HIV-1 Viral Load Assay Research Articles

Performance of point-of-care Xpert HIV-1 plasma viral load assay at a tertiary HIV care centre in Southern India.

Sustainable suppression of HIV replication forms the basis of anti-retroviral therapy (ART) medication. Thus, reliable quantification of HIV viral load has become an essential factor to monitor the effectiveness of the ART. Longer turnaround-time (TAT), batch testing and technical skills are major drawbacks of standard real-time PCR assays. The performance of the point-of-care Xpert HIV-1 viral load assay was evaluated against the Abbott RealTime PCR m2000rt system. A total of 96 plasma specimens ranging from 2.5 log10 copies ml-1 to 4.99 log10 copies ml-1 and proficiency testing panel specimens were used. Precision and accuracy were checked using the Pearson correlation co-efficient test and Bland-Altman analysis. Compared to the Abbott RealTime PCR, the Xpert HIV-1 viral load assay showed a good correlation (Pearson r=0.81; P<0.0001) with a mean difference of 0.27 log10 copies ml-1 (95 % CI, -0.41 to 0.96 log10 copies ml-1; sd, 0.35 log10 copies ml-1). Reliable and ease of testing individual specimens could make the Xpert HIV-1 viral load assay an efficient alternative method for ART monitoring in clinical management of HIV disease in resource-limited settings. The rapid test results (less than 2 h) could help in making an immediate clinical decision, which further strengthens patient care.

Comparison between Roche and Xpert in HIV-1 RNA quantitation: A high concordance between the two techniques except for a CRF02_AG subtype variant with high viral load titters detected by Roche but undetected by Xpert

BackgroundHIV-1 viral load (VL) testing is important to predict viral progression and to monitor the response to antiretroviral therapy. New HIV-1 VL tests are continuously introduced to the market. Their performance is usually compared to Abbott and/or Roche HIV-1 VL assays, as reference. The Xpert HIV-1 VL test was recently introduced, but its performance compared to Roche has not been sufficiently studied. ObjectivesTo compare the Xpert assay with Roche and to assess its use in the HIV clinical laboratory. Study designA total of 383 plasma samples of HIV-1 infected patients previously tested by Roche, were retrospectively tested by Xpert to determine concordance between the two assays. Samples included a diversity of HIV-1 subtypes and a wide range of VLs. ResultsThere was a high concordance between the two assays, except for a CRF02_AG subtype variant with high VL titters, that was detected by Roche but undetected by Xpert. The 5′ long terminal repeat gene region of this virus, targeted by the Xpert assay, was amplified and sequenced. A 25 nucleotide insert was identified, but was unmatched to any known sequences of HIV-1. This particular insert, however could not explain the false-negativity by the Xpert assay. ConclusionsThis study underlines the challenge to routine VL testing due to the high genetic diversity of HIV-1. Clinicians should, therefore be advised that a negative VL in cases where the clinical picture does not match the laboratory report, might in fact be, a false-negative result of the VL assay.

Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia.

Feasibility of HIV-1 RNA Extraction and Viral Load Testing of Bone Marrow Plasma Using the Abbott m2000 Platform and RealTime Quantitative HIV-1 Assay

Current efforts to fi nd a cure for HIV disease require that viral load assays be available to accurately detect and measure viral load in compartments other than peripheral blood. Over the years, HIV viral load assays have been adapted to measure viral presence in cerebral spinal fl uid (CSF), genital secretions and various other compartments.

Prospective evaluation of Cepheid Xpert HIV-1 Viral Load assay as a supplemental confirmatory HIV-1 test in the routine clinical laboratory setting

Prospective evaluation of Cepheid Xpert HIV-1 Viral Load assay as a supplemental confirmatory HIV-1 test in the routine clinical laboratory setting

Performance Evaluation of the New HIV-1 Quantification Assay, Xpert HIV-1 Viral Load, on a Wide Panel of HIV-1 Variants.

To evaluate the quantification performance of the new Cepheid GeneXpert HIV-1 viral load assay, on a wide panel of HIV-1 variants. Clinical performance was evaluated relative to the Abbott RealTime HIV-1 assay on 285 HIV-1 seropositive samples selected to cover the assays quantification range (40 copies/mL-10,000,000 copies/mL), and included RNA undetectable or detected seropositive samples. The panel comprised 120 subtype B, 150 non-B, and 15 nontypable clinical samples; serial dilutions of 18 viral supernatants representative of the divergent viruses of HIV-1 groups N, O, and P were also tested. Based on samples selected according to the Abbott assay viral loads (VL), the Cepheid assay detected or quantified 222/285 (78%) samples and the Abbott assay 240/285 (84%). Xpert yielded VLs for 162 (76%) of the 213 quantifiable samples with Abbott. This difference corresponded to 51 samples with VL >40 copies/mL by the Abbott assay (all below 200 copies/mL) but detected (n = 40) or undetectable (n = 11) by the Cepheid assay. VL of samples quantifiable by both assays (n = 162) showed very strong correlation, with a Spearman correlation coefficient of 0.985 and a Bland-Altman's mean of differences of -0.01. Performance for quantification of the non-M samples showed very good correlation, with significantly higher values with Cepheid for the group N and 2 group O samples. Our study showed that the Xpert HIV-1 VL assay offered very good performance for detection and quantification of the current HIV-1 genetic diversity; differences reported at the threshold could be an issue and requires further evaluations. The practicability of this new assay makes it suitable for low-income countries, where it could facilitate and improve follow-up of patients, as well as for high-income regions.

Multi-site clinical evaluation of the Xpert® HIV-1 viral load assay

BackgroundThe most important reason for measuring HIV-1 viral load (VL) is to monitor the effectiveness of antiretroviral therapy (ART), both for the initial therapeutic response and sustained responses. Maintaining low or undetectable HIV-1 VL levels can reduce both the risks of progression to AIDS and transmission of infection to others. ObjectivesTo evaluate the diagnostic accuracy of Xpert® HIV-1 Viral Load (VL) assay compared to the Abbott RealTime HIV-1 assay, including assessing specificity by testing plasma specimens from confirmed HIV-1 negative blood donors. Study designSubjects were enrolled from 4 participating sites, 2 in Europe and 2 in the USA. Fresh plasma samples were tested prospectively, while frozen plasma samples were collected prospectively, and tested retrospectively after selection of specimens to cover the assay’s quantification range (40cp/mL–10,000,000 cp/mL). Eligibility criteria included a clinician ordered HIV-1 VL test from a confirmed HIV-1 positive adult (≥18 years) with a known antiviral treatment status. Exclusion criteria included previous enrollment in this study or improper specimen collection. Human blood donor specimens determined to be HIV-1 negative by standard blood bank antibody and nucleic acid amplification methods were used to assess specificity. ResultsOf the 764 specimens collected, 752 were eligible for inclusion but 5 were not tested by the Xpert, leaving 747 specimens tested (28.2% from females and 71.8% from males). Valid results were obtained for 724/747 (96.9%) specimens tested using the Xpert HIV-1 VL assay. The Xpert HIV-1 VL assay detected or quantified 568/724 (78.5%) specimens, while the RealTime HIV-1 assay detected or quantified 559/724 (77.2%). Of the 724 specimens tested by both assays, 390 were quantified by both assays and showed strong correlation: r=0.9847, with an R2=0.9696. Bland-Altman analysis showed good agreement between the two assays (381/390; 97.7%) with a distribution within 0.5 log10 cp/mL centered around zero. Xpert yielded VLs for 393 (80%) of the 494 quantifiable samples by Abbott. VLs of those specimens quantified by one of the assays, and either detected but not quantified or not detected by the other assay were all <170cp/mL. Specificity of the Xpert assay was found to be 100% (109/109), 95% CI: 96.7–100.0. ConclusionVery good correlation was seen between the Xpert HIV-1 VL and Abbott RealTime HIV-1 assays, with added benefits for Xpert HIV-1 VL of: (1) lot-to-lot consistency traceable to WHO International Standard, (2) requiring both high and low level internal controls to be in range to have a valid result, (3) use of a single HIV-1 target for PCR and (4) faster turn-around-time for results, no need to wait to do batch testing of specimens. In summary, Xpert HIV-1 VL generated accurate VL results that if implemented could allow for actionable and timely treatment decisions during the same clinic visit. This scenario could reduce the loss to follow up often seen when these test results take days to weeks to become available to the clinician and patient.

Factors associated with HIV viral load suppression on antiretroviral therapy in Vietnam

BackgroundLimited data are available on HIV viral suppression rates among men and women on antiretroviral therapy (ART) and factors associated with HIV RNA viral load (VL) suppression in Vietnam. MethodsWe conducted a cross-sectional survey of 1255 adult patients on ART for at least 1 year across four provinces in Vietnam. Data collection included a standardised questionnaire, routine laboratory testing, and an HIV VL assay. Bivariate and logistic multivariate analyses were conducted to assess viral suppression rates and factors associated with unsuppressed HIV VL. ResultsThe median age was 34.5 years and the median time on ART was 46 months. Gender was 66% male (n=828) and 34% female (n=427). HIV viral suppression below 1000 copies/mL was 93%. Viral suppression among woman was not significantly different than among men (93.7% vs92.9%; P=0.59). On multivariate analysis, unsuppressed HIV VL was independently associated with lower CD4 cell count, social isolation, high stigma, not receiving a single-tablet daily regimen, multiple late appointments in past year, and immunological failure. ConclusionOn-treatment viral load suppression rates in Vietnam are high and already exceed the UNAIDS 90% target for viral suppression on ART. Gender does not impact viral suppression rates of patients on ART in Vietnam. Access to routine viral load testing should be improved, adherence monitoring and counselling streamlined, and ART regimens simplified to maintain viral suppression rates, as more people start ART. Psychological and social factors are also associated with unsuppressed HIV VL, necessitating treatment support interventions to address social isolation and stigma among people living with HIV in Vietnam.

Pitfalls of antiretroviral drug resistance genotyping of HIV-1 Group M and Group N from Cameroon by sequenced-based assays.

Background:HIV-1 genotyping for antiretroviral drug resistance mutations (DRMs) were developed based basically on subtype B HIV-1 Group M, which represents only 10% of HIV strains worldwide. In sub-Saharan Africa, non-B subtypes HIV-1 largely predominate and HIV-1 genetic diversity could affect the performance of drug resistance genotyping assays. We compared prospectively the performance of the ViroSeq® and Trugene® genotyping assays to detect DRM in HIV-1-infected adult patients living in Douala, Cameroun.Materials and Methods:DRM in protease (P) and reverse transcriptase (RT) genes were assessed in parallel using both ViroSeq® and Trugene® assays in plasma samples from 45 first-line antiretroviral treatment-experienced patients in Douala, Cameroon.Results:Trugene HIV-1 Genotyping Assay® (Siemens Health Care Diagnostics, NY, USA) and ViroSeq HIV-1 Genotyping System®(Celera Diagnostics, CA, USA) assessed equivalently antiretroviral DRMs in P and RT genes from non-B HIV-1 Group M in 44 Cameroonian adults in virological failure; Trugene® was slightly more sensitive than ViroSeq® (100% vs. 91%). One patient infected by HIV-1 Group N was successfully amplified only by the Trugene HIV-1 Genotyping assay®, while ViroSeq HIV-1 Genotyping System v2.0® assay could not.Conclusion:Results showed the higher performance of the Trugene® system to detected and amplify P and RT genes targeting DRM to the principal antiretroviral drugs used in sub-Saharan Africa. Discrepancies between the results of HIV viral load assays and molecular tests should alert clinicians and virologists to the possibility of infection by an atypical variant virus, especially in Central Africa where very broad HIV-1 genetic diversity exists.

Service impact of a change in HIV-1 viral load quantification assay.

IntroductionDue to discontinuation of the Siemens Versant HIV-1 RNA (bDNA) assay in the UK, our laboratory switched to the Roche Cobas Ampliprep/Taqman HIV-1 viral load (VL) assay (Roche) in April 2013. This assay has a lower cut-off of 20 RNA copies/mL (compared with <50 for the Siemens assay). Our laboratory demonstrated previously that a significant proportion (18%) of patients undetectable using bDNA HIV-1 RNA quantification exhibited low level viraemia (LLV) using the new assay. Local guidelines recommend that patients stable on therapy receive twice-yearly VLs. We evaluated the impact of the introduction of the new assay on our clinical service.MethodsA retrospective cohort analysis of treated patients with stable undetectable VL by bDNA (<50 copies/mL) followed by ≥ one low-level (<400 copies/mL) VL with the Roche assay. Demographic data were collected in addition to frequency of VL testing and genotypic resistance assays. Referrals to virtual clinic (VC) were recorded. Patients were identified using laboratory data and information collected from electronic patient records. Results were analyzed with SPSS v18.ResultsOne hundred and ninety patients were included. Demographics: 79.5% male; 60.6% homosexual; mean age of 46 years. Duration on stable treatment was 46.35 (std. dev. 38.15) months. Current treatment regimens were 43.3% PI-based; 43.3% NNRTI-based and 13.7% other. Patients were stratified into VL 20–49 copies/mL (n=109); VL 50–199 copies/mL (n=71) and VL 200–399 copies/mL (n=10). In total, there were 471 VLs measured of which 274 were additional as a result of the assay switch. This resulted in six HIV-1 genotype requests and 16 VC discussions (Table 1). Longer duration on HAART was associated with reduced frequency of VL testing. The relative risk of ongoing detectability according to drug class are: PI 1.62 (95% CI 1.18–2.21); NNRTI 0.507 (95% CI 0.30–0.85) and other 1.09 (95% CI 0.48–2.43). ConclusionsChanges in assay can result in difficulties in interpretation of patient results. The assay switch in our service had significant impact on patient and staff time and cost with an increase in patient recalls; increased frequency of VL measurement, genotypes and discussions in VC. Choice of assay is paramount to running an efficient and cost-effective clinical service.

Drug resistance is rarely the cause or consequence of long-term persistent low-level viraemia in HIV-1-infected patients on ART.

The introduction of highly sensitive HIV-1 viral load assays with a lower quantification limit of 20 copies/ml uncovered that in a number of patients on ART, the viral load systematically fluctuates around or slightly above the detection limit of the assays. This study aimed to analyse the presence or occurrence of drug resistance mutations in HIV-1-infected patients during long-term persistent low-level viraemia (PLLV) under ART. A retrospective study was carried out in which baseline and on-therapy presence of drug resistance mutations in the HIV-1 protease and reverse transcriptase genes were analysed in patients with PLLV between 20 and 250 copies/ml. For all available plasma samples collected during PLLV, resistance analysis was attempted with an ultrasensitive amplification and sequencing protocol. Resistance analysis was successful for 154 samples collected longitudinally from 23 patients over a median period of 4.7 years (IQR 3.3-5.7). Twenty of these patients were on a boosted protease inhibitor (PI)-based regimen (87%). Single drug resistance mutations were detected in isolated samples of 4 patients, 2 of the 3 patients who initiated a non-nucleoside reverse transcriptase inhibitor-based regimen and 2 of the 20 on a PI-based regimen. Only one of the detected mutations decreased susceptibility to the therapy regimen taken at the time of sample collection. Drug resistance mutations were not found in the three patients who developed virological failure (viral load >250 copies/ml) during the study. Long episodes of PLLV in patients on boosted PI-based regimens rarely result in the selection of new drug-resistant variants.

Comparing HIV viral load assays and frequency of low level virological rebound in clinical practice

Research suggests that some low-level virological rebound results occurring for no obvious clinical cause, in patients stable on antiretroviral therapy (ART), may be a consequence of the viral load assay used. We compared the relative frequency of clinically unexplained low-level virological rebound results when using the Roche HIV Taqman version-1 (CTM v1), the Roche HIV Taqman version-2 (CTM v2) and the Abbott RealTime (Abbott RT) assays in clinical practice. In all, 247 patients from our centre who had their viral loads measured by the three different assays over a period of 3 consecutive years (each assay used for a period of 1 year each) were included in the study. Low-level virological rebound was defined as <1000 copies/ml. Over similar time periods, there was significant discrepancy between the three assays when considering the proportion of clinically unexplained low-level virological rebound results in patients stable on ART: the CTM v2 assay produced the highest percentage (93%), CTM v1 much lower (65%) and Abbott RT even less (35%). There is further research required regarding what, if any, implications this has for patients who experience clinically unexplained low-level virological rebound on the more sensitive assays.

Clinical Evaluation of a Low Cost, In-House Developed Real-Time RT-PCR Human Immunodeficiency Virus Type 1 (HIV-1) Quantitation Assay for HIV-1 Infected Patients

ObjectivesHIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access.Materials and MethodsA real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays.ResultsThe assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100–1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A–H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate.ConclusionsWith a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.

Systematic Review of the Performance of HIV Viral Load Technologies on Plasma Samples

BackgroundViral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring.Methods and FindingsA search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2–26% and 9–70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0–5.1%) and 5.44% (range 1.17–30.00%) across the range of VL counts (2log10–7log10).ConclusionsThis review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same technology platform to ensure appropriate interpretation of changes in VL.Prospero registration # CD42013003603.

Identification of HIV-1 Genotypic Resistance in Patients on First-line Antiretroviral Therapy Using Polymerase Chain Reaction and Sequencing

Abstract Objective The aim of the study was to evaluate the characteristics of HIV drug-genotypic resistance among patients taking first-line ARV regimens using polymerase chain reaction and sequencing, and guide to design optimal ARV regimens for these patients. Methods HIV reverse transcriptase-encoded gene was amplified with RT-PCR and amplified PCR products were aligned and comparatively analyzed with HIV resistance database to find drug-resistance mutations. Results Twenty-eight PCR products were amplified and sequenced successfully in 30 serum samples of recruited HIV-infected patients with virologic failure. The resistance rate was 96%, mutations in NRT region were found in 26 patients (93%), while mutations in NNRT region were found in 27 patients (96%). M184V was the most common mutation (86%), K65R was selected in 14% of recruited individuals and TAMs occurred in 50% of patients, which resulted in resistance to NRTIs. Y181C and V179D were the most common mutations in NNRTIs and prevalence was 43% (12/28) and 36% (10/28), respectively, which resulted in cross-resistance to NNRTIs due to low-genetic barrier. Conclusions Virologic failure may occur in long-term administration of first-line ARV regimens, and drugresistance mutations can be found in these patients, which resulted in resistance to first-line ARV regimens. We emphasized that HIV viral load assay and resistance assay were important tools to guide healthcare workers to design an optimal second-line ARV regimens for HAART-experienced individuals with virologic failure.

Minimal sequence variability of the region of HIV-1 integrase targeted by the Abbott RealTime HIV-1 viral load assay in clinical specimens with reduced susceptibility to raltegravir

The Abbott RealTime (ART) HIV-1 assay targets the integrase region and is designed to tolerate mismatches. Variability in integrase sequences comprising the assay target regions from >1000 clinical specimens submitted for phenotypic and genotypic raltegravir resistance testing were analyzed. In this large collection of sequences from clinical specimens, the number and location of raltegravir resistance associated mutations did not differ from those tested previously and shown not to result in under-estimation of viral loads.

Comparative evaluation of the Abbott HIV-1 RealTime™ assay with the Standard Roche COBAS® Amplicor™ HIV-1 Monitor® Test, v1.5 for determining HIV-1 RNA levels in plasma specimens from Pune, India

The implementation of cost effective HIV-1 viral load assays in resource-limited settings have been an impediment for monitoring HIV-1 therapy. A study involving the comparative analytical performance of two HIV-1 viral load assays – Standard Roche COBAS® Amplicor™ HIV-1 Monitor® Test, version 1.5 (Roche Diagnostics, Basel, Switzerland) and Abbott HIV-1 RealTime™ assay (Abbott Molecular, Wiesbaden, Germany) was performed using 125 specimens in Pune, India. A strong correlation was observed between the manual endpoint reverse transcriptase polymerase chain reaction assay and the recent real time polymerase chain reaction assay (r=0.989, p value<0.0001) and agreement was 94.4%. Results of the study indicate a higher sensitivity of the Abbott HIV-1 RealTime™ assay for HIV-1 Virology Quality Assurance copy controls as compared to the Standard Roche COBAS® Amplicor™ HIV-1 Monitor® Test, version 1.5. Furthermore, features of the Abbott m2000rt RealTime™ PCR assay platform such as higher analytical sensitivity, automated/manual extraction platforms for high/low sample throughputs and ability to quantify a variety of infectious agents (Hepatitis B virus, Hepatitis C virus, Human Papillomavirus and Neisseria gonorrhoeae/Chlamydia trachomatis) justify its suitability in resource-limited Indian settings. Besides, the study also highlights utility of the precise Virology Quality Assurance validation template in performance evaluation of various quantitative viral load assays.

&lt;i&gt;In Vitro&lt;/i&gt; Testing of African Traditional Medicines for Cytotoxic, Immune Modulatory and Anti-HIV Activities

African Traditional Medicines (ATMs) serve as a major source of primary healthcare for African people. The reasons for their use range from easy access, affordability, beliefs in traditional systems and long term safety. ATMs have been used to treat individuals infected with HIV and therefore need scientific validation; a view supported by Traditional Health Practitioners (THPs). This study aimed to evaluate the in vitro cytotoxicity, immune modulatory and anti-HIV activities of traditional multiple herbal preparations from local THPs. Ugambu, Ihashi, Product Nene, Product Blue, SPNa and SDKc ATM were supplied by local THPs. Changes in adenosine triphosphate (ATP) & glutathione (GSH) over 24 hours were measured using luminometry. Changes in 12 cytokines were assayed using an ELISA-based absorbance assay. Protective effects against HIV killing of MT-4 cells were tested using the XTT assay and antiviral activity was measured using an HIV-1 viral load assay. Cyclosporine and AZT were used as positive controls. Ugambu, Ihashi, Product Nene and SDKc induced a dose dependent toxicity on treated PBMCs by reducing ATP and GSH at high doses (p< 0.001). These medicinal preparations, along with SPNa, showed immunomodulatory activity by significantly (p< 0.001) changing the secretion of pro-inflammatory cytokines. Product Blue stimulated the levels of ATP and GSH in treated PBMCs at all doses however this product did not show any immunomodulatory activity on cytokine secretion when compared to control cells. Ugambu, Ihashi, Product Nene showed promising anti-HIV activity relative to AZT (p< 0.01). This study has shown that some of these traditional medicinal preparations have at least one or all the properties of immunostimulation, immunomodulation or antiretroviral effects. The mechanism of action of the shown activities should further be investigated.

Technical and Regulatory Shortcomings of the TaqMan Version 1 HIV Viral Load Assay

BackgroundThe lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility. Sudden increases in the number of patients with detectable pVL have been reported following the introduction of the TaqMan version 1 assay.MethodsBetween October 2009 and April 2010 all routine pVL samples from British Columbia, Canada, with 40–250 copies/mL by TaqMan were re-tested by Amplicor (N = 1198). Subsequent short-term virological and resistance outcomes were followed in patients with unchanged therapy (N = 279; median 3.2 months follow-up).ResultsTaqMan and Amplicor values correlated poorly at low pVL values. Low-level pVL by TaqMan was not associated with impending short-term virological failure; only 17% of patients with 40–250 copies/mL by TaqMan had detectable pVL by Amplicor at follow-up. During the follow-up period only 20% of patients had an increase in pVL by TaqMan (median [IQR]: 80 [36–283] copies/mL). In addition, in ∼2.4% of samples pVL was dramatically underestimated by TaqMan due to poor binding of the proprietary TaqMan primers.ConclusionsThe replacement of Amplicor with the TaqMan assay has altered the previously accepted definition of HIV treatment failure without any evidence to support the clinical relevance of the new definition. Given the systematic differences in measurement in the low pVL range the British Columbia HIV treatment guidelines now use a threshold of >250 copies/mL by TaqMan to define treatment failure.

Comparison of HIV-1 viral load assay performance in immunological stable patients with low or undetectable viremia

The goal of antiretroviral therapy is reduction in morbidity and mortality via suppression of human immunodeficiency virus (HIV) viral load (VL) to undetectable levels. VL assay sensitivity has improved over time, but the reproducibility and clinical importance of VL results marginally higher than the limit of detection (LoD) are uncertain. We assessed the reproducibility and concordance of low VL results obtained with the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 version 2.0 (CAP-CTM) and the Abbott RealTime (m2000) HIV-1 assays, using longitudinal specimens from HIV-1-infected patients with low VL (<300copies/ml) and stable CD4+ cell counts. Based on replicate testing of 3 specimens, coefficients of variation for log-transformed VL results were 5-8% for m2000 and 9-10% for CAP-CTM. The concordance between assays in specimens from patients with previously undetectable, detectable but not quantifiable VL, or variable (undetectable/detectable but not quantifiable VL) results over time was 90, 56, and 56%, respectively. Correlation between results for specimens with quantifiable VL (initially 40-300copies/ml) was moderate (R (2)=0.48) with significantly higher results for CAP-CTM and a mean difference (CAP-CTM minus m2000) of 0.10 log(10)copies/ml. T-cell activation (CD8+/CD38+ percentage) in patients with low VL was initially higher than in patients with undetectable VL, and then decreased to equivalent levels over time. These results indicate that residual viremia at levels slightly above the LoD have no negative effect on T-cell activation.