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.

Efficacy and safety during formulation switch of a pasteurized VWF/FVIII concentrate: results from an Italian prospective observational study in patients with von Willebrand disease.

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by the quantitative or qualitative deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived VWF/factor VIII (FVIII) concentrates is required in patients unresponsive to desmopressin. To assess the efficacy, safety and ease of use of a new, volume-reduced (VR) formulation of VWF/FVIII concentrate Haemate(®) P in patients requiring treatment for bleeding or prophylaxis for recurrent bleeding or for invasive procedures. Pharmacoeconomic variables were also recorded. Data were analysed using descriptive statistics. This was a multicentre, prospective, observational study. Consecutively enrolled patients received Haemate(®) P VR according to their needs, and were followed for 24 months. Of the 121 patients enrolled, 25.6% had type 3 VWD and more than 40% had severe disease. All patients were followed for 2 years, for a total of 521 visits. On-demand treatment was given to 61.9% of patients, secondary long-term prophylaxis to 25.6% and prophylaxis for surgery, dental or invasive procedures to 45.5%. The response to treatment was rated as good to excellent in >93-99% of interventions. The new formulation was well tolerated by all patients with no report of drug-related adverse events. The switch to volume-reduced Haemate(®) P was easy to perform and infusion duration was decreased twofold compared with the previous formulation. Volume-reduced Haemate(®) P was at least as effective and well-tolerated as the previous formulation.

Prevalence of mutations and determinants of genotypic resistance to etravirine (TMC125) in a large Italian resistance database (ARCA).

OBJECTIVES: To evaluate whether etravirine (TMC125) might be effective in patients failing therapy with current nonnucleoside reverse transcriptase inhibitors (NNRTIs), we analysed the prevalence of TMC125 mutations and the possible determinants of genotypic resistance to this drug among sequences reported to a large database in Italy [Antiretroviral Resistance Cohort Analysis (ARCA)]. METHODS: We analysed the prevalence of TMC125 resistance-associated mutations (RAMs) and the TMC125 weighted genotypic score (WGS) together with the determinants of genotypic resistance. A total of 5011 sequences from 2955 patients failing NNRTI therapy were evaluated. RESULTS: Among the sequences in ARCA, 68% had at least one and 9.8% at least three TMC125 RAMs, whereas 31% had a WGS>2. Frequent RAMs were Y181C, G190A, K101E and A98G, whereas V179F, Y181V and G190S appeared in <5% of sequences. Multivariate analysis revealed a higher risk of developing at least three TMC125 RAMs associated with both nevirapine and efavirenz exposure, whereas CD4 counts > or = 200 cells/microL retained their protective effect. An increased risk of WGS>2 was linked to higher HIV RNA values (maximum risk at >5 log(10) copies/mL) and nevirapine exposure; CD4 counts > or = 200 cells/microL were protective. CONCLUSIONS: The prevalence of TMC125 resistance mutations in the ARCA cohort was 68%. The DUET studies showed that at least three TMC125-associated mutations were required to impair the efficacy of the drug and Y181C/V, V179F and G190S had the greatest effect on response. The prevalence of these mutations among the patients examined in our study was low. However, WGS>2 was found for one-third of our sequences. Previous nevirapine exposure was associated with an increased risk of having WGS>2 (adjusted odds ratio 1.76).

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

Human Brucella canis outbreak linked to infection in dogs.

The zoonotic risk of Brucella canis has been considered fairly high for persons who handle breeding dogs in kennels or are exposed to infected animals. Transmission to humans in other circumstances has been thought to be rare. We describe an uncommon outbreak of brucellosis caused by B. canis which, to the best of our knowledge, is the first reported in the literature. This outbreak involved six persons (three children and three adults), a bitch and three puppies which had close daily contact with the family. The clinical symptoms of the index case led to an erroneous diagnosis and the infection would have gone undiagnosed if culture had not been positive. This report aims to increase awareness of medical personnel of the need to order screening tests for children, immunodeficient persons or pregnant women presenting with fever of unknown origin, unexplained spleen or liver enlargement or other systemic signs. The emerging zoonotic potential of this disease in urban areas and the need to coordinate canine brucellosis surveillance systems should be evaluated.

The feasibility of using antigens prepared with rough Brucella strains for diagnosis of canine brucellosis.

Clinical diagnosis of canine brucellosis is not sensitive enough and a negative blood culture cannot rule out the disease. Indirect methods of serological testing such as agar gel immunodiffusion (AGID), rapid slide agglutination test (RSAT) and indirect enzyme linked immunoassay (IELISA) are preferred for routine diagnosis. Since Brucella canis shares antigenic components with the Brucella ovis and Brucella abortus RB51 strain, it would seem that either strain could be used as antigen. We present data on AGID and IELISA tests using the B. ovis antigen, RSAT and IELISA using the B. canis antigen and IELISA using the B. abortus RB51 antigen. The cut-off values were adjusted by the ROC analysis; the IELISA-B. ovis cut-off value was 23 (%P) and the IELISA-B. abortus RB51, 24 (%P), with 100% sensitivity and 98.8% specificity. RSAT detected 100% of positive cases, while AGID was less sensitive. The sera from dogs treated with antibiotic showed that %P correlated well with the clinical course. Sera from dogs presumptively infected with B. suis were negative in all tests performed with the rough Brucella strains. RSAT is a very sensitive screening test and IELISA-B. canis, B. ovis and B. abortus RB51 could be used as confirmatory tests, since they show good specificity and sensitivity.

Detection of hepatitis C virus in an exhumed body identified the origin of a nosocomial transmission that caused multiple fatal diseases.

BACKGROUND: Medico-legal conflicts arise when it is difficult to prove the cause of nosocomial infections. AIM: To report an outbreak of patient-to-patient transmission of hepatitis C virus (HCV) through the repeated use of a multi-dose saline flask during the rinsing of central venous catheters. METHODS: Blood samples were taken from each patient for the comparative analysis of their HCV RNA strains. No samples were available for one patient who died before the investigation started. Despite the known lability of HCV RNA, the body was exhumed four months after burial and postmortem samples were collected. HCV RNA was extracted successfully from liver and spleen samples. Genotyping of all the HCV strains was performed by sequence analysis of the 5'NC untranslated region, the E1 core conserved region and the E1/E2 hypervariable region. FINDINGS: Forensic investigators retraced the route used by two ward nurses, when saline catheter flushes were given to 14 patients with each nurse administering to seven patients. The comparative phylogenetic analysis of all case strains identified the deceased patient as the source of contamination to five patients. CONCLUSIONS: This study highlights the value of sequence analysis as a tool for solving medico-legal conflicts. The High Court of Justice found that a health worker's re-use of a contaminated needle resulted in the nosocomial transmission of HCV.

Italian multicentric survey on laparoscopic spleen surgery in the pediatric population.

BACKGROUND: Some technical aspects of laparoscopic spleen surgery still are debated, although efforts have been made to standardize them. The position of the patient, the approach to the spleen, vessel identification and division, and spleen extraction can vary from center to center. METHODS: This retrospective muticentric study led by the Società Italiana di Videochirurgia Infantile (SIVI) examined indications, surgical details, and complications of laparoscopic spleen surgery in the pediatric population during a 5-year period. RESULTS: The study period from January 1999 to December 2003 (5 years) involved nine centers and included 85 patients with a mean age of 10 years (range, 2-17 years). Hypersplenism or severe hemolysis in cases of hematologic disorders represented the most important indications. More than 90% of the patients underwent total laparoscopic splenectomy. Specific technical details from each center were collected. Intraoperative complications occurred in 19% of the patients (hemorrhage in 8% and technical problems in 14%), and 6% of the patients required conversion to the open approach. No deaths occurred, and no reoperations were required. Postoperative complications were experienced by 2% of the patients. CONCLUSION: Laparoscopic spleen surgery is safe, reliable, and effective in the pediatric population. On the basis of the results, some technical details for laparoscopic spleen surgery can be suggested. The patient is preferably kept supine or lateral, approaching the spleen anteriorly. Moreover, the ilar vessels should be identified selectively and individually, with initial artery division performed to achieve spleen shrinking. Any hemostatic device proved to be effective in experienced hands. Once freed, the spleen is preferably extracted via a suprapubic cosmetic transverse incision (faster, easier, and safer), although a bag can be used. Finally, the size of the spleen does not represent a contraindication for a trained and experienced surgeon. Nevertheless, this parameter must be considered when laparoscopic spleen surgery is planned.

Regional specificity of HTLV-I proviral integration in the human genome.

The location of HTLV-I (human T-cell leukemia virus type 1) proviral sequences in the genome of infected human cells was explored by hybridization of a viral probe with compositional fractions of host-cell DNAs. In the twelve cases examined, HTLV-I sequences were absent from the GC-poorest 40% of the host genome (namely, from isochores that are below 39% GC). Transcriptionally inactive proviral sequences were localized in GC-poor isochores (comprised between 39% and 42-44% GC) of the human genome, which are characterized by a constant and low gene concentration. In contrast, transcriptionally active proviral sequences were found in the GC-rich and very GC-rich isochores, which are gene rich, transcriptionally and recombinationally active, and endowed with an open chromatin structure. Since GC-rich isochores are present in R'-bands and very GC-rich isochores form T-bands, these results also provide information on the location of HTLV-I proviral sequences in human chromosomes. The results obtained with HTLV-I are in agreement with the non-random, compartmentalized integration of animal retroviral sequences that had been previously observed in other viral-host systems. They provide, however, much more detailed information on the regional location of proviral sequences in the host genome and on the correlation between their transcription and their location.

Prevalence of HTLV type I infection in Iran: a serological and genetic study.

Several publications describe the presence of the human T cell lymphotropic virus type I (HTLV-I) in Jewish individuals born in Mash-had, Iran. We report here the results of HTLV-I serological and genetic studies in the non-Jewish population of Mash-had as well as a neighboring area: Gonbad-Kavous. Seven hundred and seven serum samples from Mash-had (694 healthy individuals and 13 patients with lymphoma) and 90 from Gonbad-Kavous were tested for HTLV antibodies by gelatin particle agglutination assay (PA) and confirmatory Western blots (WBs). Seropositive rates of 3.0% (21 of 694) in Mash-had, 0% (0 of 90) in Gonbad-Kavous, and 100% (13 of 13) in lymphoma cases were observed. HTLV-I DNA sequence were amplified by polymerase chain reaction directly from the fresh PBMCs of seropositive individuals. Phylogenetic analysis of the viral DNA sequence indicated that the HTLV-I present in Mash-had belong to the HTLV-I cosmopolitan clade. Altogether, these data indicate that Mash-had, located in northeastern Iran, is a newly recognized endemic center for HTLV-I.

Anti-cell antibodies in exposed seronegative individuals with HIV type 1-neutralizing activity.

Despite repeated exposures to HIV-1, some individuals remain seronegative. This study reports that sera from a fraction of exposed seronegative (ESN) subjects showed HIV-neutralizing activity; 5 of 17 ESN sera and none of 17 controls neutralized two different HIV-1 primary isolates (range of neutralizing titers: 1/20 to 1/60). The neutralizing activity was associated with the IgG fraction of 4 of 4 neutralizing ESN sera. Moreover, in 11 of 17 and 9 of 17 ESN sera (but none of the control sera) we found antibodies against HLA class I and CD4, respectively. One of the ESN sera (EU22) neutralized efficiently the primary virus derived from the seropositive partner and showed a good broadly cross-reactive neutralization. Immunoadsorption of two IgG fractions from EU19 and EU22 on peripheral blood mononuclear cells (PBMC) removed virus-neutralizing antibodies. The correlations between the ESN status and neutralizing activity (p<0.05), anti-HLA antibodies (p<0.0002), and anti-CD4 antibodies (p<0.001) were statistically significant. However, there was no statistically significant correlation between neutralizing activity and either anti-HLA or anti-CD4 antibodies. It can therefore be said that exposure to HIV-1 without seroconversion is, in some individuals, associated with HIV-neutralizing antibodies (not directed against viral antigens) and/or with anti-cell autoantibodies, which are possibly specific for cellular antigens involved in the infection/entry process.

Effect of genotypic resistance on the virological response to highly active antiretroviral therapy in cerebrospinal fluid.

Paired plasma and cerebrospinal fluid (CSF) specimens drawn from 15 HIV-infected patients with neurological disease before and after a median 6-week duration of highly active antiretroviral therapy (HAART) were studied to assess the short-term virological response of CSF and whether this can be predicted on the basis of baseline resistance mutations. After treatment, the median plasma and CSF viral load (VL) decreased by, respectively, 2.08 log10 (p = 0.0001) and 0.91 log10 copies/ml (p = 0.007) in comparison with baseline. A plasma virological response was observed in all but one patient, whereas the posttreatment CSF VL increased, remained unchanged, or decreased at a substantial lower rate than in plasma of six "CSF non/slow responders" (40%). Direct sequencing of baseline specimens showed that none of these patients had reverse transcriptase (RT) or primary protease resistance mutations in the CSF alone, but two had RT mutations conferring high-level resistance to drugs included in the HAART regimen in both CSF and plasma. The other four patients had no RT or primary protease resistance mutations. There was no significant difference in the nucleotide diversity of the CSF and plasma RT sequences, baseline plasma or CSF VL, the CSF-to-plasma VL ratio, the number of CSF cells, the CD4+ cell counts, or the history of antiretroviral treatment between the CSF non-slow responders and the other patients. During this short-term follow-up and despite a plasma response, a significant proportion of HAART-treated patients with neurological symptoms showed a slow or absent CSF response. Most of these cases were not associated with the presence of resistant HIV strains in the CSF.

"Single step" PCR with a sensitivity similar to nested PCR for the detection of hepatitis C virus RNA.

OBJECTIVE: Evaluation of the performance of different HCV PCR detection systems for HCV RNA: A nested PCR, considered the reference assay, was compared with two single-step methods (ss-PCR): the first is based on the detection of PCR products by liquid hybridization with a 32P end-labelled probe (isotopic ss-PCR), while the second assay is a colorimetric method (colorimetric ss-PCR) using microwell plate hybridization with a specific nucleic acid probe (Amplicor HCV PCR, Roche Diagnostics Systems). METHODS: Sera from 56 patients with suspected hepatitis C infection based on reactive serology or altered liver parameters, and sera from 15 blood donors were tested for HCV RNA: After RNA extraction, the synthesized HCV cDNA was amplified in parallel using isotopic ss-PCR, colorimetric ss-PCR and nested PCR. The products were detected by autoradiography, color development and ethidium bromide fluorescence, respectively. RESULTS: In order to assess the analytical sensitivity of ss-PCR versus that of nested of PCR, experiments included serial dilutions of positive control samples. Results showed that both methods had an extinction signal at the 1:512 dilution. A comparative analysis of 71 clinical sera samples was obtained using the three protocols and the results clearly documented 100% concordance. CONCLUSIONS: Single step PCR methods for HCV RNA have a sensitivity equal to that of nested PCR and appear more suitable for diagnostic applications. Ss-PCR is safer than nested PCR in terms of both specificity and contamination problems. In particular, the Roche Amplicor HCV PCR assay minimizes sample exposure and management problems.

Comparison of protease genotype and phenotype in HIV-1 infected patients exposed to more than one protease inhibitor.

Many patterns of mutations selected by HIV-1 protease inhibitors have been described, but in most cases isolates with these patterns have been obtained from pre-clinical studies or after failures of monotherapies. We compared genotype and phenotype in HIV-1 infected patients who have failed more than one PI-including regimen. Phenotypic resistance could arise also in the absence of specific primary mutations and in the presence of different substitutions among those known to confer resistance to ritonavir, indinavir or nelfinavir. The number of secondary mutations was significantly associated with phenotypic resistance for each protease inhibitor. Thus, more study of mutational patterns in heavily pretreated patients is warranted; in the mean time treatment choices might be optimized if phenotyping could integrate genotyping within this setting.

Prevalence of mutation associated to resistance with nucleoside analogues in a cohort of naïve HIV-1 positive subjects during the period 1984-1997.

We examined samples from 98 naïve HIV-1 positive patients with the seroconversion period between 1984 and 1997, 64 of whom with a diagnosed primary infection. We observed a progressive increase in the percentage of patients harboring mutations associated with zidovudine resistance, starting from 8% during the period 1987-1994 to 20% and 36% in patients with HIV infection diagnosed during 1996 and 1997 respectively. The small number of patients analyzed in groups 3 and 4 is an important limitation to establish the real increase in the prevalence of mutations we observed during the past two years. However, it is important to underline the trend and, in our opinion, further studies to better define the relevance of such phenomenon in the clinical practice must be performed.

Characterization of GBV-C infection in HIV-1 infected patients.

BACKGROUND: GB virus C, a positive-stranded RNA virus, is classified in the family Flaviviridae. It is currently believed that persistent infection occurs in 25-50% of infected individuals, however, it still remains an "orphan" virus in search of a role in human pathology. Molecular epidemiological studies have demonstrated that GBV-C infection is present in about 1-1.4% of the healthy population in developed countries, that it shares routes of transmission with HIV and HCV and that the prevalence of GBV-C in these populations is higher than in blood donors. On the basis of the sequence variation among the isolates, GBV-C is classified into at least four major genotypes. Preliminary evidence has suggested that GBV-C is a lymphotropic virus that replicates mainly in the spleen and bone marrow. Recently, several reports have investigated the possible beneficial effect of GBV-C co-infection on HIV disease progression to AIDS, reduced mortality in HIV infected individuals and lower HIV viral loads, not leading to a definitive conclusion yet. AIM: To investigate the role of GBV virus C co-infection in two different subsets of HIV-infected patients, and to evaluate the prevalence of GBV-C genotypes in Northern Italy. METHODS: A total of 86 HIV positive patients were examined for GBV-C viremia (years after HIV sera conversion: 12 +/- 5). Control population (Group A): 46 patients (mean age 42 years) with <200CD4/ml during the observation period. Longterm non progressor population (Group B): 40 patients, (mean age 40 years) with >500 CD4/ml for at least 8 years and never treated with HAART. After extraction of viral RNA from plasma samples, amplification of a highly conserved region of 5'UTR was performed by nested RT-PCR. All positive samples were genotyped by sequencing, alignment with published sequences and phylogenetic analysis. CD4 cell count, HIV plasma levels were also evaluated. RESULTS: 9 out of 46 (19.56%) in Group A and 15 out of 40 (37.5%) in Group B had detectable GBV-C viremia (p=0.064, OR 2.47, percent confidence interval 0.94 to 6.51). No statistical difference was observed when disease stage was evaluated between the two groups. In Group B, after regression analysis for CD4 cell count decrease over the period observed, no significant difference was detected between GBV-C positive and negative patients. No significant difference was observed in Group B in HIV viremia and CD4 cell count at time of GBV-C detection between GBV-C infected patients and GBV-C negative patients. All Italian patients were genotype 2, the only African patient carried GBV-C genotype 1. CONCLUSIONS: Although previous results suggest that GBV-C virus may be a favorable marker for long term non progression of HIV disease, whether it plays a direct anti-HIV role or just takes advantage of non progessors' higher CD4 cell count to replicate more efficiently, still remains to be answered. Follow up of untreated patients and further evaluation of virological interactions, between the viruses and the host immune system, will be helpful to shed some light on these observations, offering new prognostic and eventually therapeutical tools for the management of HIV patients.

Heterogeneity in exposed uninfected individuals.

In spite of repeated exposures to HIV, some individuals remain seronegative and apparently uninfected. A variety of mechanisms potentially able to confer resistance to HIV infection, including cell-mediated and (unconventional) humoral immune responses, as well as mutations affecting receptors for virus entry have been considered and analysed. In this article, we want to discuss recent reports on specific immune responses and genetic factors potentially involved in mechanisms of protection, and to present some of our data relative to a cohort of people sexually exposed to HIV-1, but persistently seronegative. These EU (exposed uninfected) individuals can be distinguished from "normal" unexposed controls on the basis of significantly increased frequencies of a number of immunological parameters that might be considered "unconventional" correlates of HIV infection/protection. However, EU individuals are highly heterogeneous since the various unconventional immune responses considered can be present in all possible combinations. Aim of future research will be to ascertain the role of such immune responses in the maintenance of the protection state, or their secondary nature as signals of a particular kind of infection.

Phylogenetic internal control for HIV-1 genotypic antiretroviral testing.

Genotypic testing includes several steps (RNA purification, RT-PCR amplification, DNA sequencing, sequence editing and analysis) that should be individually controlled. In our laboratory, we have added to this step-by-step internal control a final phylogenetic quality control: this is performed every time a sequence is obtained from a patient previously subjected to the same test. Each sequence with this characteristic is routinely compared with sequences from previous samples of the same patient by multiple alignment and a neighbor-joining tree by using Kimura two-parameter method is constructed. To validate the quality control procedure, we have aligned and calculated the mean similarity of the reverse transcriptase (first 984 nucleotides) and protease (whole gene) sequences from 30 patients whose virus was completely wild-type for both reverse transcriptase and protease. In the same tree, we have added the sequences obtained from 5 out of the 30 patients, tested at a second time point. The wild type sequences have shown a mean inter-sample divergence of 2.9%, and all the sequence pairs from individual patients clustered together in the tree constructed with the nucleotide sequences, while the tree constructed with the inferred aminoacid sequences did not always permit to cluster the sequences from the same patients. This indicates that: 1) the phylogenetic analysis of nucleic acid sequences can be useful to rule out sample mix-up; 2) the belonging of a sequence to each individual patient can efficiently be assessed also in the cases of extreme divergence in terms of drug resistance mutations.

In vivo dynamics of the 103N mutation following the withdrawal of non-nucleoside reverse transcriptase inhibitors in HIV-infected patients: preliminary results.

Due to the preferential selection of the fittest HIV mutants, drug-resistant variants are often overgrown by wild-type virus after treatment interruption. Our objective was to investigate the dynamics of the 103N mutation (which usually does not reduce HIV fitness) following the withdrawal of non-nucleoside reverse transcriptase inhibitors (NNRTIs). Patients who were found to have the 103N mutation at or after failure of a NNRTI were selected from an observational database. Two groups of patients were identified: one which continued antiretroviral treatment without NNRTIs (group A) and one which discontinued all anti-retrovirals after failure of an NNRTI (group B). Genotype was obtained by direct sequencing of the replicating plasma virus. Sixty-two subjects tested between July 1998 and December 2002 were included in the analysis, 39 in group A and 23 in group B. At the time of the first resistance test, median (IQR) CD4+ T-lymphocytes and HIV-RNA were 269 (150-449) cells/microL and 25,000 (9,600-83,300) copies/mL. In 31 (50%), 30 (48%), and one case (2%), the 103N mutation was selected by nevirapine, efavirenz, and by delavirdine, respectively. A total of 149 tests were analyzed, with a median (IQR) of 2 (2-3) tests/patient. The median (IQR) interval between failure of NNRTIs and the last resistance test was 11 (5-22) months. Overall, a reversion to wild-type at position 103 was observed in 23/62 (37%) subjects, 14/39 (36%) in group A and 9/23 (39%) in group B. In group A, 14/23 (61%) patients tested within 12 months, 10/16 (63%) of those tested between 12 and 24 months, and 12/14 (86%) of those tested beyond 24 months from NNRTI discontinuation had the 103N mutation. In group B, 14/20 (70%) patients tested within 12 months, 3/4 (75%) of those tested between 12 and 24 months, and none out of two tested beyond 24 months from NNRTI discontinuation had the 103N mutation. In conclusion, following NNRTI discontinuation, in the majority of patients HIV variants carrying the 103N mutation are not overgrown for long by wild-type quasispecies at this position. This suggests that the 103N mutation per se influences minimally the viral fitness in vivo.

[Role of the endothelium in the control of vascular motility: physiologic aspects and physiopathologic implications in cardiovascular diseases]. / Ruolo dell'endotelio nel controllo della motilità vascolare: aspetti fisiologici ed implicazioni fisiopatologiche nelle malattie cardiovascolari.

It was recently demonstrated that vascular endothelium produces many substances which modulate the vascular tone by acting locally (endothelium derived relaxing factor, angiotensin II, endothelin, prostacyclin). The authors review the physiological regulatory role of these substances and their involvement in cardiovascular diseases. They also consider the interaction between the endothelium derived factors and some cardiovascular drugs (such as calcium antagonists and ACE-inhibitors) and also the mechanisms of the possible protective action of these drugs on the vascular wall.