The contribution of immune activation to accelerated HIV-disease progression in older individuals has not been delineated.
Prospective multicenter cohort of older (≥45 years) and younger (18–30 years) HIV-infected adults initiating 192 weeks of antiretroviral therapy (ART). Longitudinal models of CD4 cell restoration examined associations with age-group, thymic volume, immune activation, and viral load.
Forty-five older and 45 younger adults (median age 50 and 26 years, respectively) were studied. Older patients had fewer naive CD4 cells (P <0.001) and higher HLA-DR/CD38 expression on CD4 (P = 0.05) and CD8 cells (P = 0.07) than younger patients at any time on ART. The rate of naive and total CD4 cell increase was similar between age groups, but older patients had a faster mean rate of B-cell increase (by +0.7 cells/week; P = 0.01), to higher counts than healthy controls after 192 weeks (P = 0.003). Naive CD4 increases from baseline were associated with immune activation reductions (as declines from baseline of %CD8 cells expressing HLA-DR/CD38; P <0.0001), but these increases were attenuated in older patients, or in those with small thymuses. A 15% reduction in activation was associated with naive gains of 29.9 and 6.2 cells/μl in younger, versus older patients, or with gains of 25.7, 23.4, and 2.1 cells/μl in patients with the largest, intermediate, and smallest thymuses, respectively (P <0.01 for interactions between activation reduction and age-group or thymic volume).
Older patients had significant B-cell expansion, higher levels of immune activation markers, and significantly attenuated naive CD4 cell gains associated with activation reduction.
aging; immune activation; immunosenescence; thymus
BACKGROUND AND OBJECTIVE:
The impact of maternal antiretrovirals (ARVs) during pregnancy, labor, and postpartum on infant outcomes is unclear.
Infants born to HIV-infected mothers in ARV studies were followed for 18 months.
Between June 2006 and December 2008, 236 infants enrolled from Africa (n = 36), India (n = 47), Thailand (n = 152), and Brazil (n = 1). Exposure to ARVs in pregnancy included ≥3 ARVs (10%), zidovudine/intrapartum ARV (81%), and intrapartum ARV (9%). There were 4 infant infections (1 in utero, 3 late postpartum) and 4 deaths with 1.8% mortality (95% confidence interval [CI], 0.1%–3.5%) and 96.4% HIV-1–free survival (95% CI, 94.0%–98.9%). Birth weight was ≥2.5 kg in 86%. In the first 6 months, Indian infants (nonbreastfed) had lowest median weights and lengths and smallest increases in growth. After 6 months, African infants had the lowest median weight and weight-for-age z scores. Infants exposed to highest maternal viral load had the lowest height and height-for-age z scores. Serious adverse events occurred in 38% of infants, did not differ by country, and correlated with less maternal ARV exposure. Clinical diagnoses were seen in 84% of Thai, 31% of African, and 9% of Indian infants. Congenital defects/inborn errors of metabolism were seen in 18 (7.6%) infants, of which 17 were Thai (11%: 95% CI, 6.7%–17.0%); none had first trimester ARV exposure.
Infant follow-up in large international cohorts is feasible and provides important safety and HIV transmission data following maternal ARV exposure. Increased surveillance increases identification of congenital/inborn errors.
maternal ARV exposure; infant safety; ARV toxicities; A5190; P1054; MTCT; HIV
In resource-limited settings where no safe alternative to breastfeeding exists, WHO recommends that antiretroviral prophylaxis be given to either HIV-infected mothers or infants throughout breastfeeding. We assessed the effect of 28 weeks of maternal or infant antiretroviral prophylaxis on postnatal HIV infection at 48 weeks.
The Breastfeeding, Antiretrovirals, and Nutrition (BAN) Study was undertaken in Lilongwe, Malawi, between April 21, 2004, and Jan 28, 2010. 2369 HIV-infected breastfeeding mothers with a CD4 count of 250 cells per μL or more and their newborn babies were randomly assigned with a variable-block design to one of three, 28-week regimens: maternal triple antiretroviral (n=849); daily infant nevirapine (n=852); or control (n=668). Patients and local clinical staff were not masked to treatment allocation, but other study investigators were. All mothers and infants received one dose of nevirapine (mother 200 mg; infant 2 mg/kg) and 7 days of zidovudine (mother 300 mg; infants 2 mg/kg) and lamivudine (mothers 150 mg; infants 4 mg/kg) twice a day. Mothers were advised to wean between 24 weeks and 28 weeks after birth. The primary endpoint was HIV infection by 48 weeks in infants who were not infected at 2 weeks and in all infants randomly assigned with censoring at loss to follow-up. This trial is registered with ClinicalTrials.gov, number NCT00164736.
676 mother–infant pairs completed follow-up to 48 weeks or reached an endpoint in the maternal-antiretroviral group, 680 in the infant-nevirapine group, and 542 in the control group. By 32 weeks post partum, 96% of women in the intervention groups and 88% of those in the control group reported no breastfeeding since their 28-week visit. 30 infants in the maternal-antiretroviral group, 25 in the infant-nevirapine group, and 38 in the control group became HIV infected between 2 weeks and 48 weeks of life; 28 (30%) infections occurred after 28 weeks (nine in maternal-antiretroviral, 13 in infant-nevirapine, and six in control groups). The cumulative risk of HIV-1 transmission by 48 weeks was significantly higher in the control group (7%, 95% CI 5–9) than in the maternal-antiretroviral (4%, 3–6; p=0·0273) or the infant-nevirapine (4%, 2–5; p=0·0027) groups. The rate of serious adverse events in infants was significantly higher during 29–48 weeks than during the intervention phase (1·1 [95% CI 1·0–1·2] vs 0·7 [0·7–0·8] per 100 person-weeks; p<0·0001), with increased risk of diarrhoea, malaria, growth faltering, tuberculosis, and death. Nine women died between 2 weeks and 48 weeks post partum (one in maternal-antiretroviral group, two in infant-nevirapine group, six in control group).
In resource-limited settings where no suitable alternative to breastfeeding is available, antiretroviral prophylaxis given to mothers or infants might decrease HIV transmission. Weaning at 6 months might increase infant morbidity
US Centers for Disease Control and Prevention.
(See the editorial commentary by Branson and Stekler, on pages 521–4.)
Background. Most human immunodeficiency virus (HIV) point-of-care tests detect antibodies (Ab) but not p24 antigen (Ag) or RNA. In the absence of antibodies, p24 antigen and RNA typically indicate acute HIV infection. We conducted a field evaluation of the Determine® HIV-1/2 Ag/Ab Combo rapid test (Combo RT).
Methods. The antigen portion of the Combo RT (for acute HIV infection) was compared with a Roche Monitor HIV RNA polymerase chain reaction assay. The antibody portion of Combo RT (for established HIV infection) was compared with rapid test algorithms. Participants were enrolled at a sexually transmitted infection clinic and HIV testing and counseling center in Lilongwe, Malawi. Rapid testing was conducted with parallel testing in the clinic and serial testing in the center. The Combo RT was performed in clinic participants with negative or discordant antibody results and in all center participants.
Results. Of the participants 838 were HIV negative, 163 had established HIV infection, and 8 had acute HIV infection. For detecting acute HIV infection, the antigen portion had a sensitivity of 0.000 and a specificity of 0.983. For detecting established HIV infection, the antibody portion had a sensitivity of 0.994 and a specificity of 0.992.
Conclusions. Combo RT displayed excellent performance for detecting established HIV infection and poor performance for detecting acute HIV infection. In this setting, Combo RT is no more useful than current algorithms.
Characterize responses to a NNRTI-based antiretroviral treatment (ART) initiated during acute HIV infection (AHI).
This was a prospective, single-arm evaluation of once daily, co-formulated emtricitabine/tenofovir/efavirenz initiated during AHI.
The primary endpoint is the proportion of responders with HIV RNA <200 copies/mL by week 24. We examined time-to-viral-suppression and CD8 cell activation in relation to baseline participant characteristics. We compared time-to-viral-suppression and viral dynamics using linear mixed effects models between acutely infected participants and chronically-infected controls.
Between January 2005 and May 2009, 61 AHI participants were enrolled. Of participants whose enrollment date allowed 24 and 48 weeks of follow-up, 47 of 51 (92%) achieved viral suppression to <200 copies/mL by week 24, and 35 of 41 (85.4%) to <50 copies/mL by week 48. The median time from ART initiation to suppression <50 copies/mL was 93 days (range 14–337). Higher HIV RNA levels at ART initiation (p=0.02), but not time from estimated-date-of-infection to ART initiation (p=0.86), were associated with longer time-to-viral-suppression. The median baseline frequency of activated CD8+CD38+HLA-DR+ T-cells was 67% (range 40–95), and was not significantly associated with longer time to viral load suppression (p=0.15). Viremia declined to <50 copies/mL more rapidly in AHI than chronically-infected participants. Mixed model analysis demonstrated similar phase I HIV RNA decay rates between acute and chronically-infected participants, and more rapid viral decline in acutely-infected participants in phase II.
Once daily emtricitabine/tenofovir/efavirenz initiated during AHI achieves rapid and sustained HIV suppression during this highly infectious period.
Acute HIV infection; NNRTIs; antiretroviral therapy; immune activation; viral dynamics
HIV-1 RNA quantitation continues to be extremely important for monitoring patients infected with HIV-1, and a number of assays have been utilized for this purpose. Differences in assay performance with respect to log10 recovery and HIV-1 subtype specificity have been well documented for commercially available assays, although comparisons are usually limited to one or two assay platforms. Two new FDA-approved assays, the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test (RT) and the Abbott RealTime HIV-1 assay (AR), that utilize real-time PCR have replaced previous HIV-1 RNA platforms. Inadequate detection of some strains of HIV-1 resulted in the addition of a new primer/probe set and the introduction of a second version of the RT assay. In this study, comparisons of assay performance between the different FDA-approved HIV-1 RNA assay platforms (both new and existing) were performed by using validation data that included both well-characterized virus stock and locally collected clinical samples. Laboratories across diverse geographical regions performed the validation testing and submitted data to the Virology Quality Assurance program (VQA) for analysis. Correlation values for clinical sample testing varied across the assay platforms (r = 0.832 to 0.986), and average log10 recoveries for HIV-1 RNA controls (compared to the nominal value) ranged from −0.215 to 0.181. These data demonstrate the need for use of one assay platform for longitudinal patient monitoring, but the data also reinforce the notion that no one assay is superior and that testing across platforms may be required for discordance reconciliation.
Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n=13 viruses), five clinically-matched nontransmitting mothers (n=16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses).
There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants.
Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.
HIV; Mother to child transmission; Galcer; Dendritic cells; Neutralizing antibodies
(See the editorial commentary by Tossonian and Conway, on pages 10–12.)
Background. The benefits of antiretroviral therapy during early human immunodeficiency virus type 1 (HIV-1) infection remain unproved.
Methods. A5217 study team randomized patients within 6 months of HIV-1 seroconversion to receive either 36 weeks of antiretrovirals (immediate treatment [IT]) or no treatment (deferred treatment [DT]). Patients were to start or restart antiretroviral therapy if they met predefined criteria. The primary end point was a composite of requiring treatment or retreatment and the log10 HIV-1 RNA level at week 72 (both groups) and 36 (DT group).
Results. At the June 2009 Data Safety Monitoring Board (DSMB) review, 130 of 150 targeted participants had enrolled. Efficacy analysis included 79 individuals randomized ≥72 weeks previously. For the primary end point, the IT group at week 72 had a better outcome than the DT group at week 72 (P = .005) and the DT group at week 36 (P = .002). The differences were primarily due to the higher rate of progression to needing treatment in the DT group (50%) versus the IT (10%) group. The DSMB recommended stopping the study because further follow-up was unlikely to change these findings.
Conclusions. Progression to meeting criteria for antiretroviral initiation in the DT group occurred more frequently than anticipated, limiting the ability to evaluate virologic set point. Antiretrovirals during early HIV-1 infection modestly delayed the need for subsequent treatment.
Clinical Trials Registration. NCT00090779.
We evaluated the efficacy of a maternal triple-drug antiretroviral regimen or infant nevirapine prophylaxis for 28 weeks during breast-feeding to reduce postnatal transmission of human immunodeficiency virus type 1 (HIV-1) in Malawi.
We randomly assigned 2369 HIV-1–positive, breast-feeding mothers with a CD4+ lymphocyte count of at least 250 cells per cubic millimeter and their infants to receive a maternal antiretroviral regimen, infant nevirapine, or no extended postnatal antiretroviral regimen (control group). All mothers and infants received perinatal prophylaxis with single-dose nevirapine and 1 week of zidovudine plus lamivudine. We used the Kaplan–Meier method to estimate the cumulative risk of HIV-1 transmission or death by 28 weeks among infants who were HIV-1–negative 2 weeks after birth. Rates were compared with the use of the log-rank test.
Among mother–infant pairs, 5.0% of infants were HIV-1–positive at 2 weeks of life. The estimated risk of HIV-1 transmission between 2 and 28 weeks was higher in the control group (5.7%) than in either the maternal-regimen group (2.9%, P = 0.009) or the infant-regimen group (1.7%, P<0.001). The estimated risk of infant HIV-1 infection or death between 2 and 28 weeks was 7.0% in the control group, 4.1% in the maternal-regimen group (P = 0.02), and 2.6% in the infant-regimen group (P<0.001). The proportion of women with neutropenia was higher among those receiving the antiretroviral regimen (6.2%) than among those in either the nevirapine group (2.6%) or the control group (2.3%). Among infants receiving nevirapine, 1.9% had a hypersensitivity reaction.
The use of either a maternal antiretroviral regimen or infant nevirapine for 28 weeks was effective in reducing HIV-1 transmission during breast-feeding. (ClinicalTrials.gov number, NCT00164736.)
Tenofovir (TFV) is effective in preventing simian immunodeficiency virus (SIV) transmission in a macaque model, is available as the oral agent tenofovir disoproxil fumarate (TDF), and may be useful in the prevention of mother-to-child transmission of human immunodeficiency virus (HIV). We conducted a trial of TDF and TDF-emtricitabine (FTC) in HIV-infected pregnant women and their infants. Women received a single dose of either 600 mg TDF, 900 mg TDF, or 900 mg TDF-600 mg FTC at labor onset or prior to a cesarean section. Infants received no drug or a single dose of TDF at 4 mg/kg of body weight or of TDF at 4 mg/kg plus FTC at 3 mg/kg as soon as possible after birth. All regimens were safe and well tolerated. Maternal areas under the serum concentration-time curve (AUC) and concentrations at the end of sampling after 24 h (C24) were similar between the two doses of TDF; the maximum concentrations of the drugs in serum (Cmax) and cord blood concentrations were higher in women delivering via cesarean section than in those who delivered vaginally (P = 0.04 and 0.046, respectively). The median ratio of the TFV concentration in cord blood to that in the maternal plasma at delivery was 0.73 (range, 0.26 to 1.95). Without TDF administration, infants had a median TFV concentration of 12 ng/ml 12 h after birth. Following administration of a single dose of TDF at 4 mg/kg, infant TFV concentrations fell below the targeted level, 50 ng/ml, by 24 h postdose. In HIV-infected pregnant women and their infants, 600 mg of TDF is acceptable as a single dose during labor. Low concentrations at birth support infant dosing as soon after birth as possible. Rapidly decreasing TFV levels in infants suggest that multiple or higher doses of TDF will be necessary to maintain concentrations that are effective for viral suppression.
There is a worldwide need for a pediatric HIV-1 diagnostic test that has a high diagnostic accuracy, is technically simple and cost efficient. The Up24 HIV-1 assay, which requires both the HIV-1 p24 ELISA and the ELAST signal amplification kit, has previously been shown to be a robust tool to diagnose pediatric HIV-1 from dried whole blood spots (DBS) (Cachafeiro et al., JCM 2009;47:459–6213). In order to make the assay more accessible to a resource-limited clinical setting, we eliminated the ELAST system, which simplified the Up24 assay, reduced its cost, and tested the accuracy of the modified assay in a rural Malawian hospital.
In this proof of concept study, we tested the ability of a simplified Up24 antigen assay, without ELAST, to detect HIV-1 on DBS obtained via heel prick from 6-week-old Malawian infants.
A case–control study of DBS collected from 113 HIV-infected and 109 HIV-negative infants, using the HIV-1 DNA PCR assay as the reference standard.
The simplified HIV-1 Up24 assay had a sensitivity and specificity of 84% and 98%, respectively. When HIV-1 prevalence is 15%, the positive- and negative-predictive values are 89% and 97%, respectively.
The simplified Up24 assay has a good positive- and a robust negative-predictive values, is easier to perform and has a reduced cost compared to both HIV DNA PCR and Up24 assays. With additional testing, the simplified Up24 assay has the potential to increase global access to pediatric HIV-1 diagnostics.
Human immunodeficiency virus type 1; (HIV-1); Pediatrics; Diagnostics; Malawi; HIV-1 p24 antigen detection assay; Dried blood spot (DBS)
The Cavidi viral load assay and the ultra-sensitive p24 antigen assay (Up24 Ag) have been suggested as more feasible alternatives to PCR-based HIV viral load assays for use in monitoring patients infected with HIV-1 in resource-limited settings.
To describe the performance of the Cavidi ExaVir Load™ assay (version 2.0) and two versions of the Up24 antigen assay and to characterize their agreement with the Roche Monitor HIV-1 RNA assay (version 1.5).
Observational study using a convenience sample of 342 plasma specimens from 108 patients enrolled in two ACTG clinical trials to evaluate the performance characteristics of the Up24 Ag assay using two different lysis buffers and the Cavidi ExaVir Load™ assay.
In analysis of agreement with the Roche assay, the Cavidi assay demonstrated superiority to the Up24 Ag assays in accuracy and precision, as well as sensitivity, specificity, and positive and negative predictive values for HIV-1 RNA ≥400, ≥1000 and ≥5000 copies/mL. Logistic performance curves indicated that the Cavidi assay was superior to the Up24 assays for viral loads greater than 650 copies/mL.
The results suggest that the Cavidi ExaVir Load assay could be used for monitoring HIV-1 viral load in resource-limited settings.
Cavidi; p24 antigen; viral load; resource limited setting
We assessed whether 7 days of zidovudine+lamivudine postpartum with single-dose nevirapine at labor decreases nevirapine resistance in HIV-infected women in Malawi.
HIV-infected pregnant women receiving intrapartum single-dose nevirapine and 7 days of zidovudine+lamivudine (n=132), and women receiving intrapartum single-dose nevirapine alone (n=66) were followed from an antenatal visit through 6 weeks postpartum. Plasma specimens at 2 and 6 weeks postpartum were tested for genotypic resistance to nevirapine by population sequencing and sensitive real-time PCR. Poisson regression was used to determine predictors of postpartum nevirapine resistance.
Median HIV RNA was similar at entry (4.27 log vs. 4.35 log, p=0.87), differed at 2 weeks postpartum (2.67 log vs. 3.58 log, p<0.0001), but not at 6 weeks postpartum (4.49 log vs. 4.40 log, p=0.79), between single-dose nevirapine/zidovudine+lamivudine and single-dose nevirapine groups, respectively. Nevirapine resistance, measured by population sequencing and sensitive real-time PCR, was significantly less common in those receiving single-dose nevirapine/zidovudine+lamivudine compared to single-dose nevirapine, respectively, at 2 weeks (10% (4/40) vs. 74% (31/42), p<0.0001) and 6 weeks postpartum [10% (11/115) vs. 64% (41/64), p<0.0001; adjusted relative risk=0.18, 95% confidence interval (0.10–0.34)].
The significant decrease in nevirapine resistance conferred by one week of zidovudine+lamivudine should help policymakers optimize peripartum HIV prophylaxis recommendations.
Assess population attributable fractions (PAFs) for late postnatal transmission (LPT) of human immunodeficiency virus-1 (HIV-1) in a cohort of HIV-1-exposed infants.
We used data established from a risk factor analysis of LPT (negative HIV-1 results through the 4-6 week visit, but positive assays thereafter through the 12-month visit) from a perinatal clinical trial conducted in three sub-Saharan countries. PAFs were calculated as the proportions of excess LPTs attributed to identified risk factors.
For the cohort of 1317 infants, 206 (15.6%) had only low maternal CD4+ counts (< 200 cells/mm3), 332 (25.2%) had only high maternal plasma viral loads (VLs) (> 50 000 copies/mL), and 81 (6.2%) had both low CD4+ counts and high VLs. Their PAFs were 26.0% [95% confidence interval (CI), 12.0%-36.0%], 37.0% (95% CI, 22.0%-51.0%) and 16.0% (95% CI, 6.0%-25.0%), respectively.
Our PAF analysis illustrates the public health impact of the substantial proportion of LPTs accounted for by high-risk women with both low CD4+ counts and high VLs. In light of these results, access to and use of antiretroviral therapy (ART) by high-risk HIV-1-infected pregnant women is essential. Additional strategies to reduce LPT for those not meeting criteria for ART should be implemented.
Breast feeding; late postnatal transmission; prevention of mother to child transmission/vertical transmission; risk factors; viral load
There are limited data on acute HIV infection (AHI) prevalence during pregnancy.
Malawian pregnant women admitted in the third trimester and meeting eligibility criteria underwent dual HIV rapid antibody testing. AHI prevalence was retrospectively detected through HIV RNA pooling of seronegative plasma.
Among 3825 pregnant women screened, dual HIV rapid testing indicated that 30.2% were HIV positive, 69.7% were HIV negative and 0.1% were indeterminate. Sensitivity and specificity of dual rapid testing was 99.0% and 98.7%, respectively. Of 2666 seronegative specimens, 2327 had samples available for HIV RNA pooling; 5 women (0.21%) (95% CI: 0.03, 0.40%) had AHI with a median peripartum viral load of 1,324,766 copies/mL.
Pregnant women are at risk for AHI, warranting counseling of all women and their sexual partners about incident HIV during pregnancy. Dual HIV rapid tests have high sensitivity and specificity. HIV testing should be repeated in the third trimester and/or at delivery.
Acute HIV infection; pregnancy; mother to child transmission; rapid HIV testing
The clinical relevance of detecting minority drug-resistant HIV-1 variants is uncertain.
To determine the effect of pre-existing minority non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants on the risk of virologic failure (VF), we reanalyzed a case-cohort substudy of efavirenz recipients in ACTG A5095. Minority K103N or Y181C populations were determined by allele-specific PCR (ASPCR) in subjects without NNRTI resistance by population sequencing. Weighted Cox proportional hazards models adjusted for recent adherence estimated the relative risk of VF in the presence of NNRTI-resistant minority variants.
The evaluable case-cohort sample included 195 subjects from the randomly selected subcohort (51 with VF, 144 without failure [NF]), plus 127 of the remaining subjects with VF. Presence of minority K103N or Y181C mutations, or both, was detected in 8 (4.4%), 54 (29.5%) and 11 (6%), respectively, of 183 evaluable subjects in the random subcohort. Detection of minority Y181C mutants was associated with an increased risk of VF in the setting of recent adherence (HR=3.45, CI=1.90, 6.26), but not in non-adherent subjects (HR=1.39, CI=0.58, 3.29). Of note, 70% of subjects with minority Y181C achieved long-term viral suppression.
In adherent patients, pre-existing minority Y181C mutants more than tripled the risk of VF of first-line efavirenz-based ART.
HIV-1; antiretroviral therapy; antiretroviral resistance; allele-specific PCR; minority variants; quasispecies
Amplification 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.
To determine the suitability of the ExaVir™ Load and ExaVir™ Drug assays for use in patient monitoring.
Specimens 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.
HIV-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 equivalents. 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.
The 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.
Cavidi; HIV-1; phenotype assay; genotype assay; viral load
The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross–reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly-reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.
cross-reactivity; monoclonal antibody; subtype; envelope glycoprotein; HIV-1
This study compared the role of genotypic susceptibility scores (GSS) as a predictor of virologic response in a group (n = 234) of HIV-infected, protease inhibitor (PI)-experienced subjects. Two scoring methods [discrete genotypic susceptibility score (dGSS) and continuous genotypic susceptibility score (cGSS)] were developed. Each drug in the subject's regimen was given a binary susceptibility score using Stanford inferred drug resistance scores to calculate the dGSS. In contrast to the dGSS, the cGSS model was designed to reflect partial susceptibility to a drug. Both GSS were independent predictors of week 16 virologic response. We also compared the GSS to a phenotypic susceptibility score (PSS) model on a subset of subjects that had both GSS and PSS performed, and found that both models were predictive of virologic response. Genotypic analyses at enrollment showed that subjects who were virologic nonresponders at week 16 revealed enrichment of several mutated codons associated with nucleoside reverse transcriptase inhibitors (NRTI) (codons 67, 69, 70, 118, 215, and 219) or PI resistance (codons 10, 24, 71, 73, and 88) compared to subjects who were virologic responders. Regression analyses revealed that protease mutations at codons 24 and 90 were most predictive of poor virologic response, whereas mutations at 82 were associated with enhanced virologic response. Certain NNRTI-associated mutations, such as K103N, were rapidly selected in the absence of NRTIs. These data indicate that GSS may be a useful tool in selecting drug regimens in HIV-1-infected subjects to maximize virologic response and improve treatment outcomes.
HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus.
The work described in this report is directed at how HIV-1 viral RNA populations differ between the blood plasma and male genital tract in established infection. This site is of special interest since it is the proximal source of most transmissions of HIV-1. Thus, lessons learned about HIV-1 in the seminal tract are directly relevant to the mechanism of HIV-1 transmission. We have used single genome amplification to generate viral sequences from paired blood and semen samples in men with chronic HIV-1 infection. When compared to viral populations in blood plasma, we observe that virus in the seminal plasma can be equilibrated, clonally-amplified, or compartmentalized. We have also performed a characterization of the cytokine and chemokine milieu in these two compartments. We report a dramatic concentration of immune modulators in the seminal plasma relative to the blood, and these likely enhance the potential for viral replication in this compartment by creating an environment where target cells are kept in an activated state. These data define new and distinct features of virus:host interactions and represent a significant advance in our understanding of HIV-1 replication in the male genital tract.
Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi.
Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed.
Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine.
When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART.
Africa; antiretroviral failure; public health approach; resistance; resource-limited setting
HIV transmission; acute HIV infection; sexually transmitted infections; viral load; Southeastern United States
To quantitate extracellular and intracellular zidovudine (ZDV) and lamivudine (3TC) concentrations in blood and semen of HIV-1–infected men.
Nonblind, single-center, open-label pharmacokinetic (PK) study in 14 subjects receiving ZDV plus 3TC.
Paired blood and semen samples were obtained during 1 intensive visit and 3 single time point visits over 2 weeks. Extracellular ZDV and 3TC concentrations were measured in blood plasma (BP) and seminal plasma (SP), and intracellular ZDV and 3TC triphosphate (TP) concentrations were measured in isolated mononuclear cells using validated methods. HIV-1 RNA was measured in blood and semen. PK parameters were estimated using non-compartmental analysis.
Median (interquartile range [IQR]) SP/BP area under the time-concentration curve over the 12-hour dosing interval (AUC0–12h) ratios for ZDV and 3TC were 2.28 (1.48 to 2.97) and 6.67 (4.10 to 9.14), respectively, whereas individual SP/BP concentration ratios ranged from 1.9 to 91.4. Intracellular median (IQR) SP/BP AUC0–12h ratios for ZDV-TP and 3TC-TP were 0.36 (0.30 to 0.37) and 1.0 (0.62 to 1.30), respectively, whereas individual SP/BP concentration ratios ranged from 0.11 to 2.9. HIV-1 RNA was undetectable in both compartments.
ZDV and 3TC SP exposures are 2- to 6-fold greater than BP exposures. Seminal ZDV-TP exposures are ~40% of those found in peripheral blood mononuclear cells (PBMCs), whereas 3TC-TP exposures are similar to PBMC exposures. PK variability makes individual SP/BP ratios a suboptimal surrogate for genital tract exposure.
HIV; pharmacokinetics; nucleoside triphosphate; antiretroviral therapy; genital tract
Transmitted drug resistance (TDR) limits antiretroviral options, complicating management of HIV-positive patients. HIV disproportionately affects the Southern United States (US), but available national estimates of TDR prevalence principally reflect large metropolitan centers outside this region.
The Duke/UNC Acute HIV Program has collected data on acute or recent HIV infections (ARHI) in North Carolina (NC) since 1998. Acute infections represent antibody-negative, RNA-positive subjects; recent infection was determined by history of HIV testing, or concordance between detuned ELISA and antibody avidity assays. Genotypic sequence data from the earliest collected pre-treatment plasma sample were analyzed with the Stanford HIV Database and screened for Surveillance Drug Resistance Mutations (SDRMs).
253 individuals with ARHI between 1998 and May 2007 had complete genotypic sequence data for analysis; 39.5% were acute infections, 78.7% were male, 64.8% were non-white, and 53.8% were men who have sex with men. The overall prevalence of TDR was 17.8%, with SDRMs for non-nucleoside reverse transcriptase inhibitors (NNRTIs) in 9.5% of the cohort. Mutations for nucleos(t)ide RT inhibitors (NRTIs) were detected in 7.5%, and for protease inhibitors (PIs) in 3.2%. K103N was the most common mutation (7.5%). Thymidine analogue mutations were found in 4.7% of samples; the most common PI SDRM was L90M (2.4%). Dual-or triple-class antiretroviral resistance was rare, encountered in only six samples (2.4%).
The prevalence of TDR in NC is similar to estimates from US metropolitan areas. These findings have implications for initial regimen selection and secondary prevention efforts outside of large, metropolitan HIV epicenters.
HIV Infections/epidemiology; HIV Infections/transmission; North Carolina/epidemiology; Drug resistance, viral; Antiretroviral therapy, highly active
In order to evaluate strategies to reduce HIV transmission through breast milk and optimize both maternal and infant health among HIV-infected women and their infants, we designed and implemented a large, randomized clinical trial in Lilongwe, Malawi. The development of protocols for large, randomized clinical trials is a complicated and lengthy process often requiring alterations to the original research design. Many factors lead to delays and changes, including study site-specific priorities, new scientific information becoming available, the involvement of national and international human subject committees and monitoring boards, and alterations in medical practice and guidance at local, national, and international levels. When planning and implementing a clinical study in a resource-limited setting, additional factors must be taken into account, including local customs and program needs, language and socio-cultural barriers, high background rates of malnutrition and endemic diseases, extreme poverty, lack of personnel, and limited infrastructure. Investigators must be prepared to modify the protocol as necessary in order to ensure participant safety and successful implementation of study procedures. This paper describes the process of designing, implementing, and subsequently modifying the Breastfeeding, Antiretrovirals, and Nutrition, (BAN) study, a large, ongoing, randomized breastfeeding intervention trial of HIV-infected women and their infants conducted at a single site in Lilongwe, Malawi. We highlight some of the successes, challenges, and lessons learned at different stages during the conduct of the trial.
Mother-to-child transmission of HIV; breastfeeding; HIV/AIDS; nutrition; study design and management; antiretroviral drugs