Evaluation of a BioRad Avidity assay for identification of recent HIV-1 infections using dried serum or plasma spots.

Serological methods to differentiate between recently acquired and established HIV-1 infections are a useful tool in the HIV-surveillance to characterize the epidemic, identify groups at risk and assess HIV-preventive interventions. Therefore, an avidity-based, modified BioRad Genscreen™ HIV-1/2 assay (BRAEUR) was evaluated according to the avidity-based, modified BioRad HIV-1/2 Plus O protocol (BRAUSA). Overall, 692 well defined samples (82.5% B and 17.5% non-B subtypes) from recent (<180 days, n = 239), intermediate (181-364 days, n = 35) or long term infections (≥365 days, n = 419) were used to determine a 'mean duration of recent infection' (MDRI), a 'median DRI' (MdDRI), the false recent rate (FRR), and concordance between the BRAs and the Sedia BED HIV-1 Capture enzyme immunoassay (BED). The optimal avidity index cut-off was determined to be 70% resulting in an MDRI of 233 days (95% IQR: 174-351) and an MdDRI of 171 days (95% IQR: 142-212). Concordance with the BRAUSA was high with 96.4%. The FRR of 6.0% as well as the MdDRI are similar to the BED (8.4%; 170 (139-214) days). Therefore, the BRAEUR is a suitable alternative to replace the BED and trend analysis will be feasible after minimal adjustments for the MdDRI and the MDRI.

Robust and sensitive subtype-generic HIV-1 pol genotyping for use with dried serum spots in epidemiological studies.

HIV-1 genotyping of larger pol-fragments from dried serum/plasma spots (DSS/DPS) is often hindered by RNA-degradation during transportation at ambient temperature. We evaluated an in-house HIV-1 DSS/DPS-genotyping assay designed in two shorter overlapping fragments covering all resistance mutations in protease and reverse transcriptase. Validation criteria such as specificity, detection limit, accuracy, reproducibility and storage conditions were assessed using reference plasma samples prepared as DPS and clinical DSS from the German molecular HIV-1 surveillance processed under real-life transportation conditions. The specificity was 100% for both samples types, and the experimental DPS detection limit of 1000 copies/ml yielded a 98.7% (3,329/3373) success rate for DSS (including all subtypes) above this detection limit. Accuracy for DPS compared to the gold standard was 99.1% and the reproducibility was 100% for DPS replicates and 99.9% for DSS pairs. Storage of DPS at room temperature was possible for 90 or 30 days and at -20 °C for at least 180 or 90 days at viral loads of 10,000 or 1000 copies/ml, respectively. The HIV-1 pol-genotyping assay presented here is a sensitive, robust and subtype generic tool for a large-scale population-based HIV-1 drug resistance surveillance for the use of DSS/DPS.

National molecular surveillance of recently acquired HIV infections in Germany, 2013 to 2014.

To enable an up-to-date molecular analysis of human immunodeficiency virus (HIV) genotypes circulating in Germany we have established a surveillance system based on recently acquired HIV infections. New HIV infections are reported to the Robert Koch Institute as a statutory duty for anonymous notification. In 2013 and 2014, a dried serum spot (DSS) sample was received from 6,371 newly diagnosed HIV-cases; their analysis suggested that 1,797 samples originated from a recent infection. Of these, 809 were successfully genotyped in the pol region to identify transmitted drug resistance (TDR) mutations and to determine the HIV-1 subtype. Total TDR was 10.8%, comprising 4.3% with mono-resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 2.6% to non-NRTIs, 3.0% to protease inhibitors and 0.6% and 0.2%, respectively, with dual- and triple-class resistances. HIV-1 subtype B was most prevalent with 77.0%. Non-B infections were identified more often in men and women with heterosexual transmission compared with intravenous drug users or men who have sex with men (79% and 76%, 33%, 12%; all p < 0.05). Non-B subtypes were also more frequently found in patients originating from countries other than Germany (46% vs 14%; p < 0.05) and in patients infected outside of Germany (63% vs 14%; p < 0.05).

Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

BACKGROUND: Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). METHODS: Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. RESULTS: Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. CONCLUSIONS: Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

Improved testing of recent HIV-1 infections with the BioRad avidity assay compared to the limiting antigen avidity assay and BED Capture enzyme immunoassay: evaluation using reference sample panels from the German Seroconverter Cohort.

BACKGROUND: The variety and limitations of current laboratory methods for estimating HIV-incidence has driven attempts to improve and standardize the performance of serological 'Tests for Recent HIV-Infections' (TRI). Primary and follow-up HIV-1 positive plasma samples from individuals with well-defined dates of infection collected as part of the German Seroconverter Cohort provided specimens highly suitable for use in comparing the performance of three TRIs: the AWARE™ BED™ EIA HIV-1 Incidence test (BED-CEIA), Genetic systems HIV-1/HIV-2 Plus O EIA antibody avidity-based assay (BioRad Avidity) and Sedia™ HIV-1 LAg Avidity EIA (LAg Avidity). METHODS: The evaluation panel included 180 specimens: 44 from antiretroviral (ARV)-naïve individuals with recently acquired HIV-infection (≤ 130 days; 25 B and 19 non-B subtypes) and 136 from long-term (>12 months) infected individuals [101 ARV-naïve subtype B, 16 non-B subtypes, 14 ARV-treated individuals, 5 slow progressors (SLP)]. RESULTS: For long-term infected, ARV-naïve individuals the false recent rates (FRR) of both the BioRad and LAg Avidity assays were 2% (2/101 for subtype B) and 6% (1/16 for subtype 'non-B'), while the FRR of the BED-CEIA was 7% (7/101 for subtype B) and 25% (4/16 for subtype 'non-B') (all p>0.05). Misclassification of ARV-treated individuals and SLP was rare by LAg (1/14, 0/5) and BioRad Avidity assays (2/14, 1/5) but more frequent by BED-CEIA (5/14, 3/5). Among recently-infected individuals (subtype B), 60% (15/25) were correctly classified by BED-CEIA, 88% (22/25) by BioRad Avidity and significantly fewer by LAg (48%, 12/25) compared to BioRad Avidity (p = 0.005) with a higher true-recency rate among non-B infections for all assays. CONCLUSIONS: This study using well-characterized specimens demonstrated lower FRRs for both avidity methods than with the BED-CEIA. For recently infected individuals the BioRad Avidity assay was shown to give the most accurate results.

Emergence of minor drug-resistant HIV-1 variants after triple antiretroviral prophylaxis for prevention of vertical HIV-1 transmission.

BACKGROUND: WHO-guidelines for prevention of mother-to-child transmission of HIV-1 in resource-limited settings recommend complex maternal antiretroviral prophylaxis comprising antenatal zidovudine (AZT), nevirapine single-dose (NVP-SD) at labor onset and AZT/lamivudine (3TC) during labor and one week postpartum. Data on resistance development selected by this regimen is not available. We therefore analyzed the emergence of minor drug-resistant HIV-1 variants in Tanzanian women following complex prophylaxis. METHOD: 1395 pregnant women were tested for HIV-1 at Kyela District Hospital, Tanzania. 87/202 HIV-positive women started complex prophylaxis. Blood samples were collected before start of prophylaxis, at birth and 1-2, 4-6 and 12-16 weeks postpartum. Allele-specific real-time PCR assays specific for HIV-1 subtypes A, C and D were developed and applied on samples of mothers and their vertically infected infants to quantify key resistance mutations of AZT (K70R/T215Y/T215F), NVP (K103N/Y181C) and 3TC (M184V) at detection limits of <1%. RESULTS: 50/87 HIV-infected women having started complex prophylaxis were eligible for the study. All women took AZT with a median duration of 53 days (IQR 39-64); all women ingested NVP-SD, 86% took 3TC. HIV-1 resistance mutations were detected in 20/50 (40%) women, of which 70% displayed minority species. Variants with AZT-resistance mutations were found in 11/50 (22%), NVP-resistant variants in 9/50 (18%) and 3TC-resistant variants in 4/50 women (8%). Three women harbored resistant HIV-1 against more than one drug. 49/50 infants, including the seven vertically HIV-infected were breastfed, 3/7 infants exhibited drug-resistant virus. CONCLUSION: Complex prophylaxis resulted in lower levels of NVP-selected resistance as compared to NVP-SD, but AZT-resistant HIV-1 emerged in a substantial proportion of women. Starting AZT in pregnancy week 14 instead of 28 as recommended by the current WHO-guidelines may further increase the frequency of AZT-resistance mutations. Given its impact on HIV-transmission rate and drug-resistance development, HAART for all HIV-positive pregnant women should be considered.

Emergence and persistence of minor drug-resistant HIV-1 variants in Ugandan women after nevirapine single-dose prophylaxis.

BACKGROUND: Nevirapine (NVP) single-dose is still a widely used antiretroviral prophylaxis for the prevention of vertical HIV-1 transmission in resource-limited settings. However, the main disadvantage of the Non-nucleoside Reverse Transcriptase Inhibitor (NNRTI) NVP is the rapid selection of NVP-resistant virus with negative implications for subsequent NNRTI-based long-term antiretroviral therapy (ART). Here, we analysed the emergence of drug-resistant HIV-1 including minor variants in the early phase after NVP single-dose prophylaxis and the persistence of drug-resistant virus over time. METHODS AND FINDINGS: NVP-resistant HIV-1 harbouring the K103N and/or Y181C resistance mutations in the HIV-1 reverse transcriptase gene was measured from 1 week up to 18 months after NVP single-dose prophylaxis in 29 Ugandan women using allele-specific PCR assays capable of detecting drug-resistant variants representing less than 1% of the whole viral population. In total, drug-resistant HIV-1 was identified in 18/29 (62%) women; rates increased from 18% to 38% and 44% at week 1, 2, 6, respectively, and decreased to 18%, 25%, 13% and 4% at month 3, 6, 12 and 18, respectively. The proportion of NVP-resistant virus of the total viral population was significantly higher in women infected with subtype D (median 40.5%) as compared to subtype A (median 1.3%; p = 0.032, Mann-Whitney U test). 33% of resistant virus was not detectable at week 2 but was for the first time measurable 6-12 weeks after NVP single-dose prophylaxis. Three (10%) women harboured resistant virus in proportions >10% still at month 6. CONCLUSIONS: Current WHO guidelines recommend an additional postnatal intake of AZT and 3TC for one week to avoid NVP resistance formation. Our findings indicate that a 1-week medication might be too short to impede the emergence of NVP resistance in a substantial proportion of women. Furthermore, subsequent NNRTI-based ART should not be started earlier than 12 months after NVP single-dose prophylaxis.

Minor drug-resistant HIV type-1 variants in breast milk and plasma of HIV type-1-infected Ugandan women after nevirapine single-dose prophylaxis.

BACKGROUND: Nevirapine single-dose (NVP-SD) reduces mother-to-child transmission of HIV type-1 (HIV-1), but frequently induces resistance mutations in the HIV-1 genome. Little is known about drug-resistant HIV-1 variants in the breast milk of women who have taken NVP-SD. METHODS: Blood and breast milk samples of 39 HIV-1-infected Ugandan women were taken 6-12 weeks after NVP-SD intake. Samples were analysed by population sequencing and allele-specific real-time PCR (AS-PCR) with detection limits for NVP-resistant HIV-1 variants (K103N and Y181C) of < 1% of the total viral population. RESULTS: AS-PCR results for both plasma and breast milk were obtained for 19 women who constituted the final study group (HIV-1 subtype frequencies were A1 n = 11, D n = 5, G n = 2 and C n = 1). A total of 7 (37%) and 10 (53%) women carried NVP-resistant virus in breast milk and plasma, respectively. Overall, 71% (5/7) women with NVP-resistant HIV-1 in breast milk displayed >1 drug-resistant variant. Resistance in breast milk was higher at week 6 (6/13 samples [46%]) compared with week 12 (1/6 samples [17%]). In total, 10 drug-resistant populations harbouring the K103N and/or Y181C mutation were detected in the 19 breast milk samples; 7 (70%) were caused by resistant minorities (< 5% of the total HIV-1 population). In the four women with drug-resistant virus in both plasma and breast milk, the mutation patterns differed between the two compartments. CONCLUSIONS: Minor populations of drug-resistant HIV-1 were frequently found in breast milk of Ugandan women after exposure to NVP-SD. Further studies need to explore the role of minor drug-resistant variants in the postnatal transmission of (resistant) HIV-1.

Prevalence of transmitted drug resistance and impact of transmitted resistance on treatment success in the German HIV-1 Seroconverter Cohort.

BACKGROUND: The aim of this study is to analyse the prevalence of transmitted drug resistance, TDR, and the impact of TDR on treatment success in the German HIV-1 Seroconverter Cohort. METHODS: Genotypic resistance analysis was performed in treatment-naïve study patients whose sample was available 1,312/1,564 (83.9% October 2008). A genotypic resistance result was obtained for 1,276/1,312 (97.3%). The resistance associated mutations were identified according to the surveillance drug resistance mutations list recommended for drug-naïve patients. Treatment success was determined as viral suppression below 500 copies/ml. RESULTS: Prevalence of TDR was stable at a high level between 1996 and 2007 in the German HIV-1 Seroconverter Cohort (N = 158/1,276; 12.4%; CI(wilson) 10.7-14.3; p(for trend) = 0.25). NRTI resistance was predominant (7.5%) but decreased significantly over time (CI(Wilson): 6.2-9.1, p(for trend) = 0.02). NNRTI resistance tended to increase over time (NNRTI: 3.5%; CI(Wilson): 2.6-4.6; p(for trend)= 0.07), whereas PI resistance remained stable (PI: 3.0%; CI(Wilson): 2.1-4.0; p(for trend) = 0.24). Resistance to all drug classes was frequently caused by singleton resistance mutations (NRTI 55.6%, PI 68.4%, NNRTI 99.1%). The majority of NRTI-resistant strains (79.8%) carried resistance-associated mutations selected by the thymidine analogues zidovudine and stavudine. Preferably 2NRTI/1PIr combinations were prescribed as first line regimen in patients with resistant HIV as well as in patients with susceptible strains (susceptible 45.3%; 173/382 vs. resistant 65.5%; 40/61). The majority of patients in both groups were treated successfully within the first year after ART-initiation (susceptible: 89.9%; 62/69; resistant: 7/9; 77.8%). CONCLUSION: Overall prevalence of TDR remained stable at a high level but trends of resistance against drug classes differed over time. The significant decrease of NRTI-resistance in patients newly infected with HIV might be related to the introduction of novel antiretroviral drugs and a wider use of genotypic resistance analysis prior to treatment initiation.

Detection and quantification of minor human immunodeficiency virus type 1 variants harboring K103N and Y181C resistance mutations in subtype A and D isolates by allele-specific real-time PCR.

Nevirapine (single dose), commonly used to prevent the mother-to-child transmission of human immunodeficiency virus (HIV) in developing countries, frequently induces viral resistance. Even mutations which occur only in a minor population of the HIV quasispecies (<20%) are associated with subsequent treatment failure but cannot be detected by population-based sequencing. We developed sensitive allele-specific real-time PCR (ASPCR) assays for two key resistance mutations of nevirapine. The assays were specifically designed to analyze HIV-1 subtype A and D isolates accounting for the majority of HIV infections in Uganda. Assays were evaluated using DNA standards and clinical samples of Ugandan women having preventively taken single-dose nevirapine. Lower detection limits of drug-resistant HIV type 1 (HIV-1) variants carrying reverse transcriptase mutations were 0.019% (K103N [AAC]), 0.013% (K103N [AAT]), and 0.29% (Y181C [TGT]), respectively. Accuracy and precision were high, with coefficients of variation (the standard ratio divided by the mean) of 0.02 to 0.15 for intra-assay variability and those of 0.07 to 0.15 (K103N) and 0.28 to 0.52 (Y181C) for inter-assay variability. ASPCR assays enabled the additional identification of 12 (20%) minor drug-resistant HIV variants in the 20 clinical Ugandan samples (3 mutation analyses per patient; 60 analyses in total) which were not detectable by population-based sequencing. The individual patient cutoff derived from the clinical baseline sample was more appropriate than the standard-based cutoff from cloned DNA. The latter is a suitable alternative since the presence/absence of drug-resistant HIV-1 strains was concordantly identified in 92% (55/60) of the analyses. These assays are useful to monitor the emergence and persistence of drug-resistant HIV-1 variants in subjects infected with HIV-1 subtypes A and D.

Epidemiologically linked transmission of HIV-1 illustrates the impact of host genetics on virological outcome.

The diversity of HIV-1 and human genetics complicates our ability to determine the impact of treatment during primary HIV-1 infection on disease outcome. Here, we show, in a small group infected with virtually identical HIV-1 strains and treated during primary HIV-1 infection, that patients expressing protective human leucocyte antigen alleles had lower viral loads following treatment discontinuation. These data suggest that genetic factors play an important role in the outcome of HIV-1 infection despite early therapy.

Persistence of a sexually transmitted highly resistant HIV-1: pol quasispecies evolution over 33 months in the absence of treatment.

The evolution of a sexually transmitted multiresistant HIV-1 in a linked transmission chain was followed for 33 months to assess its potential to persist in the absence of treatment. The multiresistant HIV reverted slowly to wild type in reverse transcriptase (positions 44, 67, 74, 118) rendering the virus only susceptible to lamivudine/emtricitabine. Persistence of the replication-competent resistant HIV increases its potential to spread further and strengthens the importance of resistance testing in newly infected patients.