Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group.

PURPOSE: We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification. RESULTS: The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification. CONCLUSIONS: Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

Hypovitaminosis D and osteopenia/osteoporosis in a haemophilia population: a study in HCV/HIV or HCV infected patients.

Recent reports show a correlation between haemophilia and osteoporosis. HIV, HCV and their treatments are independently associated with an increased risk of osteoporosis. Vitamin D plays a pivotal role in bone mineralization. The aim of our study was to compare Vitamin D levels, bone metabolism markers and bone mineral density (BMD) in patients with haemophilia with or without co-infections. Seventy-eight adult patients with severe or moderate haemophilia A or B were subdivided into three groups of 26 patients each (HIV-HCV co-infected, HCV mono-infected and uninfected). The BMD was measured by dual energy X-ray absorptiometry (DXA) at both the femoral area (F) and lumbar spine (L). This was correlated to laboratory values and haemophilic arthropathy was assessed using validated clinical and radiological scores. The DXA showed a homogeneous F-BMD reduction in all the three groups, whereas L-BMD was significantly lower in co-infected patients (P < 0.05). The clinical score was higher in co-infected (P < 0.002) and mono-infected (P < 0.006). The radiological score was higher in mono-infected than in the other two groups (P < 0.001). Overall 25-hydroxyvitamin D (25-OH Vit D) was reduced (87%). Bone-specific alkaline phosphatase (b-ALP) and telopeptide were increased in co-infected (P < 0.001 and P < 0.01) and mono-infected (P < 0.001 and P < 0.02). The result of the homogeneous F-BMD reduction in all groups could be explained by the pivotal role of arthropathy; the lower L-BMD in co-infected and the increase of b-ALP and telopeptide in co-infected and mono-infected groups suggest faster bone metabolism in case of infections.

Key questions in antiretroviral therapy: Italian Consensus Workshop (2005).

A panel of leading Italian specialists in infectious diseases, virologists and immunologists met in Rome in 2005 to review critical data and discuss recommendations for each of the key questions in antiretroviral therapy today: When to start treatment? How to start? When to switch? What to switch to? Whether to stop or not to stop treatment, and how? The method of a nominal group meeting was used and recommendations were graded for their strength and quality using a system based on the one adopted by the Infectious Diseases Society of America. Main conclusions are summarized and critically discussed in this consensus statement, as well as some of the most recent data supporting these recommendations are provided.

[Use of thymidine-sparing regimens in first-line therapy and in switches from thymidine analogues]. / L'impiego dei regimi timidino-sparing in prima linea e nello switch da timidinici.

Therapy with two NRTIs/NtRTI represents the current basis of highly active antiretroviral therapy (HAART) and is almost always associated either with a NNRTI or a protease inhibitor. Thymidine analogues zidovudine and stavudine were largely used in the past and the use of backbones not including one of them was uncommon. However, the use of thymidine analogues today has several drawbacks, such as fragile pharmacokinetics, the need of two daily doses, unsatisfactory safety and toxicity profiles, a large number of pills and important cross-resistance phenomena. The possibility of using backbones not including thymidine analogues is thus becoming increasingly popular, provided that efficacy is not affected. Moreover, as some of the problems related to thymidine analogue toxicity are only partly reversible, the strategy supporting the use of a thymidine analogue sparing regimen appears to be the only way to prevent damage that otherwise could be potentially irreversible. However, the quality and benefit of this approach have long been acknowledged in the DHHS guidelines, positioning the association of tenofovir and emtricitabine among the choices recommended for naive patients, for a modern approach to HIV infection therapy.

Conflicting interpretations of the prevalence of mutations associated with drug resistance in antiviral naïve HIV-1 patients with acute and chronic infection.

The routine determination of drug resistance in newly HIV-1 infected individuals records a potential increase in transmissions of drug-resistant variants. Plasma samples from 38 individuals classified as newly infected (seroconversion time <12 months) and twenty four individuals with an established infection (seroconversion time ranging from 3 to 10 years) were analyzed for the presence of mutations by Trugene HIV-1 genotyping assay and Virtual phenotype. Results on the newly infected and the chronically infected individuals showed a limited number of relevant mutations associated with substantial resistance to reverse transcriptase and protease inhibitors. In particular, three patients (4.8%) carried viral major mutations (T69D and M41L) associated with resistance to reverse transcriptase inhibitors, whereas only one showed the presence of M46L, which is correlated with partial resistance to some protease inhibitors. The clinical interpretation based on different approaches to monitor resistance showed that the Virconet interpretation was less grave than Trugene, suggesting that these interpretations need standardization for the currently used sequencing methods and that they may be associated with different outcomes when eventually are used.

Mutation patterns of the reverse transcriptase genes in HIV-1 infected patients receiving combinations of nucleoside and non nucleoside inhibitors.

A genotyping assay was used to define human immunodeficiency virus type 1 (HIV-1) reverse transcriptase codons in plasma samples from 80 HIV-1 patients extensively treated with two nucleoside reverse transcriptase (zidovudine and lamivudine) and one non nucleoside reverse transcriptase (nevirapine) inhibitor. The frequencies of T215S/Y/F, M41L, D67N, L210W K70R, K219Q mutations, detectable in plasma samples, conferring resistance to zidovudine were 61.2, 56.2, 36.2, 31.5, 27.5 and 17.5%, respectively. Mutations (M184V or M184I) conferring resistance to lamivudine were detected in an extremely high percentage of patients (61%). Among mutations correlated to high (K103N, V106A, Y181C/I, Y188C/H/L, G190A/C/E/Q/S/T) or moderate (V108I, V118I) levels of nevirapine resistance, the predominant amino acid change was a substitution at 103 codon, present in 24 of 80 samples tested. Finally Q151M, the marker mutation able to confer resistance to all nucleoside analogues, was detected in seven patients with a viral load of between 1 x 10(4) and 9 x 10(4) HIV-1 RNA copies/ml. The relationship between the genotype and the viral load showed that the incidence of some specific mutations [M41L, T215Y (correlated to zidovudine resistance) and K103N (correlated to all NNRTIs drugs)] significantly (P=0.001) increased with higher viral load. Our results, albeit limited to a small cohort, showed a high frequency of mutations correlated to drugs in use, suggesting a need for therapeutic change in the near future and demonstrating that the development of genotyping tests helps to guide the therapeutic management of HIV-1 infected people. Our data highlight the dangers of selecting antiretroviral therapy without previous antiretroviral drug testing. Although the cost of these assays is a concern, prescribing inefficacious drugs could create serious problems for HIV-1 patients.

Prevalence and virologic consequences of HIV-1 genotype mutations detected in a cohort of 161 Italian patients receiving a nelfinavir-based highly active antiretroviral therapy.

A cross-sectional study was carried out in our tertiary care hospital between January 1998 and December 2001. All 161 consecutive patients naive to nelfinavir and who had received a nelfinavir-based highly active antiretroviral therapy (HAART) of at least 24-week duration were extrapolated from the 802 adult HIV-infected subjects treated with antiretroviral therapy. All cases of virologic failure were considered and viral genotyped. Virologic failure occurred in 80 out of 161 nelfinavir-treated patients, all belonging to the experienced group. On the whole, only 11 patients (7%) developed the D30N substitution, whose 6 was in association with the N88D mutation. Among the 80 failed patients, the M184V mutation was detected in 52 (65%), while only 7 patients showed simultaneously the M184V, T215Y and K103N substitutions. In our HIV-infected population receiving a nelfinavir-based HAART, the D30N mutation has shown a low absolute frequency, while the detection of M184V substitution and the simultaneous occurrence of M184V, T215Y and K103N mutations were related to a more favorable virological response.

Development of drug resistance in HIV-1 patients receiving a combination of stavudine, lamivudine and efavirenz.

The study evaluated the development of drug resistance in a group of HIV-1 patients. After failure to respond to previous therapy with two non-nucleoside reverse transcriptase inhibitors (NNRTIs), as assessed by the presence of a rebound in viral load or a constant high level of HIV plasma viraemia, the patients were treated with a combination of stavudine, lamivudine and efavirenz (EFV). Results showed that viruses carrying primary mutations, usually K103N, T215Y and M41L, presented higher levels of HIV-1 RNA, suggesting an association between a precise mutation pattern and treatment failure.

HIV-1, HAART and bone metabolism.

Much attention has been paid to the emerging complications of HIV infection in patients receiving HAART. Recently, there emerged a potentially increased risk of bone problems like osteopenia, osteoporosis and osteonecrosis as patients live longer. It could be a drug side effect, a consequence of prolonged exposure to HIV and/or activated immune cells characteristic of HIV infection, or a consequence of immune system changes that accompany suppression of virus by the drugs. Future research should focus on the etiologic mechanisms, define the incidence and prevalence prospectively, determine the relationship with HAART (especially the rule of protease inhibitors), and help to guide management. Only when the mechanism for HIV-related versus HAART-related changes can be defined, will we be much closer to designing specific interventions.

Prevalence of multiple dideoxynucleoside analogue resistance (MddNR) in a cohort of Italian HIV-1 seropositive patients extensively treated with antiretroviral drugs.

As the emergence of highly resistant virus might compromise antiretroviral regimens in HIV-1 infected patients, a constant analysis of genotypic mutations should be performed to establish the magnitude of mutation prevalence and gauge their impact in patients treated extensively with combination therapy. The frequency of multiple dideoxynucleoside analogue resistance (MddNR) was evaluated in a group of Italian HIV-1 seropositive patients who failed to respond to therapy despite a long-lasting drug treatment. Results showed the presence of one or more mutations (A62V, V75I, F77L, F116Y and Q151M) able to confer resistance to all NRTIs in a relatively high percentage (7.9%) of patients enrolled in the study. Moreover, a significantly lower HIV-1 viral replication in patients with MddNR, suggested the importance of monitoring HIV-1 subjects not only by viral load, but also by drug resistance testing, so that a correct drug regimen may be chosen.

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients.

BACKGROUND: The routine determination of drug resistance in newly HIV-1 infected individuals documents a potential increase in the transmission of drug-resistant variants. Plasma samples from twenty seven therapy naive HIV-1 infected Italian patients were analyzed by the line probe assay (LIPA) and the TruGene HIV-1 assay for the detection of mutations conferring resistance to HIV-1. RESULTS: Both tests disclosed amino-acid substitutions associated with resistance in a variable number of patients. In particular, two mutations (K70R and V118I), detectable by LIPA and by sequencing analysis respectively, revealed resistance to NRTIs in two plasma samples. At least three mutations conferring resistance to NNRTIs, not detectable by commercial LIPA, able to reveal mutations associated only with nucleoside reverse transcriptase analogues, were disclosed by viral sequence analysis. Moreover, most samples showed mutations correlated with resistance to protease inhibitors. Remarkably, a key mutation, like V82A (found as a mixture), and some "indeterminate" results (9 samples), due the absence of signal on the lines corresponding to a specific probe, was revealed only by LIPA, while a variable number of secondary mutations was detectable only by TruGene HIV-1 assay. CONCLUSION: Even if further studies are necessary to establish the impact of different tests on the evaluation of drug-resistant strains transmission, LIPA might be useful in a wide population analysis, where bulk results are needed in a short time, while sequencing analysis, able to detect mutations conferring resistance to both NRTIs and NNRTIs, might be considered a more complete assay, albeit more expensive and more technically complex.

Metabolic complications of HIV-1 antiretroviral therapy: the lipodystrophy syndrome.

The lipodystrophy syndrome is one of the complications reported with increased frequency in patients with HIV-1 infection receiving antiretroviral therapy. The wide range of prevalence estimates may be due to differing definitions, methods and patient populations. We described the various pathogenic theories and the morphological and metabolic alterations associated with this syndrome. Even if no effective treatment exists, a correct lifestyle, adequate diet and physical exercise seem to be very important. Moreover drug therapies should be used with care to avoid potentially harmful interactions with antiretroviral agents. Ideally, the future effort to define the mechanism of lipodystrophy would be multidisciplinary and would involve not only experts in AIDS research but also nutritionists, endocrinologists and cardiologists.

Development of acute psychotic disorders and HIV-1 infection.

OBJECTIVE: To gain more understanding about the relationship between human immunodeficiency virus type 1 (HIV-1) infection and new-onset psychosis, we compared clinical and immunological findings, psychiatric symptoms, global cognitive performance and, when available, computerized tomography (CT) findings between HIV-1-seropositive patients with new-onset psychosis and well-matched nonpsychotic HIV-1-seropositives. METHODS: Two groups of subjects: HIV-1-seropositives with new-onset psychosis (n = 12) and HIV-1-seropositives without psychosis (n = 15) were recruited through outpatient departments. Organic Delusional Syndrome and Organic Hallucinosis were clinically diagnosed using DSM-III-R diagnostic criteria. Of the baseline participants, twenty-two participated in the two-year follow-up examination. RESULTS: The prevalence of new-onset psychosis in HIV-1-infected subjects was 3.7 per 100 (95% C.I. = 1.6-5.7). HIV-1-seropositive persons with new-onset psychosis had more frequently a positive past psychiatric history, no antiretroviral therapy, and a lower global cognitive performance than did the nonpsychotic HIV-1-seropositives. CT was positive, showing generalized brain atrophy, in three out of nine patients. Remission of psychotic symptoms was observed only in two HIV-1-seropositive persons with new-onset psychosis. Death occurred in two psychic HIV-1-seropositives with simple loosely held delusions. Autopsy results showed that cortical sulci and ventricle size were graded as with moderate/severe enlargement. CONCLUSIONS: New-onset psychosis in HIV infected patients could raise considerable problems in deciding whether a presentation is organic or functional. An interaction of the disease or of psychologically "having" the disease with the presence of a psychotic reaction should also be considered. Interestingly, a protective effect of antiretroviral therapy for new-onset psychosis is suggested.

Viral load trend in HIV-1 seropositive patients with different CD4 cell counts before starting HAART.

BACKGROUND: The efficacy of highly active antiretroviral therapy (HAART) was prospectively monitored in HIV-1 seropositive patients by analyzing HIV-1 RNA viral load. OBJECTIVES: The aim of the study was to evaluate the viral load course in two different groups of patients (group 1: CD4>400/mm(3), group 2: CD4<200/mm(3)) during a period ranging from 9 to 25 months. STUDY DESIGN: HIV-1 viral load, at the start and during HAART, was analyzed in 117 patients who had previously been treated only with two anti-transcriptase drugs but were naive for protease inhibitors. RESULTS: The results showed that, after the beginning of therapy, high plasma HIV-1 RNA levels dropped to undetectable values (<50 copies HIV-RNA/ml) in one third of patients over a mean period of about 9 months irrespective of the initial CD4 cell count, even though a viral reduction of at least 2log(s) in a significantly shorter period of time (P<0.001) was observed only among patients who began retroviral therapy with a higher CD4 cell count. CONCLUSION: The response to HAART was not dramatically affected by the initial CD4 count. Though restricted to a small number of subjects, the data support the idea that therapeutic intervention can be effective even in an advanced stage of HIV-1 infection, when patients show a decreased number of CD4 T-lymphocytes.

Antiretroviral therapy in HIV-infected individuals in clinical practice: are the criteria for initiating and choosing the type of drug regimen based only on immunologic and virologic values?

OBJECTIVES: To determine factors associated with beginning antiretroviral therapy and with the number of drugs used. METHODS: Longitudinal study of 3169 HIV-infected individuals naïve from antiretroviral drugs at enrollment in 65 infectious disease clinics in Italy. Initiation of antiretroviral therapy and number of drugs used (i.e., < 3 vs. > or = 3 drugs) were the main outcome measures. Adjusted odds ratios were calculated by logistic models to establish cofactors of these two measures. RESULTS: From January 1997 to December 1998, 1288 (40.6%) individuals started therapy, 58.0% of whom were given a triple combination regimen. This regimen became more frequent over time. By multivariate analysis, high levels of HIV-RNA and low CD4 counts were the most important independent predictors of starting any type of therapy. A significant association was also found with HIV exposure category, reason for being antiretroviral-naïve, presence/absence of liver disease, presence/absence of a new AIDS-defining disease, and clinical centre. High levels of HIV-RNA and low CD4 counts were also the most important predictors of starting with > or = 3 drugs, compared to < 3 drugs, and men had an independent higher probability of starting with > or = 3 drugs, compared to women. The probability of starting with > or = 3 drugs significantly increased with calendar time. CONCLUSIONS: CD4 and HIV-RNA were the main cofactors of initiating both any type of therapy and therapy with > or = 3 drugs. The large variability among clinical centres suggests that clinicians are uncertain as to the exact timing of beginning therapy and the specific regimen, especially among women.

A comparison of immunocomplex-dissociated serum HIV-1 p24 antigenemia and plasma HIV-1 viral load: assessment of 3,129 paired assays.

In order to compare HIV-1 p24 antigenemia and plasma HIV-1 RNA levels as markers of viral replication, 3,129 paired determinations of alkaline immunocomplex-dissociated serum HIV-1 p24 antigenemia (performed with an immunoenzymatic assay), and plasma HIV-1 RNA levels (carried out with a branched DNA method, a reverse transcriptase-coupled polymerase chain reaction, and a nucleic acid sequence-based assay) were assessed over a two-year-period. When excluding samples with undetectable plasma HIV-1 RNA levels (which tested negative or borderline positive at serum p24 antigen assay in 97.9% of cases), immunocomplex-dissociated p24 antigenemia proved significantly less sensitive than viral load at all considered HIV-1 RNA reference levels, although the profile of positive serum p24 antigen assays (values above 10 pg/ml) paralleled the trend of plasma HIV-1 viral load, especially at higher levels. However, serum HIV-1 p24 antigenemia (even after immunocomplex dissociation) can be longer suggested as a the sole virological tool in the laboratory management of HIV-1 infection, due to its significantly lower sensitivity levels compared with viral load assessment.

[The diagnosis of new cases of HIV infection: a reversal of the trend?]. / Diagnosi di nuovi casi di infezione da HIV: un'inversione di tendenza?

In order to assess the trend of new cases of HIV infection, spontaneous requests of HIV counseling and testing made at our outpatient Center since 1993 were retrospectively evaluated. During the considered 6-year period, 7,492 subjects had voluntary HIV testing, and 225 proved HIV-positive (3.0%). While the overall number of spontaneous requests of HIV serology did not show remarkable variations over time, a tendency towards a significant increase of newly diagnosed HIV disease was observed during the past two years (1997 and 1998). In particular, among the 57 subjects with HIV infection disclosed in 1998, a predominance of young adults and a relatively elevated frequency of females compared with males was demonstrated, while heterosexual (38 cases) and homo-bisexual contacts (18 cases) accounted for the great majority of newly identified retroviral infections, and 66.1% out of the 56 patients who acquired HIV infection by sexual route were stable partners of individuals with known HIV disease. Although epidemiological estimates foresaw a progressive reduction of cases of HIV infection since 1990, a surprising increase of newly diagnosed HIV disease among patients who spontaneously asked for HIV testing was observed in our experience. The apparent temporal coincidence of this phenomenon with the remarkable advances obtained in both diagnostic and therapeutic strategies of HIV disease just since 1996, could not be a fortuitous event. The non-decline of newly diagnosed cases of HIV infection should re-orient strategies of communication and prophylaxis dealing with HIV disease, and involving the general population and people with persisting high-risk behavior.

Anti-hepatitis C virus antibodies amongst Italian homo-bisexual males.

The authors report on an anti-hepatitis C virus antibody (HCV Ab) prevalence (6.9%) in 622 homo-bisexual males from Northern Italy, voluntarily attending an HIV and STDs screening program in the period 1984-89. The anti-HCV antibody prevalence shows a significant correlation with: i) presence of serological markers for HBV (O.R. = 3.12, 95% C.I. = 1.53-6.52) and HIV (O.R. = 12.09; C.I. = 6.52-22.52) infection; ii) a stable relationship with an anti-HCV antibody positive partner (O.R. = 7.79; 95% C.I. = 2.50-23.90); iii) more than twenty different male partners per year (O.R. = 2.55; 95% C.I. = 1.17-5.66). These data demonstrate the existence of a sexual transmission of HCV among homosexuals. This route might contribute in maintaining endemic levels of HCV infection in the homo-bisexual population and it might represent an important way of spreading the virus in the general population too.