Cobas Ampliprep/Cobas TaqMan HIV-1 v2.0 Assay: Consequences at the Cohort Level

Background

High-sensitive real-time PCR assays are routinely used to monitor HIV-1 infected subjects. Inter-assay discrepancies have been described at the low viral load (VL) end, where clinical decisions regarding possible virological rebound are based.

Methods

A retrospective study was performed to analyze frequencies of viral blips after transition to the COBAS Ampliprep/COBAS TaqMan v2.0 HIV-1 assay (Taqman v2.0) in patients with prior undetectable VLs as measured with the Roche Cobas Ampliprep Amplicor HIV-1 Monitor Test, v1.5 (Amplicor) and was evaluated in comparison to a group of patients monitored with the Abbott Real-time HIV-1 assay (Abbott RT) during the same period of time.

Results

85 of 373 patients with VLs below the limit of quantification with Amplicor had VLs >50 copies/mL after transition to the TaqMan v2.0 assay. Among these 74.1% had VLs ranging from 50–499 copies/mL, 22.9% had VLs >500 copies/mL. From 22 patients with initial Taqman v2.0 based VLs exceeding 500 copies/mL, 6 patients had VLs <20 copies/mL after novel VL measurement on a next visit. In our control group with VL quantification using the Abbott RT assay, only 1 patient became detectable and showed a VL of <40 copies/mL after new measurement.

Conclusions

Transition to the Taqman v2.0 assay was accompanied by an increase of quantifiable HIV-1 VLs in patients with long term viral suppression under antiretroviral therapy that might be attributed to technical shortcomings of the Taqman v2.0 assay. A high test variability at the low VL end but also beyond was observed, making meaningful clinical interpretation of viral blips derived from different assays difficult.     

Real-Time PCR Assays for the Quantification of HCV RNA: Concordance,Discrepancies and Implications for Response Guided Therapy

Background and Aims

Monitoring of chronic Hepatitis C (CHC) treatment relies on HCV RNA quantification by means of real-time PCR methods. Assay specific analytical sensitivities may impact therapy management.

Methods

Comparative analysis between three commercial assays (Roche COBAS AmpliPrep/COBAS TaqMan Version 1 (CAP/CTM Ver. 1), Version 2 (CAP/CTM Ver. 2) and the Abbott RealTime HCV (ART) assay) was performed on 247 available samples taken at key decision time points during antiviral therapy of 105 genotype 1 patients (triple therapy: n = 70; dual therapy: n = 35).

Results

Overall concordance of HCV RNA measurements was high between the two Roche systems (89%; n = 220/247) but lower between the Roche assays and the ART (CAP/CTM Ver. 1 vs ART: 77.3%; n = 191/247 and CAP/CTM v.2 vs ART: 80.1%; n = 198/247). Most discrepancies were noted in week 4/8 samples with residual viremia (<LLOQ) detected by ART (<LLOQ: n = 45, 44.1%) but undetectable HCV RNA by CAP/CTM Ver. 1 (<LLOQ: n = 18, 17.6%) or CAP/CTM Ver. 2 (<LLOQ: n = 26, 25.5%). Based on results by CAP/CTM Ver. 1, 13 eligible patients underwent an abbreviated course of therapy (24 weeks). Only 1 patient experienced virologic breakthrough. If tested by ART, only 6/13 patients (46.2%) would have been eligible for shortened treatment. Consequently, RGT guidelines were adapted and shortening of therapy was allowed if residual viremia was detected by ART at week 4/8.

Conclusion

An abbreviated course of treatment can safely be applied in patients with residual viremia (<LLOQ) detected by ART in samples collected at week 4/8 of treatment.     

Comparison of the Cobas Ampliprep/Cobas TaqMan HBV Test versus the Cobas Amplicor HBV monitor for HBV-DNA detection and quantification during antiviral therapy

Performances of the new automatic system COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared with the endpoint PCR COBAS AMPLICOR HBV Monitor (CAHBM, Roche). Analytical evaluation with proficiency panels from UK National External Quality Assessment Scheme (UK NEQAS) over a 1-year period of distribution showed that CAP/CTM correctly measured HBV DNA levels with a close correlation between expected and observed values (r=0.995). Clinical evaluation as tested with samples from 11 HBsAg-positive patients undergoing antiviral therapy (71 serial specimens of plasma), demonstrated excellent correlation with CAHBM (r=0.958, mean difference in quantitation: 0.14 log, IU/ml), but CAP/CTM detected longer period of residual viremia. HBV DNA reduction was much higher in the combination schedule (Lamivudine+Adefovir), than in Adefovir monotherapy (5.1 vs. 3.5 logs). In conclusion, CAP/CTM allows for an accurate and standardized quantification of HBV DNA in high through-put laboratories. Due to it high sensitivity, it may further improve the detection of emerging drug resistance strains and the assessment of antiviral therapy.     

Residual Viremia Is Preceding Viral Blips and Persistent Low-Level Viremia in Treated HIV-1 Patients

Background

It has been suggested that low-level viremia or blips in HIV-infected patients on antiretroviral treatment are related to assay variation and/or increased sensitivity of new commercial assays. The 50-copy cut-off for virologic failure is, therefore, under debate.

Methods

Treated patients with low-level viremia (persistent viral loads (VL) of 50–1000 copies/mL, group A, N = 16) or a blip (single detectable VL, group B, N = 77) were compared to a control group (consistently suppressed viremia since start therapy (<50 copies/mL), N = 79). Residual viremia (detectable viral RNA <50 copies/ml) in the year preceding the first VL above 50 copies/mL (T0) was determined using Roche Cobas-Amplicor v1.5 or CAP-CTM v2.0. Subsequent virologic failure (2 consecutive VLs>500 or 1 VL>1000 copies/mL that was not followed by a VL<50 copies/mL; median follow up 34 months) was assessed.

Results

Significantly more patients in groups A and B had residual viremia in the year preceding T0 compared to controls (50% and 19% vs 3% respectively; p<0.001). Residual viremia was associated with development of low-level viremia or blips (OR 10.9 (95% CI 2.9–40.6)). Subsequent virologic failure was seen more often in group A (3/16) and B (2/77) than in the control group (0/79).

Conclusion

Residual viremia is associated with development of blips and low-level viremia. Virologic failure occurred more often in patients with low-level viremia. These results suggest that low-level viremia results from viral production/replication rather than only assay variation.     

Differential MicroRNA Expression Profile between Stimulated PBMCs from HIV-1 Infected Elite Controllers and Viremic Progressors

Background

The emerging relationship between microRNAs (miRNA) and viral-control is a topic of interest in the field of HIV. Host-genome might play an important role in the control of viremia. The aim of this study was to assess the specific miRNA profile that could contribute to the control of HIV replication in Elite Controllers

Results

After adequate normalization, expression profile of 286 human miRNAs (hsa-miR) was evaluated in phytohaemagglutinin-stimulated PBMCs from 29 individuals classified in 4 groups: 8 elite controllers (EC; viral load <50 cp/ml without treatment), 8 viremic progressors (VP; VL>5000 cp/ml without treatment), 8 patients under antiretroviral treatment (ART; VL<200 cp/ml) and 5 uninfected individuals (HIV-) through TaqMan Array Human microRNA Cards v3.0. A differential expression pattern consisting of 23 miRNAs became significantly different when comparing EC and VP. Profiling analysis segregated the population in two different blocks: while EC and HIV- clustered together in the same block (EC/HIV-_block 1), VP and ART individuals clustered together in a second block (VP/ART_block 2). Two inversely expressed miRNA patterns were determined within those two blocks: a set of 4 miRNAs (hsa-miR-221, -27a, -27b and -29b) was up-expressed in EC/HIV-_block and down-expressed in VP/ART_block while 19 miRNAs were down-expressed in block 1 and up-expressed in block 2. Differential miRNAs were successfully validated through individual RT-qPCR assays.

Conclusions

Profile in EC resembled HIV- and differentially clusters with VP and ART. Therefore, differential clustering does not rely on undetectable viremia.     

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

Background

Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India.

Results

Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p < 0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p = 0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000–25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians.

Conclusion

The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.     

Application of Polymerase Chain Reaction to Detect HIV-1 DNA in Pools of Dried Blood Spots

Nucleic acid tests that detect HIV infection at an early phase are available and have been applied on individual dried blood spot (DBS). The present study was undertaken with an aim to evaluate the feasibility of performing PCR for HIV-1 DNA on pools of DBS as an alternative to individual testing. Standardization of PCR by a modified Amplicor HIV-1 DNA assay version 1.5 (Roche molecular diagnostics, USA), on pooled DBS was performed using five confirmed HIV reactive samples with known low viral load of HIV-1 and HIV non-reactive samples in pools of 5, 10 and 20 DBS. After successful standardization of pooling procedure, a total of 183 pools (of 10 DBS each) were prepared from 1,823 DBS samples, collected from a population-based study that tested negative for HIV antibodies and p24 antigen. All these pools were screened for HIV-1 DNA by the Amplicor assay. Standardization of pooling procedure indicated that pooling of 10 DBS gave an optimum result. Out of 183 pools tested, one pool of 10 samples was positive and of these ten DBS that were tested individually to identify the positive DBS, one sample was detected to be positive for HIV-1 DNA. Our study demonstrates that PCR for HIV-1 DNA can be successfully performed on pools of DBS. However, this may be needed only on specialized studies of HIV and not for routine epidemiology studies as only a very small fraction of cases would be missed if only antibody/antigen testing were done.     

Development of an HIV-1 Subtype Panel in China: Isolation and Characterization of 30 HIV-1 Primary Strains Circulating in China

BackgroundThe complex epidemic and significant diversity of HIV-1 strains in China pose serious challenges for surveillance and diagnostic assays, vaccine development and clinical management. There is a lack of HIV-1 isolates in current canonical HIV-1 subtype panels that can represent HIV-1 diversity in China; an HIV-1 subtype panel for China is urgently needed.MethodsBlood samples were collected from HIV-1 infected patients participating in the drug-resistance surveillance program in China. The samples were isolated, cultured and stored as neat culture supernatant. The HIV-1 isolates were fully characterized. The panel was used to compare 2 viral load assays and 2 p24 assays as the examples of how this panel could be used.ResultsAn HIV-1 subtype panel for China composed of 30 HIV-1 primary strains of four subtypes (B [including Thai-B], CRF01_AE, CRF07_BC and G) was established. The samples were isolated and cultured to a high-titer (10⁶-10⁹ copies/ml)/high-volume (40ml). The HIV-1 isolates were fully characterized by the final viral load, p24 concentration, gag-pol and envC2V3 sequencing, co-receptor prediction, determination of the four amino acids at the tip of the env V3-loop, glycosylation sites in the V3 loop and the drug-resistance mutations. The comparison of two p24 assays and two viral load assays on the isolates illustrated how this panel may be used for the evaluation of diagnostic assay performance. The Pearson value between p24 assays were 0.938. The viral load results showed excellent concordance and agreement for samples of Thai-B, but lower correlations for samples of CRF01_AE.ConclusionThe current panel of 30 HIV-1 isolates served as a basis for the development of a comprehensive panel of fully characterized viral isolates, which could reflect the current dynamic and complex HIV-1 epidemic in China. This panel will be available to support HIV-1 research, assay evaluation, vaccine and drug development.     

The gp120 Protein Is a Second Determinant of Decreased Neurovirulence of Indian HIV-1C Isolates Compared to Southern African HIV-1C Isolates

Regional differences in neurovirulence have been documented among subtype/clade-C HIV-1 isolates in India and Southern Africa. We previously demonstrated that a C31S substitution in Clade-C Tat dicysteine motif reduces monocyte recruitment, cytokine induction and direct neurotoxicity. Therefore, this polymorphism is considered to be a causative factor for these differences in neurovirulence. We previously reported on the genotypic differences in Tat protein between clade-C and rest of the clades showing that approximately 90% of clade-C HIV-1 Tat sequences worldwide contained this C31S polymorphism, while 99% of non-clade C isolates lacked this Tat polymorphism at C31 residue (Ranga et al. (2004) J Virol 78∶2586–2590). Subsequently, we documented intra-clade-C differences in the frequency of Tat dicysteine variants between India and Southern Africa, as the basis for differential disease severity and showed the importance of the Tat dicysteine motif for neuropathogenesis using small animal models. We have now examined if determinants of neurovirulence besides Tat are different between the clade-C HIV-1 isolates from Southern Africa and India. Envelope glycoprotein gp120 is a well-documented contributor to neurotoxicity. We found that gp120 sequences of HIV-1 isolates from these two regions are genetically distinct. In order to delineate the contribution of gp120 to neurovirulence, we compared direct in vitro neurotoxicity of HIV-infected supernatants of a representative neurovirulent US clade-B isolate with two isolates each from Southern Africa and India using primary human neurons and SH-SY5Y neuroblastoma cells. Immunodepletion of gp120 of both US clade B and the Southern African clade C isolates revealed robust decreases in neurotoxicity, while that of the Indian isolates showed minimal effect on neurotoxicity. The gp120 as a cause of differential neurotoxicity was further confirmed using purified recombinant gp120 from HIV isolates from these regions. We conclude that gp120 is one of the key factors responsible for the decreased neurovirulence of Indian clade C HIV-1 isolates when compared to South African clade C HIV-1.     

Comparison of HIV-1 Genotypic Resistance Test Interpretation Systems in Predicting Virological Outcomes Over Time

Background

Several decision support systems have been developed to interpret HIV-1 drug resistance genotyping results. This study compares the ability of the most commonly used systems (ANRS, Rega, and Stanford''s HIVdb) to predict virological outcome at 12, 24, and 48 weeks.

Methodology/Principal Findings

Included were 3763 treatment-change episodes (TCEs) for which a HIV-1 genotype was available at the time of changing treatment with at least one follow-up viral load measurement. Genotypic susceptibility scores for the active regimens were calculated using scores defined by each interpretation system. Using logistic regression, we determined the association between the genotypic susceptibility score and proportion of TCEs having an undetectable viral load (<50 copies/ml) at 12 (8–16) weeks (2152 TCEs), 24 (16–32) weeks (2570 TCEs), and 48 (44–52) weeks (1083 TCEs). The Area under the ROC curve was calculated using a 10-fold cross-validation to compare the different interpretation systems regarding the sensitivity and specificity for predicting undetectable viral load. The mean genotypic susceptibility score of the systems was slightly smaller for HIVdb, with 1.92±1.17, compared to Rega and ANRS, with 2.22±1.09 and 2.23±1.05, respectively. However, similar odds ratio''s were found for the association between each-unit increase in genotypic susceptibility score and undetectable viral load at week 12; 1.6 [95% confidence interval 1.5–1.7] for HIVdb, 1.7 [1.5–1.8] for ANRS, and 1.7 [1.9–1.6] for Rega. Odds ratio''s increased over time, but remained comparable (odds ratio''s ranging between 1.9–2.1 at 24 weeks and 1.9–2.2 at 48 weeks). The Area under the curve of the ROC did not differ between the systems at all time points; p = 0.60 at week 12, p = 0.71 at week 24, and p = 0.97 at week 48.

Conclusions/Significance

Three commonly used HIV drug resistance interpretation systems ANRS, Rega and HIVdb predict virological response at 12, 24, and 48 weeks, after change of treatment to the same extent.     

Reliability at the Lower Limits of HIV-1 RNA Quantification in Clinical Samples: A Comparison of RT-PCR versus bDNA Assays

Introduction

To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods.

Methodology/Principal Findings

We selected patients with ≥1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads ≥1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds'' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods.

Discussion

These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results.     

Virologic Response to Tipranavir-Ritonavir or Darunavir-Ritonavir Based Regimens in Antiretroviral Therapy Experienced HIV-1 Patients: A Meta-Analysis and Meta-Regression of Randomized Controlled Clinical Trials

Background

The development of tipranavir and darunavir, second generation non-peptidic HIV protease inhibitors, with marked improved resistance profiles, has opened a new perspective on the treatment of antiretroviral therapy (ART) experienced HIV patients with poor viral load control. The aim of this study was to determine the virologic response in ART experienced patients to tipranavir-ritonavir and darunavir-ritonavir based regimens.

Methods and Findings

A computer based literature search was conducted in the databases of HINARI (Health InterNetwork Access to Research Initiative), Medline and Cochrane library. Meta-analysis was performed by including randomized controlled studies that were conducted in ART experienced patients with plasma viral load above 1,000 copies HIV RNA/ml. The odds ratios and 95% confidence intervals (CI) for viral loads of <50 copies and <400 copies HIV RNA/ml at the end of the intervention were determined by the random effects model. Meta-regression, sensitivity analysis and funnel plots were done. The number of HIV-1 patients who were on either a tipranavir-ritonavir or darunavir-ritonavir based regimen and achieved viral load less than 50 copies HIV RNA/ml was significantly higher (overall OR = 3.4; 95% CI, 2.61– 4.52) than the number of HIV-1 patients who were on investigator selected boosted comparator HIV-1 protease inhibitors (CPIs-ritonavir). Similarly, the number of patients with viral load less than 400 copies HIV RNA/ml was significantly higher in either the tipranavir-ritonavir or darunavir-ritonavir based regimen treated group (overall OR = 3.0; 95% CI, 2.15 – 4.11). Meta-regression showed that the viral load reduction was independent of baseline viral load, baseline CD4 count and duration of tipranavir-ritonavir or darunavir-ritonavir based regimen.

Conclusions

Tipranavir and darunavir based regimens were more effective in patients who were ART experienced and had poor viral load control. Further studies are required to determine their consistent viral load suppression effect as the duration of treatment is more prolonged.     

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy,Viral Evolution and Compartmentalization

Background

Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.

Methods & Results

We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.

Conclusions

The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.     

Using Plasma Viral Load to Guide Antiretroviral Therapy Initiation to Prevent HIV-1 Transmission

Background

Current WHO guidelines recommend antiretroviral therapy (ART) initiation at CD4 counts ≤350 cells/µL. Increasing this threshold has been proposed, with a primary goal of reducing HIV-1 infectiousness. Because the quantity of HIV-1 in plasma is the primary predictor of HIV-1 transmission, consideration of plasma viral load in ART initiation guidelines is warranted.

Methods

Using per-sex-act infectivity estimates and cross-sectional sexual behavior data from 2,484 HIV-1 infected persons with CD4 counts >350 enrolled in a study of African heterosexual HIV-1 serodiscordant couples, we calculated the number of transmissions expected and the number potentially averted under selected scenarios for ART initiation: i) CD4 count <500 cells/µL, ii) viral load ≥10,000 or ≥50,000 copies/mL and iii) universal treatment. For each scenario, we estimated the proportion of expected infections that could be averted, the proportion of infected persons initiating treatment, and the ratio of these proportions.

Results

Initiating treatment at viral load ≥50,000 copies/mL would require treating 19.8% of infected persons with CD4 counts >350 while averting 40.5% of expected transmissions (ratio 2.0); treating at viral load ≥10,0000 copies/mL had a ratio of 1.5. In contrast, initiation at CD4 count <500 would require treating 41.8%, while averting 48.4% (ratio 1.1).

Conclusion

Inclusion of viral load in ART initiation guidelines could permit targeting ART resources to HIV-1 infected persons who have a higher risk of transmitting HIV-1. Further work is needed to estimate costs and feasibility.     

Raltegravir Cerebrospinal Fluid Concentrations in HIV-1 Infection

Introduction

Raltegravir is an HIV-1 integrase inhibitor currently used in treatment-experienced HIV-1-infected patients resistant to other drug classes. In order to assess its central nervous system penetration, we measured raltegravir concentrations in cerebrospinal fluid (CSF) and plasma in subjects receiving antiretroviral treatment regimens containing this drug.

Methods

Raltegravir concentrations were determined by liquid chromatography tandem mass spectrometry in 25 paired CSF and plasma samples from 16 HIV-1-infected individuals. The lower limit of quantitation was 2.0 ng/ml for CSF and 10 ng/ml for plasma.

Results

Twenty-four of the 25 CSF samples had detectable raltegravir concentrations with a median raltegravir concentration of 18.4 ng/ml (range, <2.0–126.0). The median plasma raltegravir concentration was 448 ng/ml (range, 37–5180). CSF raltegravir concentrations correlated with CSF:plasma albumin ratios and CSF albumin concentrations.

Conclusions

Approximately 50% of the CSF specimens exceeded the IC₉₅ levels reported to inhibit HIV-1 strains without resistance to integrase inhibitors. In addition to contributing to control of systemic HIV-1 infection, raltegravir achieves local inhibitory concentrations in CSF in most, but not all, patients. Blood-brain and blood-CSF barriers likely restrict drug entry, while enhanced permeability of these barriers enhances drug entry.     

Recalibration of the Limiting Antigen Avidity EIA to Determine Mean Duration of Recent Infection in Divergent HIV-1 Subtypes

BackgroundMean duration of recent infection (MDRI) and misclassification of long-term HIV-1 infections, as proportion false recent (PFR), are critical parameters for laboratory-based assays for estimating HIV-1 incidence. Recent review of the data by us and others indicated that MDRI of LAg-Avidity EIA estimated previously required recalibration. We present here results of recalibration efforts using >250 seroconversion panels and multiple statistical methods to ensure accuracy and consensus.MethodsA total of 2737 longitudinal specimens collected from 259 seroconverting individuals infected with diverse HIV-1 subtypes were tested with the LAg-Avidity EIA as previously described. Data were analyzed for determination of MDRI at ODn cutoffs of 1.0 to 2.0 using 7 statistical approaches and sub-analyzed by HIV-1 subtypes. In addition, 3740 specimens from individuals with infection >1 year, including 488 from patients with AIDS, were tested for PFR at varying cutoffs.ResultsUsing different statistical methods, MDRI values ranged from 88–94 days at cutoff ODn = 1.0 to 177–183 days at ODn = 2.0. The MDRI values were similar by different methods suggesting coherence of different approaches. Testing for misclassification among long-term infections indicated that overall PFRs were 0.6% to 2.5% at increasing cutoffs of 1.0 to 2.0, respectively. Balancing the need for a longer MDRI and smaller PFR (<2.0%) suggests that a cutoff ODn = 1.5, corresponding to an MDRI of 130 days should be used for cross-sectional application. The MDRI varied among subtypes from 109 days (subtype A&D) to 152 days (subtype C).ConclusionsBased on the new data and revised analysis, we recommend an ODn cutoff = 1.5 to classify recent and long-term infections, corresponding to an MDRI of 130 days (118–142). Determination of revised parameters for estimation of HIV-1 incidence should facilitate application of the LAg-Avidity EIA for worldwide use.