Paola Corsi

Antiretroviral Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Protease from Paired Cerebrospinal Fluid and Plasma Samples

Twenty-four adults infected with human immunodeficiency virus type 1 (HIV-1) with central nervous system symptoms were studied for antiretroviral resistance mutations in HIV-1 RNA obtained from paired cerebrospinal fluid (CSF) and plasma samples. Paired sequences were obtained from 21 and 13 patients for reverse transcriptase (RT) and for protease, respectively. Mutations conferring resistance to the RT inhibitors zidovudine, lamivudine, or nevirapine were detected in 14 patients, including 11 pretreated and 3 drug-naive subjects. The mutation patterns in the 2 compartments were different in most patients. Genotypic resistance to protease inhibitors was detected in both plasma and CSF from 1 patient treated with multiple protease inhibitors. However, accessory protease inhibitor resistance mutations at polymorphic sites were different in plasma and CSF in several patients. Partially independent evolution of viral quasispecies occurs in plasma and CSF, raising the possibility that compartmentalization of drug resistance may affect response to antiretroviral treatment.

Detection of genotypically drug-resistant HIV-1 variants and non-B subtypes in recently infected antiretroviral-naive adults in Italy

Sezione di Microbiologia, Dipartimento di Biologia Molecolare, Universita di Siena, Siena, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Careggi, Firenze, Firenze, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Prato, Prato, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Grosseto, Grosseto, Italy; and Servizio di Microbiologia e Virologia, Azienda Ospedaliera Senese, Siena, Italy.

Frequency and Treatment-Related Predictors of Thymidine-Analogue Mutation Patterns in HIV-1 Isolates after Unsuccessful Antiretroviral Therapy

We investigated, in patients tested between 1991 and 2004, the patterns of mutually exclusive human immunodeficiency virus-1 thymidine-analogue mutations (TAMs) in 4039 reverse-transcriptase sequences with > or = 1 TAM. TAM pattern 1, which included M41L and L210W and excluded K70R and is coupled with more-extensive cross-resistance to drugs, became the most frequent pattern after 1996. In 1465 genotypes from 684 patients in whom highly active antiretroviral therapy (HAART) was unsuccessful, predictors of this pattern were the number of previous HAART regimens undergone (adjusted odds ratio [OR], 1.09 [95% confidence interval {CI}, 1.02-1.16]), use of stavudine/lamivudine (adjusted OR, 1.42 [95% CI, 1.05-1.99]), use of nevirapine (adjusted OR, 1.60 [95% CI, 1.14-2.24]), use of efavirenz (adjusted OR, 1.56 [95% CI, 1.08-2.27]), and use of ritonavir (adjusted OR, 1.35 [95% CI, 1.04-1.75]).

The Problem of Renal Function Monitoring in Patients Treated With the Novel Antiretroviral Drugs

Abstract Chronic kidney disease (CKD) is currently considered a major comorbidity in patients affected by HIV infection. In addition, new generation antiretroviral drugs that interact with creatinine transporters were recently introduced. Rilpivirine, dolutegravir, and cobicistat, with different mechanisms, inhibit the amount of tubular secretion of creatinine causing a slight increase in serum creatinine levels and consensual eGFRcreatreduction. This will require an unprecedented attention to renal issues, because the new drugs can also be associated to old antiretroviral drugs that may exert renal toxic effects. Owing to the interference of these drugs with creatinine secretion, an alternative way of estimating GFR would be desirable. At the moment, methods of direct GFR measurement have a high impact on the patient, are not readily available, or are not reliable in HIV patients. Consequently, use of classic formulas to estimate GFR is still recommended, considering the apparent reduction of eGFRcreat due to these drugs. Tubular function needs to be carefully monitored with simple tests such as proteinuria, phosphatemia, urinary excretion of phosphate, normoglycemic glycosuria, and excretion of uric acid. More specific and sensitive markers of tubular damage are still not readily available in all clinical labs. HIV patients treated by the novel drugs need to be monitored on a monthly basis for the first 3 months. Subsequent monitoring should be performed on a quarterly basis or guided by comorbidities.

Treatment with lopinavir/ritonavir in heavily pretreated subjects failing multiple antiretroviral regimens in clinical practice.

To the Editor: Lopinavir, coformulated with a boosting dose of ritonavir, (LPV/r) has been recently licensed as a new protease inhibitor (PI) with excellent pharmacokinetic properties (1). Due to the achievement of high blood levels, there is a high genetic barrier to clinical resistance to LPV/r. Indeed, as many as 11 mutations at HIV-1 protease codons 10, 20, 24, 46, 53, 54, 63, 71, 82, 84 and 90 have been reported to contribute to LPV/r resistance, and at least 8 of these appear to be required for significant clinical resistance (2). Although preliminary LPV/r efficacy studies have yielded promising results, especially on drug-naive subjects (3–5), the role of LPV/r as a salvage therapy for heavily experienced patients in clinical practice has not yet been fully elucidated. We report an observational study of the first 41 patients shifted to an LPV/r-including regimen and reaching at least 16 weeks (mean ± SD, 23 ± 5 weeks) of LPV/r therapy in our area. The subjects were pretreated with a median of 5 (range 4–6) nucleoside reverse transcriptase inhibitors (NRTIs), 1 (range 1–2) nonnucleoside reverse transcriptase inhibitors (NNRTIs) and 4 (range 2–5) PIs. The median number of different treatment regimens used before commencing LPV/r therapy was 8 (range 4–13), including dual NRTI treatments, and the median HIV-1 RNA load and CD4 counts at baseline were 5.29 (range 3.79–7.00) log10 copies/mL and 138 (range 4–689) cells/ L, respectively. A total of 26 (63.4%) and 30 (73.2%) subjects had >10 HIV-1 RNA copies/mL and <200 CD4 cells/ L, respectively. Genotypic antiretroviral resistance analysis (6) at baseline revealed a median number of 6 (range 0–10) NRTI resistance mutations, 1 (range 0–4) NNRTI resistance mutations, and 2 (range 0–4) and 5 (range 1–8) primary and accessory PI resistance mutations, respectively. There was only one case without resistance mutations, possibly resulting from multiple treatment interruptions before starting LPV/r. The number of patients harboring virus with at least one primary NRTI, NNRTI, and PI resistance mutations was 38 (92.7%), 28 (68.3%), and 35 (85.4%), respectively. Five (12.2%) subjects harbored virus with a 69S-XX insertion or Q151M complex NRTI class resistance, and more than two primary PI resistance mutations were present in virus from 20 (48.8%) individuals. Except for two subjects in whom LPV/r was associated with one NRTI and one NNRTI (efavirenz), the new regimen combined LPV/r with two NRTIs, mainly stavudine/didanosine (16 cases) and stavudine/lamivudine (8 cases). However, based on baseline RT genotype, only 9 (22.0%) and 18 (49.3%) patients were administered 2 and 1 reverse transcriptase inhibitors expected to retain activity against their viral isolate, respectively, according to the on-line drug resistance interpretation system available at the Stanford University web site (http://hivdb. stanford.edu/). Based on a standardized questionnaire, the selfreported adherence to treatment was optimal in this cohort. Overall, there was a significant response to LPV/r therapy both in terms of HIV-1 RNA levels (5.25 ± 0.68 log10 at baseline vs. 3.94 ± 1.27 log10 at the end of follow-up; p < .001, paired t test) and in terms of CD4 cell counts (166 ± 153 vs. 225 ± 193; p .009). Interestingly, changes in HIV-1 RNA levels and CD4 cell counts were comparable in subjects with <5 log10 HIV-1 RNA copies/mL (−1.30 log10 copies/mL and +45 cells/ L) and >5 log10 HIV-1 RNA copies/mL (−1.32 log10 copies/mL and +67 cells/ L), respectively. HIV-1 RNA levels were suppressed to <500 copies/mL and <50 copies/mL in 13 (31.7%) and 9 (21.9%) patients, respectively. Table 1 shows the evolution of PI resistance mutations under the selective pressure of LPV/r (GenBank accession numbers AF493336 to AF493415). There was a statistically significant increase in the prevalence of total PI resistance mutations (me-

HIV-1 subtype F1 epidemiological networks among Italian heterosexual males are associated with introduction events from South America.

About 40% of the Italian HIV-1 epidemic due to non-B variants is sustained by F1 clade, which circulates at high prevalence in South America and Eastern Europe. Aim of this study was to define clade F1 origin, population dynamics and epidemiological networks through phylogenetic approaches. We analyzed pol sequences of 343 patients carrying F1 subtype stored in the ARCA database from 1998 to 2009. Citizenship of patients was as follows: 72.6% Italians, 9.3% South Americans and 7.3% Rumanians. Heterosexuals, Homo-bisexuals, Intravenous Drug Users accounted for 58.1%, 24.0% and 8.8% of patients, respectively. Phylogenetic analysis indicated that 70% of sequences clustered in 27 transmission networks. Two distinct groups were identified; the first clade, encompassing 56 sequences, included all Rumanian patients. The second group involved the remaining clusters and included 10 South American Homo-bisexuals in 9 distinct clusters. Heterosexual modality of infection was significantly associated with the probability to be detected in transmission networks. Heterosexuals were prevalent either among Italians (67.2%) or Rumanians (50%); by contrast, Homo-bisexuals accounted for 71.4% of South Americans. Among patients with resistant strains the proportion of clustering sequences was 57.1%, involving 14 clusters (51.8%). Resistance in clusters tended to be higher in South Americans (28.6%) compared to Italian (17.7%) and Rumanian patients (14.3%). A striking proportion of epidemiological networks could be identified in heterosexuals carrying F1 subtype residing in Italy. Italian Heterosexual males predominated within epidemiological clusters while foreign patients were mainly Heterosexual Rumanians, both males and females, and South American Homo-bisexuals. Tree topology suggested that F1 variant from South America gave rise to the Italian F1 epidemic through multiple introduction events. The contact tracing also revealed an unexpected burden of resistance in epidemiological clusters underlying the need of public interventions to limit the spread of non-B subtypes and transmitted drug resistance.

Safety and durability in a cohort of HIV-1 positive patients treated with once and twice daily darunavir-based therapy (SCOLTA project)

OBJECTIVE To evaluate safety and durability of once-daily and twice-daily darunavir/ritonavir (DRV/r)-based treatment in HIV patients in clinical practice. METHODS The Surveillance Cohort Long-Term Toxicity Antiretrovirals (SCOLTA) project is a prospective, observational, multicenter cohort created to assess the incidence of adverse events in patients receiving new antiretroviral drugs. Twenty-five Italian infectious diseases centers enroll patients and collect their data through this on-line system. Periodical evaluations of these patients, including physical examination and laboratory tests, were performed at baseline and every 6 months. RESULTS Four hundred and twenty-nine patients were enrolled since May 2006. Eighty-five patients (19.8%) were prescribed once-daily DRV/r; 31 of them were treatment-naïve (36.5%). Among 54 (63.5%) treatment-experienced patients, 21 (38.9%) had undetectable viral load and started once-daily DRV/r as a simplification regimen. Patients on twice-daily regimen were older, more frequently lipodystrophic, HCV-coinfected, and in CDC stage C. In the following 24 months of follow-up, the viral load steadily decreased as well as the CD4 cell count rose. The reason for discontinuation did not significantly differ between groups. Mean blood glucose (BG) change from baseline did not show significant difference between groups, as well as high density lipoprotein cholesterol (HDL-C), triglycerides (TGL) and alanine transaminase (ALT). The survival curve shows that patients in the once-daily regimen withdrew treatment more frequently than those on twice-daily regimen (Log Rank Chi(2)P=0.009). CONCLUSION Our study showed that DRV/r administrated both once daily or twice daily was safe and well tolerated with few discontinuations due to adverse events.

High burden of transmitted HIV-1 drug resistance in Italian patients carrying F1 subtype

BACKGROUND Transmitted drug resistance (TDR) is mainly restricted to individuals carrying B subtype, with low prevalence among non-B subtypes when grouped together. Subtype F1 is the most frequent non-B variant found in subjects living in Italy, allowing a specific assessment of TDR associated with this clade. METHODS We analysed pol sequences of HIV-1-positive individuals carrying the F1 variant included in the Antiretroviral Resistance Cohort Analysis database in the 1998-2009 period. Mutations were analysed with the Surveillance Drug Resistance Mutation and the International AIDS Society lists for naive and treated patients, respectively. RESULTS Among 343 HIV-1-infected patients carrying an F1 subtype, resistance was evaluated in a subset of 221 patients whose treatment status was known (169 drug naive and 52 drug experienced). The prevalence of TDR was 15.4% (11.8% for nucleoside/nucleotide reverse transcriptase inhibitors, 6.5% for non-nucleoside reverse transcriptase inhibitors and 7.1% for protease inhibitors). Among the 169 naive patients, 75.1%, 10.1% and 7.1% were Italians, South Americans and Romanians, respectively. Heterosexuals were prevalent among Italians and Romanians, while men who have sex with men were predominant among South Americans. The overall frequency of TDR declined from 21.4% to 7.1% in the 1998-2009 period. Although no statistical difference was detected, the frequency of TDR was higher in South Americans (23.5%) compared with Italian and Romanian naive patients (15% and 8.3%, respectively). DISCUSSION Our study shows a remarkable frequency of TDR in the F1 subtype-infected population. The high prevalence of TDR detected in South American subjects is linked to the homosexual route of infection. However, TDR was considerably high also in Italian subjects harbouring the F1 subtype, deserving careful monitoring.

Low rate of virological failure and maintenance of susceptibility to HIV-1 protease inhibitors with first-line lopinavir/ritonavir-based antiretroviral treatment in clinical practice.

Protease inhibitor (PI)‐resistant HIV‐1 has hardly ever been detected at failed boosted PI‐based first‐line antiretroviral regimens in clinical trials. However, this phenomenon has not been investigated in clinical practice. To address this gap, data from patients starting a first‐line lopinavir/ritonavir (LPV/rtv)‐based therapy with available baseline HIV‐1 RNA load, a viral genotype and follow‐up viral load after 3 and 6 months of treatment were extracted from the Italian Antiretroviral Resistance Cohort Analysis (ARCA) observational database. Based on survival analysis, 39 (7.1%) and 43 (7.8%) of the 548 examined patient cases had an HIV‐1 RNA >500 and >50 copies/ml, respectively, after 6 months of treatment. Cox proportional hazard models detected baseline HIV‐1 RNA (RH 1.79, 95%CI 1.10–2.92 per 1 − log10 increase, P = 0.02) and resistance to the nucleoside backbone (RH 1.04, 95%CI 1.02–1.06 per 10‐point increase using the Stanford HIVdb algorithm, P < 0.001) as independent predictors of HIV‐1 RNA at >500 copies/ml, but not at the >50 copies/ml cutoff criteria. Higher baseline viral load, older patient age, heterosexual route of infection and use of tenofovir/emtricitabine were predictors of failure at month 3 using the 50‐copy and/or 500‐copy threshold. Resistance to LPV/rtv did not occur or increase in any of the available 36 follow‐up HIV‐1 genotypes. Resistance to the nucleoside backbone (M184V) developed in four cases. Despite the likely differences in patient population and adherence, both the low rate of virological failure and the lack of development of LPV/rtv resistance documented in clinical trials are thus confirmed in clinical practice. J. Med. Virol. 82:1996–2003, 2010.

Effectiveness of antiretroviral regimens containing abacavir with tenofovir in treatment-experienced patients: Predictors of virological response and drug resistance evolution in a multi-cohort study

Background:In treatment-naïve patients, a combination antiretroviral therapy (cART) containing tenofovir (TDF) and abacavir (ABC) with lamivudine leads to unacceptably high virological failure rates with frequent selection of reverse transcriptase mutations M184V and K65R. We explored the efficacy of at least 16 weeks of ABC + TDF-containing cART regimens in 307 antiretroviral-experienced HIV-1-infected individuals included in observational databases.Methods:Virological failure was defined as an HIV RNA > 400 copies/ml after at least 16 weeks of treatment. Patients had received a median of three prior cART regimens. Of these, 76% concomitantly received a potent or high genetic barrier regimen (with at least one protease inhibitor [PI]) or non-nucleoside reverse transcriptase inhibitor or thymidine analogue) while a third non-thymidine nucleoside analogue was used in the remaining patients.Results:The 1-year estimated probability of virological failure was 34% in 165 patients with HIV RNA > 400 copies/ ml at ABC + TDF regimen initiation. Independent predictors of virological failure were the absence of a potent or high genetic barrier cART, the higher number of cART regimens experienced, and the use of a new drug class. In the subset of 136 patients for whom there were genotypic resistance test results prior to ABC + TDF initiation, the virological failure (1-year estimated probability 46%) was independently predicted by the higher baseline viral load, the concomitant use of boosted PI, and the presence of reverse transcriptase mutation M41L. In 142 patients starting ABC + TDF therapy with HIV RNA £ 400 copies/ml, virological failure (1-year estimated probability 17%) was associated only with the transmission category. In a small subset of subjects for whom there were an available paired baseline and follow-up genotype (n = 28), the prevalence of most nucleoside analogue reverse transcriptase inhibitor resistance mutations decreased, suggesting a possible low adherence to treatment. No selection of K65R was detected.Conclusion:The virological response to ABC + TDF-containing regimens in this moderately-to-heavily treatmentexperienced cohort was good. Higher viral load and the presence of M41L at baseline were associated with worse virological responses, while the concomitant prescription of drugs enhancing the genetic barrier of the regimen conveyed a reduced risk of virological failure. The Appendix provides the names of other members of the MASTER cohort.