The samples were categorized in two groups based on the presence or absence of clinical malaria. Overall, thirteen out of one hundred (13/100) samples were false positives with the Bio-Rad Platelia assay and one of the same one hundred (1/100) was a false positive with the anti-Spike IgG Quanterix assay. None of the samples tested with the GenScript cPass assay were positive. False positives were more common in the clinical malaria group, 10/50 (20%) vs. the non-malaria group 3/50 (6%); p = 0.0374 using the Bio-Rad Platelia assay. Association between false positive results and parasitemia by Bio-Rad remained evident, after adjusting for age and sex in multivariate analyses. In summary, the impact of clinical malaria on assay performance appears to depend on the assay and/or antigen being used. A careful evaluation of any given assay in the local context is a prerequisite for reliable serological assessment of anti-SARS-CoV-2 humoral immunity., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Nirmatrelvir/ritonavir, the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease inhibitor, reduces the risk of hospitalization and death by coronavirus disease 2019 (COVID-19) but has been associated with symptomatic rebound after therapy completion., Methods: Six individuals with relapse of COVID-19 symptoms after treatment with nirmatrelvir/ritonavir, 2 individuals with rebound symptoms without prior antiviral therapy and 7 patients with acute Omicron infection (controls) were studied. Soluble biomarkers and serum SARS-CoV-2 nucleocapsid protein were measured. Nasal swabs positive for SARS-CoV-2 underwent viral isolation and targeted viral sequencing. SARS-CoV-2 anti-spike, anti-receptor-binding domain, and anti-nucleocapsid antibodies were measured. Surrogate viral neutralization tests against wild-type and Omicron spike protein, as well as T-cell stimulation assays, were performed., Results: High levels of SARS-CoV-2 anti-spike immunoglobulin G (IgG) antibodies were found in all participants. Anti-nucleocapsid IgG and Omicron-specific neutralizing antibodies increased in patients with rebound. Robust SARS-CoV-2-specific T-cell responses were observed, higher in rebound compared with early acute COVID-19 patients. Inflammatory markers mostly decreased during rebound. Two patients sampled longitudinally demonstrated an increase in activated cytokine-producing CD4+ T cells against viral proteins. No characteristic resistance mutations were identified. SARS-CoV-2 was isolated by culture from 1 of 8 rebound patients; Polybrene addition increased this to 5 of 8., Conclusions: Nirmatrelvir/ritonavir treatment does not impede adaptive immune responses to SARS-CoV-2. Clinical rebound corresponds to development of a robust antibody and T-cell immune response, arguing against a high risk of disease progression. The presence of infectious virus supports the need for isolation and assessment of longer treatment courses., Clinical Trials Registration: NCT04401436., Competing Interests: Potential conflicts of interest. E. G. reports grants or contracts from the National Science Foundation and NIH and a leadership or fiduciary role in other board, society, committee, or advocacy group for American Society for Microbiology (ASM, unpaid). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2022.)

Background: Levels of plasma SARS-CoV-2 nucleocapsid (N) antigen may be an important biomarker in patients with COVID-19 and enhance our understanding of the pathogenesis of COVID-19., Objective: To evaluate whether levels of plasma antigen can predict short-term clinical outcomes and identify clinical and viral factors associated with plasma antigen levels in hospitalized patients with SARS-CoV-2., Design: Cross-sectional study of baseline plasma antigen level from 2540 participants enrolled in the TICO (Therapeutics for Inpatients With COVID-19) platform trial from August 2020 to November 2021, with additional data on day 5 outcome and time to discharge., Setting: 114 centers in 10 countries., Participants: Adults hospitalized for acute SARS-CoV-2 infection with 12 days or less of symptoms., Measurements: Baseline plasma viral N antigen level was measured at a central laboratory. Delta variant status was determined from baseline nasal swabs using reverse transcriptase polymerase chain reaction. Associations between baseline patient characteristics and viral factors and baseline plasma antigen levels were assessed using both unadjusted and multivariable modeling. Association between elevated baseline antigen level of 1000 ng/L or greater and outcomes, including worsening of ordinal pulmonary scale at day 5 and time to hospital discharge, were evaluated using logistic regression and Fine-Gray regression models, respectively., Results: Plasma antigen was below the level of quantification in 5% of participants at enrollment, and 1000 ng/L or greater in 57%. Baseline pulmonary severity of illness was strongly associated with plasma antigen level, with mean plasma antigen level 3.10-fold higher among those requiring noninvasive ventilation or high-flow nasal cannula compared with room air (95% CI, 2.22 to 4.34). Plasma antigen level was higher in those who lacked antispike antibodies (6.42 fold; CI, 5.37 to 7.66) and in those with the Delta variant (1.73 fold; CI, 1.41 to 2.13). Additional factors associated with higher baseline antigen level included male sex, shorter time since hospital admission, decreased days of remdesivir, and renal impairment. In contrast, race, ethnicity, body mass index, and immunocompromising conditions were not associated with plasma antigen levels. Plasma antigen level of 1000 ng/L or greater was associated with a markedly higher odds of worsened pulmonary status at day 5 (odds ratio, 5.06 [CI, 3.41 to 7.50]) and longer time to hospital discharge (median, 7 vs. 4 days; subhazard ratio, 0.51 [CI, 0.45 to 0.57]), with subhazard ratios similar across all levels of baseline pulmonary severity., Limitations: Plasma samples were drawn at enrollment, not hospital presentation. No point-of-care test to measure plasma antigen is currently available., Conclusion: Elevated plasma antigen is highly associated with both severity of pulmonary illness and clinically important patient outcomes. Multiple clinical and viral factors are associated with plasma antigen level at presentation. These data support a potential role of ongoing viral replication in the pathogenesis of SARS-CoV-2 in hospitalized patients., Primary Funding Source: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

In response to the COVID-19 pandemic, COVID-19 vaccines have been developed, and the World Health Oraganization (WHO) has granted emergency use listing to multiple vaccines. Studies of vaccine immunogenicity data from implementing COVID-19 vaccines by national immunization programs in single studies spanning multiple countries and continents are limited but critically needed to answer public health questions on vaccines, such as comparing immune responses to different vaccines and among different populations., Competing Interests: The authors have declared that no competing interests exist.

Background: Ensovibep (MP0420) is a designed ankyrin repeat protein, a novel class of engineered proteins, under investigation as a treatment of SARS-CoV-2 infection., Objective: To investigate if ensovibep, in addition to remdesivir and other standard care, improves clinical outcomes among patients hospitalized with COVID-19 compared with standard care alone., Design: Double-blind, randomized, placebo-controlled, clinical trial. (ClinicalTrials.gov: NCT04501978)., Setting: Multinational, multicenter trial., Participants: Adults hospitalized with COVID-19., Intervention: Intravenous ensovibep, 600 mg, or placebo., Measurements: Ensovibep was assessed for early futility on the basis of pulmonary ordinal scores at day 5. The primary outcome was time to sustained recovery through day 90, defined as 14 consecutive days at home or place of usual residence after hospital discharge. A composite safety outcome that included death, serious adverse events, end-organ disease, and serious infections was assessed through day 90., Results: An independent data and safety monitoring board recommended that enrollment be halted for early futility after 485 patients were randomly assigned and received an infusion of ensovibep ( n  = 247) or placebo ( n  = 238). The odds ratio (OR) for a more favorable pulmonary outcome in the ensovibep (vs. placebo) group at day 5 was 0.93 (95% CI, 0.67 to 1.30; P  = 0.68; OR > 1 would favor ensovibep). The 90-day cumulative incidence of sustained recovery was 82% for ensovibep and 80% for placebo (subhazard ratio [sHR], 1.06 [CI, 0.88 to 1.28]; sHR > 1 would favor ensovibep). The primary composite safety outcome at day 90 occurred in 78 ensovibep participants (32%) and 70 placebo participants (29%) (HR, 1.07 [CI, 0.77 to 1.47]; HR < 1 would favor ensovibep)., Limitation: The trial was prematurely stopped because of futility, limiting power for the primary outcome., Conclusion: Compared with placebo, ensovibep did not improve clinical outcomes for hospitalized participants with COVID-19 receiving standard care, including remdesivir; no safety concerns were identified., Primary Funding Source: National Institutes of Health.

Background: A substantial proportion of persons who develop COVID-19 report persistent symptoms after acute illness. Various pathophysiologic mechanisms have been implicated in the pathogenesis of postacute sequelae of SARS-CoV-2 infection (PASC)., Objective: To characterize medical sequelae and persistent symptoms after recovery from COVID-19 in a cohort of disease survivors and controls., Design: Cohort study. (ClinicalTrials.gov: NCT04411147)., Setting: National Institutes of Health Clinical Center, Bethesda, Maryland., Participants: Self-referred adults with laboratory-documented SARS-CoV-2 infection who were at least 6 weeks from symptom onset were enrolled regardless of presence of PASC. A control group comprised persons with no history of COVID-19 or serologic evidence of SARS-CoV-2 infection, recruited regardless of their current health status. Both groups were enrolled over the same period and from the same geographic area., Measurements: All participants had the same evaluations regardless of presence of symptoms, including physical examination, laboratory tests and questionnaires, cognitive function testing, and cardiopulmonary evaluation. A subset also underwent exploratory immunologic and virologic evaluations., Results: 189 persons with laboratory-documented COVID-19 (12% of whom were hospitalized during acute illness) and 120 antibody-negative control participants were enrolled. At enrollment, symptoms consistent with PASC were reported by 55% of the COVID-19 cohort and 13% of control participants. Increased risk for PASC was noted in women and those with a history of anxiety disorder. Participants with findings meeting the definition of PASC reported lower quality of life on standardized testing. Abnormal findings on physical examination and diagnostic testing were uncommon. Neutralizing antibody levels to spike protein were negative in 27% of the unvaccinated COVID-19 cohort and none of the vaccinated COVID-19 cohort. Exploratory studies found no evidence of persistent viral infection, autoimmunity, or abnormal immune activation in participants with PASC., Limitations: Most participants with COVID-19 had mild to moderate acute illness that did not require hospitalization. The prevalence of reported PASC was likely overestimated in this cohort because persons with PASC may have been more motivated to enroll. The study did not capture PASC that resolved before enrollment., Conclusion: A high burden of persistent symptoms was observed in persons after COVID-19. Extensive diagnostic evaluation revealed no specific cause of reported symptoms in most cases. Antibody levels were highly variable after COVID-19., Primary Funding Source: Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Motivation: The existence of quasispecies in the viral population causes difficulties for disease prevention and treatment. High-throughput sequencing provides opportunity to determine rare quasispecies and long sequencing reads covering full genomes reduce quasispecies determination to a clustering problem. The challenge is high similarity of quasispecies and high error rate of long sequencing reads., Results: We developed QuasiSeq using a novel signature-based self-tuning clustering method, SigClust, to profile viral mixtures with high accuracy and sensitivity. QuasiSeq can correctly identify quasispecies even using low-quality sequencing reads (accuracy <80%) and produce quasispecies sequences with high accuracy (≥99.55%). Using high-quality circular consensus sequencing reads, QuasiSeq can produce quasispecies sequences with 100% accuracy. QuasiSeq has higher sensitivity and specificity than similar published software. Moreover, the requirement of the computational resource can be controlled by the size of the signature, which makes it possible to handle big sequencing data for rare quasispecies discovery. Furthermore, parallel computation is implemented to process the clusters and further reduce the runtime. Finally, we developed a web interface for the QuasiSeq workflow with simple parameter settings based on the quality of sequencing data, making it easy to use for users without advanced data science skills., Availability and Implementation: QuasiSeq is open source and freely available at https://github.com/LHRI-Bioinformatics/QuasiSeq. The current release (v1.0.0) is archived and available at https://zenodo.org/badge/latestdoi/340494542., Supplementary Information: Supplementary data are available at Bioinformatics online., (Published by Oxford University Press 2022.)

BackgroundThe value of the soluble receptor for advanced glycation end-products (sRAGE) as a biomarker in COVID-19 is not well understood. We tested the association between plasma sRAGE and illness severity, viral burden, and clinical outcomes in hospitalized patients with COVID-19 who were not mechanically ventilated.MethodsBaseline sRAGE was measured among participants enrolled in the ACTIV-3/TICO trial of bamlanivimab for hospitalized patients with COVID-19. Spearman's rank correlation was used to assess the relationship between sRAGE and other plasma biomarkers, including viral nucleocapsid antigen. Fine-Gray models adjusted for baseline supplemental oxygen requirement, antigen level, positive endogenous anti-nucleocapsid antibody response, sex, age, BMI, diabetes mellitus, renal impairment, corticosteroid treatment, and log2-transformed IL-6 level were used to assess the association between baseline sRAGE and time to sustained recovery. Cox regression adjusted for the same factors was used to assess the association between sRAGE and mortality.ResultsAmong 277 participants, baseline sRAGE was strongly correlated with viral plasma antigen concentration (ρ = 0.57). There was a weaker correlation between sRAGE and biomarkers of systemic inflammation, such as IL-6 (ρ = 0.36) and CRP (ρ = 0.20). Participants with plasma sRAGE in the highest quartile had a significantly lower rate of sustained recovery (adjusted recovery rate ratio, 0.64 [95% CI, 0.43-0.90]) and a higher unadjusted risk of death (HR, 4.70 [95% CI, 2.01-10.99]) compared with participants in the lower quartiles.ConclusionElevated plasma sRAGE in hospitalized, nonventilated patients with COVID-19 was an indicator of both clinical illness severity and plasma viral load. Plasma sRAGE in the highest quartile was associated with a lower likelihood of sustained recovery and higher unadjusted risk of death. These findings, which we believe to be novel, indicate that plasma sRAGE may be a promising biomarker for COVID-19 prognostication and clinical trial enrichment.Trial RegistrationClinicalTrials.gov NCT04501978.FundingNIH (5T32GM008440-24, 18X107CF6, HHSN261201500003I, R35HL140026, and OT2HL156812).

Background: In a randomized, placebo-controlled, clinical trial, bamlanivimab, a SARS-CoV-2-neutralizing monoclonal antibody, given in combination with remdesivir, did not improve outcomes among hospitalized patients with COVID-19 based on an early futility assessment., Objective: To evaluate the a priori hypothesis that bamlanivimab has greater benefit in patients without detectable levels of endogenous neutralizing antibody (nAb) at study entry than in those with antibodies, especially if viral levels are high., Design: Randomized, placebo-controlled trial. (ClinicalTrials.gov: NCT04501978)., Setting: Multicenter trial., Patients: Hospitalized patients with COVID-19 without end-organ failure., Intervention: Bamlanivimab (7000 mg) or placebo., Measurements: Antibody, antigen, and viral RNA levels were centrally measured on stored specimens collected at baseline. Patients were followed for 90 days for sustained recovery (defined as discharge to home and remaining home for 14 consecutive days) and a composite safety outcome (death, serious adverse events, organ failure, or serious infections)., Results: Among 314 participants (163 receiving bamlanivimab and 151 placebo), the median time to sustained recovery was 19 days and did not differ between the bamlanivimab and placebo groups (subhazard ratio [sHR], 0.99 [95% CI, 0.79 to 1.22]; sHR > 1 favors bamlanivimab). At entry, 50% evidenced production of anti-spike nAbs; 50% had SARS-CoV-2 nucleocapsid plasma antigen levels of at least 1000 ng/L. Among those without and with nAbs at study entry, the sHRs were 1.24 (CI, 0.90 to 1.70) and 0.74 (CI, 0.54 to 1.00), respectively (nominal P for interaction = 0.018). The sHR (bamlanivimab vs. placebo) was also more than 1 for those with plasma antigen or nasal viral RNA levels above median level at entry and was greatest for those without antibodies and with elevated levels of antigen (sHR, 1.48 [CI, 0.99 to 2.23]) or viral RNA (sHR, 1.89 [CI, 1.23 to 2.91]). Hazard ratios for the composite safety outcome (<1 favors bamlanivimab) also differed by serostatus at entry: 0.67 (CI, 0.37 to 1.20) for those without and 1.79 (CI, 0.92 to 3.48) for those with nAbs., Limitation: Subgroup analysis of a trial prematurely stopped because of futility; small sample size; multiple subgroups analyzed., Conclusion: Efficacy and safety of bamlanivimab may differ depending on whether an endogenous nAb response has been mounted. The limited sample size of the study does not allow firm conclusions based on these findings, and further independent trials are required that assess other types of passive immune therapies in the same patient setting., Primary Funding Source: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus.

Background: LY-CoV555, a neutralizing monoclonal antibody, has been associated with a decrease in viral load and the frequency of hospitalizations or emergency department visits among outpatients with coronavirus disease 2019 (Covid-19). Data are needed on the effect of this antibody in patients who are hospitalized with Covid-19., Methods: In this platform trial of therapeutic agents, we randomly assigned hospitalized patients who had Covid-19 without end-organ failure in a 1:1 ratio to receive either LY-CoV555 or matching placebo. In addition, all the patients received high-quality supportive care as background therapy, including the antiviral drug remdesivir and, when indicated, supplemental oxygen and glucocorticoids. LY-CoV555 (at a dose of 7000 mg) or placebo was administered as a single intravenous infusion over a 1-hour period. The primary outcome was a sustained recovery during a 90-day period, as assessed in a time-to-event analysis. An interim futility assessment was performed on the basis of a seven-category ordinal scale for pulmonary function on day 5., Results: On October 26, 2020, the data and safety monitoring board recommended stopping enrollment for futility after 314 patients (163 in the LY-CoV555 group and 151 in the placebo group) had undergone randomization and infusion. The median interval since the onset of symptoms was 7 days (interquartile range, 5 to 9). At day 5, a total of 81 patients (50%) in the LY-CoV555 group and 81 (54%) in the placebo group were in one of the two most favorable categories of the pulmonary outcome. Across the seven categories, the odds ratio of being in a more favorable category in the LY-CoV555 group than in the placebo group was 0.85 (95% confidence interval [CI], 0.56 to 1.29; P = 0.45). The percentage of patients with the primary safety outcome (a composite of death, serious adverse events, or clinical grade 3 or 4 adverse events through day 5) was similar in the LY-CoV555 group and the placebo group (19% and 14%, respectively; odds ratio, 1.56; 95% CI, 0.78 to 3.10; P = 0.20). The rate ratio for a sustained recovery was 1.06 (95% CI, 0.77 to 1.47)., Conclusions: Monoclonal antibody LY-CoV555, when coadministered with remdesivir, did not demonstrate efficacy among hospitalized patients who had Covid-19 without end-organ failure. (Funded by Operation Warp Speed and others; TICO ClinicalTrials.gov number, NCT04501978.)., (Copyright © 2020 Massachusetts Medical Society.)

Hygienic behaviour is a social immune response in honey bees shown to help provide resistance to honey bee pests and diseases. A survey of hygienic behaviour and brood diseases was conducted on 649 colonies in eastern Australia to initiate a selective breeding program targeting disease resistance and provide a level of resistance to Varroa (Varroa destructor Anderson and Trueman and V. jacobsoni Oudemans) mites should they become established in Australia. The test population showed a remarkably high baseline level of hygienic behaviour with 17% of colonies meeting or exceeding breeding selection thresholds. Colonies belonging to a breeding program were 5.8 times more likely to be highly hygienic and colonies headed by queens raised from hygienic queen mothers were 2.2 times more likely. Nectar availability (nectar yielding flowering plants within honey bee forage range) influenced hygienic behaviour expression but was not a significant predictor of level of hygienic behaviour. Surprisingly, hygienic behaviour was not a significant predictor of the presence of infection of the honey bee brood disease chalkbrood (Ascosphaera apis) and was not influential in predicting severity of chalkbrood infection in surveyed honey bee colonies. This study, along with reports from commercial beekeepers that chalkbrood infection is on the rise, warrants a deeper exploration of the host-pathogen relationship between Apis mellifera and Ascosphaera apis in Australia., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: R.L Dewar is head queen bee breeder of Dewar Apiaries and sells hygienic breeder queens. BeesDownunder, a commercial funder, has not had any financial, or otherwise, gain from this study. These competing interests do not alter our adherence to PLOS ONE policies on sharing data and materials. No other authors or funders have competing interests to declare.

Background: The hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development. It is unknown if the results can be influenced by sample type and anticoagulants. The purpose of this study was to evaluate the influence of different sample collection methods, in particular different anticoagulants, and choice of plasma or serum, on influenza virus serological assays., Methods: Blood samples from thirty donors previously immunized against influenza viruses were collected using six different types of blood collection tubes, two of which collect serum and four of which contain various anticoagulants for collecting plasma. Serum: (1) serum separator tubes (SST); and (2) Plus Plastic serum "red-top serum" tubes. Plasma: (3) spray-coated K2 ethylenediaminetetraacetic acid (EDTA) tubes: (4) Sodium Heparin tubes; (5) Citrate tubes with 3.2% sodium citrate solution; and (6) Glass Blood Collection tubes with acid citrate dextrose. Samples were tested against three different influenza viruses (A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012) for hemagglutination inhibition titer and virus neutralization titer via a microneutralization (MN) assay, and data compared to that obtained for standard serum sample collected in SST., Results: HAI and MN titers against type A viruses were within two dilutions compared to SST collection method over 96% of the time irrespective of sample type or anticoagulant. However, HAI titers for type B virus were more variable across different collection methods. EDTA plasma samples were greater than two dilutions higher than SST serum samples 70% (21 of 30 samples) of the time. In contrast, MN titers were within two dilutions over 96% of the time, with the highest deviation noted in acid citrate dextrose plasma samples (3 of 30 samples tested, 10%)., Conclusions: These data provide useful guidelines for sample collection and serology testing when screening: (i) influenza vaccine immunogenicity antibody response; (ii) antibody responses to newly emerging viral strains; and (iii) clinical samples for anti-influenza antibody activity.

Objective: IL-15 has been postulated to play an important role in HIV-1 infection, yet there are conflicting reports regarding its expression levels in these patients. We sought to measure the level of IL-15 in a large, well characterised cohort of HIV-1 infected patients and correlate this with well known markers of inflammation, including CRP, D-dimer, sCD163 and sCD14., Design and Methods: IL-15 levels were measured in 501 people (460 patients with HIV-1 infection and 41 uninfected controls). The HIV-1 infected patients were divided into 4 groups based on viral load: <50 copies/ml, 51-10,000 copies/ml, 10,001-100,000 copies/ml and >100,000 copies/ml. The Mann Whitney test (non-parametric) was used to identify significant relationships between different patient groups., Results: IL-15 levels were significantly higher in patients with viral loads >100,000 copies/ml (3.02 ± 1.53 pg/ml) compared to both uninfected controls (1.69 ± 0.37 pg/ml, p<0.001) or patients with a viral load <50 copies/ml (1.59 ± 0.40 pg/ml (p<0.001). There was a significant correlation between HIV-1 viremia and IL-15 levels (Spearman r = 0.54, p<0.001) and between CD4+ T cell counts and IL-15 levels (Spearman r = -0.56, p<0.001)., Conclusions: IL-15 levels are significantly elevated in HIV-1 infected patients with viral loads >100,000 copies/ml compared to uninfected controls, with a significant direct correlation noted between IL-15 and HIV-1 viremia and an inverse correlation between IL-15 levels and CD4+ T cell counts. These data support a potential role for IL-15 in the pathogenesis of HIV-associated immune activation., Competing Interests: The authors have declared that no competing interests exist.

Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5'LTR-to-3'LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of "defective" proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not "silent," but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient's HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients' HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.

We describe a patient with two recent episodes of tenofovir disoproxil fumarate (TDF)-associated acute kidney injury and six-class drug-resistant HIV infection who achieved and maintained viral suppression without worsening kidney function on a regimen including tenofovir alafenamide (TAF) through 48 weeks of therapy. The safety and efficacy of TAF in patients with TDF-associated renal tubulopathy and multiple drug resistant HIV has not yet been described. TAF may represent a useful option to maximally suppress HIV in patients with these complications.

Objective: IL-27 is an immunomodulatory cytokine with potent anti-HIV properties in PBMCs, CD4+ T cells, macrophages and immature dendritic cells. Previous smaller studies have suggested that HIV-1 infection may alter IL-27 and influence HIV-1 pathogenesis. The aim of this study was to examine the relationship between plasma IL-27 levels in a well-characterised cohort of HIV-1 infected patients., Methods: Patients were stratified into four groups based on HIV-1 viral load and matched according to age, gender and those receiving antiretroviral treatment. IL-27 levels and C-reactive protein (CRP) were measured using electrochemiluminescence assays. D-dimer and CD4+ T cell counts were measured using an Enzyme Linked Fluorescence Assay and FACS, respectively. sCD14 and sCD163 were measured using ELISA. HIV-1 viral load was measured by bDNA or qRT-PCR assays., Results: Plasma IL-27 levels were measured in 505 patients (462 HIV+, 43 controls). The mean level (±SEM) of IL-27 in controls was 2990.7±682.1 pg/ml, in the <50 copies/ml group it was 2008.0±274.8 pg/ml, in the 51-10,000 copies group it was 1468.7±172.3 pg/ml, in the 10,001-100,000 copies/ml group it was 1237.9±127.3 pg/ml and in the >100,000 copies/ml group it was 1590.1±223.7 pg/ml. No statistically significant difference in IL-27 levels between groups were seen. There were no correlations noted between IL-27 and HIV-1 viral load or CD4+ T cell counts. There was a small correlation noted between D-dimer and IL-27 (Spearman r = 0.09, p = 0.03) and sCD163 and IL-27 (Spearman r = 0.12, p = 0.005). No correlation was observed between IL-27 and CRP or sCD14 levels., Conclusions: This is the largest study examining the levels of plasma IL-27 in HIV-1 infection. While IL-27 levels are not significantly altered in HIV-1 infection compared to uninfected controls there may be a small association between IL-27 and D-dimer levels and IL-27 and sCD163 levels.

We report a case of AIDS presenting as varicella-zoster virus (VZV) meningomyeloradiculitis associated with human immunodeficiency virus (HIV) quasispecies compartmentalization within the cerebrospinal fluid (CSF), and a CSF viral load that was 1 log higher than in peripheral blood. Prolonged antiviral therapy for both VZV and HIV type 1 was associated with partial resolution.

We show that the self-organized single-helical-axis (SHAx) and double-axis (DAx) states in reversed field pinches can be reproduced in a minimally constrained equilibrium model using only five parameters. This is a significant reduction on previous representations of the SHAx which have required an infinite number of constraints. The DAx state, which has a nontrivial topology, has not previously been reproduced using an equilibrium model that preserves this topological structure. We show that both states are a consequence of transport barrier formation in the plasma core, in agreement with experimental results. We take the limit of zero pressure in this work, although the model is also valid for finite pressure.

The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin β nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1-mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27-treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.

True long-term nonprogressors (LTNPs)/elite controllers (ECs) maintain durable control over HIV replication without antiretroviral therapy. Herein we describe 4 unique persons who were distinct from conventional LTNPs/ECs in that they had extraordinarily low HIV burdens and comparatively weak immune responses. As a group, typical LTNPs/ECs have unequivocally reactive HIV-1 Western blots, viral loads below the lower threshold of clinical assays, low levels of persistent viral reservoirs, an over-representation of protective HLA alleles, and robust HIV-specific CD8(+) T-cell responses. The 4 unique cases were distinguished from typical LTNPs/ECs based on weakly reactive Western blots, undetectable plasma viremia by a single copy assay, extremely low to undetectable HIV DNA levels, and difficult to isolate replication-competent virus. All 4 had at least one protective HLA allele and CD8(+) T-cell responses that were disproportionately high for the low antigen levels but comparatively lower than those of typical LTNPs/ECs. These unique persons exhibit extraordinary suppression over HIV replication, therefore, higher-level control than has been demonstrated in previous studies of LTNPs/ECs. Additional insight into the full spectrum of immune-mediated suppression over HIV replication may enhance our understanding of the associated mechanisms, which should inform the design of efficacious HIV vaccines and immunotherapies.

Nonnucleoside reverse transcriptase inhibitor-drug resistance mutations (DRM) are increasingly reported in Africans failing their first antiretroviral regimen. The Phidisa II trial randomized treatment-naive participants to lopinavir/ritonavir or efavirenz with stavudine + lamivudine or zidovudine + didanosine. We report the prevalence of DRM in subjects who achieved HIV RNA <400 copies per milliliter at 6 months, but subsequently had 2 consecutive HIV RNA >1000 copies per milliliter. Sixty-eight participants fulfilled the inclusion criteria. nonnucleoside reverse transcriptase inhibitor-DRM were found in 17 of 36 (47.2%) efavirenz recipients, and M184V/I mutation in 14 of 40 (35.0%) lamivudine recipients. No protease inhibitor mutation was identified in 38 lopinavir/ritonavir recipients. This is one of the first studies in Africa confirming the paucity of protease inhibitor-associated DRM despite virologic failure.

Compartmental differences in human immunodeficiency virus type 1 (HIV-1) between the gut and peripheral blood and within the gut were examined. Biopsy specimens from the colon and ileum and peripheral blood samples were collected from chronically HIV-1-infected individuals. HIV-1 envelope sequences were examined from cell-associated DNA and RNA and virion RNA. Phylogenetic analysis revealed no evidence of compartmentalization of HIV-1 between the gut and peripheral blood and within the gut (colon and ileum). HIV-1 sequences detected in the gut were transcriptionally active and were also found in peripheral blood from matching time points, providing evidence of ongoing virus production in the gut and equilibrium of HIV-1 between the gut and peripheral blood compartments.

Study Objective: To evaluate the effect of a previous single dose of nevirapine given to prevent mother-to-child transmission of human immunodeficiency virus (HIV) on virologic and immunologic measures after months of an antiretroviral regimen containing either efavirenz or lopinavir-ritonavir., Design: Retrospective subgroup analysis of data from the Phidisa II trial., Setting: Six South African research clinics. Patients. A total of 394 women with HIV who completed 6 months of combination antiretroviral regimen containing either efavirenz or lopinavirritonavir as part of the Phidisa II trial., Measurements and Main Results: During the screening process for the Phidisa II study, 478 women were asked about previous nevirapine use: 392 women (82%) were nevirapine naïve, and 86 (18%) had received nevirapine. During the study, patients received either an efavirenz-based or lopinavir-ritonavir- based antiretroviral regimen. After 6 months of treatment, virologic (HIV RNA levels) and immunologic (CD4(+) cell count) responses were measured. These data were compared between women with or without previous nevirapine exposure, and between women who received efavirenz versus lopinavirritonavir. After 6 months of treatment, 394 women (324 nevirapine naïve, 70 exposed to nevirapine) had follow-up HIV RNA results. Two hundred twenty-seven (70.1%) of the nevirapine-naïve patients and 48 (68.6%) of the nevirapine-exposed patients achieved HIV RNA levels lower than 400 copies/ml (p=0.89), with CD4(+) cell count increases of 115.5 and 120.4 cells/mm(3), respectively (p=0. 7). Among the nevirapine-exposed women, 27 (75%) of 36 efavirenz-treated and 21 (61.8%) of 34 lopinavir-ritonavir-treated patients had HIV RNA levels lower than 400 copies/ml at months (p=0.31)., Conclusion: In this retrospective analysis of a small cohort, previous exposure to a single dose of nevirapine did not affect virologic outcomes after 6 months of either an efavirenz-based or lopinavir-ritonavir-based antiretroviral regimen. As efavirenz is one of the first-line combination antiretroviral therapies administered in Africa, it remains an option for women who received single-dose nevirapine.

Method: In this study we sought to characterize the relationship between several pharmacokinetic and pharmacodynamic parameters and virologic responses among HIV/hepatitis C virus genotype-1 co-infected patients receiving pegylated interferon-alpha-2b (peg-IFN2b) and ribavirin. We also tried to establish the underlying mechanisms that lead to poor sustained virologic responder rates observed with African-Americans against Caucasians and compared their results with those observed in a cohort of hepatitis C virus mono-infected patients., Results: Among our studied population, a viral decline of more than 1.0 log at day 3 combined with viral load of less than 5.0 log IU/ml at day 28 predicted sustained virologic responders with negative predictive value 100% and positive predictive value 100%. African-Americans had significantly (P < 0.01) slower hepatitis C virus viral kinetics as compared to Caucasians. However, peg-IFN2b concentrations and pharmacokinetic parameters, peg-IFN2b(max) and peg-IFN2b half-life, were similar in both groups and did not predict sustained virologic responders. Nevertheless, the pharmacodynamic parameter EC(50), estimated from nonlinear fitting of the viral kinetics together with peg-IFN2b concentration data, showed that HIV/ hepatitis C virus co-infected African-Americans have lower sensitivity to interferon-alpha thus giving rise to slower viral decline. The combined pharmacokinetic/pharmacodynamic parameter IFN(max)/EC(90) was an excellent predictor of sustained virologic responders, thus showing the importance of maintaining peg-IFN2b levels above EC(90) to achieve successful treatment., Conclusion: Further studies are needed to evaluate whether these pharmacodynamic predictions are a result of differential host response to peg-IFN2b or other viral factors conferring relative resistance to peg-IFN2b.

Abstract Multiple HIV-1 subtypes and circulating recombinant forms (CRFs) are known to cocirculate in Africa. In West Africa, the high prevalence of CRF02&#95;AG, and cocirculation of subtype A, CRF01&#95;AE, CRF06&#95;cpx, and other complex intersubtype recombinants has been well documented. Mali, situated in the heart of West Africa, is likely to be affected by the spread of recombinant subtypes. However, the dynamics of the spread of HIV-1 recombinant subtypes as well as nonrecombinant HIV-1 group M subtypes in this area have not been systematically assessed. Herein, we undertook genetic analyses on full-length env sequences derived from HIV-1-infected individuals living in the capital city of Mali, Bamako. Of 23 samples we examined, 16 were classified as CRF02&#95;AG and three had a subsubtype A3. Among the remaining HIV-1 strains, CRF06&#95;cpx and CRF09&#95;cpx were each found in two patients. Comparison of phylogenies for six matched pol and full-length env sequences revealed that two strains had discordant subtype/CRF designations between the pol and env regions: one had A3(pol)CRF02&#95;AG(env) and the other had CRF02&#95;AG(pol)A3(env). Taken together, our study demonstrated the high prevalence of CRF02&#95;AG and complexity of circulating HIV-1 strains in Mali. It also provided evidence of ongoing virus evolution of CRF02&#95;AG, as illustrated by the emergence of more complex CRF02&#95;AG/A3 intersubtype recombinants in this area.

Patient management frequently involves quantitative evaluation of a patient's attributes. For example, in HIV studies, a high viral load can be a trigger to initiate or modify an antiretroviral therapy. At times, a new method of evaluation may substitute for an established one, provided that the new method does not result in different clinical decisions compared with the old method. Traditional measures of agreement between the two methods are inadequate for deciding if a new method can replace the old. Especially, when the data are censored by a detection limit, estimates of agreement can be biased unless the distribution for the censored data is correctly specified; this is usually not feasible in practice. We propose a nonparametric likelihood test that seamlessly handles censored data. We further show that the proposed test is a generalization of the test on nominal measurement concordance to continuous measurement. An exact permutation procedure is proposed for implementing the test. Our application is an HIV study to determine whether one method of processing plasma samples can safely substitute for the other. The plasma samples are used to determine viral load and a large portion of data are left censored due to a lower detection limit., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Viral breakthroughs (VB), defined as having detectable HCV VL while on anti-HCV therapy after achieving maximal suppression, have not yet been characterized with the use of PEG-IFN in HIV/HCV-coinfected patients. We evaluated possible mechanisms for VB among HIV/HCV-coinfected patients receiving PEG-IFN/RBV. Thirty HIV/HCV coinfected patients were treated with PEG-IFN (1.5 mug/kg sc qwk) and RBV (1-1.2 g daily) for 48 weeks. Liver chemistry, HCV VL, genotyping, DNA microarray, and sequencing of HCV E-2 envelope were performed before and during treatment. VB had lower baseline HCV VL but higher ALT and AST than relapsers (ETR) (p < 0.05) and lower CD4+ T lymphocytes (%) than patients with sustained virological responses (SVR), but similar first and second phase HCV viral kinetics (vs. ETR and SVR; p > 0.05). HCV genotypes and envelope sequences were similar for patients with VB pretreatment and at break-through. VB had higher levels of interferon-induced gene (IFIG) expression pretreatment than patients with ETR (p < 0.01). HIV/HCV-coinfected patients have a high rate of VB on PEG-IFN/RBV therapy characterized by higher levels of IFIG expression, immunodeficiency, and hepatic inflammation. Novel strategies are required for the treatment of persons with VB.

A human immunodeficiency virus type 1 (HIV-1) derivative (HIV(NL-DT5R)) containing sequences encoding a 7-amino-acid segment of CA and the entire vif gene from simian immunodeficiency virus (SIV) was previously shown to establish spreading infections in cultured macaque peripheral blood mononuclear cells. To assess its replicative and disease-inducing properties in vivo, HIV(NL-DT5R) was inoculated into pig-tailed macaques. HIV(NL-DT5R) generated plasma viremia in all five of the monkeys and elicited humoral responses against all of the HIV-1 structural proteins but did not cause CD4(+) T-lymphocyte depletion or clinical disease. Additional adaptation will be required to optimize infectivity in vivo.

Objective: The major antiviral effect of interferon (IFN)-alpha on hepatitis C virus (HCV) is blocking of virion production from infected cells. We now investigate the previously unknown mechanism of action of IFN-alpha against HIV., Methods: HIV kinetics in parallel to HCV kinetics and IFN pharmacokinetics during pegylated-IFN-alpha-2b (1.5 microg/Kg q.w., PEG-IFN) and ribavirin (1-1.2 g daily) treatment in nine HIV patients co-infected with HCV genotype 1 were analyzed. In vivo modeling predictions of suppression of HIV replication by PEG-IFN in CD8-depleted peripheral blood mononuclear cells were verified by in vitro experiments., Results: HCV and HIV show different viral decline patterns after administration of PEG-IFN. Unlike the bi-phasic decline shown by HCV, HIV shows a slow continuous decline during the first week, with no rebound when PEG-IFN levels decline. Fitting of HIV kinetics with known half-lives of free virus and infected cells indicates that the major effect of IFN on HIV is to block de novo infection rather than to block virion production. The magnitude of the antiviral effect is similar (mean 1.1 log10 decline at 7 days) to those of direct anti-HIV drugs, but shows an inverse correlation with baseline viremia. In vitro studies show that preincubation with IFN renders a suppression of HIV replication superior to that of treatment postinfection, thus corroborating the mathematical analysis in vivo., Conclusion: The complimentary antiviral properties of IFN-alpha and antiretroviral therapy suggest a role for pharmacokinetically improved formulations of IFN as part of salvage therapy for HIV-infected individuals.

This study compared two commercially available assays for the measurement of hepatitis C virus (HCV) RNA levels, the Bayer HCV RNA (version 3.0) branched DNA assay and the Abbott HCV analyte-specific reagent real-time PCR assay, to assess their quantitative relationships, ease of performance, and time to completion. The study group consisted of randomly selected patients from the NIAID human immunodeficiency virus (HIV) outpatient clinic who were infected with HIV type 1 and HCV. One hundred eighty-four samples from 66 patients coinfected with HIV and HCV receiving treatments under various protocols were analyzed for the correlation and agreement of the results. The results indicated that the two assays correlate well in the overlapping linear ranges of the assays and show good agreement. From the results obtained, we have derived a mathematical formula to compare the viral load results between the two assays, which is given as log(10) Abbott assay measure = 0.032 + 1.01 log(10) Bayer assay measure. Although it is preferable to use the same quantitation assay throughout the course of a patient's treatment, valid comparisons of the HCV RNA levels may be made between the results obtained by either of these assays in the overlapping linear range (615 to 7,700,000 IU/ml).

Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1' position (Leu to Pro) and the P5' position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.

Background: Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy., Objective: To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy., Methods: HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10 copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis., Results: Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21-0.48%, which was 7-16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy., Conclusions: Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.

To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis.

A Suydam-unstable circular cylinder of plasma with periodic boundary conditions in the axial direction is studied within the approximation of linearized ideal magnetohydrodynamics (MHD). The normal mode equations are completely separable, so both the toroidal Fourier harmonic index n and the poloidal index m are good quantum numbers. The full spectrum of eigenvalues in the range 1< or = m < or = m(max) is analyzed quantitatively, using asymptotics for large m, numerics for all m, and graphics for qualitative understanding. The density of eigenvalues scales like m(2)(max) as m(max) -->infinity . Because finite-m corrections scale as 1/ m(2)(max) , their inclusion is essential in order to obtain the correct statistics for the distribution of eigenvalues. Near the largest growth rate, only a single radial eigenmode contributes to the spectrum, so the eigenvalues there depend only on m and n as in a two-dimensional system. However, unlike the generic separable two-dimensional system, the statistics of the ideal-MHD spectrum departs somewhat from the Poisson distribution, even for arbitrarily large m(max) . This departure from Poissonian statistics may be understood qualitatively from the nature of the distribution of rational numbers in the rotational transform profile.

More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10(6) per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

Objective: To determine the longitudinal response of HIV in the cerebrospinal fluid (CSF) to highly active antiretroviral therapy (HAART) and to investigate the levels of indinavir penetrating into the CSF., Design: Open study of HIV-infected subjects naive to therapy with protease inhibitors., Setting: Tertiary care referral center., Subjects: Twenty-five participants were begun on indinavir, nevirapine, zidovudine, and lamivudine., Interventions: Lumbar punctures were performed prior to therapy and 2 and 6 months after beginning therapy. Plasma and CSF were assayed for routine cell counts, chemistries, HIV load and indinavir levels., Results: Twenty-two subjects had CSF HIV RNA level data available at all three time points, three others at baseline and 2 months. At month 2 of therapy, nine of 25 (36%) subjects had CSF HIV RNA levels > 50 HIV RNA copies/ml. By 6 months, all 22 subjects had CSF HIV RNA levels < 50 HIV RNA copies/ml. CSF white blood cell counts fell from a baseline mean of 5.3 x 10(6)/l to 1.9 x 10(6)/l (P = 0.013) at 6 months. Plasma indinavir levels declined rapidly while CSF levels remained stable throughout the 8-h dosing interval. The median CSF indinavir level was 71 ng/ml, approximating the upper limit of the 95% inhibitory concentration for indinavir against HIV-1., Conclusions: CSF HIV RNA levels cannot be expected to fall below 50 HIV RNA copies/ml even after 2 months of therapy on HAART. Prolonged therapy may be required to suppress HIV levels within the central nervous system.

The structural properties of an economical model for a confined plasma turbulence governor are investigated through bifurcation and stability analyses. A close relationship is demonstrated between the underlying bifurcation framework of the model and typical behavior associated with low- to high-confinement transitions such as shear-flow stabilization of turbulence and oscillatory collective action. In particular, the analysis evinces two types of discontinuous transition that are qualitatively distinct. One involves classical hysteresis, governed by viscous dissipation. The other is intrinsically oscillatory and nonhysteretic, and thus provides a model for the so-called dithering transitions that are frequently observed. This metamorphosis, or transformation, of the system dynamics is an important late side-effect of symmetry breaking, which manifests as an unusual nonsymmetric transcritical bifurcation induced by a significant shear-flow drive.

Treatment advances have led to dramatic clinical improvements for patients with HIV-1 infection. These clinical improvements reflect treatment-related improvements in immune function, which are most striking in individuals who develop exaggerated immune inflammatory responses to occult opportunistic infections. The mechanisms accounting for these exaggerated immune responses are unknown. To gain insight into these mechanisms, we intensively studied a subject untreated for disseminated tuberculosis and HIV-1 coinfection who then began treatment for both diseases. We examined the changing frequencies of Mycobacterium tuberculosis (MTB)-specific CD4(+) T cells that produced interferon gamma (IFN-gamma) after short-term stimulation with MTB antigen, and we compared these frequencies with those in HIV-1-seronegative subjects with and without prior exposure to MTB antigens. For the HIV-1/MTB-coinfected subject, the proportion of peripheral blood CD4(+) T cells expressing MTB-specific IFN-gamma was 8.6% at 11 days, 11% at 33 days, and 33% at 95 days after starting treatment for HIV-1. CD4(+) IFN-gamma(+) T cells had a CD45RA(-)CD62L(-) (effector memory) phenotype and most coexpressed interleukin 2. Median frequencies of CD4(+) IFN-gamma(+) T cells from six subjects without and nine subjects with prior exposure to MTB antigens were 0.06 and 0.46%, respectively. We conclude that individuals starting treatment for disseminated tuberculosis and HIV-1 coinfection can accumulate remarkably large numbers of MTB-specific CD4(+) T cells in the peripheral blood. The rapid expansion of antigen-specific effector CD4(+) T cells is one mechanism to explain immediate improvements in clinical immunity after HIV-1 treatment. This mechanism provides a theoretical framework to understand the unusual inflammatory responses recently reported to occur after starting HIV-1 treatment.

The chemokine and HIV receptor CXCR4 has been shown to play a role in chemotaxis and HIV-1 entry into T cells. Dibutyryl cAMP (DcAMP), an analog of cAMP, has been shown to increase CXCR4 cell surface expression and HIV-1 infectivity, but the molecular mechanism(s) responsible is unknown. Here we show that DcAMP treatment of purified human T lymphocytes increased transcription of CXCR4 mRNA as well as cell surface and intracellular CXCR4 protein expression. DcAMP-mediated stimulation of human PBL increased T-trophic HIV-1 (X4) fusion and viral replication as measured by syncytia formation and p24 levels, respectively. To determine the region(s) of the CXCR4 promoter required for cAMP responsiveness, truncations and point mutations of the CXCR4 promoter (nucleotides -1098 to +59) fused to luciferase were constructed and transiently transfected into human PBL. Deletional analysis demonstrated that the -1098 to -93 region of the CXCR4 promoter construct could be eliminated; the residual (-93 to +59) promoter retained cAMP responsiveness. Site-directed mutagenesis of a putative cAMP-responsive element (CRE) in the 5' UTR (+41 to +49) significantly and specifically attenuated the ability of DcAMP to drive the minimal CXCR4 promoter. Electrophoretic mobility shift assays demonstrated the formation of a complex between the CREB transcription factor and the putative CXCR4 CRE site. Our findings demonstrate a CRE element within the CXCR4 promoter that regulates CXCR4 transcription in response to changes in cAMP signaling. The cAMP-dependent up-regulation of CXCR4 mRNA results in increased CXCR4 intracellular and cell surface protein expression as well as increased HIV infectivity.