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1.  A Nonparametric Likelihood Test for Detecting Discordance Between Two Measurements with Application to Censored Viral Load Determinations 
Statistics in medicine  2008;27(22):4489-4501.
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 as compared to 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 which 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.
doi:10.1002/sim.3298
PMCID: PMC4326688  PMID: 18465838
Detection limit; likelihood ratio; maximum likelihood estimation; McNemar test; nonparametric likelihood
2.  Matched Longitudinal Analysis of Biomarkers Associated with Survival 
The identification of host or pathogen factors linked to clinical outcome is a common goal in many animal studies of infectious diseases. When the disease is fatal, statistical analysis of such factors may be biased from missing observations due to deaths. For example, when observations of a subject are censored before completing the intended study period, the complete trajectory will not be observed. Even if the factor is not associated with outcome, comparisons of data from survivors with those from nonsurvivors may lead to the wrong conclusions regarding associations with survival. Comparisons between subjects must account for differing observation lengths for those who survive relative to those who do not. Analyzing data over an interval common to all subjects provides one solution but requires eliminating data, some of which may be informative about the differences between groups. Here, we present a novel approach, matched longitudinal analysis (MLA), for analyzing such data based on matching biomarker intervals for survivors and nonsurvivors. We describe the results from simulation studies and from a study of monkeypox virus infection in nonhuman primates. In our application, MLA identified low monocyte chemoattractant protein-1 (MCP-1) levels as having a statistically significant association with survival, whereas the alternative methods did not identify an association. The method has general application to longitudinal studies that seek to find associations of biomarker changes with survival.
doi:10.1128/CVI.00252-14
PMCID: PMC4135908  PMID: 24943381
3.  Mycobacterial Antigen Driven Activation of CD14++CD16− Monocytes Is a Predictor of Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome 
PLoS Pathogens  2014;10(10):e1004433.
Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14++CD16− monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment.
Author Summary
Tuberculosis and HIV majorly impact host immune responses, resulting in immune deregulation and inflammation-driven tissue damage. Initiation of anti-retroviral therapy in patients with HIV-TB co-infection may result in immune reconstitution inflammatory syndrome (TB-IRIS), a disorder associated with increased immunopathology due to unfettered inflammation after CD4+ T-cell reconstitution. Monocytes are critical to the innate immune system and play an important role in several inflammatory conditions associated with chronic infections. Immunopathogenesis of TB-IRIS has been linked to activation of the adaptive immune response against opportunistic infection, yet the role of monocytes is still unknown. Here we investigated associations between soluble markers of monocyte activation, differential activation of monocyte subsets and TB-IRIS prospectively in two geographically distinct HIV-TB co-infected patient cohorts. Prior to ART initiation, patients who developed IRIS displayed a biosignature of elevated soluble monocyte activation markers, which were closely related to the mycobacterial antigen load in sputum samples. Amongst monocyte subsets, we observed that pre-ART circulating CD14++CD16− cell frequency independently predicted TB-IRIS and expanded during IRIS events. This monocyte subset was tightly associated with systemic markers of inflammation, and was found to produce inflammatory cytokines. Identification of this monocyte subset and its link with inflammation may lead to conception of novel therapies reducing immunopathology in TB-IRIS.
doi:10.1371/journal.ppat.1004433
PMCID: PMC4183698  PMID: 25275318
4.  Enhanced Effector Function of CD8+ T Cells From Healthy Controls and HIV-Infected Patients Occurs Through Thrombin Activation of Protease-Activated Receptor 1 
The Journal of Infectious Diseases  2012;207(4):638-650.
Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4+ and CD8+ T lymphocytes expressed PAR-1 and that expression was increased in CD8+ T cells from human immunodeficiency virus (HIV)–infected patients. Thrombin enhanced cytokine secretion in CD8+ T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8+ T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.
doi:10.1093/infdis/jis730
PMCID: PMC3549602  PMID: 23204166
PAR-1; T cells; thrombin; HIV pathogenesis; coagulation; inflammation
5.  Discordant MIC Analysis: A New Path to Licensure for Anti-infective Drugs 
Clinical trials (London, England)  2013;10(6):10.1177/1740774513507503.
Summary
Background
Evaluation of anti-infective drugs for licensure often relies on a noninferiority (NI) design where new drug B is noninferior to comparator drug A if the difference in success rates is reliably not worse than some fixed margin. The margin must be based on historical studies that estimate the magnitude of the benefit of drug A over placebo. This approach hampers drug development because the obligatory evidence for margin determination is often nonexistent.
Purpose
To develop a new method for licensure of anti-infective drugs when there is no historical evidence for reliable construction of the NI margin.
Methods
The MIC measures the minimum amount of drug that it takes to inhibit growth of bacteria in vitro. Patients who are infected with bacteria that have a low MIC to a given drug are expected to have good outcome when treated with that drug. Thus a differential effect of drug B versus drug A, if it exists, is likely to occur in patients whose pathogens have discordant MICs (e.g. low MIC for drug B, high MIC for drug A, or vice versa). A new paradigm for licensure of anti-infective drugs is proposed where a clinically acceptable NI margin is selected and licensure supported if the NI margin is met and B is reliably demonstrated superior to A in a subset of patients whose paired MICs favor B. The requirement for some evidence of superiority encourages a study that is carefully designed and executed.
Results
Simulations indicate the approach shows promise in realistic settings provided adequate data are available. A simulated example illustrates use of the methods.
Limitations
If the data have small sample size, weak MIC/success relationship, or high correlation between MIC-A, MIC-B, this procedure will have poor power.
Conclusion
Discordant MIC analysis may offer a novel path to licensure for certain anti-infective drugs.
doi:10.1177/1740774513507503
PMCID: PMC3845416  PMID: 24287133
AUC:MIC ratio; Interaction Test; Logistic Regression; Licensure
6.  Linezolid for Treatment of Chronic Extensively Drug-Resistant Tuberculosis 
The New England journal of medicine  2012;367(16):10.1056/NEJMoa1201964.
BACKGROUND
Linezolid has antimycobacterial activity in vitro and is increasingly used for patients with highly drug-resistant tuberculosis.
METHODS
We enrolled 41 patients who had sputum-culture–positive extensively drug-resistant (XDR) tuberculosis and who did not have a response to any available chemotherapeutic option during the previous 6 months. Patients were randomly assigned to linezolid therapy that started immediately or after 2 months, at a dose of 600 mg per day, without a change in their background regimen. The primary end point was the time to sputum-culture conversion on solid medium, with data censored 4 months after study entry. After confirmed sputum-smear conversion or 4 months (whichever came first), patients underwent a second randomization to continued linezolid therapy at a dose of 600 mg per day or 300 mg per day for at least an additional 18 months, with careful toxicity monitoring.
RESULTS
By 4 months, 15 of the 19 patients (79%) in the immediate-start group and 7 of the 20 (35%) in the delayed-start group had culture conversion (P = 0.001). Most patients (34 of 39 [87%]) had a negative sputum culture within 6 months after linezolid had been added to their drug regimen. Of the 38 patients with exposure to linezolid, 31 (82%) had clinically significant adverse events that were possibly or probably related to linezolid, including 3 patients who discontinued therapy. Patients who received 300 mg per day after the second randomization had fewer adverse events than those who continued taking 600 mg per day. Thirteen patients completed therapy and have not had a relapse. Four cases of acquired resistance to linezolid have been observed.
CONCLUSIONS
Linezolid is effective at achieving culture conversion among patients with treatment-refractory XDR pulmonary tuberculosis, but patients must be monitored carefully for adverse events. (Funded by the National Institute of Allergy and Infectious Diseases and the Ministry of Health and Welfare, South Korea; ClinicalTrials.gov number, NCT00727844.)
doi:10.1056/NEJMoa1201964
PMCID: PMC3814175  PMID: 23075177
7.  Enhancing effects of adjuvanted 2009 pandemic H1N1 influenza A vaccine on memory B-cell responses in HIV-infected individuals 
AIDS (London, England)  2011;25(3):295-302.
Objective
To assess the humoral immune response to low-dose AS03-adjuvanted and standard-dose nonadjuvanted 2009 pandemic H1N1 influenza A vaccine in HIV-infected aviremic individuals receiving antiretroviral therapy and in uninfected individuals.
Design
A three-arm study.
Setting
Two clinics: one at the National Institutes of Health in Bethesda, Maryland, USA; and the other at the Maple Leaf Medical Clinic in Toronto, Ontario, Canada.
Participants
HIV-infected and HIV-uninfected adults.
Intervention
Single intramuscular 15µg dose of the monovalent inactivated 2009 pandemic H1N1 influenza A vaccine without adjuvant or 3.75µg dose of the same strain with adjuvant AS03.
Main outcomes
Immunogenicity, as measured by hemagglutination inhibition (HAI) antibody titers and vaccine-specific memory B-cell responses.
Results
A total of 74 participants were enrolled. Twenty-one HIV-infected individuals received the low-dose adjuvanted 2009 pandemic H1N1 influenza A vaccine. Twenty-nine HIV-infected and 24 HIV-uninfected individuals received the standard-dose nonadjuvanted vaccine. There were no significant differences in antibody responses at 9 weeks postvaccination among the three groups studied. However, the IgG memory B-cell response against the vaccine was significantly higher in the HIV-infected group that received the low-dose adjuvanted vaccine when compared to the HIV-infected and uninfected groups that received the standard-dose nonadjuvanted vaccine. Conclusions remained unchanged after regression adjustment for age, gender, CD4+ T-cell count, and baseline HAI titer.
Conclusion
These data suggest that adjuvants could be used to expand coverage through dose sparing and improve humoral immune responses in immunocompromised individuals.
doi:10.1097/QAD.0b013e328342328b
PMCID: PMC3791488  PMID: 21157297
adjuvants; antibody response; HIV infection; memory B-cell response; pandemic influenza; vaccination
8.  A valid formulation of the analysis of noninferiority trials under random effects meta-analysis 
Biostatistics (Oxford, England)  2012;13(4):637-649.
A noninferiority (NI) trial is sometimes employed to show efficacy of a new treatment when it is unethical to randomize current patients to placebo because of the established efficacy of a standard treatment. Under this framework, if the NI trial determines that the treatment advantage of the standard to the new drug (i.e. S−N) is less than the historic advantage of the standard to placebo (S−P), then the efficacy of the new treatment (N−P) is established indirectly. We explicitly combine information from the NI trial with estimates from a random effects model, allowing study-to-study variability in k historic trials. Existing methods under random effects, such as the synthesis method, fail to account for the variability of the true standard versus placebo effect in the NI trial. Our method effectively uses a prediction interval for the missing standard versus placebo effect rather than a confidence interval of the mean. The consequences are to increase the variance of the synthesis method by incorporating a prediction variance term and to approximate the null distribution of the new statistic with a t with k−1 degrees of freedom instead of the standard normal. Thus, it is harder to conclude NI of the new to (predicted) placebo, compared with traditional methods, especially when k is small or when between study variability is large. When the between study variances are nonzero, we demonstrate substantial Type I error rate inflation with conventional approaches; simulations suggest that the new procedure has only modest inflation, and it is very conservative when between study variances are zero. An example is used to illustrate practical issues.
doi:10.1093/biostatistics/kxs006
PMCID: PMC3658490  PMID: 22467938
Active control trial; Clinical trial; Meta-analysis; Noninferiority trial; Random effects; Synthesis method
9.  Anthrax Vaccine Induced Antibodies Provide Cross-Species Prediction of Survival to Aerosol Challenge 
Science translational medicine  2012;4(151):151ra126.
Because clinical trials to assess the efficacy of vaccines against anthrax are not ethical or feasible, licensure for new anthrax vaccines will likely involve the Food and Drug Administration’s “Animal Rule,” a set of regulations that allow approval of products based on efficacy data only in animals combined with immunogenicity and safety data in animals and humans. US government sponsored animal studies have shown anthrax vaccine efficacy in a variety of settings. We examined data from 21 of those studies to determine if an immunological bridge based on lethal toxin neutralization activity assay (TNA) can predict survival against an inhalation anthrax challenge within and across species and genera. The 21 studies were classified into 11 different settings, each of which had the same animal species, vaccine type and formulation, vaccination schedule, time of TNA measurement, and challenge time. Logistic regression models determined the contribution of vaccine dilution dose and TNA on prediction of survival. For most settings, logistic models using only TNA explained more than 75% of the survival effect of the models with dose additionally included. Cross species survival predictions using TNA were compared to the actual survival and shown to have good agreement (Cohen’s κ ranged from 0.55 to 0.78). In one study design, cynomolgus macaque data predicted 78.6% survival in rhesus macaques (actual survival 83.0%) and 72.6% in rabbits (actual survival, 64.6%). These data add support for the use of TNA as an immunological bridge between species to extrapolate data in animals to predict anthrax vaccine effectiveness in humans.
doi:10.1126/scitranslmed.3004073
PMCID: PMC3668972  PMID: 22972844
animal rule; anthrax vaccine adsorbed; correlate of protection; recombinant protective antigen; toxin neutralizing activity assay
10.  Empirical Likelihood-Based Estimation of the Treatment Effect in a Pretest–Posttest Study 
The pretest–posttest study design is commonly used in medical and social science research to assess the effect of a treatment or an intervention. Recently, interest has been rising in developing inference procedures that improve efficiency while relaxing assumptions used in the pretest–posttest data analysis, especially when the posttest measurement might be missing. In this article we propose a semiparametric estimation procedure based on empirical likelihood (EL) that incorporates the common baseline covariate information to improve efficiency. The proposed method also yields an asymptotically unbiased estimate of the response distribution. Thus functions of the response distribution, such as the median, can be estimated straightforwardly, and the EL method can provide a more appealing estimate of the treatment effect for skewed data. We show that, compared with existing methods, the proposed EL estimator has appealing theoretical properties, especially when the working model for the underlying relationship between the pretest and posttest measurements is misspecified. A series of simulation studies demonstrates that the EL-based estimator outperforms its competitors when the working model is misspecified and the data are missing at random. We illustrate the methods by analyzing data from an AIDS clinical trial (ACTG 175).
doi:10.1198/016214508000000625
PMCID: PMC3666595  PMID: 23729942
Auxiliary information; Biased sampling; Causal inference; Observational study; Survey sampling
11.  A maximum pseudo-profile likelihood estimator for the Cox model under length-biased sampling 
Biometrika  2012;99(1):199-210.
This paper considers semiparametric estimation of the Cox proportional hazards model for right-censored and length-biased data arising from prevalent sampling. To exploit the special structure of length-biased sampling, we propose a maximum pseudo-profile likelihood estimator, which can handle time-dependent covariates and is consistent under covariate-dependent censoring. Simulation studies show that the proposed estimator is more efficient than its competitors. A data analysis illustrates the methods and theory.
doi:10.1093/biomet/asr072
PMCID: PMC3667656  PMID: 23843659
Approximate likelihood; Cross-sectional sampling; Product-limit estimator; Random truncation; Screening trials
12.  A test of location for exchangeable multivariate normal data with unknown correlation 
Journal of multivariate analysis  2012;104(1):115-125.
We consider the problem of testing whether the common mean of a single n–vector of multivariate normal random variables with known variance and unknown common correlation ρ is zero. We derive the standardized likelihood ratio test for known ρ and explore different ways of proceeding with ρ unknown. We evaluate the performance of the standardized statistic where ρ is replaced with an estimate of ρ and determine the critical value cn that controls the type I error rate for the least favorable ρ in [0,1]. The constant cn increases with n and this procedure has pathological behavior if ρ depends on n and ρn converges to zero at a certain rate. As an alternate approach, we replace ρ with the upper limit of a (1 − βn) confidence interval chosen so that cn = c for all n. We determine βn so that the type I error rate is exactly controlled for some ρ in [0,1]. We also investigate a simpler approach where we bound the type I error rate. The former method performs well for all n while the less powerful bound method may be a useful in some settings as a simple approach. The proposed tests can be used in different applications, including within-cluster resampling and combining exchangeable p-values.
doi:10.1016/j.jmva.2011.07.003
PMCID: PMC3224074  PMID: 22125347
Confidence interval; Within Cluster Resampling; Likelihood Ratio Test
13.  Genotypic Resistance at Viral Rebound among Patients Who Received Lopinavir/ritonavir- or Efavirenz-Based First Antiretroviral Therapy in South Africa 
NNRTI drug resistance mutations (DRM) are increasingly reported in Africans failing their first antiretroviral regimen. The Phidisa II trial randomized treatment-naïve participants to LPV/r or EFV with d4T+3TC or ZDV+ddI. We report the prevalence of DRM in subjects who achieved HIV RNA < 400 copies/mL at 6-months but subsequently had 2 consecutive HIV RNA >1000 copies/mL. Sixty-eight participants fulfilled the inclusion criteria. NNRTI-DRM were found in 17/36 (47.2%) EFV-recipients, and M184V/I mutation in 14/40 (35.0%) 3TC-recipients. No PI mutation was identified in 38 LPV/r-recipients. This is one of the first studies in Africa confirming the paucity of PI-associated DRM despite virologic failure.
doi:10.1097/QAI.0b013e3182278c29
PMCID: PMC3197956  PMID: 21694608
genotypic resistance; non-nucleoside reverse transcriptase inhibitors; protease inhibitors; HIV; South Africa; antiretroviral naïve
14.  An Augmented Probit Model for Missing Predictable Covariates in Quantal Bioassay With Small Sample Size 
Biometrics  2011;67(3):1127-1134.
Quantal bioassay experiments relate the amount or potency of some compound; e.g. poison, antibody, or drug to a binary outcome such as death or infection in animals. For infectious diseases, probit regression is commonly used for inference and a key measure of potency is given by the IDP, the amount that results in P% of the animals being infected. In some experiments, a validation set may be used where both direct and proxy measures of the dose are available on a subset of animals with the proxy being available on all. The proxy variable can be viewed as a messy reflection of the direct variable, leading to an errors-in-variables problem. We develop a model for the validation set and use a constrained seemingly unrelated regression (SUR) model to obtain the distribution of the direct measure conditional on the proxy. We use the conditional distribution to derive a pseudo-likelihood based on probit regression and use the parametric bootstrap for statistical inference. We re-evaluate an old experiment in twenty-one monkeys where neutralizing antibodies to HIV were measured using an old (proxy) assay in all monkeys and with a new (direct) assay in a validation set of eleven who had sufficient stored plasma. Using our methods, we obtain an estimate of the ID1 for the new assay, an important target for HIV vaccine candidates. In simulations we compare the pseudo-likelihood estimates with regression calibration and a full joint likelihood approach.
doi:10.1111/j.1541-0420.2010.01536.x
PMCID: PMC3116946  PMID: 21210769
Assay; Errors-in-variables; HIV Vaccine; ID50; Measurement Error; Probit Regression; Regression Calibration; Validation Set
15.  Dimension reduced kernel estimation for distribution function with incomplete data 
SUMMARY
This work focuses on the estimation of distribution functions with incomplete data, where the variable of interest Y has ignorable missingness but the covariate X is always observed. When X is high dimensional, parametric approaches to incorporate X — information is encumbered by the risk of model misspecification and nonparametric approaches by the curse of dimensionality. We propose a semiparametric approach, which is developed under a nonparametric kernel regression framework, but with a parametric working index to condense the high dimensional X — information for reduced dimension. This kernel dimension reduction estimator has double robustness to model misspecification and is most efficient if the working index adequately conveys the X — information about the distribution of Y. Numerical studies indicate better performance of the semiparametric estimator over its parametric and nonparametric counterparts. We apply the kernel dimension reduction estimation to an HIV study for the effect of antiretroviral therapy on HIV virologic suppression.
doi:10.1016/j.jspi.2011.03.030
PMCID: PMC3127551  PMID: 21731174
curse of dimensionality; dimension reduction; distribution function; ignorable missingness; kernel regression; quantile
16.  A model checking method for the proportional hazards model with recurrent gap time data 
Biostatistics (Oxford, England)  2010;12(3):535-547.
Recurrent events are the natural outcome in many medical and epidemiology studies. To assess covariate effects on the gaps between consecutive recurrent events, the Cox proportional hazards model is frequently employed in data analysis. The validity of statistical inference, however, depends on the appropriateness of the Cox model. In this paper, we propose a class of graphical techniques and formal tests for checking the Cox model with recurrent gap time data. The building block of our model checking method is an averaged martingale-like process, based on which a class of multiparameter stochastic processes is proposed. This maneuver is very general and can be used to assess different aspects of model fit. Numerical simulations are conducted to examine finite-sample performance, and the proposed model checking techniques are illustrated with data from the Danish Psychiatric Central Register.
doi:10.1093/biostatistics/kxq071
PMCID: PMC3138070  PMID: 21138876
Correlated failure times; Induced-dependent censoring; Kaplan–Meier estimator; Renewal processes
17.  Pre-ART Levels of Inflammation and Coagulation Markers Are Strong Predictors of Death in a South African Cohort with Advanced HIV Disease 
PLoS ONE  2012;7(3):e24243.
Background
Levels of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and D-dimer predict mortality in HIV patients on antiretroviral therapy (ART) with relatively preserved CD4+ T cell counts. We hypothesized that elevated pre-ART levels of these markers among patients with advanced HIV would be associated with an increased risk of death following the initiation of ART.
Methods
Pre-ART plasma from patients with advanced HIV in South Africa was used to measure hsCRP, IL-6 and D-dimer. Using a nested case-control study design, the biomarkers were measured for 187 deaths and two controls matched on age, sex, clinical site, follow-up time and CD4+ cell counts. Odds ratios were estimated using conditional logistic regression. In addition, for a random sample of 100 patients, biomarkers were measured at baseline and 6 months following randomization to determine whether ART altered their levels.
Results
Median baseline biomarkers levels for cases and controls, respectively, were 11.25 vs. 3.6 mg/L for hsCRP, 1.41 vs. 0.98 mg/L for D-dimer, and 9.02 vs. 4.20 pg/mL for IL-6 (all p<0.0001). Adjusted odds ratios for the highest versus lowest quartile of baseline biomarker levels were 3.5 (95% CI: 1.9–6.7) for hsCRP, 2.6 (95%CI 1.4–4.9) for D-dimer, and 3.8 (95% CI: 1.8–7.8) for IL-6. These associations were stronger for deaths that occurred more proximal to the biomarker measurements. Levels of D-dimer and IL-6, but not hsCRP, were significantly lower at month 6 after commencing ART compared to baseline (p<0.0001).
Conclusions
Among patients with advanced HIV disease, elevated pre-ART levels of hsCRP, IL-6 and D-dimer are strongly associated with early mortality after commencing ART. Elevated levels of inflammatory and coagulation biomarkers may identify patients who may benefit from aggressive clinical monitoring after commencing ART. Further investigation of strategies to reduce biomarkers of inflammation and coagulation in patients with advanced HIV disease is warranted.
Trial Registration
Parent Study: ClinicalTrials.gov NCT00342355
doi:10.1371/journal.pone.0024243
PMCID: PMC3308955  PMID: 22448211
18.  High Dose Atorvastatin Decreases Cellular Markers of Immune Activation Without Affecting HIV-1 RNA Levels: Results of a Double-blind Randomized Placebo Controlled Clinical Trial 
The Journal of Infectious Diseases  2011;203(6):756-764.
(See the editorial commentary by Carr, on pages 751–2.)
Background. 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) exhibit antiviral activity against human immunodeficiency virus type 1 (HIV-1) in vitro and may modulate the immune response to HIV infection. Studies evaluating the antiviral activity of statins have yielded conflicting results.
Methods. We conducted a randomized, double-blind, placebo-controlled crossover trial to investigate the effect of atorvastatin on HIV-1 RNA (primary objective) and cellular markers of immune activation (secondary objective). HIV-infected individuals not receiving antiretroviral therapy were randomized to receive either 8 weeks of atorvastatin (80 mg) or placebo daily. After a 4–6 week washout phase, participants switched treatment assignments. The study had 80% power to detect a 0.3 log10 decrease in HIV-1 RNA level. Expression of CD38 and HLA-DR on CD4+ and CD8+ T cells was used to measure immune activation.
Results. Of 24 randomized participants, 22 completed the study. Although HIV-1 RNA level was unaffected by the intervention (–0.13 log10 copies/mL; P = .85), atorvastatin use resulted in reductions in circulating proportions of CD4+ HLA-DR+ (–2.5%; P = .02), CD8+ HLA-DR+ (–5%; P = .006), and CD8+ HLA-DR+ CD38+ T cells (–3%; P = .03). Reductions in immune activation did not correlate with declines in serum levels of low-density lipoprotein cholesterol.
Conclusions. Short-term use of atorvastatin was associated with modest but statistically significant reductions in the proportion of activated T lymphocytes.
doi:10.1093/infdis/jiq115
PMCID: PMC3071124  PMID: 21325137
19.  Development of Functional and Molecular Correlates of Vaccine-Induced Protection for a Model Intracellular Pathogen, F. tularensis LVS 
PLoS Pathogens  2012;8(1):e1002494.
In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rβ2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.
Author Summary
Diseases such as tuberculosis (caused by Mycobacterium tuberculosis) or tularemia (caused by Francisella tularensis) result from infections by microbes that live within cells of a person's body. New vaccines are being developed against such intracellular pathogens, but some will be difficult to test, because disease takes a long time to develop (e.g., tuberculosis) or because outbreaks are unpredictable (e.g., tularemia). Usually such infections are controlled by activities of T cells. However, there are no accepted measures of T cell function that reliably predict vaccine-induced protection. We studied two new ways to do so. We used a group of vaccine candidates against tularemia that stimulated good, fair, or poor protection of mice against Francisella challenge. We then measured whether Francisella–immune cells from vaccinated mice controlled the growth of bacteria inside cells, and/or whether the expression of immune genes in Francisella–immune cells was increased. We found that the degree of protection was matched by the degree of the cells' function in controlling intramacrophage bacterial growth. Further, the degree was predicted by relative amounts of gene expression for several immune mediators. Thus the two new options explored here may help predict protection, without waiting for the onset of disease.
doi:10.1371/journal.ppat.1002494
PMCID: PMC3262015  PMID: 22275868
20.  Interferon-α Produces Significant Decreases in HIV Load 
A randomized, controlled clinical trial was started in the pre-HAART era to compare the efficacy of zidovudine (AZT) and interferon-alpha (IFN-α) either alone or in combination to reduce HIV viremia, maintain CD4+ cell count, and decrease time to AIDS progression and death. The purpose of the current study was to compare the effects of AZT and IFN on HIV load using modern technology. One hundred and eighty patients with CD4+ counts above 500 cells/mm3 were randomized to receive AZT alone, IFN-α alone, or AZT and IFN-α in combination. CD4+ cell count and HIV load at baseline and at the last follow-up visit were compared, and time to AIDS or death was calculated by treatment group. At a mean follow-up of 45 weeks, the mean change in log HIV RNA was −0.06 for the AZT alone group, −0.47 for the AZT plus IFN-α group (P = 0.01 versus AZT group), and −0.35 for the IFN-α alone group (P = 0.02 versus AZT group). There was no significant difference among groups in change in total CD4+ count or in time to AIDS or death. Since treatment with IFN-α produces significant decreases in HIV load, additional studies of IFN-α as part of a combination regimen are warranted.
doi:10.1089/jir.2009.0090
PMCID: PMC2964361  PMID: 20235638
21.  Semiparametric dimension reduction estimation for mean response with missing data 
Biometrika  2010;97(2):305-319.
Summary
Model misspecification can be a concern for high-dimensional data. Nonparametric regression obviates model specification but is impeded by the curse of dimensionality. This paper focuses on the estimation of the marginal mean response when there is missingness in the response and multiple covariates are available. We propose estimating the mean response through nonparametric functional estimation, where the dimension is reduced by a parametric working index. The proposed semiparametric estimator is robust to model misspecification: it is consistent for any working index if the missing mechanism of the response is known or correctly specified up to unknown parameters; even with misspecification in the missing mechanism, it is consistent so long as the working index can recover E(Y  |   X), the conditional mean response given the covariates. In addition, when the missing mechanism is correctly specified, the semiparametric estimator attains the optimal efficiency if E(Y  |   X) is recoverable through the working index. Robustness and efficiency of the proposed estimator is further investigated by simulations. We apply the proposed method to a clinical trial for HIV.
doi:10.1093/biomet/asq005
PMCID: PMC3412576  PMID: 23049121
Dimension reduction; Inverse probability weighting; Kernel regression; Missing at random; Robustness to model misspecification
22.  Lack of Effect from a Previous Single Dose of Nevirapine on Virologic and Immunologic Responses After 6 Months of Antiretroviral Regimens Containing Efavirenz or Lopinavir-ritonavir 
Pharmacotherapy  2011;31(2):158-163.
Study Objective
To evaluate the effect of prior single dose nevirapine (sd-NVP) use on HIV RNA and CD4+ T-cell responses after 6 months of efavirenz - or lopinavir/ritonavir -based antiretroviral regimens.
Design
This is a retrospective analysis of a subset of participants in the Phidisa II trial, a randomized controlled trial enrolled HIV+ participants 14 years or older with no or < 7 day history of antiretroviral use. At screening, subjects were asked about prior nevirapine nevirapine use, a positive response in a woman was used as a surrogate for prior sd-NVP. Virologic and CD4 responses at 6-month were compared between women with or without prior nevirapine exposure, and amongst women who received efavirenz vs. lopinavir/ritonavir.
Setting
Six South African Medical Health Services’ Phidisa research clinics.
Subjects
478 women responded to the question regarding prior nevirapine use.
Measurements and Main Results
392 women were nevirapine-naïve (NVP-), 86 had prior nevirapine (NVP+). At 6-month, 396 women (324 NVP-, 70 NVP+) had follow-up HIV-RNA results. 69.9% of NVP- and 68.6% of NVP+ subjects achieved HIV-RNA <400 copies/mL (p=0.35), with CD4 changes of +115.5 and +120.4 cells/mm3 respectively (p=0.67). Among the NVP+ women, 75% efavirenz-treated and 61.8% lopinavir/ritonavir-treated subjects had HIV-RNA < 400 copies/mL at 6 months (p=0.31).
Conclusion
In this retrospective analysis, 75% of participants with self-reported prior sd-NVP use achieved HIV RNA <400 copies/mL with efavirenz-based regimen at 6-month follow-up. Prior exposure to sd-NVP per self report did not affect virologic outcome at 6-month in this small cohort.
doi:10.1592/phco.31.2.158
PMCID: PMC3058317  PMID: 21275494
single-dose nevirapine; South Africa; efavirenz; HIV; response; antiretroviral; women
23.  Neutralizing Antibody Titers Conferring Protection to Macaques from a Simian/Human Immunodeficiency Virus Challenge Using the TZM-bl Assay 
Abstract
We previously reported that passive transfer of polyclonal neutralizing antibodies (NAbs) sufficient to generate a titer of 1:38 in the plasma would confer sterilizing protection to 99% of macaques challenged intravenously with 75 TCID50 of SHIVDH12. Neutralizing activity in that study was measured in an MT4 cell assay in which infection was completely blocked (EC100). In the current study, the TZM-bl system was used to measure EC50 neutralizing titers in several of the same macaque plasma samples and the relationship between these titers and in vivo protection was determined. The antiviral EC50 NAb titers measured in individual plasma samples were higher than those previously obtained in the MT4 system. Furthermore, the geometric mean EC50 NAb titers against pseudotyped SHIVDH12 were 33-fold greater than the EC100 titers measured in the MT4 cell assay against the replication-competent SHIVDH12 inoculated into animals. An augmented probit regression model was used to generate curves relating TZM-bl EC50 NAb titers and protection from a virus challenge; estimated titers conferring various levels of protection were then determined. In TZM-bl assays using pseudotyped SHIVDH12, representative percent in vivo protection/estimated EC50 titers were 99%/1:4467, 90%/1:1175, 80%/1:676, 50%/1:234, and 33%/1:141. Because it is likely that contributions from other arms of the immune system will contribute to vaccine-induced control, the range of EC50 NAb titers we have derived may be more informative for evaluating the protective value of NAb activity from TZM-bl assays.
doi:10.1089/aid.2009.0144
PMCID: PMC2858899  PMID: 20059398
24.  CCR5 Deficiency is a Risk Factor for Early Clinical Manifestations of West Nile Virus Infection, but not for Infection per se 
The Journal of infectious diseases  2010;201(2):178-185.
Background
West Nile virus (WNV) is a neurotropic flavivirus transmitted to humans by mosquito vectors. Homozygosity for CCR5Δ32, a complete loss-of-function mutation in the chemokine receptor CCR5, has been previously associated with severe symptomatic WNV infection in patients presenting with clinical disease, however whether it acts at the level of initial infection or in promoting clinical progression is unknown.
Methods
Here we address this gap in knowledge by comparing CCR5Δ32 distribution in US blood donors identified through a comprehensive blood supply screening program (n=34,766,863 donations from 2003–2008) as either WNV true positive (WNV+ cases; n=634) or false positive (WNV− controls; n=422).
Results
No difference was observed in CCR5Δ32 homozygous frequency between the WNV+ cases and WNV− controls, suggesting that CCR5 deficiency is not a risk factor for initial infection. However, CCR5Δ32 homozygosity was associated with worse clinical outcome, with these individuals developing more clinical symptoms as determined by a standardized questionnaire (P=0.0016).
Conclusions
Thus CCR5 deficiency is not a risk factor for WNV infection per se, but is a risk factor for both early and late clinical manifestations after infection. Our study also illustrates the power of large scale databases to answer important clinical questions in man.
doi:10.1086/649426
PMCID: PMC2934858  PMID: 20025530
CCR5; polymorphism; West Nile virus; genetic risk; blood donations; clinical outcome; early symptomatology
25.  Immunological and Virological Events in Early HIV Infection Predict Subsequent Rate of Progression 
The Journal of infectious diseases  2010;201(2):272-284.
Background
Variability in HIV disease progression cannot fully be predicted by CD4 T-cell counts or viral load. Because central memory T cells (TCM) play a critical role in the pathogenesis of SIV disease, we hypothesized that quantifying these cells in early HIV-infection could provide prognostic information.
Methods
We measured expression of CD45RO, CCR5, CCR7, CD27, and CD28 to enumerate naïve and memory subsets in samples from recently-infected individuals. We also quantified proliferation, CD127 expression, and cell-associated viral load. Disease progression was compared across subgroups defined by these measurements, using Kaplan-Meier survival curves and multivariate Cox proportional hazards regression.
Results
466 subjects contributed 101 events. The proportion or absolute count of TCM did not correlate with disease progression, defined as the time to AIDS or death. However, significant associations were observed for: proliferation within CD4 or CD8 T cells, loss of naïve or CD127+ CD8 T cells, and CD4 cell-associated proviral load.
Conclusion
Our results demonstrate that the extent of the immunopathogenesis established early in HIV infection predicts the course of future disease. Since antiretroviral drug treatment reverses such defects in part, our study provides mechanistic clues to why early use of antiretrovirals may prove beneficial.
doi:10.1086/649430
PMCID: PMC2939466  PMID: 20001854
Central memory; immune activation; cell associated viral load; disease progression; survival

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