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.

Successful treatment of Cryptococcal meningitis with a combination of liposomal amphotericin B, flucytosine and posaconazole: two case reports.

Cryptococcus neoformans CNS infection frequently affects HIV-infected patients and is often lethal, despite antifungal therapy. The most recent treatment guidelines for Cryptococcal meningitis recommend therapy with lyposomal amphotericin B and possible association with flucitosine. However, clinical response rates in HIV-infected patients are not satisfactory, with a persistent high mortality rate and long term therapy is affected by a high risk of major side effects. Posaconazole, the latest broad-spectrum azole, with both in vitro- and in vivo-documented potent activity against C. neoformans, clearly showed no antagonism with amphotericin B, echinocandins or flucytosine and it has both in vitro and in vivo agonistic activity with flucytosine against C. neoformans. We report two cases of successful salvage therapy based on the addition of posaconazole to a standard treatment based on liposomal amphotericin B and Flucytosine. In addition we used posaconazole also in a maintenance therapeutic regimen with no evidence of recurrences in the follow up of these patients. Our report confirms that posaconazole has clinical activity in the CNS against C. neoformans infection. In addition posaconazole showed no antagonism with any other currently available antifungal agent, and was in fact synergistic to some of them (flucytosine); consequently, it seems to be an ideal candidate for antimicrobial combination salvage therapies. Finally posaconazole represents a good alternative to parenteral therapy and an ideal candidate for long-term maintenance therapy due to its competent toxicity profile and oral bioavailability.

The Management of Geriatric and Frail HIV Patients. A 2017 Update from the Italian Guidelines for the Use of Antiretroviral Agents and the Diagnostic Clinical Management of HIV-1 Infected Persons.

OBJECTIVE: This article deals with the attempt to join HIV and geriatric care management in the 2017 edition of the Italian guidelines for the use of antiretrovirals and the diagnostic-clinical management of HIV-1 infected persons. METHODS: The outlined recommendations are based on evidence from randomized clinical trials and observational studies published in peer-reviewed journals and/or presented at international scientific conferences in recent years. The principles of starting antiretroviral therapy in elderly patients and the viro-immunological goals are the same as in the general HIV population. However, there are some specificities to consider, related to the host as well as the therapy itself. HIV care in elderly patients must shift from a combined AntiRetroviral Therapy specific approach to a more comprehensive management, and from a disease-based model (list of co-morbidities) to a multi-morbidity and frailty standpoint. The implementation of a geriatric approach, based on the Comprehensive Geriatric Assessment, is essential and consists of a broader evaluation of health status. This multidimensional and multidisciplinary evaluation is focused on the development of a tailored intervention plan. Polypharmacy is a frequent condition in the older population and an independent risk factor for negative health-related outcomes. This can be overcome with a multidisciplinary and cooperative approach involving HIV specialists, geriatricians and primary care physicians. CONCLUSION: The inclusion of geriatric care becomes necessary due to the novel needs of an evolving patient population. It is important to underline that the HIV specialist will continue to lead multidimensional interventions and optimize quality of care for HIV-positive people.

Analysis of HIV-1 load in blood, semen and saliva: evidence for different viral compartments in a cross-sectional and longitudinal study.

OBJECTIVES: To quantify the HIV-1 load (measured as copies of viral RNA/ml using competitive reverse transcription-polymerase chain reaction) in blood, semen and saliva and to look for relationships between the viral burden, the clinical and immunological status and antiretroviral therapy. METHODS: Peripheral blood, semen and whole saliva samples were collected from 26 anti-HIV-1-seropositive patients selected for a cross-sectional study. Nine of the 26 patients provided samples of the three biological fluids for a longitudinal study. RESULTS: HIV-1 RNA was detected in 26 out of 26 samples of plasma, in 25 out of 26 samples of semen and in 24 out of 25 samples of saliva. The median number of HIV-1 copies in plasma was 14 817/ml (range: 167-254 880), in semen was 515/ml (range: 0-196 050) and in saliva was 162/ml (range: 0-72 080). The viral load in semen and in saliva was significantly lower than in plasma (P < 0.0001). The HIV-1 RNA levels in plasma and in saliva were correlated (P < 0.05), but levels in semen were not correlated with either plasma or saliva levels. The HIV-1 copy number in plasma was significantly higher in symptomatic patients than in asymptomatic subjects (P < 0.05). Plasma and saliva HIV-1 RNA levels were higher in subjects with a CD4+ cell count < 200 x 10(6)/l than in subjects with a CD4+ cell count > 200 x 10(6)/l (P < 0.05). The HIV-1 RNA load in either plasma, semen or saliva is not related to antiretroviral therapy. CONCLUSIONS: The absence of a correlation between plasma and semen loads suggests that semen and blood are distinct viral compartments. Viral load in semen is not related to the clinical stage of HIV infection or to the CD4+ lymphocyte count. Consequently, HIV-1-infected subjects are potentially infectious at all stages of immuno-deficiency and adequate precautions must always be taken to prevent the sexual transmission of HIV.

Rare mutations in a domain crucial for V3-loop structure prevail in replicating HIV from long-term non-progressors.

OBJECTIVE: To evaluate the role of the selective forces exerted by the host on the HIV-1 structures involved in viral entry. DESIGN AND METHODS: The V3 region of the env gene was analysed in cell-free HIV-1 RNA from 17 infected subjects: 11 long-term non-progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV-1 molecular clone. RESULTS: The intrapatient divergence of the V3-loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non-synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3-loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3-loop structure were more prevalent in LTNP (P = 0.0012). The pNL4-3-derived mutant reproducing a V3-loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte-derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. CONCLUSIONS: The results indicate that host factors impose selective constraints on the evolution of the HIV-1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.

Premature lesions of the carotid vessels in HIV-1-infected patients treated with protease inhibitors.

OBJECTIVES: To evaluate the presence of premature atherosclerotic lesions of epiaortic vessels in HIV-1-infected protease inhibitor-(PI) treated patients compared with PI-naive patients and healthy individuals. DESIGN: One-hundred and two HIV-1-positive patients, including 55 treated with PI for at least 12 months and 47 either naive or treated with PI-sparing regimens, were subjected to epiaortic vessel ultrasonography. These data were compared with those obtained from 104 healthy individuals. METHODS: Intima characteristics, pulsation and resistance indexes, and minimal, peak and mean speed were evaluated using a colour power doppler. Atherosclerotic plaques were described. Independent risk factors and values for glycaemia, cholesterolaemia and triglyceridaemia were considered. Statistical analysis included the chi-square test, Mantel-Haenszel test, odds ratio and logistic regression analysis. RESULTS: Of the PI-treated patients, 29 out of 55 (52.7%) presented acquired lesions of the vascular wall at ultrasonography, whereas similar lesions were found in seven out of 47 (14.9%) PI-naive patients. Of the 104 healthy individuals, seven cases (6.7%) of intimal medial thickness were noted. A slightly significant correlation was found between carotid lesions and age, male sex and hypercholesterolaemia, whereas cigarette smoking, hypertriglyceridaemia and Centers for Disease Control and Prevention stage significantly increased the risk of vascular lesions (P= 0.022, P= 0.017 and P= 0.079 respectively). However, the highest significance regarded use of PI (P= 0.011). These results were confirmed by logistic regression analysis. CONCLUSIONS: These data demonstrate a higher than expected prevalence of premature carotid lesions in the PI-treated compared with PI-naive patients. If confirmed, a periodic ultrasonographic study of the vascular wall should be included in the follow-up of HIV infected patients.

A combination of nucleoside analogues and a protease inhibitor reduces HIV-1 RNA levels in semen: implications for sexual transmission of HIV infection.

Direct contact with semen is the major route of sexual acquisition of human immunodeficiency virus (HIV) in homosexual and heterosexual partners of seropositive men. In this study, we show that concentrations of HIV-1 RNA molecules in plasma and semen of seropositive patients are related to the duration and type of antiretroviral agents used in treatment. In patients treated with zidovudine alone, 1, 3 and 6 months after the start of therapy, the mean HIV-1 load in plasma was reduced by 0.57, 0.38 and 0.21 log10 and in semen by 0.66, 0.50 and 0.15 log10, respectively. In patients treated with zidovudine plus didanosine at months 1, 3 and 6, the mean decrease in plasma HIV-1 RNA was 1.40, 1.25 and 1.12 log10 and in semen 1.10, 1.41 and 1.32 log10, respectively. In patients treated with a combination of a protease inhibitor and two nucleoside analogues the mean log10 decrease was 1.77, 1.83, 1.71 and 2.38 log10 in plasma and 1.17, 1.74, 2.19 and 3.02 log10 in semen at 1, 2, 3 and 4 months, respectively. Treatment with a combination of a protease inhibitor and two nucleoside analogues caused a dramatic decrease in cell-free HIV-1 RNA in semen, which is a reliable measure of viral load. These findings could have implications for the sexual transmission of HIV-1.

Comparison of once and twice daily dosing of didanosine in combination with stavudine for the treatment of HIV-1 infection. AI 454-146 Team.

OBJECTIVE: To compare the antiviral activity, safety and tolerability of didanosine dosed once and twice daily when administered in combination with stavudine dosed twice daily in human immunodeficiency virus type 1 (HIV-1)-infected individuals with little or no previous exposure to antiretroviral drugs. DESIGN: Comparative, multicentre, randomized, open-label, short-term study. PATIENTS AND METHODS: Eighty-four HIV-1-infected adults with qualifying baseline CD4 cell counts of 200 to 500 cells/mm3 were included in the study. Of these, 43 patients received once daily didanosine plus twice daily stavudine (group A) and 41 subjects received twice daily didanosine plus twice daily stavudine (group B). The primary efficacy analysis used was the time-averaged difference (TAD) between treatment regimens of variations in plasma HIV-1 RNA levels from baseline over the first 12 weeks of therapy. Plasma HIV-1 RNA levels, CD4 cell counts and adverse events were monitored. RESULTS: At week 12, median HIV-1 RNA variations were -1.18 log10 copies/ml in group A and -0.88 log10 copies/ml in group B. For patients who were followed up to week 24, median variations of HIV-1 RNA levels from baseline were -1.21 log10 copies/ml in group A and -0.78 log10 copies/ml in group B. The TAD between the two treatment groups for variations from baseline plasma HIV-1 RNA levels over the first 12 weeks was 0.10 log10 copies/ml (95% confidence interval, -0.19 to 0.40), indicating equivalence. CONCLUSION: Once daily didanosine plus twice daily stavudine and twice daily didanosine plus twice daily stavudine are equally effective in reducing plasma HIV-1 RNA levels and increasing CD4 cell counts. Both regimens are safe and well tolerated.

Primary HIV-1 resistance in recently and chronically infected individuals of the Italian Cohort Naive for Antiretrovirals.

The risk of acquiring HIV-1 drug resistance at time of infection has become a public health problem following the widespread use of antiretroviral drugs in developed countries. Although a number of studies have reported data regarding the prevalence of HIV-1 primary resistance in developed countries over the past years, limited knowledge is available regarding the proportion of mutations related to drug resistance in antiretroviral naive subjects with chronic HIV-1 disease. In this study, we evaluated the prevalence of mutations in the reverse-transcriptase (RT) and protease region both in a representative group of recently HIV-1 infected subjects (n=68) and a cohort of chronically-infected HIV-positive patients (n=347) enrolled in the Italian Cohort of Antiretroviral Naive patients (I.CO.NA.). In recently infected individuals, the overall prevalence of mutations for nucleoside RTI (NRTIs) was 10/68 (14.7%). The distribution of mutations by calendar year were 0, 1 in 1996, 9, 3 in 1997 and 1, 0 in 1998 for NRTIs and protease inhibitors (PIs) respectively. Thymidine associated mutations were identified in six subjects (8.8%), five of whom had one mutation [41L, 70K (n=2), 215Y] and one had two mutations (67N+219Q). Four subjects (5.9%) showed the changes associated with resistance to lamivudine (184V or 118I). No non nucleoside-RTI (NNRTI) mutations were present in the study period. Primary PIs mutations (two 46L and two 82I) were present in four subjects (5.9%). Of note, mutations related to resistance to more than one class of antiretrovirals were present in one (1.5%). Among patients with chronic infection a large proportion (88.5%) carried no mutations in RT region, 11.5% individuals carried one or more mutations associated with resistance to NRTI (7.8%), or NNRTI (4.9%), with 4 patients carrying mutations to both classes. Among mutations associated with high-level resistance to RTI, T215Y was found in only 2 patients, M184V in 2 cases, T69D in another case, and K103N in only 1 patient, for a total of 6 patients (one carrying both T215Y and M184V) (1.7%). Primary mutations associated with substantial resistance to PIs were found in only 5/347 patients (1.4%); all the other patients carried only secondary mutations. Prevalence of mutations associated with high-level resistance to antiretroviral drugs is stable in recently infected individuals and low in patients with established HIV infection. The potential impact of transmitted mutations on the response to first regimen in individuals carrying transmitted mutations needs to be assessed by prospective studies.

90K (Mac-2 BP) predicts CD4 decline in human immunodeficiency virus-infected patients with CD4 counts above 200 x 10(6) cells/L.

OBJECTIVE: To evaluate the ability of serum levels of 90K, previously reported as a progression marker of human immunodeficiency virus infection, to predict the future rate of CD4 lymphocyte decline. DESIGN: Retrospective analysis of data from outpatients enrolled in a multi-institutional study. PATIENTS: One hundred five human immunodeficiency virus-positive intravenous drug users who had at least six serial CD4 lymphocyte measurements and starting CD4 levels of 200 x 10(6) cells/L or higher. MAIN OUTCOME MEASURE: Rate of CD4 lymphocyte decline. RESULTS: During a median follow-up of 28 months (range, 20-36 months), the estimated loss of CD4 cells in the whole patient population was 3.4 x 106 cells/L per month (P = .0045). Subjects who were on zidovudine treatment at study entry showed an average loss of 3.8 x 10(6) cells/L per month, significantly higher than in untreated subjects (P = .02), but similar to the loss observed for those requiring initiation of treatment during the course of the study. At baseline, 56 subjects had 90K levels of 10 microg/mL or less, and 49 had more than 10 microg/mL. The rate of CD4 decline in the high-90K group was approximately 5 x 10(6) cells/L per month (P < .0015), whereas in the low-90K group it was not different from zero (P = ns). No difference emerged in the rate of CD4 decline when subjects were stratified according to baseline 90K levels and zidovudine treatment, beta2-microglobulin, or neopterin serum levels. CONCLUSION: 90K serum levels are predictive of CD4 decline.

Immunohistochemical expression of cyclin T1 in a case of AIDS-related cachexia.

The expression of cyclin T1 in an autoptic case of AIDS-related cachexia was investigated by immunohistochemistry. When contrasted with normal human tissues, a very similar pattern of expression was found. However, a peculiar distribution of cyclin T1 was noticed in the brown fat and in lymph nodes affected by AIDS-associated lymphadenopathy.

[Oral hairy leukoplakia]. / Oral hairy leukoplakia.

We report our experience of oral hairy leukoplakia in HIV seropositive patients. Etiopathogenesis and relationship between oral hairy leukoplakia and HIV infection are briefly discussed, as well as its prognostic value.

Penetration of didanosine in semen of HIV-1-infected men.

OBJECTIVES: The disposition of antiretroviral agents into genital tissue and fluids is one of the factors implicated in the control of viral replication within the male genital tract and should be an objective of highly active antiretroviral therapy. We have investigated didanosine penetration in seminal plasma of 16 HIV-infected patients. PATIENTS AND METHODS: A total of 16 patients on didanosine (200 mg every 12 h or 400 mg once daily) participated in the pharmacokinetic study. After the didanosine morning dose, peripheral blood plasma and semen plasma were collected within the intervals 0-4, 4-8 and 8-12 h in the twice-daily regimen and 0-4, 4-12 and 12-24 h in the once-daily regimen. RESULTS: Within each sampling time interval didanosine concentrations in seminal plasma were higher than in blood. The interquartile range of concentrations in seminal plasma was 292-1217 ng/mL, compared with 50-150 ng/mL in blood plasma. Didanosine could be detected in 14 of the 16 semen samples analysed and in 8 of the 16 blood samples. CONCLUSIONS: We have demonstrated that didanosine penetrates into the seminal plasma in higher concentrations than in blood plasma.

[Aspecificity of the immunodiffusion reaction for the diagnosis of non-A non-B viral hepatitis]. / Aspecificità della reazione di immunodiffusione per la diagnosi di epatite virale non-A, non-B.

A precipitating antigen-antibody system was detected by immunodiffusion using acute and convalescent sera from patients with non-A, non-B (NANB) viral hepatitis in which the diagnosis was made by exclusion. The system showed identity with reference sera. Positivity for antigen was found in 5 out 13 patients with acute NANB hepatitis, but also in 3 out 8 patients with acute A hepatitis, in 6 out of 15 with acute B hepatitis and in 95 out of 221 voluntary blood donors. Antibody was detected in all the above mentioned categories of patients and also in normal subjects in percentages ranging from 9.1 to 25.0 percent. The lack of specificity observed is in contrast with results of other reported serological NANB related systems.

[Effect of methadone administration on MHV-3 virus-induced experimental hepatitis in the mouse]. / Effetto della somministrazione di metadone sulla epatite sperimentale del topo da virus MHV-3.

Mice treated with 15 mg/Kg/day methadone and infected with MHV-3 virus after 7 days did not show increased susceptibility to MHV-3 virus infection, did not develop more serious forms of hepatitis and not mortality did not increase with respect to the controls. Drug administration was continued for the duration of the experiment.

[Effect of heroin administration on MHV-3 virus experimental hepatitis in mice]. / Effetto della somministrazione di eroina sulla epatite sperimentale del topo da virus MHV-300.

Administration of heroin (5mg/Kg/day) in mice for a period of time sufficient to induce dependence and continued during the experiment did not increase susceptibility to MHV-3 virus infection, did not cause more serious forms of hepatitis and did not increase mortality with respect to the controls.

[Effect of pentazocine administration on MHV3 virus experimental hepatitis in mice]. / Effetto della somministrazione di pentazocina sulla epatite sperimentale del topo da virus MHV-3.

Mice treated with 15mg/kg/day pentazocin and infected with MHV-3 virus after 7 days did not show increased susceptibility to MHV-3 virus infection, did not develop more serious forms of hepatitis and mortality did not increase with respect to the controls. Drug administration was continued for the duration of the experiment.