Bayesian network analysis of resistance pathways against HIV-1 protease inhibitors.

Interpretation of Human Immunodeficiency Virus 1 (HIV-1) genotypic drug resistance is still a major challenge in the follow-up of antiviral therapy in infected patients. Because of the high degree of HIV-1 natural variation, complex interactions and stochastic behaviour of evolution, the role of resistance mutations is in many cases not well understood. Using Bayesian network learning of HIV-1 sequence data from diverse subtypes (A, B, C, F and G), we could determine the specific role of many resistance mutations against the protease inhibitors (PIs) nelfinavir (NFV), indinavir (IDV), and saquinavir (SQV). Such networks visualize relationships between treatment, selection of resistance mutations and presence of polymorphisms in a graphical way. The analysis identified 30N, 88S, and 90M for nelfinavir, 90M for saquinavir, and 82A/T and 46I/L for indinavir as most probable major resistance mutations. Moreover we found striking similarities for the role of many mutations against all of these drugs. For example, for all three inhibitors, we found that the novel mutation 89I was minor and associated with mutations at positions 90 and 71. Bayesian network learning provides an autonomous method to gain insight in the role of resistance mutations and the influence of HIV-1 natural variation. We successfully applied the method to three protease inhibitors. The analysis shows differences with current knowledge especially concerning resistance development in several non-B subtypes.

Cost effectiveness and delivery study for future HIV vaccines.

Research teams from five countries, Brazil, China, Kenya, Peru and Thailand, have initiated a policy-maker survey on vaccine delivery, cost studies for future HIV vaccination programmes, and associated simulation modeling exercises analysing the relative cost-effectiveness of potential HIV vaccination strategies. The survey assesses challenges and opportunities for future country-level HIV vaccination strategies, providing data on the vaccine characteristics (e.g. vaccine efficacies for susceptibility, infectiousness and disease progression) and vaccination programme strategies to be considered in the cost-effectiveness modeling analyses. The study will provide decision-makers with modeling data on vaccination policy considerations that will assist in developing country-level capacities for future HIV vaccine policy adoption and effective delivery systems, and will help delineate the long-term financial requirements for sustainable HIV vaccination programmes. The WHO-UNAIDS HIV Vaccine Initiative and the collaborating researchers welcome comments or questions from policy makers, health professionals and other stakeholders in the public and private sectors about this effort to help advance policy and capacity related to future potential HIV vaccines.

Distribution of HIV-1 subtypes seen in an AIDS clinic in Sao Paulo City, Brazil.

OBJECTIVE: To determine the distribution of HIV-1 subtypes in Sao Paulo, Brazil. METHODS: Samples were obtained from 80 consecutive HIV-1-infected individuals attending the Immunodeficiency Clinic at the University of Sao Paulo in 1993. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque gradient and a portion was used for routine CD4 counts; the remainder were frozen. PBMC were proteinase-K-digested and DNA-purified by organic extraction. Samples were amplified for the env region of HIV, and envelope sequence subtypes determined by heteroduplex mobility analysis using prototypic subtypes as references. A subset of these were also sequenced through the C2-V3 region of env. RESULTS: A total 69 of 80 samples yielded env polymerase chain reaction product enabling subtype determination; samples that did not amplify were those with low DNA yields. Among 12 injecting drug users (IDU) or sexual partners of IDU, four were typed as clade F and eight as clade B. Forty-three homosexual men or female sexual partners of bisexual men were typed as clade B. The 14 additional cases without known risk factors were typed as clade B. CONCLUSION: These data suggest that subtype F is related to injecting drug use in Brazil.

PIP: Serum samples from 80 consecutive HIV-1-infected individuals presenting to the Immunodeficiency Clinic at the University of Sao Paulo in 1993 were analyzed to determine the distribution of HIV-1 subtypes in the city. Peripheral blood mononuclear cells (PBMC) were separated using Ficoll-Hypaque gradient, a portion was used for routine CD4 counts, and the rest were frozen. PBMC were proteinase-K-digested and DNA-purified by organic extraction. The samples were amplified for the env region of HIV, and envelope sequence subtypes determined by heteroduplex mobility analysis using prototypic subtypes as references. A subset was also sequenced through the C2-V3 region of env. 69 samples yielded env polymerase chain reaction product enabling subtype determination. The samples which did not amplify had low DNA yields. Among 12 IV-drug users or their sex partners, four were typed as clade F and eight as clade B. 43 homosexual men or female sex partners of bisexual men were typed as clade B. The 14 additional cases with no known risk factor were typed as clade B.

AIDS incidence and survival in a hospital-based cohort of asymptomatic HIV seropositive patients in São Paulo, Brazil.

BACKGROUND: In spite of the high incidence of AIDS in Brazil, few studies have tried to evaluate the prognosis of asymptomatic HIV seropositive Brazilian patients. METHODS: A hospital outpatient facility-based cohort of HIV seropositive asymptomatic subjects was followed to determine their probability of remaining AIDS-free at 2 and 4 years of follow-up, as well as the one-year estimated cumulative probability of survival for the AIDS incident cases. The cohort was made up of all asymptomatic HIV seropositive subjects referred to the Immunology Branch of a large university hospital in São Paulo, Brazil, between 1985 and June 1997. RESULTS: The cumulative probability of remaining free from AIDS was 79% (+/- 3.7% SE) at 2 years, and 64.4% (+/- 5.1% SE) at 4 years after first known positive anti-HIV serology. Women had a marginally significant better probability of remaining AIDS-free after both 2 and 4 years of known seropositivity, as compared with men. There were no significant differences in the prognosis of the infection by age; the only single parameter associated with better prognosis was an initial CD4+ count > or =350/microl. The probability of survival one year after the diagnosis of AIDS was 78%, and the 50% estimated probability of survival was 19 months. Older patients (aged > or =35 years) had a better prognosis, as suggested by their longer survival estimates (P = 0.06). CONCLUSIONS: The probability of survival with AIDS observed in this study was higher than in the few previously published estimates for Brazil. However, since the time frame was so wide, it may not be entirely comparable with earlier studies. Some likely explanations for this possibly better prognosis could include more efficient prophylaxis for opportunistic diseases, as well as an increase in the availability of anti-retroviral drugs. The 8% incidence of AIDS at 2 years observed in this study for those individuals whose initial CD4+ count was > or =350/ml was close to that found in a large international epidemiological study of seroconverters.

The value of the lymphocyte proliferation test with phytohemagglutinin in the immune evaluation of Brazilian HIV-infected patients.

We evaluated phytohemagglutinin (PHA) lymphocyte proliferation and delayed-type hypersensitivity tests in 130 Brazilian HIV-infected patients with the objective of assessing the value of these tests in staging HIV infection. Patients were divided into three groups according to their CD4+ cell counts (cells/mm3): < 200 (n = 28); 200 to 499 (n = 50); and > or = 500 (n = 52). An additional 114 individuals, who had come to the same institution for elective surgeries, were enrolled as controls. Results showed a significant decrease in PHA responses when CD4+ cell counts fell below 500 cells/mm3. This decrease was, however, indistinguishable from that of patients with < 200 cells/mm3. In contrast, skin test anergy to common antigenic preparations was only evident in the group of patients with less than 200 CD4+ cells/mm3. Decreases in PHA responses and in CD4+ cell counts were significantly correlated. Since the introduction of antiretroviral therapy is recommended when CD4+ cell counts are below 500 cells/mm3, the PHA proliferation test, in our conditions, could be useful as an additional parameter to initiate antiretroviral therapy. Further prospective studies are needed to establish the value of this test in HIV-infected patients.

PIP: During 1993-94, in Sao Paulo, Brazil, clinical investigators used the phytohemagglutinin (PHA) lymphocyte proliferation and delayed-type hypersensitivity tests to determine the stage of HIV infection among 130 adult HIV-infected patients. They aimed to evaluate the value of these tests. 28 (21.5%) patients had CD4+ cell counts of less than 200 cells/sq m. 50 had 200-499 CD4+ cells/sq m. The remaining 52 had at least 500 CD4+ cells/sq m. The researchers compared data on the HIV-positive adults with data on 111 HIV-negative controls. When patients had a CD4+ count of less than 500 cells/sq m, PHA responses were significantly lower than those for a CD4+ cell count of more than 500 cells/sq m and the controls (p 0.01 and p 0.001; respectively). Reductions in PHA responses and in CD4+ cell counts had a significant association. HIV-positive patients with less than 200 CD4+ cells/sq m were less likely to exhibit skin test anergy to common antigenic preparations than other HIV-positive patients (5% vs. 46%; p = 0.003). These findings suggest that the PHA proliferation test could help providers determine at what point in an individual's progression of HIV disease to initiate antiretroviral therapy (i.e., 500 cells/sq m). Further prospective studies are warranted to confirm the value of this test in HIV-positive individuals.

Two variants of HIV-1 B serotype are transmitted heterosexually in São Paulo, Brazil.

HIV-1 variability may have an important impact on transmission and pathogenicity. Better characterization of the HIV epidemic in Brazil is necessary for the development of vaccine trials in this country. We analyzed sera from 108 HIV-1-infected volunteers from São Paulo City to determine serotype and reactivity for V3 motifs of HIV in this population, and the relationship to transmission mode. We concluded that the HIV-1 B serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major V3 motifs, GPGR and GWGR, had similar prevalence among women (48% and 52%, respectively) and men (56% and 44%, respectively). We also observed an equal distribution of these strains regardless of their CD4+ T cell counts, clinical status, and mode of transmission. Even though V3 serology for HIV-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct DNA sequencing, should be included to determine HIV-1 genetic diversity.

Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity.

The main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the V3 region of HIV-1 gp120, which is the main target for neutralizing antibodies. Results from an enzyme immunoassay (EIA) employing a panel of synthetic peptides of HIV-1 subtypes and using urea washes to detect high avidity antibodies (AAV3) were compared with those obtained by the heteroduplex mobility assay and DNA sequencing. The EIA correctly typed 100% of subtype B (sensitivity = 1.0; specificity = 0.95), 100% of HIV-1 E samples (sensitivity = 1.0; specificity = 1.0), and 95% of subtype C specimens (sensitivity = 0.95; specificity = 0.94). In contrast, only 50% of subtype A (sensitivity = 0.5; specificity = 0.95), 60% of subtype D (sensitivity = 0.6; specificity = 1.0), and 28% of subtype F samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. This approach was also able to discriminate in a few samples antibodies from patients infected with B variants circulating in Brazil and Thailand that reacted specifically. The assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. Moreover, the classification in subtypes (genotypes) may overestimate HIV-1 diversity and a classification into serotypes, based on antigenic V3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants.

Impact of adherence to antiretroviral therapy in HIV-1-infected patients at a university public service in Brazil.

The objective of this study was to assess if a simple evaluation, adherence to antiretroviral therapy, would correlate to clinical and laboratory outcomes. We followed an open cohort of patients from a public teaching hospital AIDS outpatient clinic. Patients were categorized according to adherence as: regular (Reg), optimal, all doses all days, tolerating only irregular timing (+/- 2 hours) of intake; quasi-regular (qReg), those missing up to four doses or 1 full day during a month; irregular (Irreg), all other irregular regimens, and ignored (Ign), those without information. The results from a simple questionnaire were compared to CD4+ cell counts and human immunodeficiency virus type 1 (HIV-1) RNA plasma viremia. One hundred eighty-two HIV-1-infected patients (126 males, 69%; 56 females, 31%) were analyzed. Information on adherence was available for 168 (90%). Reg adherence was reported by 75 (41%) patients, qReg adherence by 35 (19%), and Irreg by 53 (29%) of patients. The main reasons for nonadherence were forgetfulness, intolerance, use of alcohol, and misunderstanding of prescription. A significant increase of CD4+ T-cell counts and absolute gain were only observed among Reg and qReg users (p < 0.001). The median viral RNA load log10 decreases were -1.68, -1.45, -0.9 log, respectively, for Reg, qReg, and Irreg patients (p = 0.043, Kruskal-Wallis). Development of and death from AIDS occurred almost exclusively among those with Ign or Irreg adherence. Previous use of antiretroviral therapy may have had an impact in treatment response. Individuals who were treatment-naive were more likely to be Reg users (41%). Although more refined methods to assess adherence should be implemented when available, the inability to do so should not prevent simple, albeit subjective measurements that also correlate with favorable outcome. Mechanisms to improve adherence should be considered an integral part of antiretroviral therapy.

Southern Brazil HIV type 1 C expansion into the state of São Paulo, Brazil.

Abstract HIV diversity reflects multifactorial evolutionary forces, but monitoring subtype prevalence may provide clues to understanding the epidemic. In the Americas HIV-1 C is present at significant levels only in the southern states of Brazil. We describe in this study the presence of the HIV-1 C pol genome in 11.6% (95 CI 6-21%) of antiretroviral-naive individuals from São Paulo, the major city of South America, and 6.8% (95 CI 4-12%) from the second metropolitan area of the State of São Paulo, Brazil. Moreover, a significant growth trend of this subtype was documented among cases failing therapy in the area. Sequences were obtained by direct nested PCR from cDNA retrotranscribed from plasma RNA. Phylogenetic and amino acid signatures support an expansion from variants previously identified in southern Brazil. The evaluation of additional genomic regions (partial gag, envelope, and/or integrase) in samples with HIV-1 C at pol showed extensive recombination with clade B, observed in 47% of ARV-naive cases. The spread of HIV-1 C locally and to other areas of South America should be monitored as it may influence the dynamics of the epidemic.

High frequency of dolutegravir resistance in patients failing a raltegravir-containing salvage regimen

OBJECTIVES: Dolutegravir is a second-generation integrase strand transfer inhibitor (InSTI) that has been recently approved by the FDA to treat antiretroviral therapy-naive as well as treatment-experienced HIV-infected individuals, including those already exposed to the first-generation InSTI. Despite having a different mutational profile, some cross-resistance mutations may influence its susceptibility. The aim of this study was to evaluate the impact of a raltegravir-containing salvage regimen on dolutegravir activity. PATIENTS AND METHODS: Blood samples of 92 HIV-infected individuals with virological failure (two or more viral loads >50 copies/mL after 6 months of treatment) using raltegravir with optimized background therapy were sequenced and evaluated according to the Stanford University HIV Drug Resistance Database algorithm. RESULTS: Among the 92 patients analysed, 32 (35%) showed resistance to dolutegravir, in most cases associated with the combination of Q148H/R/K with G140S/A mutations. At genotyping, patients with resistance to dolutegravir had viral load values closer to the highest previously documented viral load. CONCLUSIONS: Changes in viraemia during virological failure may indicate the evolution of raltegravir resistance and may predict the emergence of secondary mutations that are associated with a decrease in dolutegravir susceptibility. Early discontinuation of raltegravir from failing regimens might favour subsequent salvage with dolutegravir, but further studies are necessary to evaluate this issue.

Transmission of a multidrug-resistant HIV-! from an accupational exposure, in São Paulo, Brazil

We documented the first transmission of a multidrug-resistant HIV from an occupational exposure in Sao Paulo, Brazil, albeit with antiretroviral prophylaxis instituted within 1 h after the accident. A 27-year-old female healthcare worker (HCW) sustained an index finger needle stick injury with a 20-gauge needle while puncturing the forearm of an HIV-infected patient. The putative source (index) patient was a 44-year-old homeless female, on irregular use of zidovudine (AZT), lamivudine (3TC) and ritonavir boosted lopinavir(LPV/r). She was hepatitis C virus (HCV) coinfected and had been prescribed different regimens including nucleos(t)ide reverse transcriptase inhibitors (NRTI), non-nucleos(t)ide reverse transcriptase inhibitors (NNRTI) or protease inhibitors since 2011. Around the time of the accident, she had a HIV viral load of 4.56 log10, HCV viral load of 5.9 log10 (Abbott Real Time HIV and HCV, USA) and CD4+ cell count (BD Biosciences FACSCalibur Flow Cytometer, USA) of 143 cells/µl. After the HCW tested negative by rapid test, AZT/3TC/LPV/r was instituted, as suggested by current guidelines [1,2], within 1 h of the accident.

[Immunologic changes associated with human immunodeficiency virus (HIV)]. / Alterações imunológicas associadas ao vírus da imunodeficiencia humana (HIV).

Infection by HIV usually leads to dysfunction of the immune system which, facilitates the development of opportunistic infections or neoplasia in which almost always are fatal. The pathological process associated with this infection involves, characteristically, the destruction or malfunction of helper T lymphocytes (CD4+), due to, besides other factors, direct parasitism of the virus or to the secondary immune response, which seems to include autoimmune mechanisms. These pathological changes, along with the action of the virus on other components of the immune system, disturb the production of trophic factors and also the proliferation, differentiation and effector ability of different cells of the immune system. The long latent period, when a large number of infected individuals remain asymptomatic or with only minor clinical and/or laboratory disturbances suggest the possible role of some as yet uncharacterized cofactors, either inherent to the host or to other environmental agents; these cofactors may play a role in the modulation of the progression to this syndrome (AIDS).

[Common variable immunodeficiency (hypogammaglobulinemia of late onset or acquired hypogammaglobulinemia): initial follow-up of 11 cases]. / Imunodeficiência comum variável (hipogamaglobulinemia de início tardio ou hipogamaglobulinemia adquirida): seguimento inicial de 11 casos.

The present paper describes the clinical and laboratory follow-up of 11 patients with the diagnosis of common variable immunodeficiency. Their age varied from 8 to 45 years. The mean disease time was 12.6 years and mean diagnosis time 4.3 years. Infectious manifestations, mainly of the respiratory and digestive tracts, occurred in all patients. Polyadenomegaly was noted in seven, hepatomegaly in six, splenomegaly in five and arthralgia in four patients. All of them presented serum IgG less than 250 mg/dl. IgA less than 33 mg/dl and IgM less than 31 mg/dl, except one with IgM = 176 mg/dl. The isohaemagglutinin titers were less than 1/20 in all but one patient. The determination of the number of B lymphocytes in the peripheral blood revealed normal counts in three, elevated in one and decreased in five patients. The CD-4/CD-8 ratio was less than 1 in 8 and greater than 1 in three of them. Five patients had positive cutaneous late reactions to at least one of the following antigens: PPD, SK-SD (Varidase), Trichophytin and Levedurin (Candidin). A decrease of the proliferative activity of peripheral blood mononuclear cells stimulated by lectins (PHA, Con-A, PWM) was also noted. Natural killer function was decreased. The association a possible role of regulatory lymphocytes in the immunopathogenesis of this disease. The data presented here emphasize the diversity of clinical and immunological manifestations of this disease, which could be noted between diverse patients and in the follow-up of a single one. In our cases the disease had an evolutive character, with a primarily humoral dysfunction followed by cellular immunity disturbances that determined poorer prognosis and progressive difficulties in the therapeutics. We suggest a conceptual reevaluation of this condition and a new denomination, for instance "Late-Onset Combined Immunodeficiency". The long delay between the initial clinical manifestations of the disease and its diagnosis was a handicap for an adequate treatment. Early intervention could certainly decrease the morbidity and mortality of the disease.

HIV-1 diversity in Brazil: genetic, biologic, and immunologic characterization of HIV-1 strains in three potential HIV vaccine evaluation sites. Brazilian Network for HIV Isolation and Characterization.

The Brazilian Network for HIV Isolation and Characterization was established for the surveillance of HIV variability in Brazil. Here, we report characterization of HIV strains and virus-specific immune responses from 35 clinical samples collected from three potential HIV vaccine sites. Three genetic subtypes of HIV-1 were identified by heteroduplex mobility assay (HMA) B (in 82.9% of the samples), F (14.3%), and C (2.9%). Phylogenetic analysis based on the C2V3/env DNA sequence from all 25 specimens examined was 100% concordant with HMA results. Four variants of subtype B with different tetrapeptides at the tip of the V3 loop were found: the GPGR motif (North American), GWGR motif (Brazilian B"), and two minor variants, GFGR and GPGS, as previously detected. No significant association was found between HIV-1 subtypes and the mode of transmission or biologic properties of HIV-1 isolates (derived from 88.6% of the specimens). Only 5 of 16 isolates studied were neutralized by the autologous sera. Consistent with previous results, no relation between viral subtype and peptide enzyme-linked immunosorbent assay (ELISA) seroreactivity or neutralization was evident. This study also demonstrated the effectiveness of the collaborative approach followed by Brazilian scientists when addressing a complex subject such as HIV variability.

Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity

The main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the V3 region of HIV-1 gp120, which is the main target for neutralizing antibodies. Results from an enzyme immunoassay (EIA) employing a panel of synthetic peptides of HIV-1 subtypes and using urea washes to detect high avidity antibodies (AAV3) were compared with those obtained by the heteroduplex mobility assay and DNA sequencing. The EIA correctly typed 100 percent of subtype B (sensitivity = 1.0; specificity = 0.95), 100 percent of HIV-1 E samples (sensitivity = 1.0; specificity = 1.0), and 95 percent of subtype C specimens (sensitivity = 0.95; specificity = 0.94). In contrast, only 50 percent of subtype A (sensitivity = 0.5; specificity = 0.95), 60 percent of subtype D (sensitivity = 0.6; specificity = 1.0), and 28 percent of subtype F samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. This approach was also able to discriminate in a few samples antibodies from patients infected with B variants circulating in Brazil and Thailand that reacted specifically. The assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. Moreover, the classification in subtypes (genotypes) may overestimate HIV-1 diversity and a classification into serotypes, based on antigenic V3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants

Two variants of HIV-1 B serotype are transmitted heterosexually in Säo Paulo, Brazil

HIV-1 variability may have an important impact on transmission and pathogenicity. Better characterization of the HIV epidemic in Brazil is necessary for the development of vaccine trials in this country. We analyzed sera from 108 HIV-1-infected volunteers from Säo Paulo City to determine serotype and reactivity for V3 motifs of HIV in this population, and the relationship to transmission mode. We concluded that the HIV-1 B serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major V3 motifs, GPGR and GWGR, had similar prevalence among women (48 per cent and 52 per cent, respectively) and men (56 per cent and 44 per cent, respectively). We also observed an equal distribution of these strains regardless of their CD4+ T cell counts, clinical status, and mode of transmission. Even though V3 serology for HIV-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct DNA sequencing, should be included to determine HIV-1 genetic diversity.