Carmen Vidal

Dynamics of viral load rebound and immunological changes after stopping effective antiretroviral therapy

BACKGROUND This study addresses the dynamic of viral load rebound and immune system changes in a cohort of eight consecutive HIV-1-infected patients in very early stages [all the patients were taking highly active antiretroviral therapy (HAART} and were recruited in the coordinating center from a larger study] who decided to discontinue HAART after 1 year of treatment and effective virologic response. The safety of this procedure and the outcome with reintroduction of the same treatment was also investigated. METHODS Plasma, cerebrospinal fluid (CSF), and lymphatic tissue viral loads were measured at baseline; lymphocyte immunophenotyping and CD4 lymphocyte proliferative responses to mitogens and specific antigens were assessed. The same antiretroviral therapy was reintroduced as soon as plasma viral load became detectable (above 200 copies/ml). RESULTS At day 0, plasma viral load was below 20 copies/ml in all eight patients (and below 5 copies/ml in five of eight patients). A rebound in plasma viral load was detected in all patients from day 3 to day 31 with a mean doubling time of 2.01 (SE 0.29) days. Three out of eight patients achieved a peak plasma viral load at least 0.5 log10 above baseline, pretreatment values. Mutations associated with resistance to reverse transcriptase or protease inhibitors were not detected. After 31 days off therapy, CD4 lymphocytes decreased [mean 45% (SE 4) to 37% (SE 3); P = 0.04], CD8+CD28+ lymphocytes decreased [mean 59% (SE 5) to 43% (SE 4); P = 0.03], and CD8+CD38+ lymphocytes increased [mean 55% (SE 3) to 66% (SE 4); P = 0.009]. Mean stimulation indices of lymphocytes treated with phytohemagglutinin (PHA) and CD3 decreased from day 0 to day 31 from 34% (SE 8) to 17% (SE 9) (P = 0.06) and from 24% (SE 8) to 5% (SE 2) (P = 0.02), respectively. These changes were mainly contributed by the group of five patients with plasma viral load below 5 copies/ml at day 0. Viral load dropped below 20 copies/ml in all patients after 1 month of restarting the same antiretroviral regimen. CONCLUSIONS Discontinuation of HAART after 1 year of successful treatment is followed by a rapid rebound of viral load; this rapidly returns to undetectable levels following reintroduction of the same treatment. In patients with more effective control of virus replication (viremia below 5 copise/ml), discontinuation of treatment was associated with more severe impairment of immunologic parameters.

The virological and immunological consequences of structured treatment interruptions in chronic HIV-1 infection

BackgroundSome individuals with chronic HIV-1 infection have discontinued their drug therapy with consequent plasma virus rebound. In a small number of patients, a delayed or absent rebound in plasma virus load has been noted after drug cessation, apparently associated with prior drug interruptions and autologous boosting of HIV-1 specific immune responses. We hypothesized that cyclic structured treatment interruptions structured treatment interruptions (STI) could augment HIV-1 specific immune responses in chronic HIV-1 infection, which might help to control HIV-1 replication off therapy. MethodsWe initiated an STI pilot study in 10 antiretroviral treatment-naive HIV-1 chronically infected subjects with baseline CD4 T-cell counts > 500 × 106 cells/l and plasma viral load > 5000 copies/ml who received highly active antiretroviral therapy (HAART) for 1 year with good response (plasma viral load < 20 copies/ml for at least 32 weeks). Three cycles of HAART interruption were performed. ResultsIn all of the patients viral load rebounded, but doubling times increased significantly between the first and third stops (P = 0.008), and by the third stop, six out of nine subjects had a virological set-point after a median 12 months off therapy that was lower than baseline before starting HAART (ranging from 0.6 log10 to 1.3 log10 lower than baseline) and in four it remained stable below 5000 copies/ml. Those subjects who controlled viral replication developed significantly stronger HIV-1 specific cellular immune responses than subjects lacking spontaneous decline (P < 0.05). During viral rebounds no genotypic or phenotypic changes conferring resistance to reverse trancriptase inhibitors or protease inhibitors was detected, but mean absolute CD4 T-cell counts declined significantly, although never below 450 × 106/l and the mean value at 12 months off therapy was significantly higher than the pre-treatment level (P = 0.004). ConclusionsOur findings suggest that STI in chronic HIV-1 infection might augment HIV-1-specific cellular immune responses associated with a spontaneous and sustained drop in plasma viral load in some subjects but at the potential cost of lower CD4 T-cell counts.

Immunological benefits of antiretroviral therapy in very early stages of asymptomatic chronic HIV-1 infection

ObjectivesTo assess whether an almost complete restoration of immune system can be achieved when antiretroviral therapy is initiated at very early stages of asymptomatic chronic HIV-1 infection. DesignT cell subsets and cell-mediated responses were analysed at baseline and after 12 months of either a double or a triple antiretroviral therapy in 26 asymptomatic HIV-1-infected patients with CD4 T cell counts > 500 × 106 cells/l and a baseline plasma viral load > 10 000 copies/ml. ResultsTriple therapy was significantly more effective in reducing plasma HIV RNA to undetectable levels, in returning CD4:CD8 ratio to nearly normal levels, in reducing activated cells (CD38) and in increasing naive (CD45RA+CD45RO−−) and memory (CD45RA−−CD45RO+) CD4 cells. Both double and triple therapies caused a clear decrease in memory (CD45RA−−CD45RO+) CD8 cells as well as a significant increase in the CD28 subset of CD8 cells. At baseline, there was an important increase in cells producing interferon-γ (IFNγ) with no significant abnormalities in T lymphocytes producing interleukin 2 (IL-2), tumour necrosis factor α and interleukin 4. Both types of therapy reduced IFNγ- and IL2-producing CD4 T lymphocytes while IFNγ-producing CD8 cells remained increased. Even before therapy, these HIV-1-positive patients lacked significant abnormalities in the T cell responsiveness to polyclonal stimuli as well as in the secretion of CCR5 chemokines by peripheral blood mononuclear cells. ConclusionsInitiating highly active antiretroviral therapy at very early stages of chronic HIV-1 infection allows rapid and almost complete normalization of T cell subsets and preservation of T cell functions. These early-treated patients could be excellent candidates for receiving additional HIV-specific immune-based therapies, which might be essential for the control of HIV infection.

Cerebrospinal fluid HIV-1 RNA levels in asymptomatic patients with early stage chronic HIV-1 infection: support for the hypothesis of local virus replication.

OBJECTIVE To assess HIV-1 RNA levels in cerebrospinal fluid (CSF) and their potential correlation with plasma viral load and central nervous system (CNS) HIV-1 infection markers in stable asymptomatic patients with a CD4 T cell count >500x10(6) cells/l. PATIENTS AND METHODS Consecutive patients screened for two trials were eligible for lumbar puncture assessment. At day 0, simultaneous samples of CSF and plasma were obtained and levels of total proteins, albumin, IgG, antibodies against HIV-1 p24 antigen, HIV-1 RNA (using the polymerase chain technique) and white cells were measured. RESULTS The integrity of the blood-brain barrier was preserved (albumin index > or =7) in 59 out of 70 patients (84%). Intrathecal production of antibodies against HIV-1 p24 antigen was demonstrated in 55 out of 70 individuals (78%). Viral load in CSF was significantly lower than plasma values (3.13+/-0.95 versus 4.53+/-0.53, P = 0.0001). HIV-1 RNA was not detected in CSF in only three of the 70 patients (4%). Overall, there was a significant correlation between plasma and CSF HIV-1 RNA levels (r = 0.43, P = 0.0001); however, in 29 patients (41%) there were significant differences (>1.5 log10 copies/ml) between the viral loads in plasma and CSF. In the multivariate analysis, a high level of protein and white cells in CSF, but not the HIV-1 RNA plasma level, were factors independently associated with a higher level of HIV-1 RNA in CSF (P = 0.0001). CONCLUSIONS HIV-1 RNA can be detected almost always in CSF of asymptomatic patients in early stages of HIV-1 infection including those with a preserved integrity of the blood-brain barrier. The important discrepancies between plasma and CSF viral load, and the independent association between CSF abnormalities and CSF viral load, support the hypothesis of local production of HIV-1.

Viral load in asymptomatic patients with Cd4+ lymphocyte counts above 500 × 106/l

Background:HIV-1-infected patients with a CD4+ lymphocyte count ≥500×106/l may be selected for antiretroviral treatment when viral load is above a given cut-off point. Objectives:To assess the stability of viral load measurement at CD4+ T-cell counts above 500×106/l, and the proportion of patients selected for treatment if a cut-off point of 10 000 or 30 000 RNA copies/ml is used. Design and methods:Seventy-eight consecutive asymptomatic antiretroviral-naive HIV-1-infected patients with CD4+ lymphocyte counts ≥500 × 106/l, presenting for previously scheduled medical visits as outpatients, were enrolled. None of the patients had suffered from symptomatic primary infection or seroconverted within 6 months before enrolment. Two blood samples separated by a 1-month interval [day −30 (screening) and day 0 (enrolment)] were collected in an EDTA tube. Plasma was separated and frozen at −70°C within 4 h of collection. HIV-1 RNA was quantified by polymerase chain reaction. CD4+ T cells were measured by flow cytometry. Results:Viral load was fairly stable, and only four (13%) out of 30 pairs had a variation ≥0.5 log10. At day −30 and day 0, log10 HIV RNA levels (mean ± SD) were 4.24 ± 0.7 and 4.35 ± 0.87 log10 copies/ml plasma (P= 0.23). The difference of the mean was −0.11 (95% confidence interval, −0.28 to 0.07). At day 0 (n = 78) mean ± SD value was 35730 ± 73700 RNA copies/ml (range, <200–438480; median, 9331; 25th and 75th percentiles, 1518 and 37193, respectively). In 13 patients (16%) the viral load was <200 copies RNA/ml. Seven out of 10 patients, who fulfilled the criteria of long-term non-progressors (LTNP), had viral load >10 000 RNA copies/ml, and two patients had >30 000 RNA copies/ml. Only two of the 13 patients with CD4+ T-cell counts >750 × 106/l had viral load >10 000 copies/ml. Conclusions:A single-point viral load assessment is enough in asymptomatic patients with CD4+ lymphocytes counts ≥500 × 106/l since plasma HIV RNA measurements obtained 1 month apart are fairly stable. Approximately 25% of these patients (including some patients with LTNP criteria) will be selected for treatment if 30 000 RNA copies/ml is used as cut-off point, and approximately 50% if the cut-off point is 10 000 RNA copies/ml. Viral load ≥10 000 is very unusual in patients with CD4+ T-cell counts >750 × 106/l.

Residual Low-Level Viral Replication Could Explain Discrepancies between Viral Load and CD4+ Cell Response in Human Immunodeficiency Virus—Infected Patients Receiving Antiretroviral Therapy

We report the evolution of chronic infection with human immunodeficiency virus type 1 (HIV-1) in a patient treated with stavudine plus didanosine, whose CD4+ lymphocyte count progressively decreased, despite a sustained plasma viral load <20 copies/mL. After 12 months of therapy, treatment was switched to zidovudine plus lamivudine plus nelfinavir. CD4+ T cell count decreased from 559 x 10(6)/L at month 0 to 259 x 10(6)/L at month 12. Plasma viral load decreased from 21,665 HIV-1 RNA copies/mL at baseline (month 0) to <20 copies/mL after 1 month of therapy with stavudine plus didanosine, and remained below 20 copies/mL until month 12, but always >5 copies/mL. Viral load in tonsilar tissue at month 12 was 125,000 copies/mg of tissue. After the change to triple-drug therapy, the plasma viral load decreased to 5 copies/mL, the CD4+ T cell count increased to 705 x 10(6)/L, and the viral load in tonsilar tissue decreased to <40 copies/mg of tissue at month 24. A low level of HIV-1 replication could explain the lack of immunologic response in patients with apparent virological response.

Immunologic reconstitution after 1 year of highly active antiretroviral therapy, with or without protease inhibitors.

Objectives: To assess the effectiveness of two triple antiretroviral combinations (2 nucleoside reverse transcriptase inhibitors [NRTIs] + 1 protease inhibitors [PI] vs. 2 NRTIs + 1 nonnucleoside reverse transcriptase inhibitor [NNRTI]) to correct T‐cell subsets abnormalities and to restore immune functions in asymptomatic antiretroviralnaive HIV‐1‐infected patients with a baseline CD4 T‐cell counts >500/mm3 and plasma viral load >5000 copies/mL. Design and Methods: Twenty randomized patients from 2 cohort studies receiving either stavudine (d4T) + lamivudine (3TC) + indinavir (n = 9), or d4T + didanosine (ddI) + nevirapine (NVP) (n = 11) were studied. Viral load, T‐cell subsets and T‐cell functions were analyzed at baseline and after 1 year of treatment. Results: After 1 year of follow‐up, the PI regimen was significantly more effective in reducing plasma and lymphoid tissue VL to undetectable levels. A significant increase in CD4+ T cells was observed in patients treated with PI (p = .0007) compared with those treated with NVP. Percentages of CD8+ T‐cells and of activated CD8+ T‐cells (CD38+ and DR+ as well as memory CD45RO+) decreased in all patients. An increase of the CD28+ subset of CD8+ T‐cells also occurred in both groups of treatment. Naive T cells were maintained in the CD4+ subset and augmented in the CD8+ subset in all patients. In both PI and NVP groups, memory CD4+ T‐cells increased significantly (p = .03). Peripheral blood mononuclear cell responsiveness to polyclonal stimuli and to tetanus toxoid and cytomegalovirus (CMV) antigen was similar in both groups of treatment. HIV‐infected patients treated for 1 year with both triple combinations lacked significant T‐cell responsiveness to HIV‐1 proteins. Conclusions: These data suggest that immune reconstitution achieved after 1 year of therapy with PI‐containing or PI‐sparing regimens is similar, despite the higher effectiveness of PI‐containing regimens in reducing viral load. Additional therapeutic approaches should be designed to restore HIV‐1‐specific responses.

Lack of evidence of a stable viral load set-point in early stage asymptomatic patients with chronic HIV-1 infection

Objective:To address the question of whether individuals with chronic HIV-1 infection have a stable viral load set-point and to assess the influence of host and viral factors on the evolution of viral load in a subset of stable asymptomatic patients with a baseline viral load below 5000 copies/ml and CD4+ T-cell count above 500 × 106/l. Methods:Medical visits were performed at least every 6 months including routine blood analysis, viral load and CD4+ T-cell count. HIV-1 RNA was measured in frozen (−70°C) plasma samples using PCR. Patients were classified into three groups according to baseline viral load: group A, < 200 copies/ml (undetectable); group B, 201–2000 copies/ml; group C, 2001–5000 copies/ml. A survival analysis and a Cox regression model were performed to assess the influence of viral and host factors in the increase of baseline viral load. The endpoint was the time to increase viral load to a stable level > 0.5 log10 copies/ml above baseline viral load in groups B and C and to a stable detectable viral load (> 200 copies/ml) in group A. Results:A cohort of 114 patients with viral load below 5000 copies/ml was followed for a median of 12 months (6–42 months). Overall, 22 (19%) out of 114 patients had an increase > 0.5 log10 copies/ml of baseline viral load. Baseline viral load increased in two (5%) out of 37 patients in group A, four (12%) out of 33 patients in group B, and 16 (36%) out of 44 patients in group C (survival analysis, P < 0.002). Patients of group C had a eightfold higher risk of increasing baseline viral load than patients in the other two groups pooled together (hazards ratio, 8.28; 95% confidence interval, 1.78–38; P = 0.006). Patients with an increase of viral load to the virological endpoint had a threefold higher risk of decreasing baseline CD4+ T-cell counts > 100 × 106/l than patients with stable viral load (hazards ratio, 2.78; 95% confidence interval, 1.12–14; P = 0.03). Conclusions:In our cohort of chronically HIV-1-infected asymptomatic patients with a baseline viral load < 5000 copies/ml and CD4+ cell count > 500 × 106/l, a true viral load set-point did not seem to exist. Patients with baseline viral load of 2000–5000 copies/ml had an eightfold higher risk of increasing the level of viral load than patients with a baseline viral load below 2000 copies/ml.

Diet quality and related factors among Spanish female participants in breast cancer screening programs

Objective A healthy diet is particularly important during menopause, a period in which the risk of a number of health problems increases. This study analyzed diet quality as measured by two indices, namely, the Alternate Healthy Eating Index (AHEI) and the Alternate Mediterranean Diet (aMED) index, which measures adherence to a Mediterranean diet, and examined the factors associated with lower diet quality. Methods This was a cross-sectional study covering 3,564 women aged 45 to 68 years who underwent breast cancer screening at 7 centers (Corunna, Barcelona, Burgos, Palma de Mallorca, Pamplona, Valencia, and Zaragoza). Data on diet were collected using a food frequency questionnaire validated for the Spanish population. We calculated the AHEI out of a total of 80 points and the aMED out of a total of 9 points. Ordinal logistic regression models were fitted, taking diet quality (tertiles of the AHEI and the aMED) as dependent variables. The following were included in the final multivariate models as explanatory variables: sociodemographic characteristics, chronic diseases, and lifestyles that were associated with diet quality, with a P value <0.100 in an initial simple model (adjusted solely for calorie intake and screening center). Interaction between menopause status and the other explanatory variables was checked. Results The median score for AHEI was 40 of a maximum of 80 points. Lower diet quality was registered by the youngest women (P for trend < 0.001), premenopausal and perimenopausal women (odds ratio [OR], 1.25; 95% confidence interval [CI], 1.01-1.56; and OR, 1.48; CI, 1.20-1.83, respectively), obese women (OR, 1.18; CI, 0.99-1.41), those with a diagnosis of diabetes (OR, 1.35; CI, 1.01-1.79), smokers (OR, 1.41; CI, 1.21-1.66), and women reporting lower daily physical activity (OR, 1.31; CI, 1.12-1.53). Better diet quality was shown by women with higher education (OR, 0.74; CI, 0.62-0.88) and ex-smokers (OR, 0.82; CI, 0.69-0.98). Nulliparity was associated with higher AHEI scores, but only among premenopausal women (OR, 0.50; CI, 0.32-0.78). aMED index varied between 0 and 9 (median 5). Lower scores were associated with younger age (P for trend < 0.001), low socioeconomic level (OR, 1.13; CI, 0.96-1.33), lower educational level (P for trend = 0.008), and low level of daily physical activity (OR, 1.27, CI, 1.08-1.50). Conclusions The youngest women, the most sedentary women, and those who had a lower educational level and socioeconomic status registered worse diet quality. Ex-smokers and postmenopausal women obtained better scores, probably reflecting a keener concern about leading a healthy life.

Calorie intake, olive oil consumption and mammographic density among Spanish women

High mammographic density (MD) is one of the main risk factors for development of breast cancer. To date, however, relatively few studies have evaluated the association between MD and diet. In this cross‐sectional study, we assessed the association between MD (measured using Boyds semiquantitative scale with five categories: <10%, 10–25%, 25–50%, 50–75% and >75%) and diet (measured using a food frequency questionnaire validated in a Spanish population) among 3,548 peri‐ and postmenopausal women drawn from seven breast cancer screening programs in Spain. Multivariate ordinal logistic regression models, adjusted for age, body mass index (BMI), energy intake and protein consumption as well as other confounders, showed an association between greater calorie intake and greater MD [odds ratio (OR) = 1.23; 95% confidence interval (CI) = 1.10‐1.38, for every increase of 500 cal/day], yet high consumption of olive oil was nevertheless found to reduce the prevalence of high MD (OR = 0.86;95% CI = 0.76‐0.96, for every increase of 22 g/day in olive oil consumption); and, while greater intake of whole milk was likewise associated with higher MD (OR = 1.10; 95%CI 1.00‐1.20, for every increase of 200 g/day), higher consumption of protein (OR = 0.89; 95% CI 0.80‐1.00, for every increase of 30 g/day) and white meat (p for trend 0.041) was found to be inversely associated with MD. Our study, the largest to date to assess the association between diet and MD, suggests that MD is associated with modifiable dietary factors, such as calorie intake and olive oil consumption. These foods could thus modulate the prevalence of high MD, and important risk marker for breast cancer.