Dara A. Lehman

Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification

ABSTRACT Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes. IMPORTANCE Diagnosis of HIV-1 infection in infants cannot rely on the antibody-based tests used in adults because of the transfer of maternal HIV-1 antibodies from mother to child. Therefore, infant diagnostics rely on detection of the virus itself. However, current infant HIV-1 diagnostic methods require a laboratory setting with complex equipment. Here we describe the initial development of an HIV-1 diagnostic for infants that may be performed at the point of care in rural health clinics. We utilize a method that can amplify and detect HIV-1 DNA at an incubation temperature within the range of 25 to 42°C, eliminating the need for thermocycling equipment. HIV-1 diagnostics are challenging to develop due to the high diversity seen in HIV-1 strains worldwide. Here we show that this method detects the major HIV-1 strains circulating globally. Diagnosis of HIV-1 infection in infants cannot rely on the antibody-based tests used in adults because of the transfer of maternal HIV-1 antibodies from mother to child. Therefore, infant diagnostics rely on detection of the virus itself. However, current infant HIV-1 diagnostic methods require a laboratory setting with complex equipment. Here we describe the initial development of an HIV-1 diagnostic for infants that may be performed at the point of care in rural health clinics. We utilize a method that can amplify and detect HIV-1 DNA at an incubation temperature within the range of 25 to 42°C, eliminating the need for thermocycling equipment. HIV-1 diagnostics are challenging to develop due to the high diversity seen in HIV-1 strains worldwide. Here we show that this method detects the major HIV-1 strains circulating globally.

Lumenal transmission of decapentaplegic in Drosophila imaginal discs.

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.

HIV-1 persists in breast milk cells despite antiretroviral treatment to prevent mother-to-child transmission.

Background:The effects of short-course antiretrovirals given to reduce mother-to-child transmission (MTCT) on temporal patterns of cell-associated HIV-1 RNA and DNA in breast milk are not well defined. Methods:Women in Kenya received short-course zidovudine (ZDV), single-dose nevirapine (sdNVP), combination ZDV/sdNVP or short-course highly active antiretroviral therapy (HAART). Breast milk samples were collected two to three times weekly for 4–6 weeks. HIV-1 DNA was quantified by real-time PCR. Cell-free and cell-associated RNA levels were quantified by the Gen-Probe HIV-1 viral load assay. Results:Cell-free HIV-1 RNA levels in breast milk were significantly suppressed by sdNVP, ZDV/sdNVP or HAART therapy compared with ZDV between day 3 and week 4 postpartum (P ≤ 0.03). Breast milk HIV-1 DNA levels (infected cell levels) were not significantly different between treatment arms at any timepoint during the 4–6-week follow-up. At 3 weeks postpartum, when the difference in cell-free RNA levels was the greatest comparing HAART directly with ZDV (P = 0.0001), median log10 HIV-1 DNA copies per 1 × 106 cells were 2.78, 2.54, 2.69, and 2.31 in the ZDV, sdNVP, ZDV/sdNVP and HAART arms, respectively (P = 0.23). Cell-associated HIV-1 RNA levels were modestly suppressed in HAART versus ZDV/sdNVP during week 3 (3.37 versus 4.02, P = 0.04), as well as over time according to a linear mixed-effects model. Conclusion:Cell-free and, to a lesser extent, cell-associated HIV-1 RNA levels in breast milk were suppressed by antiretroviral regimens used to prevent MTCT. However, even with HAART, there was no significant reduction in the reservoir of infected cells, which could contribute to breast milk HIV-1 transmission.

Risk of Drug Resistance Among Persons Acquiring HIV Within a Randomized Clinical Trial of Single- or Dual-Agent Preexposure Prophylaxis

BACKGROUND Preexposure prophylaxis (PrEP) with emtricitabine plus tenofovir disoproxil fumarate (FTC/TDF) or TDF alone reduces the risk of human immunodeficiency virus (HIV) acquisition. Understanding the risk of antiretroviral resistance selected by PrEP during breakthrough infections is important because of the risk of treatment failure during subsequent antiretroviral use. METHODS Within the largest randomized trial of FTC/TDF versus TDF as PrEP, plasma samples were tested for HIV with resistance mutations associated with FTC (K65R and M184IV) and TDF (K65R and K70E), using 454 sequencing. RESULTS Of 121 HIV seroconverters, 25 received FTC/TDF, 38 received TDF, and 58 received placebo. Plasma drug levels in 26 individuals indicated PrEP use during or after HIV acquisition, of which 5 had virus with resistance mutations associated with their PrEP regimen. Among those with PrEP drug detected during infection, resistance was more frequent in the FTC/TDF arm (4 of 7 [57%]), compared with the TDF arm (1 of 19 [5.3%]; P = .01), owing to the FTC-associated mutation M184IV. Of these cases, 3 had unrecognized acute infection at PrEP randomization, and 2 were HIV negative at enrollment. CONCLUSIONS These results suggest that resistance selected by PrEP is rare but can occur both with PrEP initiation during acute seronegative HIV infection and in PrEP breakthrough infections and that FTC is associated with a greater frequency of resistance mutations than TDF.

Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.

Breast milk HIV-1 suppression and decreased transmission: a randomized trial comparing HIVNET 012 nevirapine versus short-course zidovudine

Objective:To compare the effect of perinatal regimens of short-course nevirapine (HIVNET 012) and zidovudine [Thai-Centers for Disease Control and Prevention (CDC) regimen] on breast milk viral shedding and perinatal transmission during the first 6 weeks postpartum in a randomized clinical trial. Design:Randomized clinical trial. Methods:Pregnant HIV-1 seropositive women in Nairobi, Kenya who planned to breastfeed were randomized to HIVNET 012 or Thai-CDC regimens. Two to four breast milk samples were collected each week between delivery and 6 weeks postpartum. Breast milk HIV-1 RNA was quantified using the Gen-Probe TMA assay. Infants were tested for HIV-1 DNA at birth and 6 weeks. Results:From March to October 2003, 76 women were enrolled and 795 breast milk samples were collected from 60 women who were randomized and followed after delivery. Between 3 and 21 days postpartum, nevirapine was associated with significantly greater suppression of breast milk log10 HIV-1 RNA: days 3 to 7 (1.98 versus 2.42, P = 0.1); days 8 to 14 (1.78 versus 2.48, P = 0.005); days 15 to 21 (1.90 versus 2.97, P = 0.003). At 6 weeks, the HIV-1 perinatal transmission rate was significantly lower among those who took nevirapine than zidovudine (6.8% versus 30.3%, P = 0.02). Conclusions:Compared to a peripartum zidovudine regimen, nevirapine was significantly more likely to decrease HIV-1 RNA in breast milk during the first week and through the third week postpartum following single-dose administration, and corresponded with decreased transmission risk at 6 weeks. Sustained breast milk HIV-1 suppression may contribute to the ability of nevirapine to decrease perinatal transmission of HIV-1.

Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care

Recombinase Polymerase Amplification (RPA) can be used to detect pathogen-specific DNA or RNA in under 20 min without the need for complex instrumentation. These properties enable its potential use in resource limited settings. However, there are concerns that deviations from the manufacturers protocol and/or storage conditions could influence its performance in low resource settings. RPA amplification relies upon viscous crowding agents for optimal nucleic acid amplification, and thus an interval mixing step after 3-6 min of incubation is recommended to distribute amplicons and improve performance. In this study we used a HIV-1 RPA assay to evaluate the effects of this mixing step on assay performance. A lack of mixing led to a longer time to amplification and inferior detection signal, compromising the sensitivity of the assay. However lowering the assay volume from 50 μL to 5 μL showed similar sensitivity with or without mixing. We present the first peer-reviewed study that assesses long term stability of RPA reagents without a cold chain. Reagents stored at -20 °C, and 25 °C for up to 12 weeks were able to detect 10 HIV-1 DNA copies. Reagents stored at 45 °C for up to 3 weeks were able to detect 10 HIV-1 DNA copies, with reduced sensitivity only after >3 weeks at 45 °C. Together our results show that reducing reaction volumes bypassed the need for the mixing step and that RPA reagents were stable even when stored for 3 weeks at very high temperatures.

Lower Risk of Resistance After Short-Course HAART Compared With Zidovudine/Single-Dose Nevirapine Used for Prevention of HIV-1 Mother-to-Child Transmission

Background:Antiretroviral resistance after short-course regimens used to prevent mother-to-child transmission has consequences for later treatment. Directly comparing the prevalence of resistance after short-course regimens of highly active antiretroviral therapy (HAART) and zidovudine plus single-dose nevirapine (ZDV/sdNVP) will provide critical information when assessing the relative merits of these antiretroviral interventions. Methods:In a clinical trial in Kenya, pregnant women were randomized to receive either ZDV/sdNVP or a short-course of HAART through 6 months of breastfeeding. Plasma samples were collected 3-12 months after treatment cessation, and resistance to reverse transcriptase inhibitors was assessed using both a sequencing assay and highly sensitive allele-specific polymerase chain reaction assays. Results:No mutations associated with resistance were detectable by sequencing in either the ZDV/sdNVP or HAART arms at 3 months posttreatment, indicating that resistant viruses were not present in >20% of virus. Using allele-specific polymerase chain reaction assays for K103N and Y181C, we detected low levels of resistant virus in 75% of women treated with ZDV/sdNVP and only 18% of women treated with HAART (P = 0.007). Y181C was more prevalent than K103N at 3 months and showed little evidence of decay by 12 months. Conclusions:Our finding provides evidence that compared with ZDV/sdNVP, HAART reduces but does not eliminate nevirapine resistance.

Long-term virologic response and genotypic resistance mutations in HIV-1 infected Kenyan children on combination antiretroviral therapy.

Background:HIV-infected children may require the use of combination antiretroviral treatment (cART) into adulthood. However, regimens are limited to first line and second line in many African settings. Therefore, understanding the long-term rate of virologic failure and drug resistance during prolonged antiretroviral treatment is important for establishing treatment strategies in African pediatric cohorts. Methods:Children aged 18 months to 12 years initiated first-line cART and were followed every 1–3 months, for up to 5.5 years. Treatment was switched to second-line cART based on clinical and immunologic criteria according to national guidelines. Virologic failure was determined retrospectively as defined by ≥2 viral loads >5000 copies per milliliter. Drug resistance was assessed during viral failure by population-based sequencing. Results:Among 100 children on first-line cART followed for a median of 49 months, 34% children experienced virologic failure. Twenty-three (68%) of the 34 children with viral failure had detectable resistance mutations, of whom 14 (61%) had multiclass resistance. Fourteen (14%) children were switched to second-line regimens and followed for a median of 28 months. Retrospective analysis revealed that virologic failure had occurred at a median of 12 months before switching to second line. During prolonged first-line treatment in the presence of viral failure, additional resistance mutations accumulated; however, only 1 (7%) of 14 children had persistent viremia during second-line treatment. Discussion:Virologic suppression was maintained on first-line cART in two-thirds of HIV-infected children for up to 5 years. Switch to second line based on clinical/immunologic criteria occurred ∼1 year after viral failure, but the delay did not consistently compromise second-line treatment.

Low-frequency nevirapine resistance at multiple sites may predict treatment failure in infants on nevirapine-based treatment

Background:Resistance commonly arises in infants exposed to single-dose nevirapine (sdNVP) for prevention of mother to child transmission. Although K103N and Y181C are common following sdNVP, multiple other mutations also confer NVP resistance. It remains unclear whether specific NVP resistance mutations or combinations of mutations predict virologic failure in infants when present at low frequencies before NVP-based treatment. Methods:Twenty sdNVP-exposed infants who were subsequently treated with NVP-based highly active antiretroviral therapy were examined. Pretreatment plasma samples were tested for the presence of NVP resistance mutations by allele-specific polymerase chain reaction for K103N and Y181C and ultradeep pyrosequencing (UDPS) for all primary NVP mutations. Viral levels were determined every 3 months for up to 24 months on NVP–highly active antiretroviral therapy. Cox proportional hazard models were used to determine correlates of viral failure. Results:The NVP resistance mutations K103N or Y181C were detected in pretreatment plasma samples in 6 infants by allele-specific polymerase chain reaction. NVP resistance at these or other sites was detectable by UDPS in 10 of 20 infants tested. Virologic failure occurred in 50% of infants with any NVP resistance mutations detected, whereas only 20% of infants without resistance experienced viral failure, but the difference was not significant (P = 0.19). An increase in the number of NVP resistance mutations detectable by UDPS in an infant was significantly associated with an increased risk of virologic failure [HR = 1.79 (95% confidence interval: 1.07 to 2.99), P = 0.027]. Conclusions:Low frequencies of multiple NVP resistance mutations, in addition to K103N and Y181C, present in infants before NVP-based treatment may predict treatment outcome.