Development and optimization of thermal contrast amplification lateral flow immunoassays for ultrasensitive HIV p24 protein detection.

Detection of human immunodeficiency virus (HIV) p24 protein at a single pg/ml concentration in point-of-care (POC) settings is important because it can facilitate acute HIV infection diagnosis with a detection sensitivity approaching that of laboratory-based assays. However, the limit of detection (LOD) of lateral flow immunoassays (LFAs), the most prominent POC diagnostic platform, falls short of that of laboratory protein detection methods such as enzyme-linked immunosorbent assay (ELISA). Here, we report the development and optimization of a thermal contrast amplification (TCA) LFA that will allow ultrasensitive detection of 8 pg/ml p24 protein spiked into human serum at POC, approaching the LOD of a laboratory test. To achieve this aim, we pursued several innovations as follows: (a) defining a new quantitative figure of merit for LFA design based on the specific to nonspecific binding ratio (BR); (b) using different sizes and shapes of gold nanoparticles (GNPs) in the systematic optimization of TCA LFA designs; and (c) exploring new laser wavelengths and power regimes for TCA LFA designs. First, we optimized the blocking buffer for the membrane and running buffer by quantitatively measuring the BR using a TCA reader. The TCA reader interprets the thermal signal (i.e., temperature) of GNPs within the membrane when irradiated by a laser at the plasmon resonance wavelength of the particle. This process results in higher detection and quantitation of GNPs than in traditional visual detection (i.e., color intensity). Further, we investigated the effect of laser power (30, 100, 200 mW), GNP size and shape (30 and 100 nm gold spheres, 150 nm gold-silica shells), and laser wavelength (532, 800 nm). Applying these innovations to a new TCA LFA design, we demonstrated that 100 nm spheres with a 100 mW 532 nm laser provided the best performance (i.e., LOD = 8 pg/ml). This LOD is significantly better than that of the current colorimetric LFA and is in the range of the laboratory-based p24 ELISA. In summary, this TCA LFA for p24 protein shows promise for detecting acute HIV infection in POC settings.

Drug resistance emergence in macaques administered cabotegravir long-acting for pre-exposure prophylaxis during acute SHIV infection.

A long-acting injectable formulation of the HIV integrase inhibitor cabotegravir (CAB-LA) is currently in clinical development for PrEP. Although the long plasma half-life of CAB-LA is an important attribute for PrEP, it also raises concerns about drug resistance emergence if someone becomes infected with HIV, or if PrEP is initiated during undiagnosed acute infection. Here we use a macaque model of SHIV infection to model risks of drug resistance to CAB-LA PrEP. Six macaques infected with SHIV received CAB-LA before seroconversion. We show integrase mutations G118R, E92G/Q, or G140R in plasma from 3/6 macaques as early as day 57, and identify G118R and E92Q in viruses from vaginal and rectal fluids. G118R and G140R confer > 800-fold resistance to CAB and cross-resistance to all licensed integrase inhibitors. Our results emphasize the need for appropriate HIV testing strategies before and possibly shortly after initiating CAB LA PrEP to exclude acute infection.

Incident Infection and Resistance Mutation Analysis of Dried Blood Spots Collected in a Field Study of HIV Risk Groups, 2007-2010.

OBJECTIVE: To assess the utility of cost-effective dried blood spot (DBS) field sampling for incidence and drug resistance surveillance of persons at high risk for HIV infection. METHODS: We evaluated DBS collected in 2007-2010 in non-clinical settings by finger-stick from HIV-positive heterosexuals at increased risk of HIV infection (n = 124), men who have sex with men (MSM, n = 110), and persons who inject drugs (PWID, n = 58). Relative proportions of recent-infection findings among risk groups were assessed at avidity index (AI) cutoffs of ≤25%, ≤30%, and ≤35%, corresponding to an infection mean duration of recency (MDR) of 220.6, 250.4, and 278.3 days, respectively. Drug resistance mutation prevalence was compared among the risk groups and avidity indices. RESULTS: HIV antibody avidity testing of all self-reported ARV-naïve persons (n = 186) resulted in 9.7%, 11.3% and 14.0% with findings within the 221, 250, and 278-day MDRs, respectively. The proportion of ARV-naïve MSM, heterosexuals, and PWID reporting only one risk category who had findings below the suggested 30% AI was 23.1%, 6.9% and 3.6% (p<0.001), respectively. MSM had the highest prevalence of drug resistance and the only cases of transmitted multi-class resistance. Among the ARV-experienced, MSM had disproportionately more recent-infection results than did heterosexuals and PWID. CONCLUSIONS: The disproportionately higher recent-infection findings for MSM as compared to PWID and heterosexuals increased as the MDR window increased. Unreported ARV use might explain greater recent-infection findings and drug resistance in this MSM population. DBS demonstrated utility in expanded HIV testing; however, optimal field handling is key to accurate recent-infection estimates.

Sensitive tenofovir resistance screening of HIV-1 from the genital and blood compartments of women with breakthrough infections in the CAPRISA 004 tenofovir gel trial.

The Centre for the AIDS Programme of Research in South Africa 004 (CAPRISA 004) study demonstrated that vaginally applied tenofovir gel is a promising intervention for protecting women from sexually acquiring human immunodeficiency virus (HIV). However, the potential for emergence of tenofovir resistance remains a concern in women who seroconvert while using the gel despite the lack of plasma virus resistance as assessed by population sequencing during the trial. We applied highly sensitive polymerase chain reaction-based assays to screen for tenofovir resistance in plasma and vaginal swab specimens. The absence of mutation detection suggested little immediate risk of tenofovir-resistant HIV-1 emergence and forward transmission in settings in which gel users are closely monitored for HIV seroconversion.

Demographic but not geographic insularity in HIV transmission among young black MSM.

OBJECTIVE: To understand patterns of HIV transmission among young black MSM and others in Mississippi. DESIGN: Phylogenetic analysis of HIV-1 polymerase (pol) sequences from 799 antiretroviral-naive persons newly diagnosed with HIV infection in Mississippi during 2005-2008, 130 (16%) of whom were black MSM aged 16-25 years. METHODS: We identified phylogenetic clusters and used surveillance data to evaluate demographic attributes and risk factors of all persons in clusters that included black MSM aged 16-25 years. RESULTS: We identified 82 phylogenetic clusters, 21 (26%) of which included HIV strains from at least one young black MSM. Of the 69 persons in these clusters, 59 were black MSM and seven were black men with unknown transmission category; the remaining three were MSM of white or Hispanic race/ethnicity. Of these 21 clusters, 10 included residents of one geographic region of Mississippi, whereas 11 included residents of multiple regions or outside of the state. CONCLUSION: Phylogenetic clusters involving HIV-infected young black MSM were homogeneous with respect to demographic and risk characteristics, suggesting insularity of this population with respect to HIV transmission, but were geographically heterogeneous. Reducing HIV transmission among young black MSM in Mississippi may require prevention strategies that are tailored to young black MSM and those in their sexual networks, and prevention interventions should be delivered in a manner to reach young black MSM throughout the state. Phylogenetic analysis can be a tool for local jurisdictions to understand the transmission dynamics in their areas.

Detection of low-level K65R variants in nucleoside reverse transcriptase inhibitor-naive chronic and acute HIV-1 subtype C infections.

To substantiate reports of greater emergence of the K65R nucleoside reverse transcriptase inhibitor (NRTI) mutation in human immunodeficiency virus type 1 (HIV-1) subtype C, we examined natural low-level K65R expression in subtype C relative to subtypes B and AE. We used allele-specific polymerase chain reaction to screen HIV-1 amplified by reverse-transcription high-fidelity polymerase chain reaction from subtype C-infected South African women and infants and CRF01(subtype AE) from Thailand; all subjects were NRTI naive. We found low-level K65R of unknown clinical significance in NRTI-naive subtype C-infected women and infants at frequencies above the natural occurrence in subtypes B and AE. The frequent appearance of subtype C frameshift deletions at codon 65 supports a propensity for transcription error in this region.

Minority HIV mutation detection in dried blood spots indicates high specimen integrity and reveals hidden archived drug resistance.

BACKGROUND: Dried blood spots (DBS) could serve as an attractive, cost-effective alternative to plasma for HIV drug resistance testing. OBJECTIVES: To assess the utility and potential gain in genotypic information with sensitive testing of DBS compared to conventional bulk plasma genotyping, and examine the correlation of majority and minority-level resistance mutations in DBS with treatment history. STUDY DESIGN: Evaluate nucleic acids from the DBS of 33 antiretroviral-experienced subtype B-infected subjects for minority M41L, K65R, K70R, K103N, Y181C, M184V, and T215Y/F mutations by real-time PCR. Compare minority resistance mutations in DBS with bulk genotypes from the same DBS cards and available plasma specimens. RESULTS: All but one (50/51, 98%) mutation from the original plasma bulk sequencing were still detectable in the DBS after three years of storage. The one mutation not identified in DBS was also no longer detectable by bulk sequencing. Furthermore, sensitive testing found 12 additional drug resistance mutations at minority levels in the DBS of 11 (33%) patients. Six minority mutations were in the RNA compartment and six were detected only in the DNA compartment. Resistance was detected in the DBS RNA compartment only in cases where the associated drug was in use within one year of sample collection. CONCLUSIONS: Our ability to identify majority and additional minority-level resistance mutations demonstrated that DBS, if stored properly, is a high-integrity specimen type for conventional and sensitive drug resistance testing. Our data further support the global utility of DBS for drug resistance surveillance and clinical monitoring.

Development of two avidity-based assays to detect recent HIV type 1 seroconversion using a multisubtype gp41 recombinant protein.

Current laboratory methods to detect recent HIV-1 infection for the estimation of incidence have various limitations, including varying performance in different subtypes or populations. Therefore, new methods are needed to detect recent infections with increased specificity. We developed a recombinant protein, rIDR-M, that covered divergent sequences from the immunodominant region (IDR) of gp41 from all major subtypes and recombinants of HIV-1 group M and expressed in Escherichia coli. The rIDR-M protein was highly reactive with HIV antibodies in sera from different subtypes and equivalently detected antibodies to divergent subtypes B and AE from Thailand, in contrast to individual gp41 peptides derived from respective subtypes, suggesting that it can be used for incidence assays. The protein was used in two different assay formats to measure antibody avidity: (1) a two-well avidity index assay (AI-EIA) and (2) a new one-well limiting antigen avidity assay (LAg-avidity EIA), both with a pH 3.0 buffer to dissociate low-avidity antibodies present during early infection. Limiting the amount of antigen allowed detection of recent HIV-1 infection, with or without dissociation buffer, but the detection was most efficient when the pH 3.0 dissociation buffer was included. When a well-characterized 41-member seroincidence panel (20 recent and 21 long-term) was used, both the two-well AI-EIA and one-well LAg-avidity EIA efficiently distinguished recent and long-term infections. The new avidity-based assays using rIDR-M antigen may improve the accuracy of detecting recent HIV-1 infection and allow a better estimation of incidence in diverse HIV-1 subtypes.

Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-naïve populations and associate with reduced treatment efficacy.

BACKGROUND: Transmitted HIV-1 drug resistance can compromise initial antiretroviral therapy (ART); therefore, its detection is important for patient management. The absence of drug-associated selection pressure in treatment-naïve persons can cause drug-resistant viruses to decline to levels undetectable by conventional bulk sequencing (minority drug-resistant variants). We used sensitive and simple tests to investigate evidence of transmitted drug resistance in antiretroviral drug-naïve persons and assess the clinical implications of minority drug-resistant variants. METHODS AND FINDINGS: We performed a cross-sectional analysis of transmitted HIV-1 drug resistance and a case-control study of the impact of minority drug resistance on treatment response. For the cross-sectional analysis, we examined viral RNA from newly diagnosed ART-naïve persons in the US and Canada who had no detectable (wild type, n = 205) or one or more resistance-related mutations (n = 303) by conventional sequencing. Eight validated real-time PCR-based assays were used to test for minority drug resistance mutations (protease L90M and reverse transcriptase M41L, K70R, K103N, Y181C, M184V, and T215F/Y) above naturally occurring frequencies. The sensitive real-time PCR testing identified one to three minority drug resistance mutation(s) in 34/205 (17%) newly diagnosed persons who had wild-type virus by conventional genotyping; four (2%) individuals had mutations associated with resistance to two drug classes. Among 30/303 (10%) samples with bulk genotype resistance mutations we found at least one minority variant with a different drug resistance mutation. For the case-control study, we assessed the impact of three treatment-relevant drug resistance mutations at baseline from a separate group of 316 previously ART-naïve persons with no evidence of drug resistance on bulk genotype testing who were placed on efavirenz-based regimens. We found that 7/95 (7%) persons who experienced virologic failure had minority drug resistance mutations at baseline; however, minority resistance was found in only 2/221 (0.9%) treatment successes (Fisher exact test, p = 0.0038). CONCLUSIONS: These data suggest that a considerable proportion of transmitted HIV-1 drug resistance is undetected by conventional genotyping and that minority mutations can have clinical consequences. With no treatment history to help guide therapies for drug-naïve persons, the findings suggest an important role for sensitive baseline drug resistance testing.

Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

BACKGROUND: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. METHODOLOGY: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. SIGNIFICANCE: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.