HIV-1 Assay Research Articles

A highly sensitive aptamer-based HIV reverse transcriptase detection assay

Although many new assays for HIV have been developed, several labs still use simple and reliable radioactivity-based reverse transcriptase (RT) nucleotide incorporation assays for detection and quantification. We describe here a new assay for detection and quantitation of HIV RT activity that is based on a high affinity DNA aptamer to RT. The aptamer is sequestered on 96-well plates where it can bind to RT and other constituents can be removed by extensive washing. Since the aptamer mimics a primer-template, upon radiolabeled nucleotide addition, bound RT molecules can extend the aptamer and the radioactive signal can be detected by standard methods. In addition to being procedurally simple, the assay demonstrated high sensitivity (detection limits for RT and virions were ≤6400 molecules (∼4 × 10−8 units) and ∼100–300 virions, respectively) and was essentially linear over a range of at least 104. Both wild type and drug-resistant forms of HIV-1 RT were detectable as was HIV-2 RT, although there were some modest differences in sensitivity.

ARCHITECT HIV Combo Ag/Ab and RealTime HIV-1 Assays Detect Diverse HIV Strains in Clinical Specimens

Periodic evaluation of the impact of viral diversity on diagnostic tests is critical to ensure current technologies are keeping pace with viral evolution. To determine whether HIV diversity impacts the ARCHITECT HIV Combo Ag/Ab (HIV Combo) or RealTime HIV-1 (RT) assays, a set of N = 199 HIV clinical specimens from Cameroon, Senegal, Saudi Arabia, and Thailand were sequenced and tested in both assays. The panel included historical groups N and P specimens and a newly identified group N specimen. These and specimens classified as H, U (unclassified)/URF (unique recombinant form), CRF (circulating recombinant form) 01, 02, 06, 09, 11, 13, 18, 22, 37, and 43 were detected by both the RT assay (1.75–6.84 log copies/ml) and the HIV Combo assay (3.26–1121.96 sample to cutoff ratios). Sequence alignment identified 3 or fewer mismatches to the RT assay oligos in 82.4% of samples. Altogether, these data demonstrate the HIV Combo and RT assays detect diverse strains of HIV in clinical specimens.

What resources are used in emergency departments in rural sub-Saharan Africa? A retrospective analysis of patient care in a district-level hospital in Uganda

ObjectivesTo determine the most commonly used resources (provider procedural skills, medications, laboratory studies and imaging) needed to care for patients.SettingA single emergency department (ED) of a district-level hospital in rural...

SALMONELLA TYPHIMURIUM MENINGITIS IN AN IMMUNOCOMPETENT INFANT

Meningitis due to Salmonella is rare and associated with high morbidity, mortality, and relapse rates despite adequate treatment.Here, we report a case of meningitis due to Salmonella typhimurium in an immunocompetent infant. The child was brought to thepediatric emergency department with complaints of high-grade fever, loose motions, vomiting, and irritability of 3-day duration.Cerebrospinal fluid (CSF) analysis was suggestive of acute bacterial meningitis, and the child was started on intravenous ceftriaxone.The CSF and blood cultures grew S. typhimurium and the treatment was accordingly modified. His serum immunoglobulin levelswere normal, and enzyme-linked immunosorbent assay for HIV was negative. Clinical course, antibiotic treatment, and outcomeof the case are discussed here.

Multicountry Validation of SAMBA - A Novel Molecular Point-of-Care Test for HIV-1 Detection in Resource-Limited Setting.

Early diagnosis of HIV-1 infection and the prompt initiation of antiretroviral therapy are critical to achieving a reduction in the morbidity and mortality of infected infants. The Simple AMplification-Based Assay (SAMBA) HIV-1 Qual Whole Blood Test was developed specifically for early infant diagnosis and prevention of mother-to-child transmission programs implemented at the point-of-care in resource-limited settings. We have evaluated the performance of this test run on the SAMBA I semiautomated platform with fresh whole blood specimens collected from 202 adults and 745 infants in Kenya, Uganda, and Zimbabwe. Results were compared with those obtained with the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 assay as performed with fresh whole blood or dried blood spots of the same subjects, and discrepancies were resolved with alternative assays. The performance of the SAMBA and CAP/CTM assays evaluated at 5 laboratories in the 3 countries was similar for both adult and infant samples. The clinical sensitivity, specificity, positive predictive value, and negative predictive value for the SAMBA test were 100%, 99.2%, 98.7%, and 100%, respectively, with adult samples, and 98.5%, 99.8%, 99.7%, and 98.8%, respectively, with infant samples. Our data suggest that the SAMBA HIV-1 Qual Whole Blood Test would be effective for early diagnosis of HIV-1 infection in infants at point-of-care settings in sub-Saharan Africa.

Adalimumab-Inducing Reactivation of Tuberculosis With Previously Negative QuantiFERON Test

Intestinal tuberculosis (TB) is a very rare disease in the 21st Century. This case highlights the necessity of including TB enteritis as a differential diagnosis in patients who had previously negative interferon-gamma release assay (Quantiferron) who presents with non-specific abdominal symptoms while on immunosuppressive therapy. A 37 year old Bangladeshi male with a remote history of treated pulmonary TB and Psoriasis, presented with fever and loose stools for one month. Fever was intermittent, associated with night sweats and mild RLQ discomfort. He denied sick contacts or recent travel history. Three months prior to admission, dermatology had initiated Adalimumab (ADA) for refractory Psoriasis. Prior to starting ADA, sputum and interferon-gamma release assay was negative. Physical exam was notable for temperature of 103 degrees F and mild RLQ tenderness without a discrete mass. CBC, serum transaminases, lipase, viral hepatitis panel, and HIV assay were all negative. ESR was elevated at 110 and CRP at 220. Sputum culture for AFB, stool culture and blood culture were negative. Chest x-ray and CT chest was unremarkable. CT abdomen and pelvis showed short segment mural thickening of the terminal ileum with bowel distention, right retroperitoneal lymphadenopathy and 4.2 cm right retroperitoneal lymph node conglomerate. Pt underwent CT guided biopsy of mesenteric lymph node which showed patchy necrosis, multinucleated giant cells, few granulomas and rare acid fast bacilli. Pt was started on TB therapy with improvement in symptoms. Extra-pulmonary TB (EPTB) is found in approximately 20% of the patients with TB in the U.S. The 6th most common extra pulmonary site is the ileocecal region. Clinical and radiological findings of GI TB often mimic diseases such as Crohn's disease and malignancy. Therefore, this can lead to delays in accurate diagnosis and treatment. This is particularly true for a case like ours, where a prior negative Quantiferron may ‘rule-out' TB as a diagnostic possibility. Our case points out that under circumstances where a high index of suspicion for EPTB exists, particularly in patients with negative PPD, Quantiferron, and sputum, lymph node biopsy can be a very important supplement to our diagnostic armamentarium. An algorithm of these diagnostic methods leads to considerably higher precision in the diagnosis of this insidious disease which primarily necessitates a clinical awareness of this serious health problem.FigureFigure

Comparison of Roche TaqMan v.2.0, Abbott RealTime and Modified Abbott Residual Viremia Assays in Testing of Routine Samples for Viremia Monitoring in HIV Care

BackgroundRoutine HIV RNA clinical monitoring frequently reports virermia (VL) as detectable, quantifiable <50 or blips >50 c/mL. To assess whether the results represent true increases or just assay variability, we designed comparisons of the Roche COBAS AmpliPrep / COBAS TaqMan HIV-1 Test version 2.0 (TM) and the Abbott RealTime HIV-1 assay (RT) to a residual viremia (RV) assay with a limit of detection of 3 c/mL, the Modified Abbott Residual Viremia assay (MARV).MethodsHIV suppressed patients on stable therapy presenting for routine visits at the HIV clinic in Henry Ford Hospital (HFH) were prospectively enrolled. Blood samples and questionnaires were obtained at each visit. Paired plasma samples were processed and analyzed per manufacturer instructions; HFH clinical laboratory processed the samples using the TM assay and the McKinnon Research Laboratory performed the FDA approved RT assay and RV testing using the MARV assay. Parametric and non-parametric analyses were conducted as indicated.Results124 HIV patients are reported with a mean age of 51, mostly male (84%) and African American (64%). Mean CD4 cell counts were 661 cells/mm3. TM assay results for 196 plasma samples were not detectable (ND) 119 (61%), detectable (DT) 61 (31%) and quantifiable viremia (QV) >20 c/mL 16 (8%). 5 patients had QV over 50 c/mL. 60 tested patients (49.5%) had at least one sample either DT or QV. TM results were not correlated to CD4 cell counts, antiretrovirals, medical conditions or reported adherence (avg. 97%). 174 RT and 187 MARV paired tests were ND on 142 (81%) / 121 (65%), DT on 34 (17%) RT, and quantifiable in 0% / 34 (33%) of the samples respectively. Higher VL detection by the TM was shown compared with the RT (P = 0.005) and MARV (P < 0.001) assays. Mean QV by the MARV was 5.0 vs.. 55 c/mL on the TM assay (P < 0.001). VL for all assays trends correlated when compared for ND, DT and >20 copies/mL (P < 0.001). Bland-Altman plots show higher VL detected by TM as compared with MARV (P < 0.001) and good correlation between RT and MARV assays (P = 0.6).ConclusionHIV viremia is more frequently reported by TM assay as compared with the RT assay and was significantly higher than VL detected by the MARV assay. Over detection of VL by the TM assay may impact clinical decision making and increase cost of care.Disclosures J. Mckinnon, Abbott Molecular: Speaker’s Bureau, Grant recipient, Research grant and Speaker honorarium D. Lucic, Abbott Molecular: Employee, Salary

Discordant rapid HIV tests: lessons from a low-resource community.

HIV rapid antibody tests are widely used in Africa, but dual testing sometimes produces discordant results. It is not clear if discordant rapid HIV tests should always heighten suspicion by frontline health workers that early HIV infection is present. Some studies have reported that discordant rapid tests have value for identifying early HIV infection in high HIV prevalence populations. It is not known if rapid test performance influenced this conclusion, or if this observation will hold true for low HIV prevalence populations. We therefore explored the occurrence of discordant rapid HIV tests in a low-resource community. A cross-sectional sample of HIV status-unaware adults with recent exposure to unsafe sex was assessed using a validated risk-based tool (University of North Carolina (UNC)-Malawi Risk Screening Score) for acute HIV infection. Participants received rapid testing with Determine™ HIV 1/2 and Uni-Gold™ HIV assays, plus plasma HIV-1 antigen testing with the COBAS® Ampliprep/COBAS® Taqman® HIV-1 assay, followed by western blot in those with detected HIV-1 antigen. Of 408 participants, 1.0% were confirmed to have established HIV infection. The discordance between rapid tests at initial screening was 2.45 and 2.94% when the two assays were used sequentially and simultaneously, respectively. Discordant rapid tests were strongly associated with risk scores > 2 [odds ratio (OR) 10.88; 95% confidence interval (CI) 2.35-50.43], and with detected HIV-1 RNA (OR 26.06; 95% CI 3.91-173.60). When the sample occurrence of discordance between the first and second tests is below 5%, discordant rapid tests in an adult with sexual risk behaviour should trigger strong suspicion of early HIV infection in low HIV prevalence populations.

A retrospective analysis of the application of the Elecsys® HIV combi PT assay in southern China.

Fourth-generation HIV assays have been implemented worldwide as a screening test for many years. Understanding the performance of fourth-generation assay in low HIV prevalence region is pivotal to interpret the test result correctly. In this study, retrospective analysis was used to evaluate application of the Elecsys® HIV combi PT assay. A total of 85043 specimens from a low prevalence setting were detected between June 2013 and October 2015. We evaluated the false-positive rate (FPR), specificity, and positive predictive value (PPV). The specificity between male and female were 99.85% and 99.82%, respectively. The PPV on male (50.75%) was higher than female (17.05%) significantly, while the FPR was 0.15% and 0.18%. The gap between false-positive (median: 1.83, [IQR]: 1.30, 3.38) and confirmed-positive (median: 407.5, [IQR]: 184.2, 871.7) is enormous. The highest s/co ratio for false-positive cases was 85.45, while the lowest s/co ratio for confirmed-positive cases was 59.68. Various reasons were attributed to false-positive cases. Optimal cutoff value is needed to be set to reduce the false-positive cases and predict the final status of HIV infection reliably. Retrospective analysis will help us to understand more about diagnosis of HIV.

Development of a web-based application and multicountry analysis framework for assessing interdicted infections and cost-utility of screening donated blood for HIV, HCV and HBV.

Most countries test donations for HIV, HCV and HBV using serology with or without nucleic acid testing (NAT). Cost-utility analyses provide information on the relative value of different screening options. The aim of this project was to develop an open access risk assessment and cost-utility analysis web-tool for assessing HIV, HCV and HBV screening options (http://www.isbtweb.org/working-parties/transfusion-transmitted-infectious-diseases/). An analysis for six countries (Brazil, Ghana, the Netherlands, South Africa, Thailand and USA) was conducted. Four strategies; (1) antibody assays (Abs) for HIV and HCV + HBsAg, (2) antibody assays that include antigens for HIV and HCV (Combo) + HBsAg, (3) NAT in minipools of variable size (MP NAT) and (4) individual donation (ID) NAT can be evaluated using the tool. Country-specific data on donors, donation testing results, recipient outcomes and costs are entered using the online interface. Results obtained include the number infections interdicted using each screening options, and the (incremental and average) cost-utility of the options. In each of the six countries evaluated, the use of antibody assays is cost effective or even cost saving. NAT has varying cost-utility depending on the setting, and where adopted, the incremental cost-utility exceeds any previously defined or proposed threshold in each country. The web-tool allows an assessment of infectious units interdicted and value for money of different testing strategies. Regardless of gross national income (GNI) per capita, countries appear willing to dedicate healthcare resources to blood supply safety in excess of that for other sectors of health care.

HIV-1 viral load measurement in venous blood and fingerprick blood using Abbott RealTime HIV-1 DBS assay

BackgroundHIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings. ObjectivesTo evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol. Study designLimit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol. ResultsThe Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×107 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×107 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was −0.07 log copies/mL between DBS-fingerprick and plasma and −0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%. ConclusionThe results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an alternative sample collection and transfer option in resource-limited settings and expands the utility of a viral load test to monitor HIV-1 ART treatment for infected patients.

Cost-effective HIV-1 virological monitoring in resource-limited settings using a modified commercially available qPCR RNA assay

Virological monitoring through plasma viral load (PVL) quantification is essential for clinical management of HIV patients undergoing antiretroviral treatment (ART), and for detecting treatment failure. Quantitative PCR (qPCR)-based tests are the gold standard for measuring PVL. Largely because of their high cost, however, implementation of these tests in low- and middle-income countries fails to cover the testing demand. In this study, we aimed at reducing the running cost of the commercially available Abbott RealTime™ HIV-1 assay by minimizing the reagent consumption. To this end, a modified version of the assay was obtained by reducing the assay’s reagents volume to about a half, and validated using a panel of 104 plasma samples. Compared to the standard version, the modified Abbott assay allowed for a 50% reduction in running costs. At the same time, it showed a 100% concordance in identifying samples with detectable viral load, strong correlation (Pearson’s r=0.983, P<0.0001), and a high agreement between PVL values (mean percent difference between PVL values±standard deviation=0.76±3.18%). In detecting viral failure (PVL>1000copiesmL−1), the modified assay showed a sensitivity of 94.6%, a specificity of 93.8%, and a negative and positive predictive values of 93.8% and 94.6%, respectively. The modified assay therefore reliably quantifies PVL, predicts viral failure, and allows for a ca. 50% reduction in the assay’s running costs. It may thus be implemented as an ART monitoring tool in resource-limited settings and for research purposes.

A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay

BackgroundViral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System.¥ ObjectivesEvaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Study designMethod comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. ResultsBland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log10cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log10cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. ConclusionsThe VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 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

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

Estimation of measurement error in plasma HIV-1 RNA assays near their limit of quantification.

BackgroundPlasma HIV-1 RNA levels (pVLs), routinely used for clinical management, are influenced by measurement error (ME) due to physiologic and assay variation.ObjectiveTo assess the ME of the COBAS HIV-1 Ampliprep AMPLICOR MONITOR ultrasensitive assay version 1.5 and the COBAS Ampliprep Taqman HIV-1 assay versions 1.0 and 2.0 close to their lower limit of detection. Secondly to examine whether there was any evidence that pVL measurements closest to the lower limit of quantification, where clinical decisions are made, were susceptible to a higher degree of random noise than the remaining range.MethodsWe analysed longitudinal pVL of treatment-naïve patients from British Columbia, Canada, during their first six months on treatment, for time periods when each assay was uniquely available: Period 1 (Amplicor): 08/03/2000–01/02/2008; Period 2 (Taqman v1.0): 07/01/2010–07/03/2012; Period 3 (Taqman v2.0): 08/03/2012–30/06/2014. ME was estimated via generalized additive mixed effects models, adjusting for several clinical and demographic variables and follow-up time.ResultsThe ME associated with each assay was approximately 0.5 log10 copies/mL. The number of pVL measurements, at a given pVL value, was not randomly distributed; values ≤250 copies/mL were strongly systematically overrepresented in all assays, with the prevalence decreasing monotonically as the pVL increased. Model residuals for pVL ≤250 copies/mL were approximately three times higher than that for the higher range, and pVL measurements in this range could not be modelled effectively due to considerable random noise of the data.ConclusionsAlthough the ME was stable across assays, there is substantial increase in random noise in measuring pVL close to the lower level of detection. These findings have important clinical significance, especially in the range where key clinical decisions are made. Thus, pVL values ≤250 copies/mL should not be taken as the “truth” and repeat pVL measurement is encouraged to confirm viral suppression.

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.

Centrifugal Microfluidic Platform for Rapid, Multiplexed Detection of TB and HIV Biomarkers in Whole Blood Samples

Infection with Mycobacterium Tuberculosis represents a significant threat to people with immune disorders, such as HIV-positive individuals, and can result in significant health complications or death if not diagnosed and treated early. We present a centrifugal microfluidic platform for multiplexed detection of tuberculosis and HIV biomarkers in human whole blood with minimal sample preparation and a sample-to-answer time of 30 minutes. This multiplexed assay was developed for the detection of two M.tuberculosis secreted proteins, whose secretion represents an active and ongoing infection, as well as detection of HIV p24 protein and human anti-p24 antibodies. The limit of detection for this multiplex assay is in the pg/mL range for both HIV and M.tuberculosis proteins, making this assay potentially useful in the clinical diagnosis of both HIV and Tuberculosis proteins indicative of active infection. Antigen detection for the HIV assay sensitivity was 89%, the specificity 85%. Serological detection had 100% sensitivity and specificity for the limited sample pool. The centrifugal microfluidic platform presented here offers the potential for a portable, fast and inexpensive multiplexed diagnostic device that can be used in resource-limited settings for diagnosis of TB and HIV.

Field evaluation of an open and polyvalent universal HIV-1/SIVcpz/SIVgor quantitative RT-PCR assay for HIV-1 viral load monitoring in comparison to Abbott RealTime HIV-1 in Cameroon

With the increasing demand of HIV viral load (VL) tests in resource-limited countries (RLCs) there is a need for assays at affordable cost and able to quantify all known HIV-1 variants. VLs obtained with a recently developed open and polyvalent universal HIV-1/SIVcpz/SIVgor RT-qPCR were compared to Abbott RealTime HIV-1 assay in Cameroon. On 474 plasma samples, characterized by a wide range of VLs and a broad HIV-1 group M genetic diversity, 97.5% concordance was observed when using the lower detection limit of each assay. When using the threshold of 3.00 log10 copies/mL, according to WHO guidelines to define virological failure (VF) in RLCs, the concordance was 94.7%, 360/474 versus 339/474 patients were identified with VF with the new assay and Abbott RealTime HIV-1, respectively. Higher VLs were measured with the new assay, +0.47 log10 copies/mL (95% CI; 0.42-0.52) as shown with Bland-Altman analysis. Eleven samples from patients on VF with drug resistance were not detected by Abbott RealTime HIV-1 versus two only with the new assay. Overall, our study showed that the new assay can be easily implemented in a laboratory in RLCs with VL experience and showed good performance on a wide diversity of HIV-1 group M variants.

Structure and Glycan Binding of a New Cyanovirin-N Homolog

The HIV-1 envelope glycoprotein gp120 is heavily glycosylated and bears numerous high mannose sugars. These sugars can serve as targets for HIV-inactivating compounds, such as antibodies and lectins, which bind to the glycans and interfere with viral entry into the target cell. We determined the 1.6 Å x-ray structure of Cyt-CVNH, a recently identified lectin from the cyanobacterium Cyanothece(7424), and elucidated its glycan specificity by NMR. The Cyt-CVNH structure and glycan recognition profile are similar to those of other CVNH proteins, with each domain specifically binding to Manα(1-2)Manα units on the D1 and D3 arms of high mannose glycans. However, in contrast to CV-N, no cross-linking and precipitation of the cross-linked species in solution was observed upon Man-9 binding, allowing, for the first time, investigation of the interaction of Man-9 with a member of the CVNH family by NMR. HIV assays showed that Cyt-CVNH is able to inhibit HIV-1 with ∼4-fold higher potency than CV-N(P51G), a stabilized version of wild type CV-N. Therefore, Cyt-CVNH may qualify as a valuable lectin for potential microbicidal use.