Insights into HIV-1 Transmission Dynamics Using Routinely Collected Data in the Mid-Atlantic United States.

Background: Molecular epidemiological approaches provide opportunities to characterize HIV transmission dynamics. We analyzed HIV sequences and virus load (VL) results obtained during routine clinical care, and individual's zip-code location to determine utility of this approach. Methods: HIV-1 pol sequences aligned using ClustalW were subtyped using REGA. A maximum likelihood (ML) tree was generated using IQTree. Transmission clusters with ≤3% genetic distance (GD) and ≥90% bootstrap support were identified using ClusterPicker. We conducted Bayesian analysis using BEAST to confirm transmission clusters. The proportion of nucleotides with ambiguity ≤0.5% was considered indicative of early infection. Descriptive statistics were applied to characterize clusters and group comparisons were performed using chi-square or t-test. Results: Among 2775 adults with data from 2014−2015, 2589 (93%) had subtype B HIV-1, mean age was 44 years (SD 12.7), 66.4% were male, and 25% had nucleotide ambiguity ≤0.5. There were 456 individuals in 193 clusters: 149 dyads, 32 triads, and 12 groups with ≥ four individuals per cluster. More commonly in clusters were males than females, 349 (76.5%) vs. 107 (23.5%), p < 0.0001; younger individuals, 35.3 years (SD 12.1) vs. 44.7 (SD 12.3), p < 0.0001; and those with early HIV-1 infection by nucleotide ambiguity, 202/456 (44.3%) vs. 442/2133 (20.7%), p < 0.0001. Members of 43/193 (22.3%) of clusters included individuals in different jurisdictions. Clusters ≥ four individuals were similarly found using BEAST. HIV-1 viral load (VL) ≥3.0 log10 c/mL was most common among individuals in clusters ≥ four, 18/21, (85.7%) compared to 137/208 (65.8%) in clusters sized 2−3, and 927/1169 (79.3%) who were not in a cluster (p < 0.0001). Discussion: HIV sequence data obtained for HIV clinical management provide insights into regional transmission dynamics. Our findings demonstrate the additional utility of HIV-1 VL data in combination with phylogenetic inferences as an enhanced contact tracing tool to direct HIV treatment and prevention services. Trans-jurisdictional approaches are needed to optimize efforts to end the HIV epidemic.

Whole-Genome Analysis of Cervical Human Papillomavirus Type 35 from rural Zimbabwean Women.

Human papillomavirus (HPV) types differ by geographic location and the ethnicity of the human host, which may have implications for carcinogenicity. HPV35 is one of the least frequently identified high-risk types in North America and Europe but was the most common high-risk HPV (hrHPV) infection in a cohort in rural Zimbabwe. Whole genome analysis is limited for HPV35; no such studies have been performed in Zimbabwe. Of 648 women in the initial cohort in Zimbabwe, 19 (19/648, 2.9%) tested positive for HPV35, and eight samples were successfully sequenced for HPV35. The maximum number of sequence variants for the whole genome was 58 nucleotides (0.7%) compared to the prototype (58/7879). The maximum number of sequence variants in E6 and E7 was 3 (3/450, 0.7%) 2 (2/300, 0.7%), respectively. These are the first HPV35 whole genome sequences from Zimbabwe, and these data further lend support to the carcinogenicity of HPV35 despite limited sequence heterogeneity. Further studies to determine carcinogenic effects and impact of HPV vaccinations are warranted, especially in sub-Saharan Africa.

Persistence of Human Immunodeficiency Virus-1 Drug Resistance Mutations in Proviral Deoxyribonucleic Acid After Virologic Failure of Efavirenz-Containing Antiretroviral Regimens.

BACKGROUND: Efavirenz (EFV)-based regimens select broad drug resistance to nonnucleoside reverse-transcriptase inhibitors (NNRTIs), limiting the effectiveness of EFV and other NNRTIs. The duration, persistence, and decay of drug resistance mutations (DRMs) in the proviral reservoir is not well defined. METHODS: Participants with virologic failure of EFV-based regimens and drug-resistant viremia with the K103N mutation in plasma ribonucleic acid (RNA) were identified from AIDS Clinical Trials Group (ACTG) studies A364 and A5095. These individuals received a second-line, boosted protease inhibitor-based regimen with suppression of viremia for up to10 years during long-term follow-up (median = 3.6 years; interquartile range, 2.1-6.9 years). Proviral deoxyribonucleic acid (DNA) from cryopreserved peripheral blood mononuclear cells was sequenced to identify the persistence of DRM. RESULTS: Twenty-eight participants from ACTG 364 and ACTG 5095 were evaluated. Sanger sequencing of proviral DNA detected K103N as well as additional reverse-transcriptase inhibitor (RTI) mutations. Ultradeep sequencing confirmed persistence of K103N in 71% of participants with minimal decay over time. In an adjusted model including years since suppression, persistent proviral K103N was 2.6 times more likely (95% confidence interval, 1.0-6.4) per log10 higher human immunodeficiency virus RNA at EFV failure. CONCLUSIONS: Persistence of RTI mutations in proviral DNA after virologic failure has implications for the effectiveness of future drug regimens and the recycling of RTI drugs.

hrHPV prevalence and type distribution in rural Zimbabwe: A community-based self-collection study using near-point-of-care GeneXpert HPV testing.

OBJECTIVES: High-risk human papilloma viruses (hrHPV) are the causative agents of cervical cancer, the leading cause of cancer deaths among Zimbabwean women. The objective of this study was to describe the hrHPV types found in Zimbabwe for consideration in cervical cancer screening and vaccination efforts. DESIGN AND METHODS: To determine hrHPV prevalence and type distribution in Zimbabwe we implemented a community-based cross-sectional study of self-collected cervicovaginal samples with hrHPV screening using near-point-of-care Cepheid GeneXpert HPV. RESULTS: The hrHPV prevalence was 17% (112/643); 33% (41/123) vs. 14% (71/520) among HIV-1-positive and -negative participants, respectively (p=2.3E-07). Typing via Xpert HPV showed very good overall agreement (77.2%, kappa=0.698) with the Seegene Anyplex II HPV HR Detection kit. The most common types were HPV16, HPV18, HPV35, HPV52, HPV58, HPV68, HPV18, and HPV51, each of which appeared in 14-20% of infections. 37% (28/76) of women with positive cytology results (ASCUS+) had a type not included in the basic vaccine and 25% (19/76) had a type not currently in the nine-valent vaccine. CONCLUSIONS: hrHPV type distribution includes less common high-risk types in rural Zimbabwe. The distribution and carcinogenicity of hrHPV type distribution should be considered during screening assay design, program development, as well as vaccine distribution and design.

Evolution of gag and gp41 in Patients Receiving Ritonavir-Boosted Protease Inhibitors.

Several groups have proposed that genotypic determinants in gag and the gp41 cytoplasmic domain (gp41-CD) reduce protease inhibitor (PI) susceptibility without PI-resistance mutations in protease. However, no gag and gp41-CD mutations definitively responsible for reduced PI susceptibility have been identified in individuals with virological failure (VF) while receiving a boosted PI (PI/r)-containing regimen. To identify gag and gp41 mutations under selective PI pressure, we sequenced gag and/or gp41 in 61 individuals with VF on a PI/r (n = 40) or NNRTI (n = 20) containing regimen. We quantified nonsynonymous and synonymous changes in both genes and identified sites exhibiting signal for directional or diversifying selection. We also used published gag and gp41 polymorphism data to highlight mutations displaying a high selection index, defined as changing from a conserved to an uncommon amino acid. Many amino acid mutations developed in gag and in gp41-CD in both the PI- and NNRTI-treated groups. However, in neither gene, were there discernable differences between the two groups in overall numbers of mutations, mutations displaying evidence of diversifying or directional selection, or mutations with a high selection index. If gag and/or gp41 encode PI-resistance mutations, they may not be confined to consistent mutations at a few sites.

Collaborative update of a rule-based expert system for HIV-1 genotypic resistance test interpretation.

INTRODUCTION: HIV-1 genotypic resistance test (GRT) interpretation systems (IS) require updates as new studies on HIV-1 drug resistance are published and as treatment guidelines evolve. METHODS: An expert panel was created to provide recommendations for the update of the Stanford HIV Drug Resistance Database (HIVDB) GRT-IS. The panel was polled on the ARVs to be included in a GRT report, and the drug-resistance interpretations associated with 160 drug-resistance mutation (DRM) pattern-ARV combinations. The DRM pattern-ARV combinations included 52 nucleoside RT inhibitor (NRTI) DRM pattern-ARV combinations (13 patterns x 4 NRTIs), 27 nonnucleoside RT inhibitor (NNRTI) DRM pattern-ARV combinations (9 patterns x 3 NNRTIs), 39 protease inhibitor (PI) DRM pattern-ARV combinations (13 patterns x 3 PIs) and 42 integrase strand transfer inhibitor (INSTI) DRM pattern-ARV combinations (14 patterns x 3 INSTIs). RESULTS: There was universal agreement that a GRT report should include the NRTIs lamivudine, abacavir, zidovudine, emtricitabine, and tenofovir disoproxil fumarate; the NNRTIs efavirenz, etravirine, nevirapine, and rilpivirine; the PIs atazanavir/r, darunavir/r, and lopinavir/r (with "/r" indicating pharmacological boosting with ritonavir or cobicistat); and the INSTIs dolutegravir, elvitegravir, and raltegravir. There was a range of opinion as to whether the NRTIs stavudine and didanosine and the PIs nelfinavir, indinavir/r, saquinavir/r, fosamprenavir/r, and tipranavir/r should be included. The expert panel members provided highly concordant DRM pattern-ARV interpretations with only 6% of NRTI, 6% of NNRTI, 5% of PI, and 3% of INSTI individual expert interpretations differing from the expert panel median by more than one resistance level. The expert panel median differed from the HIVDB 7.0 GRT-IS for 20 (12.5%) of the 160 DRM pattern-ARV combinations including 12 NRTI, two NNRTI, and six INSTI pattern-ARV combinations. Eighteen of these differences were updated in HIVDB 8.1 GRT-IS to reflect the expert panel median. Additionally, HIVDB users are now provided with the option to exclude those ARVs not considered to be universally required. CONCLUSIONS: The HIVDB GRT-IS was updated through a collaborative process to reflect changes in HIV drug resistance knowledge, treatment guidelines, and expert opinion. Such a process broadens consensus among experts and identifies areas requiring further study.

Evaluation of the Aptima HIV-1 Quant Dx Assay Using Plasma and Dried Blood Spots.

HIV-1 RNA quantitation in plasma, or virus load testing, is the primary method by which the response to antiretroviral therapy is monitored. Here we describe evaluation of the Aptima HIV-1 Quant Dx assay (Aptima) performed on the automated Panther system. The clinical performance of Aptima was compared to that of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test v2.0 (CAP/CTM) using 162 EDTA plasma samples collected from patients undergoing HIV-1 monitoring. Overall agreement was 84.0% (136/162), with a kappa statistic of 0.723 (standard error, 0.047; 95% confidence interval [CI], 0.630 to 0.815), indicating substantial agreement. Using the 86 clinical samples quantifiable by both methods, Passing-Bablok regression revealed a regression line of Y = (1.069 × X) - 0.346 (95% CI of the slope [1.003 to 1.139] and intercept [-0.666 to -0.074]), and Bland-Altman analysis demonstrated a mean difference (Aptima-CAP/CTM) of -0.075 log10 copies/ml (95% limits of agreement of -0.624 to 0.475), consistent with negative bias. Comparison of Aptima results for paired dried blood spot (DBS) and plasma specimens archived from participants in the Peninsula AIDS Research Cohort Study (PARC) demonstrated an overall agreement of 94.7% (90/95) when 1,000 copies/ml was used as the threshold. In conclusion, the Aptima HIV-1 Quant Dx assay provides a suitable alternative for HIV-1 monitoring in plasma and DBS.

Global Comparison of Drug Resistance Mutations After First-Line Antiretroviral Therapy Across Human Immunodeficiency Virus-1 Subtypes.

Background. Human immunodeficiency virus (HIV)-1 drug resistance mutations (DRMs) often accompany treatment failure. Although subtype differences are widely studied, DRM comparisons between subtypes either focus on specific geographic regions or include populations with heterogeneous treatments. Methods. We characterized DRM patterns following first-line failure and their impact on future treatment in a global, multi-subtype reverse-transcriptase sequence dataset. We developed a hierarchical modeling approach to address the high-dimensional challenge of modeling and comparing frequencies of multiple DRMs in varying first-line regimens, durations, and subtypes. Drug resistance mutation co-occurrence was characterized using a novel application of a statistical network model. Results. In 1425 sequences, 202 subtype B, 696 C, 44 G, 351 circulating recombinant forms (CRF)01_AE, 58 CRF02_AG, and 74 from other subtypes mutation frequencies were higher in subtypes C and CRF01_AE compared with B overall. Mutation frequency increased by 9%-20% at reverse transcriptase positions 41, 67, 70, 184, 215, and 219 in subtype C and CRF01_AE vs B. Subtype C and CRF01_AE exhibited higher predicted cross-resistance (+12%-18%) to future therapy options compared with subtype B. Topologies of subtype mutation networks were mostly similar. Conclusions. We find clear differences in DRM outcomes following first-line failure, suggesting subtype-specific ecological or biological factors that determine DRM patterns.

The cryptococcal antigen lateral flow assay: A point-of-care diagnostic at an opportune time.

Cryptococcal meningitis is a devastating HIV-related opportunistic infection, affecting nearly 1 million individuals and causing over 500 000 deaths each year. The burden of disease is greatest in sub-Saharan Africa and Southeast Asia, where cryptococcal disease is the most common cause of meningitis. Rapid, accurate and affordable diagnosis of cryptococcal disease has been lacking in many of the most heavily affected areas. Here, we review a point-of-care assay for cryptococcal disease, the dipstick-formatted cryptococcal antigen lateral flow assay (LFA) (IMMY, Norman, OK). In comparison to culture, the assay is 99.5% sensitive and 98% specific. In comparison to other commercially available tests for cryptococcal antigen, the LFA has equal or superior sensitivity and specificity in CSF, plasma and serum samples. We discuss potential applications for the use of the assay in resource-limited settings, including what is likely to be an important role of the LFA in screening for early cryptococcal infection before clinical disease and in evaluating pre-emptive treatment.

Lopinavir/Ritonavir Monotherapy as Second-line Antiretroviral Treatment in Resource-Limited Settings: Week 104 Analysis of AIDS Clinical Trials Group (ACTG) A5230.

BACKGROUND: The AIDS Clinical Trials Group (ACTG) A5230 study evaluated lopinavir/ritonavir (LPV/r) monotherapy following virologic failure (VF) on first-line human immunodeficiency virus (HIV) regimens in Africa and Asia. METHODS: Eligible subjects had received first-line regimens for at least 6 months and had plasma HIV-1 RNA levels 1000-200 000 copies/mL. All subjects received LPV/r 400/100 mg twice daily. VF was defined as failure to suppress to <400 copies/mL by week 24, or confirmed rebound to >400 copies/mL at or after week 16 following confirmed suppression. Subjects with VF added emtricitabine 200 mg/tenofovir 300 mg (FTC/TDF) once daily. The probability of continued HIV-1 RNA <400 copies/mL on LPV/r monotherapy through week 104 was estimated with a 95% confidence interval (CI); predictors of treatment success were evaluated with Cox proportional hazards models. RESULTS: One hundred twenty-three subjects were enrolled. Four subjects died and 2 discontinued prematurely; 117 of 123 (95%) completed 104 weeks. Through week 104, 49 subjects met the primary endpoint; 47 had VF, and 2 intensified treatment without VF. Of the 47 subjects with VF, 41 (33%) intensified treatment, and 39 of 41 subsequently achieved levels <400 copies/mL. The probability of continued suppression <400 copies/mL over 104 weeks on LPV/r monotherapy was 60% (95% CI, 50%-68%); 80%-85% maintained levels <400 copies/mL with FTC/TDF intensification as needed. Ultrasensitive assays on specimens with HIV-1 RNA level <400 copies/mL at weeks 24, 48, and 104 revealed that 61%, 62%, and 65% were suppressed to <40 copies/mL, respectively. CONCLUSIONS: LPV/r monotherapy after first-line VF with FTC/TDF intensification when needed provides durable suppression of HIV-1 RNA over 104 weeks. CLINICAL TRIALS REGISTRATION: NCT00357552.

Association between latent proviral characteristics and immune activation in antiretrovirus-treated human immunodeficiency virus type 1-infected adults.

UNLABELLED: Generalized immune activation during HIV infection is associated with an increased risk of cardiovascular disease, neurocognitive disease, osteoporosis, metabolic disorders, and physical frailty. The mechanisms driving this immune activation are poorly understood, particularly for individuals effectively treated with antiretroviral medications. We hypothesized that viral characteristics such as sequence diversity may play a role in driving HIV-associated immune activation. We therefore sequenced proviral DNA isolated from peripheral blood mononuclear cells from HIV-infected individuals on fully suppressive antiretroviral therapy. We performed phylogenetic analyses, calculated viral diversity and divergence in the env and pol genes, and determined coreceptor tropism and the frequency of drug resistance mutations. Comprehensive immune profiling included quantification of immune cell subsets, plasma cytokine levels, and intracellular signaling responses in T cells, B cells, and monocytes. These antiretroviral therapy-treated HIV-infected individuals exhibited a wide range of diversity and divergence in both env and pol genes. However, proviral diversity and divergence in env and pol, coreceptor tropism, and the level of drug resistance did not significantly correlate with markers of immune activation. A clinical history of virologic failure was also not significantly associated with levels of immune activation, indicating that a history of virologic failure does not inexorably lead to increased immune activation as long as suppressive antiretroviral medications are provided. Overall, this study demonstrates that latent viral diversity is unlikely to be a major driver of persistent HIV-associated immune activation. IMPORTANCE: Chronic immune activation, which is associated with cardiovascular disease, neurologic disease, and early aging, is likely to be a major driver of morbidity and mortality in HIV-infected individuals. Although treatment of HIV with antiretroviral medications decreases the level of immune activation, levels do not return to normal. The factors driving this persistent immune activation, particularly during effective treatment, are poorly understood. In this study, we investigated whether characteristics of the latent, integrated HIV provirus that persists during treatment are associated with immune activation. We found no relationship between latent viral characteristics and immune activation in treated individuals, indicating that qualities of the provirus are unlikely to be a major driver of persistent inflammation. We also found that individuals who had previously failed treatment but were currently effectively treated did not have significantly increased levels of immune activation, providing hope that past treatment failures do not have a lifelong "legacy" impact.

Prototypical Recombinant Multi-Protease-Inhibitor-Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type 1.

The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI-resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI-resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activities of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most if not all clinically relevant PI-resistant viruses.

Comparing Peripheral Blood Mononuclear Cell DNA and Circulating Plasma viral RNA pol Genotypes of Subtype C HIV-1.

INTRODUCTION: Drug resistance mutations (DRM) in viral RNA are important in defining to provide effective antiretroviral therapy (ART) in HIV-1 infected patients. Detection of DRM in peripheral blood mononuclear cell (PBMC) DNA is another source of information, although the clinical significance of DRMs in proviral DNA is less clear. MATERIALS AND METHODS: From 25 patients receiving ART at a center in Zimbabwe, 32 blood samples were collected. Dideoxy-sequencing of gag-pol identified subtype and resistance mutations from plasma viral RNA and proviral DNA. Drug resistance was estimated using the calibrated population resistance tool on www.hivdb.stanford.edu database. Numerical resistance scores were calculated for all antiretroviral drugs and for the subjects' reported regimen. Phylogenetic analysis as maximum likelihood was performed to determine the evolutionary distance between sequences. RESULTS: Of the 25 patients, 4 patients (2 of which had given 2 blood samples) were not known to be on ART (NA) and had exclusively wild-type virus, 17 had received Protease inhibitors (PI), 18, non-nucleoside reverse transcriptase inhibitors (NNRTI) and 19, two or more nucleoside reverse transcriptase inhibitors (NRTI). Of the 17 with history of PI, 10 had PI mutations, 5 had minor differences between mutations in RNA and DNA. Eighteen samples had NNRTI mutations, six of which demonstrated some discordance between DNA and RNA mutations. Although NRTI resistance mutations were frequently different between analyses, mutations resulted in very similar estimated phenotypes as measured by resistance scores. The numerical resistance scores from RNA and DNA for PIs differed between 2/10, for NNRTIs between 8/18, and for NRTIs between 17/32 pairs. When calculated resistance scores were collapsed, 3 pairs showed discordance between RNA and DNA for at least one PI, 6 were discordant for at least one NNRTI and 11 for at least one NRTI. Regarding phylogenetic evolutionary analysis, all RNA and DNA sequence pairs clustered closely in a maximum likelihood tree. CONCLUSION: PBMC DNA could be useful for testing drug resistance in conjunction with plasma RNA where the results of each yielded complementary information about drug resistance. Identification of DRM, archived in proviral DNA, could be used to provide for sustainable public health surveillance among subtype C infected patients.

Global analysis of sequence diversity within HIV-1 subtypes across geographic regions.

AIMS: HIV-1 sequence diversity can affect host immune responses and phenotypic characteristics such as antiretroviral drug resistance. Current HIV-1 sequence diversity classification uses phylogeny-based methods to identify subtypes and recombinants, which may overlook distinct subpopulations within subtypes. While local epidemic studies have characterized sequence-level clustering within subtypes using phylogeny, identification of new genotype - phenotype associations are based on mutational correlations at individual sequence positions. We perform a systematic, global analysis of position-specific pol gene sequence variation across geographic regions within HIV-1 subtypes to characterize subpopulation differences that may be missed by standard subtyping methods and sequence-level phylogenetic clustering analyses. MATERIALS #ENTITYSTARTX00026; METHODS: Analysis was performed on a large, globally diverse, cross-sectional pol sequence dataset. Sequences were partitioned into subtypes and geographic subpopulations within subtypes. For each subtype, we identified positions that varied according to geography using VESPA (viral epidemiology signature pattern analysis) to identify sequence signature differences and a likelihood ratio test adjusted for multiple comparisons to characterize differences in amino acid (AA) frequencies, including minority mutations. Synonymous nonsynonymous analysis program (SNAP) was used to explore the role of evolutionary selection witihin subtype C. RESULTS: In 7693 protease (PR) and reverse transcriptase (RT) sequences from untreated patients in multiple geographic regions, 11 PR and 11 RT positions exhibited sequence signature differences within subtypes. Thirty six PR and 80 RT positions exhibited within-subtype geography-dependent differences in AA distributions, including minority mutations, at both conserved and variable loci. Among subtype C samples from India and South Africa, nine PR and nine RT positions had significantly different AA distributions, including one PR and five RT positions that differed in consensus AA between regions. A selection analysis of subtype C using SNAP demonstrated that estimated rates of nonsynonymous and synonymous mutations are consistent with the possibility of positive selection across geographic subpopulations within subtypes. CONCLUSION: We characterized systematic genotypic pol differences across geographic regions within subtypes that are not captured by the subtyping nomenclature. Awareness of such differences may improve the interpretation of future studies determining the phenotypic consequences of genetic backgrounds.

Panel of prototypical recombinant infectious molecular clones resistant to nevirapine, efavirenz, etravirine, and rilpivirine.

We created a panel of 10 representative multi-nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant recombinant infectious molecular HIV-1 clones to assist researchers studying NNRTI resistance or developing novel NNRTIs. The cloned viruses contain most of the major NNRTI resistance mutations and most of the significantly associated mutation pairs that we identified in two network analyses. Each virus in the panel has intermediate- or high-level resistance to all or three of the four most commonly used NNRTIs.

Lopinavir/ritonavir monotherapy after virologic failure of first-line antiretroviral therapy in resource-limited settings.

OBJECTIVE: To evaluate virologic response rates of lopinavir/ritonavir (LPV/r) monotherapy as second-line antiretroviral treatment (ART) among adults in resource-limited settings (RLSs). DESIGN: An open-label pilot study of LPV/r monotherapy in participants on first-line nonnucleoside reverse transcriptase inhibitor three-drug combination ART with plasma HIV-1 RNA 1000-200 000  copies/ml. METHODS: Participants were recruited from five sites in Africa and Asia within the AIDS Clinical Trials Group (ACTG) network. All participants received LPV/r 400/100  mg twice daily. The primary endpoint was remaining on LPV/r monotherapy without virologic failure at week 24. Participants with virologic failure were offered addition of emtricitabine and tenofovir (FTC/TDF) to LPV/r. RESULTS: Mutations associated with drug resistance were encountered in nearly all individuals screened for the study. One hundred and twenty-three participants were enrolled, and 122 completed 24 weeks on study. A high proportion remained on LPV/r monotherapy without virologic failure at 24 weeks (87%). Archived samples with HIV-1 RNA levels less than 400  copies/ml at week 24 (n=102) underwent ultrasensitive assay. Of these individuals, 62 had levels less than 40  copies/ml and 30 had levels 40-200  copies/ml. Fifteen individuals experienced virologic failure, among whom 11 had resistance assessed and two had emergent protease inhibitor mutations. Thirteen individuals with virologic failure added FTC/TDF and one individual added FTC/TDF without virologic failure. At study week 48, 11 of 14 adding FTC/TDF had HIV-1 RNA levels less than 400  copies/ml. CONCLUSION: In this pilot study conducted in diverse RLS, LPV/r monotherapy as second-line ART demonstrated promising activity.

Viral sequence analysis from HIV-infected mothers and infants: molecular evolution, diversity, and risk factors for mother-to-child transmission.

Great progress has been made in understanding the pathogenesis, treatment, and transmission of HIV and the factors influencing the risk of mother-to-child transmission (MTCT). Many questions regarding the molecular evolution and genetic diversity of HIV in the context of MTCT remain unanswered. Further research to identify the selective factors governing which variants are transmitted, how the compartmentalization of HIV in different cells and tissues contributes to transmission, and the influence of host immunity, viral diversity, and recombination on MTCT may provide insight into new prevention strategies and the development of an effective HIV vaccine.

Genetic analyses of HIV-1 env sequences demonstrate limited compartmentalization in breast milk and suggest viral replication within the breast that increases with mastitis.

The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na(+)) concentration. The association between milk Na(+) and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P = 0.621), but Na(+) was correlated with greater genetic distance between milk and blood HIV-1 populations (P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.

Detection of HIV-1 in Saliva: Implications for Case-Identification, Clinical Monitoring and Surveillance for Drug Resistance.

BACKGROUND: Saliva tests that detect antibodies are used to diagnose HIV infection. The goal of this study was to determine whether saliva could be used for nucleic acid-based tests to measure HIV-1 virus load (VL) and detect drug resistance. METHODS: 69 HIV infected individuals provided 5-10 ml of saliva and blood samples. Viral RNA was isolated from saliva and dried blood spots using the Nuclisens extraction. Saliva VL was measured using a modified Amplicor assay, and genotyping was performed using an in-house RT-PCR/sequencing protocol. Plasma VLs were obtained from concurrently drawn clinical tests. RESULTS: Thirty-six of 47 (77%) plasma viremic patients had measurable saliva HIV-1 RNA. Paired plasma and saliva HIV RNA levels were significantly correlated (Spearman's correlation = .6532, p<.0001), but saliva VL was typically lower. Three of 22 patients with undetectable plasma VL (<50 copies/ml) had detectable saliva HIV RNA. Eleven of 30 patients with undetectable saliva RNA had detectable plasma HIV-1 RNA. Comparison of the protease and reverse transcriptase gene sequences from paired saliva and plasma of 20 patients showed less than 1% difference overall, and few resistance-related amino acid differences CONCLUSIONS: Most patients with plasma virus >50 copies/mL had detectable saliva HIV RNA, and the genotypic data was highly concordant between saliva and plasma. In patients with high levels of plasma HIV RNA, saliva might be useful in identifying viremia and evaluating drug resistance.