Detection and differentiation of HIV-2 using the point-of-care Alere q HIV-1/2 Detect nucleic acid test

BackgroundThe Alere q HIV-1/2 Detect test (Alere Detect) is a rapid point-of-care (POC) nucleic acid test (NAT) that can detect and differentiate HIV-1 and HIV-2 in 25-μL whole blood or plasma samples. The Alere Detect test has been validated for early infant diagnosis of HIV-1 infection, and it is the only POC NAT device currently known to detect HIV-2, which is endemic in West Africa.ObjectivesTo evaluate the sensitivity detecting HIV-2 RNA and the differential performance of the Alere Detect.Study designPlasma samples from non-HIV (n = 4), HIV-1 (n = 22), HIV-2 (n = 111; 29 Group A, 2 Group B) and HIV-1/HIV-2 dually-seropositive (n = 8) participants in Senegal and the United States and HIV-2 reference strains (3 Group A, 1 Group B) were tested by Alere Detect, Abbott RealTime HIV-1 and the University of Washington HIV-2 RNA quantitative (UW HIV-2) assays.ResultsThe Alere Detect correctly differentiated between HIV-1 and HIV-2 in all 80 (100%) patient samples with detectable HIV RNA (n = 20 HIV-1, 60 HIV-2). The overall HIV-2 detection concordance between Alere Detect and the UW HIV-2 assay was 68% (54/80); the concordance improved to 100% (30/30) for samples with HIV-2 RNA >300copies/mL. Neither assay detected HIV-2 RNA in 31 of 111 HIV-2 seropositive samples.ConclusionsThe Alere Detect test is a novel device detecting HIV RNA in clinical samples, and differentiating HIV-1 and HIV-2 with a high level of specificity. It has the potential for use as a rapid HIV-2 NAT-based diagnosis tool in resource-limited settings and to confirm HIV-2 infection for the CDC 4th generation HIV-1/2 diagnostic algorithm.     

Comparative evaluation of three FDA-approved HIV Ag/Ab combination tests using a genetically diverse HIV panel and diagnostic specimens

BackgroundHIV Ag/Ab combination assays are recommended by CDC for routine screening and several HIV Ag/Ab combination tests are now FDA-approved. Maintaining high specificity and consistent sensitivity across diverse HIV strains is critical for these assays to accurately detect HIV infection and expedite delivery of patient results.ObjectivesTo evaluate performance of three FDA-approved HIV tests: ARCHITECT HIV Combo (Abbott), ADVIA Centaur HIV Combo (Siemens) and BioPlex HIV Ag-Ab (Bio-Rad).Study designSensitivity and specificity were evaluated using an extensive panel of 28 HIV infected human specimens and 17 cultured virus isolates representing multiple genotypes, 6 seroconversion panels, 4 human samples with acute infection, WHO p24 standard and 4020 clinical specimens.ResultsThe p24 limit of detection (LOD) for the WHO standard was 0.19 IU/ml, 0.70 IU/ml, and 1.77 IU/ml in BioPlex, ARCHITECT, and Centaur respectively. The distribution of LODs across 15 HIV-1 isolates was substantially narrower in ARCHITECT (5–33 pg/ml) than in BioPlex (11–198 pg/ml) and Centaur (6–384 pg/ml). All assays detected antibodies to the majority of HIV-1 and HIV-2 variants. However, reduced sensitivity was observed for Centaur in detection of antibodies to HIV-1 group M (CRF02_AG), O and N variants. BioPlex and ARCHITECT showed better seroconversion sensitivity than Centaur, detecting one bleed (3–7 days) earlier in 4 (BioPlex) and 3 (ARCHITECT) of 6 seroconversion panels. ARCHITECT demonstrated the highest specificity (99.90–100%) compared to BioPlex (99.80%) and Centaur (99.42%).ConclusionsThe overall performance of ARCHITECT and BioPlex was superior to Centaur, especially for detection of acute HIV infection.     

Comparative performance of the Geenius™ HIV-1/HIV-2 supplemental test in Florida's public health testing population

BackgroundThe Centers for Disease Control and Prevention (CDC) published updated guidelines in 2014 for the laboratory diagnosis of HIV in the United States, which recommend use of a supplemental immunoassay (IA) that differentiates HIV-1 from HIV-2 after a repeatedly reactive HIV-1/2 antigen/antibody “Combo” screening test. In October 2014, Bio-Rad Laboratories introduced the FDA-cleared Geenius HIV-1/HIV-2 Supplemental assay and in July 2016, it replaced the Multispot HIV-1/HIV-2 differentiation rapid test as the second test in the HIV diagnostic algorithm.ObjectiveTo compare performance of the new FDA-cleared Bio-Rad Geenius HIV-1/HIV-2 Supplemental assay and the Bio-Rad Multispot HIV-1/HIV-2 differentiation assay for use as the primary supplemental test in the 2014 CDC/APHL HIV Diagnostic Algorithm.Study designTwo sets of specimens were used to assess the performance of Geenius; 340 select retrospective specimens, obtained through routine clinical submissions from individuals seeking HIV serostatus determinations and 10 known HIV-2 antibody reactive specimens provided by Bio-Rad Laboratories. Panels were created and characterized solely by in-house laboratory results. The panels consisted of: algorithm-defined “established HIV-1 infections” (n = 250), “acute HIV-1 infections” (n = 20), “early HIV-1 infections” (n = 10) and “false positive Combo specimens” (n = 60).Results

• •Geenius results for the 250 specimens classified as HIV-1 established infections were HIV-1 Positive (246) or HIV Positive Untypable (4), a 100% concordance with HIV positivity observed with Multispot results (HIV-1 Positive, HIV-2 Negative).
• •Of the 20 HIV-1 algorithm-defined acute infection specimens (Multispot Negative), 85% (17/20) were Geenius HIV-1 Negative and 15% (3/20) HIV-1 Indeterminate.
• •Similarly, of the 10 algorithm-defined early infection specimens (MS Indeterminate), 30% (3/10) were Geenius HIV-1 Indeterminate, 60% (6/10) HIV-1 Positive and 10% (1/10) HIV Negative.
• •Of the 60 false positive HIV-1/2 Combo panel, 98.3% (59/60) were HIV-1 Negative by Geenius and 95% (57/60) were HIV-1 Negative by Multispot.
• •Geenius was concordant with all HIV-2 antibody positive specimens.

ConclusionsThe Geenius assay provides significant advantages over Multispot as an appropriate replacement for the primary supplemental test in the HIV Diagnostic Algorithm. In this retrospective study, Geenius was highly concordant with Multispot, reclassified some acute and early algorithm-defined HIV-1 positive specimens and demonstrated a potential decrease in the number HIV-1 RNA nucleic acid amplification tests needed to complete the diagnostic algorithm.     

Does HIV-1 co-receptor tropism correlate with fibrosis progression in HIV/HCV co-infected patients?

BackgroundIn HIV/HCV co-infected patients, HIV-1 gp120 activates human hepatic stellate cells (HSCs) which play a key role in fibrosis pathogenesis. It is still unclear whether pro-fibrogenic effects are more attributable to X4 or R5 strains in vivo.ObjectiveTo assess if HIV-1 X4 or R5 variants are associated with a different progression of fibrosis.Study designA total of 105 HIV/HCV co-infected patients were submitted to gp120 sequencing on proviral DNA and classified as X4 or R5 based on g2p (20% false positive rate). The fibrosis evolution was retrospectively determined by means of APRI and FIB-4 scores at 3-month intervals from the first anti-HCV-positive test. The association of co-receptor tropism with increased fibrosis scores was evaluated by linear mixed models.ResultsX4 variants were found in 41 patients (39%). The median observation period was similar in X4 and R5 patients (17 years). No difference was observed between the two groups of patients, except for nadir CD4 which was lower in X4 compared to R5 (percentage, p = 0.005, and absolute number, p = 0.005). X4 and R5 patients did not significantly differ for FIB-4 and APRI score over time (p = 0.5, and p = 0.1, respectively). No association between HCV-RNA levels over time and co-receptor tropism was noted (p = 0.9). Conversely, a significant correlation of fibrosis scores with gamma-glutamyl transferase levels, lower current CD4 count, HIV viremia and use of antiretrovirals was observed.ConclusionsThis retrospective analysis of fibrosis evolution did not support the evidence of a differing pro-fibrogenic activity for X4 and R5 HIV-1 variants in HIV/HCV co-infected patients.     

The fourth generation AlereTM HIV Combo rapid test improves detection of acute infection in MTN-003 (VOICE) samples

BackgroundEarly and accurate detection of HIV is crucial when using pre-exposure prophylaxis (PrEP) for HIV prevention to avoid PrEP initiation in acutely infected individuals and to minimize the risk of drug resistance in individuals with breakthrough infection.ObjectiveTo determine if fourth-generation antigen/antibody (Ag/Ab) rapid diagnostic tests (RDT) would have detected HIV infection earlier than the third-generation RDT used in MTN-003 (VOICE).Study design5029 VOICE participants were evaluated with third-generation Alere Determine™ HIV-1/2, OraQuick ADVANCE^® Rapid HIV-1/2, Uni-Gold™ Recombigen^® HIV-1/2 and Bio-Rad GS HIV-1/2 + O EIA; and fourth-generation Alere Determine™ HIV-1/2 Ag/Ab Combo, Conformité Européene (CE)-Marked Alere™ HIV Combo and Bio-Rad HIV Combo Ag/Ab EIA. Multispot^®, GS HIV-1 Western Blot (WB) and Geenius™ (Bio-Rad) were also evaluated.ResultsOf 57 antibody-negative pre-seroconversion plasma samples with HIV RNA >20 copies/mL identified, 16 (28%) were reactive by CE-Marked Alere™ HIV Combo (1 Ab; 9 Ag; 6 Ag/Ab reactive) and 4 (7%) by Alere Determine™ HIV-1/2 Ag/Ab Combo (2 Ab; 2 Ag; 0 Ag/Ab reactive) (p = 0.0005). Multispot^® confirmed only 1 of 16 acute infections while WB and Geenius™ confirmed none. GS HIV Combo Ag/Ab EIA identified 27 of 57 (47%) pre-seroconversion RNA-positive samples.ConclusionIn VOICE, 28% of infections missed by current third-generation RDT would have been identified with the use of CE-Marked Alere™ HIV Combo. Geenius™, Multispot^® and WB were all insensitive ( < 10%) in confirming infections detected by fourth-generation assays. An improved diagnostic algorithm that includes a fourth-generation RDT with HIV RNA testing will be essential for efficiently identifying seroconverters on PrEP.     

Performance comparison of the 4th generation Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA on the EVOLIS™ automated system versus Abbott ARCHITECT HIV Ag/Ab Combo,Ortho Anti-HIV 1 + 2 EIA on Vitros ECi and Siemens HIV-1/O/2 enhanced on Advia Centaur

BackgroundA multisite study was conducted to evaluate the performance of the Bio-Rad 4th generation GS HIV Combo Ag/Ab EIA versus Abbott 4th generation ARCHITECT HIV Ag/Ab Combo. The performance of two 3rd generation EIAs, Ortho Diagnostics Anti-HIV 1 + 2 EIA and Siemens HIV 1/O/2 was also evaluated.ObjectiveStudy objective was comparison of analytical HIV-1 p24 antigen detection, sensitivity in HIV-1 seroconversion panels, specificity in blood donors and two HIV false reactive panels.Study designAnalytical sensitivity was evaluated with International HIV-1 p24 antigen standards, the AFFSAPS (pg/mL) and WHO 90/636 (IU/mL) standards; sensitivity in acute infection was compared on 55 seroconversion samples, and specificity was evaluated on 1000 negative blood donors and two false reactive panels.ResultsGS HIV Combo Ag/Ab demonstrated better analytical HIV antigen sensitivity compared to ARCHITECT HIV Ag/Ab Combo: 0.41 IU/mL versus 1.2 IU/mL (WHO) and 12.7 pg/mL versus 20.1 pg/mL (AFSSAPS); GS HIV Combo Ag/Ab EIA also demonstrated slightly better specificity compared to ARCHITECT HIV Ag/Ab Combo (100% versus 99.7%). The 4th generation HIV Combo tests detected seroconversion 7–11 days earlier than the 3rd generation HIV antibody only EIAs.ConclusionBoth 4th generation immunoassays demonstrated excellent performance in sensitivity, with the reduction of the serological window period (7–11 days earlier detection than the 3rd generation HIV tests). However, GS HIV Combo Ag/Ab demonstrated improved HIV antigen analytical sensitivity and slightly better specificity when compared to ARCHITECT HIV Ag/Ab Combo assay, with higher positive predictive values (PPV) for low prevalence populations.     

Real-world performance of the new US HIV testing algorithm in medical settings

BackgroundOur medical center laboratory recently adapted its 24/7, two-hourly testing program to use an ARCHITECT-Multispot-viral load (AR-MS-VL) algorithm in place of a previous rapid test-immunofluorescence (RT-IF) algorithm.ObjectivesWe evaluated screening test performance, acute case detection, turnaround time and ability to resolve HIV status under the new algorithm.Study designWe considered consecutive HIV tests from January to November 2015. AR-MS-VL results at Zuckerberg San Francisco General Hospital and Trauma Center (ZSFG) were compared with RT-IF results at ZSFG and also with AR-MS-VL results in the recently completed CDC Screening Targeted Populations to Interrupt On-going Chains of HIV Transmission with Enhanced Partner Notification (STOP) Study for targeted testing of MSM at publicly funded testing sites in San Francisco.ResultsAmong 21,985 HIV tests performed at ZSFG, 16,467 were tested by RT-IF and 5518 by AR-MS-VL. There were 321 HIV infections detected, of which 274 (84%) were known HIV+ cases, and 47 were newly identified HIV infections. Considering only patients of HIV-negative or -unknown status, prevalence was 0.22%. Under the AR-MS-VL algorithm, turnaround times for screening results and full algorithm results were 3 and 21 h; status-unresolved cases were reduced (from 47% to 22%) compared with the RT-IF algorithm. The positive predictive value (PPV) of a new-positive AR screening test was low (0.44) at ZSFG, where no acute infections were detected. At STOP Study sites where HIV prevalence was higher and acute infection was more common, the AR PPV was higher (0.93). All 24 false-positive AR screening tests at ZSFG had a signal/cutoff (S/CO) ratio of <15 and all 88 true-positive tests had S/CO ratio >15. Of 62 acute infections in the STOP Study, 23 (37%) had an S/CO < 15.DiscussionAn AR-MS-VL algorithm is feasible and can return rapid results in a large medical center. In this setting, reactive 4th generation assay tests that are negative for HIV antibodies are typically false-positive with low S/CO ratios.     

Evaluation of dried blood spot protocols with the Bio-Rad GS HIV Combo Ag/Ab EIA and Geenius™ HIV 1/2 Supplemental Assay

ObjectiveFDA-approved antigen/antibody combo and HIV-1/2 differentiation supplemental tests do not have claims for dried blood spot (DBS) use. We compared two DBS-modified protocols, the Bio-Rad GS HIV Combo Ag/Ab (BRC) EIA and Geenius™ HIV-1/2 (Geenius) Supplemental Assay, to plasma protocols and evaluated them in the CDC/APHL HIV diagnostic algorithm.MethodsBRC-DBS p24 analytical sensitivity was calculated from serial dilutions of p24. DBS specimens included 11 HIV-1 seroconverters, 151 HIV-1-positive individuals, including 20 on antiretroviral therapy, 31 HIV-2-positive and one HIV-1/HIV-2-positive individuals. BRC-reactive specimens were tested with Geenius using the same DBS eluate. Matched plasma specimens were tested with BRC, an IgG/IgM immunoassay and Geenius. DBS and plasma results were compared using the McNemar's test. A DBS-algorithm applied to 348 DBS from high-risk individuals who participated in surveillance was compared to HIV status based on local testing algorithms.ResultsBRC-DBS detects p24 at a concentration 18 times higher than in plasma. In seroconverters, BRC-DBS detected more infections than the IgG/IgM immunoassay in plasma (p = 0.0133), but fewer infections than BRC-plasma (p = 0.0133). In addition, the BRC/Geenius-plasma algorithm identified more HIV-1 infections than the BRC/Geenius-DBS algorithm (p = 0.0455). The DBS protocols correctly identified HIV status for established HIV-1 infections, including those on therapy, HIV-2 infections, and surveillance specimens.ConclusionsThe DBS protocols exhibited promising performance and allowed rapid supplemental testing. Although the DBS algorithm missed some early infections, it showed similar results when applied to specimens from a high-risk population. Implementation of a DBS algorithm would benefit testing programs without capacity for venipuncture.     

Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection

BackgroundIn the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing.ObjectivesTo evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection.Study designA series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients > 18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children ≤ 18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay.ResultsMultispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot.ConclusionsMultispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2.     

Microbial translocation is associated with residual viral replication in HAART-treated HIV+ subjects with <50 copies/ml HIV-1 RNA

BackgroundRecent data have shown that plasma levels of lipopolysaccharide (LPS) are a quantitative indicator of microbial translocation in HIV infected individuals.ObjectivesTo assess the impact of residual viral replication on plasma LPS in HAART-treated HIV+ subjects with <50 copies/ml HIV-1 RNA and to evaluate LPS changes during repeated HAART interruptions not exceeding 2-month duration.Study designLPS was measured in 44 HIV+ subjects at T0 (during HAART) and at day 15 of the first and fourth HAART interruption. Ten uninfected, healthy donors were studied as well. Residual plasma HIV-1 RNA was measured at T0 by an ultra-ultrasensitive method with limit of detection of 2.5 copies HIV-1 RNA/ml. Subjects with less than 2.5 copies/ml (fully suppressed – FS) were compared to those with 2.5–50 copies/ml (partially suppressed – PS).ResultsAt T0, plasma LPS levels were comparable in FS and uninfected subjects, whereas in PS they were higher than in uninfected subjects (p = 0.049). After 4 HAART interruptions, they did not change significantly. However, LPS values were lower in FS than in PS (p = 0.020). An inverse correlation was found between CD4 and LPS levels (p = 0.044) in PS group only.ConclusionsA reduced degree of microbial translocation was seen in subjects with a more complete suppression of viral replication. Repeated HAART interruptions had no significant impact on plasma LPS levels.     

Evaluation of the performance of a new automated HIV combination assay

BackgroundMore and more countries test for HIV infection using combination assays that simultaneously detect p24 antigen and HIV antibodies.ObjectiveTo assess the performance of a new HIV combo assay: LIAISON^® XL murex HIV Ab/Ag HT.Study designThe assays were examined with a total of 3090 samples that included 769 selected HIV antibody-negative samples, 1849 unselected HIV samples, 15 HIV-1 p24 Ag reference samples, 90 primary HIV-1 infection (PHI) samples, 167 HIV-1 antibody-positive samples (well characterized of groups M and O), 95 HIV-1 antibody-positive samples and 105 HIV-2 antibody-positive samples.ResultsThe specificity of the LIAISON^® XL murex HIV Ab/Ag HT was 99.7%. The analytical sensitivity of Ag p24 of the LIAISON^® XL murex HIV Ab/Ag HT was 0.58 IU/mL and 9.93 pg/mL when using WHO and French national standards, respectively. All screened HIV subtypes was identified by this assay. Also, 90 PHI specimens were detected by this screening assay.ConclusionThe sensitivity and specificity of the LIAISON^® XL murex HIV Ab/Ag HT assay are high. Hence the assay offers automated high-throughput screening with ability to detect primary infection.     

Performances of fourth generation HIV antigen/antibody assays on filter paper for detection of early HIV infections

BackgroundPoint-of-care testing and diagnosis of HIV acute infections play important roles in preventing transmission, but HIV rapid diagnosis tests have poor capacity to detect early infections. Filter paper can be used for capillary blood collection and HIV testing using 4th generation immunoassays.ObjectivesAntigen/antibody combined immunoassays were evaluated for their capacity to identify early HIV infections using filter paper in comparison with rapid test.Study designThirty nine serum samples collected from HIV seroconverters were spotted onto filter paper and tested by the Roche Elecsys^® HIV Combi PT test and the DiaSorin Liaison XL Murex HIV Ab/Ag assay.ResultsFourth generation immunoassays identified 34 out of 39 HIV early infections using dried serum spot, whereas the Determine™ HIV-1/2 rapid test detected 24 out of 39 HIV positive serum (87.2% vs 61.5% respectively, p = 0.009). p24 antigen was detected by the Liaison XL in 19 dried serum samples (48.7%). In the group characterized by a negative western blot, 7 out of 8 (87.5%) and 6 out of 8 (75.0%) samples were found positive for HIV using the Elecsys and the Liaison XL, respectively. None of these eight samples classified in this group of early acute infections were found positive by the rapid test.ConclusionFourth generation Ag/Ab immunoassays performed on dried serum spot had good performance for HIV testing during the early phases of HIV infection. This method may be useful to detect HIV early infections in hard-to-reach populations and individuals living in remote areas before rapid tests become positive.     

Performance of Determine Combo and other point-of-care HIV tests among Seattle MSM

Background and objectiveThe rapid test study was a real-time comparison of point-of-care (POC) HIV tests to determine their abilities to detect early HIV infection.Study designMen and transgender persons reporting sex with men in the prior year were recruited at the Public Health—Seattle & King County STD Clinic, Gay City Health Project, and University of Washington Primary Infection Clinic. Study tests included the OraQuick ADVANCE Rapid HIV-1/2 Antibody Test performed on oral fluids and tests performed on fingerstick whole blood specimens including OraQuick, Uni-Gold Recombigen HIV test, Determine HIV-1/2 Ag/Ab Combo, and INSTI HIV-1 Rapid Antibody Test. Specimens from subjects with negative results were sent for EIA and nucleic acid amplification testing. McNemar's exact tests compared the numbers of HIV-infected subjects detected.ResultsBetween February 2010 and August 2014, there were 3438 study visits. Twenty-four subjects had discordant POC results with at least one reactive and one non-reactive test, including one subject with a reactive Determine p24 antigen. OraQuick performed on oral fluids identified fewer persons compared to all fingerstick tests. OraQuick performed on fingerstick whole blood detected fewer persons compared to the Determine Combo antibody component (p = .008) and Combo overall (p = .004), and there was a trend when compared to INSTI (p = .06). The Determine Combo specificity was 98.99%.ConclusionsAs reported by others, Determine Combo underperforms compared to laboratory-based testing, but it did detect one acute infection. If these results are validated, the specificity of Determine Combo may limit its usefulness in populations with lower HIV incidence.     

Microbial translocation is associated with residual viral replication in HAART-treated HIV+ subjects with <50 copies/ml HIV-1 RNA

BackgroundRecent data have shown that plasma levels of lipopolysaccharide (LPS) are a quantitative indicator of microbial translocation in HIV infected individuals.ObjectivesTo assess the impact of residual viral replication on plasma LPS in HAART-treated HIV+ subjects with <50 copies/ml HIV-1 RNA and to evaluate LPS changes during repeated HAART interruptions not exceeding 2-month duration.Study designLPS was measured in 44 HIV+ subjects at T0 (during HAART) and at day 15 of the first and fourth HAART interruption. Ten uninfected, healthy donors were studied as well. Residual plasma HIV-1 RNA was measured at T0 by an ultra-ultrasensitive method with limit of detection of 2.5 copies HIV-1 RNA/ml. Subjects with less than 2.5 copies/ml (fully suppressed – FS) were compared to those with 2.5–50 copies/ml (partially suppressed – PS).ResultsAt T0, plasma LPS levels were comparable in FS and uninfected subjects, whereas in PS they were higher than in uninfected subjects (p = 0.049). After 4 HAART interruptions, they did not change significantly. However, LPS values were lower in FS than in PS (p = 0.020). An inverse correlation was found between CD4 and LPS levels (p = 0.044) in PS group only.ConclusionsA reduced degree of microbial translocation was seen in subjects with a more complete suppression of viral replication. Repeated HAART interruptions had no significant impact on plasma LPS levels.     

Evaluation of Xpert HIV-1 Qual assay for resolution of HIV-1 infection in samples with negative or indeterminate Geenius HIV-1/2 results

BackgroundDiagnosis of HIV infection is a multistage algorithm. Following screening with 4^th generation combination immunoassay, confirmation of HIV infection is performed with an antibody assay that differentiates HIV-1 from HIV-2 infection. In the newly updated algorithm, samples that are nonreactive or indeterminate in the differentiation assay are to be tested with an HIV-1 nucleic acid amplification (NAAT) test for resolution. Xpert HIV-1 Qual is a new NAAT assay approved for the identification of HIV infection in whole and dried blood.ObjectivesTo assess the performance of Xpert HIV-1 Qual supplementary assay in resolving the clinical status of serum samples reactive by 4^th generation immunoassays and indeterminate or negative by Geenius HIV-1/2 confirmatory assay.Study designIn a retrospective study, samples from 97 individuals for whom the true HIV-1 status was already known (by follow-up samples) and which were negative or indeterminate by HIV-1/2 Geenius assay were tested with Xpert Qual HIV-1 assay.ResultsXpert Qual assay correctly classified all 97 samples from HIV-1 positive (n = 49) and negative (n = 48) individuals. The sensitivity and specificity of Xpert Qual when using the true HIV status as a reference were 100% (92.7–100% at 95% confidence interval [CI] and 92.6–100% at 95% CI, respectively).ConclusionsApplying Xpert Qual HIV-1 assay in the new HIV multi-stage diagnostic algorithm correctly classified 100% of HIV-1 infections including 49 from HIV-1 carriers who have not yet seroconverted. With this assay the total time required for acute HIV diagnosis could be significantly reduced.     

Viral load assay sensitivity and low level viremia in HAART treated HIV patients

BackgroundThe recent introduction of highly sensitive viral load assays resulted in a significant increase in number of treated HIV-infected patients with a detectable viral load. The significance of a viral load between 20 and 50 copies/mL remains unclear.ObjectivesTo compare the performance of three viral load assays, with special attention for specificity and sensitivity at the lowest level of quantification.Study designSamples (n = 181) were selected from 62 HIV-positive individuals that experience viral blips or episodes of low but detectable viremia under antiretroviral treatment, and from 216 HIV-negative individuals. Each sample was tested in at least two of three assays: the Cobas Amplicor HIV-1 Monitor (CAP/CA), the Cobas Ampliprep/Cobas TaqMan HIV-1 version 1 (CAP/CTM1) and the Cobas Ampliprep/Cobas TaqMan HIV-1 version 2 (CAP/CTM2).ResultsNo false positive results were recorded. Kappa statistics revealed fair to moderate agreement between the results of the three assays, but important differences in sensitivity were observed, with the highest sensitivity reported for CAP/CTM2 followed by CAP/CTM1 and CAP/CA. The differences in sensitivity remained after equalization of the detection limit for all assays at 50 copies/mL. Analysis of samples collected over time showed that patients with single blips in CAP/CA present with recurrent blips in CAP/CTM1 and persistent detectable viremia in CAP/CTM2.ConclusionsViral load results between 20 and 50 copies/mL in either CAP/CTM1 or CAP/CTM2, indicate true viremia. The availability of highly sensitive assays force reconsideration of the terms ‘undetectable’ viral load and ‘virological success’ of antiretroviral treatment.     

HIV-1 viral load and phenotypic antiretroviral drug resistance assays based on reverse transcriptase activity in comparison to amplification based HIV-1 RNA and genotypic assays

BackgroundAmplification based HIV-1 viral load and genotypic resistance assays are expensive, technologically complex and may be difficult to implement in resource limited settings. Inexpensive, simpler assays are urgently needed.ObjectivesTo determine the suitability of the ExaVir? Load and ExaVir? Drug assays for use in patient monitoring.Study designSpecimens from 108 adults were used to compare ExaVir? Load HIV-1 RT to Amplicor HIV-1 Monitor^® HIV-1 RNA, and ExaVir? Drug phenotype to HIV GenoSure? genotype.ResultsHIV-1 RT and HIV-1 RNA levels were comparable (Pearson correlation coefficient 0.83). Most (94%) had detectable results in both assays. The mean difference (HIV-1 RT minus HIV-1 RNA) was ?0.21 log₁₀ cps/mL equiv. Relationship between HIV-1 RT and HIV-1 RNA was not affected by RT mutations, CD4 cell count, or efavirenz (EFV) or nevirapine (NVP) use. Phenotypes were generally consistent with genotype findings for EFV, but not for NVP. Most patients (93.9%) with phenotypic EFV resistance had at least one EFV mutation, while 78.0% of patients with phenotypic NVP resistance had at least one NVP mutation. Eleven of 49 samples tested for EFV susceptibility were found resistant (n = 2) or with reduced susceptibility (n = 9) despite the absence of genotypic resistance. Eleven of 45 samples tested for NVP susceptibility were found resistant (n = 9) or with reduced susceptibility (n = 2) with no evidence of genotypic mutations.ConclusionsThe ExaVir? Load assay performed well and may be an alternative to amplification based techniques for HIV-1 RNA quantification. The ExaVir? Drug assay for phenotypic resistance testing requires further evaluation, especially for NVP.     

Comparative effectiveness of dried plasma HIV-1 viral load testing in Brazil using ViveST for sample collection

BackgroundUtilization of dried plasma for HIV-1 viral load testing would significantly decrease sample shipping costs.ObjectivesTo describe the precision and reproducibility of ViveST^® (ST) as a transportation method for shipping specimens for HIV-1 viral load (VL) testing.Study designThirty clinical plasma samples were used to generate replicate samples with HIV VL values of 4 log₁₀, 3 log₁₀ and 2 log₁₀ copies/mL for reproducibility testing and an additional 299 samples with HIV VL <50 copies/mL (99); 1.7 log₁₀ to 3.99 log₁₀ (100); and 4 log₁₀ to 5.99 log₁₀/mL (100) were used to compare ViveST to frozen plasma samples using the VERSANT^® HIV-1 RNA 3.0 Assay. Results were compared using Student t-test, Pearson correlation and Bland–Altman analyses.ResultsMean intra-assay variance among frozen and dried plasma triplicates was 0.15 log₁₀ and 0.09 log₁₀ copies/mL respectively (n = 10, P = NS). Compared to frozen plasma, there was a mean reduction of 0.3 log₁₀, 0.27 log₁₀, and 0.35 log₁₀ copies/mL at the 4 log₁₀, 3 log₁₀, and 2 log₁₀ copy/mL samples respectively (n = 30, all comparisons, P < 0.01). Overall correlation between 299 frozen and ViveST samples was r = 0.97, where 12 of 99 undetectable frozen VL were positive with ST, and 12 of 200 frozen detectable VL were undetectable with ViveST (mean VL 2.1, 1.9 log₁₀ copies/mL respectively).ConclusionsHIV-1 viral load results using ViveST were reproducible, correlated well with frozen plasma, though yielding minimally lower values. Our data suggest that dried plasma for HIV-1 VL testing using ViveST has promise for use in HIV clinical practice.     

Association of plasma viremia with HIV-1 RNA levels in cervicovaginal lavage secretions in HIV-1 seropositive ART naïve women from Pune,India

BackgroundCoherent drug/microbicide/vaccine development research would benefit through a precise knowledge of HIV dissemination and its persistence in the female genital tract. Understanding relationship between plasma viremia and cervicovaginal HIV shedding may help to unveil mechanisms underlying transmission, compartmentalization and pathogenesis.ObjectivesTo study the association between HIV-1 RNA levels in the plasma and CVL specimens.Study designWhole blood, plasma and CVL specimens were collected from 36 ART naïve HIV-1 seropositive women qualifying the study inclusion criteria. Absolute CD4 counts, plasma and CVL HIV-1 RNA levels were estimated using commercially available kits (BD MultiSET™ Kit, Becton Dickinson, US and Abbott RT, Abbott Molecular, Germany). Correlation between plasma and CVL viral load was estimated by the Spearman's rank correlation coefficient. Additionally, the correlation between CVL viral load and absolute CD4 counts was studied.ResultsHIV-1 viral load in the CVL specimens was successfully quantified using the Abbott RT. Twenty-seven of 36 women (75%) had detectable HIV-1 RNA levels in plasma and CVL specimens. The CVL viral load did not show any correlation with plasma viral load (ρ = 0.281, p = 0.096) and showed a ‘moderate correlation’ (ρ = −0.563, p = 0.0004) with absolute CD4 counts.ConclusionsAlbeit, the Abbott RT is designed for estimating plasma HIV-1 RNA levels, the study reports its use for estimating HIV-1 RNA levels in the CVL specimens as well. In accordance with the previous studies, our results suggest that plasma and CVL viral load are not correlated and plasma viremia might not solely predict cervico vaginal HIV shedding.     

Detection and quantification of HIV-1 RNA with a fully automated transcription-mediated-amplification assay

BackgroundNucleic acid testing is the major method used to monitor HIV viral load. Commercial systems based on real-time PCR assays are available for high-volume centralized laboratory testing, but they are not fully automated.Objectives and study designWe have compared the diagnostic performance of the Hologic Aptima HIV-1 Quant Dx assay (Aptima) (based on real-time TMA) on the Panther instrument, a fully-automated random access platform, to that of, the Roche Cobas Ampliprep Cobas TaqMan (CAP/CTM) HIV-1 version 2.0 (based on real-time PCR).ResultsProbit analysis of replicate dilutions of NIBSC WHO International HIV-1 Standard, gave LODs of 8.6 c/ml for Aptima and 15.2 c/ml for CAP/CTM. The agreement between the assays was excellent when measuring HIV RNA in a calibrated reference (κ = 0.90, p < 0.001) and good when measuring clinical samples (κ = 0.62, p < 0.001). The correlation among the samples quantified by the two methods was very good (r = 0.95, p< 0.001) and the mean difference between the values obtained with the two assays was 0.02 log c/ml for B and non-B subtypes. The vast majority of results showed <0.5 log variance between the two assays (89%); only one sample showed results that differed by over 1.0 log c/ml.ConclusionThe performance of the new fully automated Aptima assay is adequate for clinical monitoring of HIV-1 RNA during infections and treatment. The Aptima assay is well suited for routine laboratory use.