Trends in drug resistance-associated mutations in a real-life cohort of Italian patients infected with HIV-1.

Recent studies support the idea that human immunodeficiency virus type 1 (HIV-1) drug resistance is declining in developed countries. To help assess the current situation in Italy, the dynamics of drug resistance mutations in pol and integrase genes in plasma samples from HIV-1-positive patients attending Sapienza University Hospital, Rome, from 2003 to 2014 were analysed. In total, 1730 genotype resistance tests (GRTs) were retrospectively analysed. The prevalence of major drug resistance mutations (DRMs) was evaluated over time in the global population and in patients with antiretroviral therapy (ART) failure. Population dynamics, changes in ART administration, and HIV-1 RNA levels were analysed in combination with DRM trends. The global population showed a strong reduction in major DRMs to all drug classes. Over the 2003-2014 decade, resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) declined from 80.0% to 18.7%, from 42.8% to 20.1% and from 74.2% to 8.3%, respectively (P<0.005 for all comparisons). However, only PI-associated mutations showed a significant decrease in patients experiencing ART failure. Interestingly, analysis of the integrase gene disclosed an increased resistance to integrase inhibitors, mainly regarding N155H, detected in 32.6% of raltegravir-treated patients in 2012-2014. In conclusion, in line with previous findings, this study shows that drug resistance is declining in Italy. However, the persistence of DRMs to NRTIs and NNRTIs suggests that despite adherence and treatment optimisation, some patients still experience therapy failure, emphasising the need for GRTs both in naïve and ART-failed patients.

Short communication: analysis of the integrase gene from HIV type 1-positive patients living in a rural area of West Cameroon.

Major mutations associated with HIV-I integrase inhibitors (INI) resistance are rare in INI-naive patients. However, polymorphisms at positions that may influence the genetic barrier and/or drive the selection of specific INI resistance pathways are common in HIV non-B subtypes. The aim was to evaluate the presence of natural polymorphisms and/or INI resistance mutations in HIV-1 non-B subtype samples obtained from INI-naive patients living in rural west Cameroon. Thirty-three HIV-1 non-B samples were obtained from INI-naive African women and, as controls, 15 samples of HIV-1 subtype B were obtained from antiretroviral-naive Italian patients. The integrase gene was amplified and sequenced using Trugene Core Reagents. Several amino acid positions in B and non-B subtypes were found to be polymorphic. Interestingly, two patients infected with the CRF02_AG subtype had the resistance mutations N155H and E157Q/E and 12% of African samples had an amino acid substitution at position 143. Silent mutations leading to a higher increment of genetic barriers were detected at 140 and 151 positions in non B-subtypes. Although most polymorphisms may have little effect on INI susceptibility, the IN gene variations found in the present study should be taken into consideration as they may facilitate or delay the emergence of variants fully resistant to INIs.

Mutational resistance pattern of HIV type 1 in CD14+ monocytes, CD4+ T cells, and plasma from treated patients.

It is necessary to understand the molecular nature of the virus population that persists in cellular reservoirs. To achieve this we planned to characterize the patterns of resistance of HIV-1 in CD14(+) monocytes, CD4(+) T cells, and plasma. Blood samples were collected from 42 patients treated for HIV: 32 were in virological failure and in 10 viremia was undetectable. CD14(+) and CD4(+) T cells were isolated using magnetic beads. Genotyping of the reverse transcriptase and protease gene of HIV-1 was undertaken using the fluorescent dideoxy-terminator method. Of the 32 patients in virological failure, 24 (75%) had resistance mutations in at least one compartment. The numbers and types of mutations from monocytes were the same as those detected in both CD4(+) T cell-associated virus and plasma in only 8% whereas in 71% monocytes exhibited a different mutation pattern. In 21% of patients, the profile of drug-resistant mutations in the virus from blood monocytes was identical to that in plasma but differed from that in CD4. In the 71% of patients with virological suppression, the genotypic resistance pattern differed between monocytes and CD4(+) T cells. Circulating monocytes may harbor a viral dominant population different from those viruses circulating in blood and archived in CD4(+) T cells. Hence, monocytes and other cellular reservoirs might serve as an indirect source of a drug-resistant viral variant.

Human Papillomaviruses and genital co-infections in gynaecological outpatients.

BACKGROUND: High grade HPV infections and persistence are the strongest risk factors for cervical cancer. Nevertheless other genital microorganisms may be involved in the progression of HPV associated lesions. METHODS: Cervical samples were collected to search for human Papillomavirus (HPV), bacteria and yeast infections in gynaecologic outpatients. HPV typing was carried out by PCR and sequencing on cervical brush specimens. Chlamydia trachomatis was identified by strand displacement amplification (SDA) and the other microorganisms were detected by conventional methods. RESULTS: In this cross-sectional study on 857 enrolled outpatients, statistical analyses revealed a significant association of HPV with C. trachomatis and Ureaplasma urealyticum (at high density) detection, whereas no correlation was found between HPV infection and bacterial vaginosis, Streptococcus agalactiae, yeasts, Trichomonas vaginalis and U. urealyticum. Mycoplasma hominis was isolated only in a few cases both in HPV positive and negative women and no patient was infected with Neisseria gonorrhoeae. CONCLUSION: Although bacterial vaginosis was not significantly associated with HPV, it was more common among the HPV positive women. A significant association between HPV and C. trachomatis was found and interestingly also with U. urealyticum but only at a high colonization rate. These data suggest that it may be important to screen for the simultaneous presence of different microorganisms which may have synergistic pathological effects.

Type-specific human papillomavirus-DNA load in anal infection in HIV-positive men.

OBJECTIVE: To characterize anal human papillomavirus (HPV) infections in terms of genotype prevalence and type-specific DNA load in HIV-positive men. DESIGN: HIV-positive men attending the colo-proctological clinic of a University Hospital in Rome were recruited prospectively from November 2004 to July 2007. HIV-negative outpatients attending the same clinic over the same period were used as a control group. METHODS: Anal brushings were tested for HPV-DNA using polymerase chain reactions and direct sequencing; type-specific HPV-DNA copies were measured in most positive samples. HPV data were correlated with patient HIV status and risk factors. RESULTS: HPV-DNA infection was detected in 81% of HIV-positive men. Almost all homosexual men were HPV-infected. The infection rate in low-risk HPV types was higher than in high-risk types. The spectrum of HPV genotypes was comparable between HIV-positive and HIV-negative men. Numbers of HPV-DNA copies varied greatly between samples but did not differ significantly between HIV-positive and HIV-negative men. In many samples, low-risk (HPV 6, 61, 70, and 74) viral loads were comparable with those of high-risk HPVs. CONCLUSION: Type-specific HPV-DNA copies at baseline appear to be independent of patient immune status and of HPV genotype. HPV genotype risk and viral load should be further evaluated for their potential predictive role in persistence and progression.

Genotypic resistance of archived and circulating viral strains in the blood of treated HIV-infected individuals.

The aim of this study was to evaluate patterns of antiretroviral resistance of HIV-1 in peripheral blood mononuclear cells (PBMCs) and in the plasma of patients whose therapeutic regimen is failing. Plasma and PBMC samples were collected from 95 HIV-infected patients undergoing long-term treatment. Genotyping of the reverse transcriptase (RT) and protease genes of HIV-1 was undertaken using the fluorescent dideoxy-terminator method. Comparison of the amino acid sequence of the RT and protease genes in cell-associated variants of HIV-1 with that of the plasma revealed that 62 of the 95 patients' samples tested exhibited different genotypic resistance patterns (discordant samples [DSs]). In 27% of samples, the patterns of resistance detected were concordant in both compartments. In 51% of DSs, the greatest number of mutations was found in plasma; however, in 37% of DSs, greater numbers of mutations were found in PBMC DNA. The HIV mutation patterns detected in plasma do not necessarily reflect those found in the cell-associated compartment. The observation that the cellular compartment may contain an archive of the resistance variant makes this reservoir an interesting substrate for analysis of the "resistance potential" in a given patient.

Direct sequencing of HPV DNA detected in gynaecologic outpatients in Rome, Italy.

In order to assess the frequency of different human papillomavirus (HPV) types in Rome and the association between HPV and behavioural characteristics, we tested cervical scrapes of a population of sexually active women referring to university clinics for routine gynaecologic care. The presence of HPV DNA was revealed by polymerase chain reaction on two genome regions (L1 and E6/E7) followed by sequencing. Thirty different HPV types were identified; HPV 16 was the most prevalent (14.18%), followed by HPV 53 (9.21%), HPV 58 (7.80%), HPV 6 and 66 (both 5.67%) whereas all the other genotypes ranged below 5%. In univariate analysis the characteristics significantly associated with HPV DNA detection were the youngest age (P<0.01), the high number of lifetime partners (P<0.001) and the smoking habit (P<0.01). In multiple logistic regression analyses, the characteristics significantly associated with HPV DNA detection remained the younger age and the higher number of lifetime sexual partners. This study may be interesting in order to evaluate the circulation of HPV genotypes in Italy and to add a contribution to anti-cancer vaccine development.

Human immunodeficiency virus infection in vitro activates naturally integrated human papillomavirus type 18 and induces synthesis of the L1 capsid protein.

Human papillomavirus (HPV) infections are prevalent in human immunodeficiency virus (HIV)-positive individuals. To highlight the effect of HIV on HPV expression, HPV-18-positive HIV-permissive HeLa-T4 cells were either infected with HIV-1 or treated with Tat or with the cytokines IL-1alpha, IL-1beta, IL-6 and TNF-alpha. The presence of HPV-18 E1 (early) and L1 (late) transcripts was then determined by dot-blot or Northern blot hybridization with E1 and L1 or with genomic HPV-18 DNA probes, respectively. Protein extracts from parallel cultures were challenged by Western blotting with an antiserum raised against an L1-beta-galactosidase hybrid protein. Results indicated that HeLa-T4 cells constitutively express E1 and L1 transcripts. When cells were infected with HIV, the amounts of E1 and L1 RNAs increased with time, followed by the de novo appearance of L1 protein. E1 and L1 transcripts were also increased, in a dose-dependent manner, by treatment of uninfected cultures with Tat or with IL-6, but were not affected by IL-1alpha, IL-1beta and TNF- alpha. Neither Tat nor IL-6 could induce L1 translation. These findings raise the hypothesis that the increase of HPV shedding and of HPV-associated diseases in HIV-infected individuals could be due in part to a direct or cytokine-mediated action of HIV, in addition to the HIV-induced immunodeficiency.