HIV DNA In Blood Research Articles

2071. HIV reservoir evaluation in GALT and PBMCs of virologically suppressed patients

Abstract Background The extension of HIV reservoir in the rectal gut associated lymphoid tissue (GALT) and the ART influence on this compartment are still poorly studied. We aimed to evaluate the extent of HIV-DNA in GALT and peripheral blood of virologically suppressed patients (VS) Methods 40 HIV-1+ VS pts were enrolled. Blood and GALT HIV-DNA levels and residual plasma viremia (RV) were quantified by droplet digital-PCR. Rectal biopsies were collected during high resolution anoscopy. Lymphocyte (L) phenotype and antiretroviral drug concentrations were analysed in tissue and peripheral blood Results Pts median age (IQR) was 37 (32-45) yrs, 39 (97.5%) were males. At HIV-1 diagnosis, median (IQR) viremia and CD4+ were 4.7 (4.3-5) Log10cps/mL and 469 (299-627) cells/µL, respectively. 27 (67.5%) pts had HIV-1 B subtype. At the enrolment, median (IQR) CD4+ was 720 (583-1041) cells/µL and RV was 3 (2-6) cps/mL. All pts were on 2 NRTI-regimen with a 3rd drug (20 INSTI, 15 NNRTI, 5 PI). A positive correlation emerged among HIV-DNA levels in the GALT and PBMC (Rho= 0.432; p=0.005), CD8+ L counts (Rho=0.367; p=0.02), and a negative correlation emerged with blood CD3+ L percentage (Rho=-0.489; p=0.006). Peripheral blood HIV-DNA values were inversely correlated with the CD4+ nadir (Rho= -0.379; p=0.017) and blood CD4+/CD8+ ratio (Rho= -0.362; p=0.022), and directly correlated with GALT CD4+CD38+ (Rho= 0.414; p=0.028) and blood CD8+CD38+ L (%) (Rho= 0.400; p=0.026). A positive correlation among GALT and peripheral blood CD4+CD38+ (Rho= 0.555; p=0.005), CD8+CD38+ (Rho= 0.417; p=0.030), and CD19+CD38+ L (Rho= 0.498; p=0.007) was found. There was no difference in HIV-DNA levels in the GALT and PBMCs according to the ART 3rd drug. In a subgroup of 20 pts the analysis of rectal tissue and blood drug concentrations showed that tissue/plasma ratio was higher in pts on Elvitegravir (EVG) and Rilpivirine (RPV) (median ratio 2.8 and 1.7, respectively) GALT and Blood HIV-DNA levels Tissue-Plasma Drug concentration Conclusion HIV-DNA from rectal GALT is a valid marker to characterize viral reservoir. The correlation of higher GALT HIV-DNA levels, CD8+ T L counts and CD4 and CD8 activation levels suggests that a greater amount of virus in the anorectal reservoir can perpetuate the inflammatory state characteristic of HIV+ pts. EVG and RPV showed higher tissue exposure than other ART regimens Disclosures All Authors: No reported disclosures.

Tissue-specific differences in HIV DNA levels and mechanisms that govern HIV transcription in blood, gut, genital tract and liver in ART-treated women.

IntroductionSex‐specific differences affect multiple aspects of HIV infection, yet few studies have quantified HIV levels in tissues from women. Since an HIV functional cure will likely require a major reduction of infected cells from most tissues, we measured total and intact HIV DNA and the HIV transcription profile in blood, gut, genital tract and liver from HIV‐positive antiretroviral therapy (ART) ‐treated women.MethodsPeripheral blood mononuclear cells (PBMC) and biopsies from the gastrointestinal (ileum, colon, rectosigmoid +/‐ liver) and genital (ectocervix, endocervix and endometrium) tracts were collected from 6 ART‐treated (HIV RNA < 200 copies/mL) women. HIV DNA (total and intact) and levels of read‐through, initiated (total), 5’elongated, polyadenylated and multiply spliced HIV transcripts were measured by droplet digital PCR. Immunophenotyping of cells was performed using Cytometry by time of flight (CyTOF).ResultsWe detected total HIV DNA in all tissues and intact HIV DNA in blood, ileum, colon, rectosigmoid and ectocervix. Initiated HIV transcripts per provirus were higher in PBMC and endometrium than in ileum, colon, rectosigmoid, ectocervix or endocervix, and higher in the rectum than either ileum or colon. 5’Elongated HIV transcripts per provirus were comparable in PBMC and endometrium, but higher than in gut or cervical samples. Polyadenylated and multiply spliced HIV transcripts were detected in PBMC (6/6 and 3/6 individuals respectively), but rarely in the tissues.ConclusionsThese results suggest tissue‐specific differences in the mechanisms that govern HIV expression, with lower HIV transcription in most tissues than blood. Therapies aimed at disrupting latency, such as latency‐reversing or latency‐silencing agents, will be required to penetrate into multiple tissues and target different blocks to HIV transcription.

Safety, Immune, and Antiviral Effects of Pegylated Interferon Alpha 2b Administration in Antiretroviral Therapy-Suppressed Individuals: Results of Pilot Clinical Trial

In the pilot NCT01935089 trial, we tested whether pegylated interferon alpha2b (Peg-IFN-α2b) with antiretroviral therapy (ART) was safe and could impact HIV and immune measures in blood and in gut-associated lymphoid tissue (GALT). Twenty HIV-1+ ART-suppressed individuals received 1 μg/kg/week Peg-IFN-α2b with ART for 20 weeks, with intermediate 4-week analytical ART interruption (ATI). Safety, immune activation, HIV viral load and integrated HIV DNA in blood, and HIV RNA and DNA in gut biopsies were measured. A total of 7/20 participants experienced grade 3-4 adverse events, while 17/20 participants completed the study. Of the 17 participants who completed the study, 8 remained suppressed during ATI, while all 17 were suppressed at end of treatment (EoT). As expected, treatment increased activation of T and natural killer (NK) cells and IFN-stimulated molecule expression on monocytes in periphery. While circulating CD4+ T cells showed a trend for a decrease in integrated HIV DNA, GALT showed a significant decrease in HIV-1 RNA+ cells as measured by in situ hybridization along with a reduction in total HIV DNA and cell-associated RNA by EoT. The observed decrease in HIV-1 RNA+ cells in GALT was positively associated with the decrease in activated NK cells and macrophages. This study documents for the first time that 20 weeks of immunotherapy with Peg-IFN-α2b+ART (inclusive of a 4-week ATI) is safe and results in an increase in blood and GALT immune activation and in a significant decrease in HIV-1 RNA+ cells in GALT in association with changes in innate cell activation.

Increasing contribution of integrated forms to total HIV DNA in blood during HIV disease progression from primary infection.

BackgroundIn the current context of research on HIV reservoirs, offering new insights into the persistence of HIV DNA in infected cells, which prevents viral eradication, may aid in identifying cure strategies. This study aimed to describe the establishment of stable integrated forms among total HIV DNA during primary infection (PHI) and their dynamics during the natural history of infection.MethodsTotal and integrated HIV DNA were quantified in blood from 74 PHI patients and 97 recent seroconverters (<12 months following infection, “progression cohort”). The evolution of both markers over six years was modelled (mixed-effect linear models). Their predictive values for disease progression were studied (Cox models).FindingsFor most patients during PHI, stable integrated forms were a minority among total HIV DNA (median: 12%) and became predominant thereafter (median at AIDS stage: 100%). Both total and integrated HIV DNA increased over a six-year period. Patients from the progression cohort who reached clinical AIDS during follow-up (n = 34) exhibited higher total and integrated HIV DNA levels at seroconversion and a higher percentage of integrated forms than did slower progressors (n = 63) (median: 100% vs 44%). The integrated HIV DNA load was strongly associated with the risk of developing AIDS (aRR = 2.63, p = 0.002).InterpretationThe profile of “rapid” or “slower” progression in the natural history of HIV infection appears to be determined early in the course of HIV infection. The strong predominance of unstable unintegrated forms in PHI may explain the great benefit of this early treatment, which induces a sharp decrease in total HIV DNA.FundFrench National Agency for Research on AIDS and Viral Hepatitis.

Impact of early cART on HIV blood and semen compartments at the time of primary infection

BackgroundHIV-infected cells in semen facilitate viral transmission. We studied the establishment of HIV reservoirs in semen and blood during PHI, along with systemic immune activation and the impact of early cART.MethodsPatients in the ANRS-147-OPTIPRIM trial received two years of early cART. Nineteen patients of the trial were analyzed, out of which 8 had acute PHI (WB ≤1 Ab). We quantified total cell-associated (ca) HIV-DNA in blood and semen and HIV-RNA in blood and semen plasma samples, collected during PHI and at 24 months of treatment.ResultsAt enrollment, HIV-RNA load was higher in blood than in semen (median 5.66 vs 4.22 log10 cp/mL, p<0.0001). Semen HIV-RNA load correlated strongly with blood HIV-RNA load (r = 0.81, p = 0.02, the CD4 cell count (r = -0.98, p<0.0001), and the CD4/CD8 ratio (r = -0.85, p<0.01) in acute infection but not in later stages of PHI. Median blood and seminal cellular HIV-DNA levels were 3.59 and 0.31 log10cp/106 cells, respectively. HIV-DNA load peaked in semen later than in blood and then correlated with blood IP10 level (r = 0.62, p = 0.04). HIV-RNA was undetectable in blood and semen after two years of effective cART. Semen HIV-DNA load declined similarly, except in one patient who had persistently high IP-10 and IL-6 levels and used recreational drugs.ConclusionsHIV reservoir cells are found in semen during PHI, with gradual compartmentalization. Its size was linked to the plasma IP-10 level. Early treatment purges both the virus and infected cells, reducing the high risk of transmission during PHI.Clinical trials registrationNCT01033760

The Benefits of Early Antiretroviral Therapy for HIV Infection: How Early is Early Enough?

The case of the “Berlin Patient” is well-known. In 2007, an HIV-infected adult with leukemia underwent allogeneic hematopoietic stem cell transplant and was cured of his cancer (Hutter et al., 2009). As the donor was homozygous for the CCR5Δ32 deletion (which makes CD4+ T cells difficult to infect with HIV), the repopulated immune system lacked any detectable HIV and the patient has been apparently been cured. Two subsequent allogeneic transplants performed under potent ART (which protected the donor cells from de novo infection) resulted in a dramatic reduction in the frequency of cells harboring replication-competent HIV (generally referred to as the HIV “reservoir”) (Henrich et al., 2014). This was not enough to cure these two patients, however, as both individuals experienced a delayed but robust rebound in viremia after stopping therapy. A true cure will likely require complete eradication of the entire reservoir of HIV. This is a formidable challenge.

Compartmentalized HIV rebound in the central nervous system after interruption of antiretroviral therapy.

To design effective eradication strategies, it may be necessary to target HIV reservoirs in anatomic compartments other than blood. This study examined HIV RNA rebound following interruption of antiretroviral therapy (ART) in blood and cerebrospinal fluid (CSF) to determine whether the central nervous system (CNS) might serve as an independent source of resurgent viral replication.Paired blood and CSF samples were collected longitudinally from 14 chronically HIV-infected individuals undergoing ART interruption. HIV env (C2-V3), gag (p24) and pol (reverse transcriptase) were sequenced from cell-free HIV RNA and cell-associated HIV DNA in blood and CSF using the Roche 454 FLX Titanium platform. Comprehensive sequence and phylogenetic analyses were performed to search for evidence of unique or differentially represented viral subpopulations emerging in CSF supernatant as compared with blood plasma.Using a conservative definition of compartmentalization based on four distinct statistical tests, nine participants presented a compartmentalized HIV RNA rebound within the CSF after interruption of ART, even when sampled within 2 weeks from viral rebound. The degree and duration of viral compartmentalization varied considerably between subjects and between time-points within a subject. In 10 cases, we identified viral populations within the CSF supernatant at the first sampled time-point after ART interruption, which were phylogenetically distinct from those present in the paired blood plasma and mostly persisted over time (when longitudinal time-points were available). Our data suggest that an independent source of HIV RNA contributes to viral rebound within the CSF after treatment interruption. The most likely source of compartmentalized HIV RNA is a CNS reservoir that would need to be targeted to achieve complete HIV eradication.

Methamphetamine Use in HIV-infected Individuals Affects T-cell Function and Viral Outcome during Suppressive Antiretroviral Therapy.

We investigated the associations between methamphetamine (meth) use, immune function, and the dynamics of HIV and cytomegalovirus [CMV] in the blood and genital tract of HIV-infected ART-suppressed subjects. Self-reported meth use was associated with increased CD4+ and CD8+ T-cell proliferation (Ki67+, p < 0.005), CD4+ T-cell activation (CD45RA–CD38+, p = 0.005) and exhaustion (PD-1+, p = 0.0004) in blood, compared to non-meth users. Meth use was also associated with a trend towards higher blood HIV DNA levels (p = 0.09) and more frequent shedding of CMV in seminal plasma (p = 0.002). To explore possible mechanisms, we compared ex vivo spontaneous and antigen-specific proliferation in PBMC collected from subjects with and without positive meth detection in urine (Utox+ vs. Utox-). Despite higher levels of spontaneous proliferation, lymphocytes from Utox+ meth users had a significantly lower proliferative capacity after stimulation with a number of pathogens (CMV, candida, mycobacterium, toxoplasma, HIV, p < 0.04 in all cases), compared to Utox- participants. Our findings suggest that meth users have greater proliferation and exhaustion of the immune system. Meth use is also associated with a loss of control of CMV replication, which could be related to loss of immune response to pathogens. Future studies should consider meth use as a potential modulator of T-cell responses.

HIV-1 Tat immunization restores immune homeostasis and attacks the HAART-resistant blood HIV DNA: results of a randomized phase II exploratory clinical trial.

BackgroundThe phase II multicenter, randomized, open label, therapeutic trial (ISS T-002, Clinicaltrials.gov NCT00751595) was aimed at evaluating the immunogenicity and the safety of the biologically active HIV-1 Tat protein administered at 7.5 or 30 μg, given 3 or 5 times monthly, and at exploring immunological and virological disease biomarkers. The study duration was 48 weeks, however, vaccinees were followed until the last enrolled subject reached the 48 weeks.Reported are final data up to 144 weeks of follow-up. The ISS T-002 trial was conducted in 11 clinical centers in Italy on 168 HIV positive subjects under Highly Active Antiretroviral Therapy (HAART), anti-Tat Antibody (Ab) negative at baseline, with plasma viremia <50 copies/mL in the last 6 months prior to enrollment, and CD4+ T-cell number ≥200 cells/μL. Subjects from a parallel observational study (ISS OBS T-002, Clinicaltrials.gov NCT0102455) enrolled at the same clinical sites with the same criteria constituted an external reference group to explore biomarkers of disease.ResultsThe vaccine was safe and well tolerated and induced anti-Tat Abs in most patients (79%), with the highest frequency and durability in the Tat 30 μg groups (89%) particularly when given 3 times (92%). Vaccination promoted a durable and significant restoration of T, B, natural killer (NK) cells, and CD4+ and CD8+ central memory subsets. Moreover, a significant reduction of blood proviral DNA was seen after week 72, particularly under PI-based regimens and with Tat 30 μg given 3 times (30 μg, 3x), reaching a predicted 70% decay after 3 years from vaccination with a half-life of 88 weeks. This decay was significantly associated with anti-Tat IgM and IgG Abs and neutralization of Tat-mediated entry of oligomeric Env in dendritic cells, which predicted HIV-1 DNA decay. Finally, the 30 μg, 3x group was the only one showing significant increases of NK cells and CD38+HLA-DR+/CD8+ T cells, a phenotype associated with increased killing activity in elite controllers.ConclusionsAnti-Tat immune responses are needed to restore immune homeostasis and effective anti-viral responses capable of attacking the virus reservoir. Thus, Tat immunization represents a promising pathogenesis-driven intervention to intensify HAART efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0151-y) contains supplementary material, which is available to authorized users.

Cytomegalovirus replication in semen is associated with higher levels of proviral HIV DNA and CD4+ T cell activation during antiretroviral treatment.

Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4(+) T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4(+) T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4(+) (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. Importance: Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in blood, and the size of the HIV DNA reservoir in 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. In support of our primary hypothesis, shedding of CMV DNA in semen was associated with increased activation and proliferation of T cells in blood and also significantly higher levels of HIV DNA in blood cells. These results suggest that CMV reactivation might play a role in the maintenance of the HIV DNA reservoir during suppressive ART and that it could be a target of pharmacologic intervention in future studies.

Peripheral blood HIV DNA is associated with atrophy of cerebellar and subcortical gray matter

We evaluated regional brain volumes and cerebral metabolite levels as correlates of HIV DNA in peripheral blood mononuclear cells (PBMCs). In this cross-sectional study, 35 HIV+ subjects aged ≥40 years (25 with detectable PBMC HIV DNA; 10 with HIV DNA <10 copies/10(6) cells, the threshold of detection) and 12 seronegative controls underwent structural brain MRI and magnetic resonance spectroscopy at 3 T. HIV+ subjects were on combination antiretroviral therapy ≥1 year; all but 1 had plasma HIV RNA <50 copies/mL. We used logistic regression to evaluate relationships of likely predictor variables to the outcome of PBMC HIV DNA detectability in the HIV+ subjects. Effects of serostatus and HIV DNA on regional brain volumes (normalized to intracranial volume) and on metabolite ratios over creatine were evaluated by analyses of covariance, controlling for age. Relative to the HIV+ group with undetectable HIV DNA, subjects with detectable HIV DNA demonstrated decreased volumes of cerebellar (-14%, p = 0.020) and total subcortical (-10%, p = 0.024) gray matter. Compared to healthy controls, only the detectable HIV DNA group showed significant (p < 0.05) enlargement of lateral ventricles and volumetric reductions of caudate, putamen, thalamus, hippocampus, nucleus accumbens, brainstem, total cortical gray matter, and cerebral white matter. Detectable HIV DNA was not associated with significantly altered cerebral metabolite levels. Inability to clear peripheral blood of HIV DNA is associated with regional brain atrophy in well-controlled HIV infection, supporting the involvement of peripheral viral reservoirs in the neuropathogenesis of persistent HIV-related neurocognitive disorders.

Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection.

BackgroundLimited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy.Methods and FindingsWe prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24.At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm3. HIV RNA was 5.5 log10 copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/106 PBMC) vs. Fiebig I (8 copy/106 PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008).After 24 weeks of megaHAART, HIV RNA levels of <50 copies were achieved in 14/15 in blood and 13/13 in gut. Total blood HIV DNA at week 0 predicted reservoir size at week 24 (p<0.001). Total HIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02).ConclusionsGut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission.

Greater diversity of HIV DNA variants in the rectum compared to variants in the blood in patients without HAART

The gut-associated lymphoid tissue represents the largest reservoir of HIV-1. Improving knowledge of this reservoir by studying the diversity of viral population is a key step towards understanding the pathogenesis and dynamics of HIV. Obtaining samples is difficult and little information is available on gut viral quasispecies during the course of infection in humans. The aim of this study was to characterize rectal viral strains and their diversity and to investigate the relationships between the rectal tissue reservoir and viral variants in the blood. Phylogenetic analyses were performed on the env sequences for rectal HIV DNA, blood HIV DNA, and HIV RNA clones, with maximum-likelihood and neighbor-joining methods on seven patients. Genetic diversity was assessed. Higher diversity of HIV DNA clones was noted in the rectum compared to blood in four out of five patients without HAART. Viral diversity was present in the rectum from time of the primary infection. Similar degrees of diversity were observed in the rectum and blood during HAART. Rectal and blood HIV variants were interspersed partially or totally in the seven patients. A certain number of rectal HIV DNA clones were clustered together in six patients. These results suggest that variants in the rectum were more heterogeneous than variants in the blood from patients without HAART, probably because the activated milieu of gut-associated lymphoid tissue may provide an improved environment for viral replication, and indicate exchange of viral populations between blood and rectal tissues, reflecting the dynamics of HIV during course of infection.

Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication

Sustained suppression of plasma viremia in HIV-infected individuals is attainable with antiretroviral therapy (ART); however, eradication of virus that would allow discontinuation of ART has been hampered by the persistence of HIV reservoirs. It is of great interest to identify individuals who had received ART for prolonged periods of time with extremely low or undetectable HIV reservoirs and monitor plasma viremia following discontinuation of therapy. We measured the size of HIV reservoirs in CD4(+) T cells of individuals on long-term ART and monitored plasma viremia following cessation of ART in one individual with an exceptionally low viral burden after a decade of therapy. We demonstrated undetectable levels of HIV DNA in the blood of eight of 45 infected individuals on long-term ART. Among those eight individuals, the frequency of cells carrying infectious virus was significantly lower in those who initiated ART during the early versus the chronic phase of infection. One individual with undetectable HIV DNA in both blood and tissue and a profoundly low level of infectious virus experienced plasma viral rebound 50 days following discontinuation of ART. Our data suggest that a significant reduction in the size of viral reservoirs may be achievable in selected individuals who initiate standard ART early in infection. However, given re-emergence of plasma viremia in an individual with an extraordinarily low viral burden, therapeutic strategies aimed at specifically targeting these extremely rare HIV-infected cells with novel interventions may be necessary in order to achieve eradication of virus.

Early diagnosis of human immunodeficiency virus-1 infection in infants with the NucliSens EasyQ assay on dried blood spots

Background More routine laboratories in South Africa are equipped to perform quantitative than qualitative HIV viral detection assays. The accessibility of early infant diagnosis would be improved if a quantitative viral load (VL) assay performed on dried blood spots (DBS) could accurately diagnose HIV-infection. The VL assay routinely used in the country has not previously been assessed on DBS for early infant diagnosis. Objectives To determine the accuracy of the NucliSens EasyQ assay (bioMerieux, Lyon, France) on DBS for early infant diagnosis of HIV in a subtype C-infected population. Study design Stored DBS samples collected from children presenting for HIV testing were analyzed. DBS EasyQ VL results were compared to the child's HIV status as determined by a whole blood HIV DNA PCR result. Results The EasyQ assay was performed on 235 stored DBS samples from 71 HIV-infected and 164 HIV-uninfected children. Of the 216 infants (children aged 12 months or less) tested, all 52 HIV-infected infants were detected (sensitivity of 100%). Six of 164 HIV-uninfected infants yielded false positive results (specificity 96.3%). All false positive and six of the true positive infants had VL < 3.7 log IU/ml. These 12 (5.6%) infants would require repeat testing to differentiate true from false positives. Using a threshold above 3.7 log IU/ml (equivalent to 4 log copies/ml) to define a positive result would yield an accurate diagnosis in 204 (94.4%) infants. Conclusions DBS EasyQ VL assays provide an accurate option for early infant diagnosis in low resource settings.

HIV-DNA in rectal cells is well correlated with HIV-DNA in blood in different groups of patients, including long-term non-progressors

Most of the body's lymphoid tissue is in gut and constitutes an immense HIV reservoir. We quantified HIV-DNA in rectum and compared it with blood levels for 27 HIV-infected adults from different groups. We observed a large range of rectal and blood HIV-DNA levels. They were positively correlated (r = 0.841, P<0.0001). Long-term nonprogressors and patients in 'remission' after antiretroviral treatment interruption had the lowest blood and mucosal HIV-DNA levels.

HIV-1 DNA for the measurement of the HIV reservoir is predictive of disease progression in seroconverters whatever the mode of result expression is

Background HIV-1 DNA levels, reported as copies/10 6 peripheral blood mononuclear cells (PBMC), are very predictive of disease progression in seroconverters, independently of CD4 +T cell count and HIV-RNA. Previously, HIV-DNA levels have sometimes been reported by other means: copies/10 6 CD4 +T cells, reflecting the proportion of infected cells; or copies/mL whole blood, reflecting the global blood reservoir size. Objectives We investigated if the predictive value over the natural course of the disease depends on how the results are reported. Study design Results reported as HIV-DNA copies/10 6 PBMC were converted to copies/10 6 CD4 +T cells or to copies/mL whole blood for 422 seroconverters included in the French SEROCO cohort (ANRS). Results The three methods for reporting HIV-DNA levels yielded different ranges, but these values were highly correlated. The level of HIV-DNA during the seroconversion period was strongly associated with disease progression in all three reporting methods. Conclusions This reinforces the value of HIV-DNA quantification in physiopathological and therapeutical studies, particularly in an era of research aimed at diminishing the HIV reservoir. Even if blood represents a small part of this reservoir, HIV-DNA in blood is a simple marker that provides an informative picture of the global reservoir and is strongly predictive of disease progression.

Multiple measures of HIV burden in blood and tissue are correlated with each other but not with clinical parameters in aviremic subjects.

To determine the levels of residual HIV DNA and RNA in blood and gut reservoirs in aviremic patients, assess correlations among compartmental measurements of HIV burden, and evaluate association with clinical parameters. Cross-sectional analysis of baseline data only, on 40 patients enrolled in phase II study evaluating efficacy of autologous gene-modified CD4+ and CD8+ T cells. All patients were on stable antiretroviral regimen with undetectable plasma HIV RNA (< 50 copies/ml). Measurements repeatedly performed over 8-12 weeks pre-intervention: blood HIV DNA, analysis of rectal mucosa-associated lymphoid tissue for both HIV RNA and HIV DNA, and quantitative co-culture of HIV from CD8-depleted peripheral blood mononuclear cells (PBMC). Quantifiable levels of HIV detected in compartments despite undetectable levels of plasma HIV RNA: HIV co-culture of PBMC (88%), blood HIV DNA (95%), rectal biopsy HIV DNA (95%), rectal biopsy HIV RNA (65%). A significant correlation existed among various measures of HIV burden (HIV co-culture, blood HIV DNA, rectal biopsy HIV RNA and DNA) but not between assays and clinical parameters [duration of highly active antiretroviral therapy (HAART), type of HAART]. All assays had comparable or less variability than in plasma viral load assays; HIV co-culture had the highest coefficient of variability whereas the blood HIV DNA assay had the lowest and was considered the most reliable assay. The data support safety, feasibility and high compliance of quantifying reservoirs of residual HIV in treated subjects with undetectable plasma HIV RNA. Lack of correlation between levels of HIV in residual reservoirs and duration of HAART suggests treatment-mediated viral suppression alone does not lead to reproducible decay in HIV reservoirs.