Angela Gonnelli

Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression.

In a preliminary cross‐sectional analysis of 109 human immunodeficiency virus type 1 (HIV‐1)‐infected subjects the presence of 2‐long terminal repeat (LTR) unintegrated circular HIV‐1 DNA in peripheral blood mononuclear cells (PBMC) was found to be associated with both symptomatic infection (P = 0.0037) and low CD4 counts (P = 0.0004). To investigate the prognostic significance of the presence of 2‐LTR HIV‐1 DNA, a subset of 23 2‐LTR‐negative and 25 2‐LTR‐positive asymptomatic individuals were followed up for 12–24 months. The two groups did not differ in terms of baseline CD4 counts, zidovudine (ZDV) therapy, and duration of HIV‐1 infection. Longitudinal analysis of CD4 values did not indicate a significantly different CD4 outcome between the two groups. However, when only ZDV‐treated subjects were considered, a significant (P = 0.042) decrease in CD4 counts was found at month 24 with respect to baseline in 2‐LTR‐positive (n = 12) but not in 2‐LTR‐negative (n = 11) patients. Moreover, when >40% CD4 loss from baseline and/or development of CDC stage B or C symptoms were considered as indicators of disease progression, there was a significantly higher number of events in the whole 2‐LTR‐positive group than in the whole 2‐LTR‐negative group (P = 0.0197 at month 12, P = 0.0299 at month 18, P = 0.0373 at month 24). Thus, the presence of 2‐LTR‐HIV‐1 DNA in PBMC merits further investigation as a simple, qualitative, molecular predictor of disease progression and decreased response to antiretroviral therapy. J. Med. Virol. 52:20–25, 1997.

Rules-based HIV-1 genotypic resistance interpretation systems predict 8 week and 24 week virological antiretroviral treatment outcome and benefit from drug potency weighting

OBJECTIVES To test retrospectively the ability of four freely available rules-based expert systems to predict short- and medium-term virological outcome following an antiretroviral treatment switch in pre-treated HIV-1 patients. METHODS The HIV-1 genotype interpretation systems (GISs) HIVdb, ANRS, Rega and AntiRetroScan were tested for their accuracy in predicting response to highly active antiretroviral therapy using 8 week (n = 765) and 24 week (n = 634) follow-up standardized treatment change episodes extracted from the Italian Antiretroviral Resistance Cohort Analysis (ARCA) database. A genotypic sensitivity score (GSS) was derived for each genotype-treatment pair for the different GISs and tested as a predictor of virological treatment outcome by univariable and multivariable logistic regression as well as by receiver operating characteristic curve analysis. The two systems implementing drug potency weights (AntiRetroScan and Rega) were evaluated with and without this correction factor. RESULTS All four GSSs were strong predictors of virological treatment outcome at both 8 and 24 weeks after adjusting for baseline viro-immunological parameters and previous drug exposure (odds ratios ranging from 2.04 to 2.43 per 1 unit GSS increase; P < 0.001 for all the systems). The accuracy of AntiRetroScan and Rega was significantly increased by drug potency weighting with respect to the unweighted versions (P <or= 0.001). HIVdb and ANRS also increased their performance with the same drug potency weighting adopted by AntiRetroScan and Rega, respectively (P < 0.001 for both analyses). CONCLUSIONS Currently available GISs are valuable tools for assisting antiretroviral treatment choices. Drug potency weighting can increase the accuracy of all systems.

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]).

Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010

Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviral-naïve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR.

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-

Zidovudine resistance mutations and human immunodeficiency virus type 1 DNA burden: Longitudinal evaluation of six patients under treatment

SummaryZidovudine (ZDV) is by far the most widely used drug to counteract human immunodeficiency virus type 1 (HIV-1) infection, both in monotherapy and in combination therapy regimens. However, the majority of patients under prolonged ZDV therapy have been shown to harbour HIV-1 mutant genomes displaying reduced sensitivity to the drugin vitro. In order to investigate the pathogenic role ofin vitro resistance to ZDV, six HIV-1-infected ZDV-treated subjects were evaluated longitudinally (mean follow-up 28.5 months, range 12–39 months) for HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) and for the presence of HIV-1pol gene mutations responsible for ZDV resistance. Quantitation of HIV-1 DNA was performed by competitive polymerase chain reaction (cPCR) and thepol genotype was determined by direct sequencing of PCR products. All of the six patients developed one or more of the HIV-1pol mutations known to confer resistance to ZDVin vitro (Met41→Leu, Asp67→Asn, Lys70→Arg, Thr215→Phe/Tyr, Lys219→Gln/Glu). A temporal association was found between HIV-1 DNA burden and the level of ZDV resistance, as predicted on the basis of thepol genotype (genotypic resistance). Both virus load and ZDV resistance were inversely correlated with CD4+ cell counts. These results are compatible with a directin vivo pathogenetic role forpol gene mutations shown to be involved in resistance to ZDVin vitro. Monitoring the degree of genotypic resistance to ZDV and to other antiretroviral drugs should be considered in designing protocols for the management of treated patients.ZusammenfassungZidovudin (ZDV) ist das Medikament, das weitaus am häufigsten in Monotherapie oder Kombinationstherapie zur Behandlung von Infektionen durch HIV-1 eingesetzt wird. Unter Langzeitbehandlung mit ZDV wurden jedoch bei der Mehrzahl der Patienten HIV-1-Mutanten mit reduzierterIn-vitro-Empfindlichkeit gegen das Medikament nachgewiesen. Um die pathogene Bedeutung derIn vitro-Resistenz gegen ZDV zu untersuchen, wurden die HIV-1 DNA-Last in mononukleären Zellen des peripheren Blutes und das Vorliegen von für die ZDV-Resistenz verantwortlichen HIV-1pol-Genmutationen bei sechs HIV-infizierten Patienten unter ZDV-Therapie in einer Längsschnittstudie (mit mittlerer Beobachtungszeit von 28.5 Monaten, Bereich 12–39 Monate) gemessen. Die quantitative Bestimmung der HIV-1 DNA erfolgte durch kompetitive Polymerasekettenreaktion (cPCR). Derpol-Genotyp wurde durch direkte Sequenzierung der PCR-Produkte ermittelt. Alle sechs Patienten entwickelten eine oder mehrere der HIV-1 Mutationen, deren Assoziation mitIn vitro-Resistenz gegen ZDV bekannt ist (Met41→Leu, Asp67→Asn, Lys70→Arg, Thr215→Phe/Tyr, Lys219→Gln/Glu). Zwischen dem auf der Basis derpol-Genotypen (Genotypresistenz) vorhergesagten Spiegel der ZDV-Resistenz und der HIV-1 DNA-Last fand sich eine zeitliche Assoziation. Sowohl die Viruslast wie auch die ZDV-Resistenz standen in umgekehrter Beziehung zu den CD4+-Zellzahlen. Diese Ergebnisse sind mit direkten pathogenenin vivo-Effekten der für die ZDV-Resistenzin vitro verantwortlichenpol-Genmutationen vereinbar. Das Monitoring des Ausmaßes genotypischer Resistenz gegen ZDV und andere antiretrovirale Substanzen sollte bei der Entwicklung von Behandlungsprotokollen berücksichtigt werden.

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.

Genotypic resistance to zidovudine as a predictor of failure of subsequent therapy with human immunodeficiency virus type-1 nucleoside reverse-transcriptase inhibitors

Abstract To define factors predictive of failure to respond to nucleoside reverse-transcriptase inhibitors in human immunodeficiency virus type-1 (HIV-1)-infected subjects pretreated with zidovudine (ZDV), three groups of subjects shifted to double therapy with ZDV plus didanosine (ddI, n=13), zalcitabine (ddC, n=14), or lamivudine (3TC, n=12) were retrospectively evaluated, with respect to addition of the second NRTI, at week 0 and week 24. Factors considered included duration of ZDV pretreatment, CD4+ cell counts, plasma HIV-1 RNA load, peripheral blood mononuclear cell HIV-1 DNA load, and HIV-1 DNA genotypic resistance to nucleoside reverse-transcriptase inhibitors. The three groups were well matched for baseline characteristics and did not differ significantly in virological and immunological response to the different combination treatments. Drug-specific resistance mutations were selected in more than half the cases by 3TC, but not by ddI and ddC. Low-level and substantial genotypic resistance to ZDV was detected 13 (33.3%) and in 19 (48.7%) patients at baseline, respectively, and evolved through week 24 in several patients. When subjects were divided into responders and nonresponders to the second nucleoside reverse-transcriptase inhibitor on the basis of a decrease of more than 0.5 log10 (n=15) or less than 0.5 log10 (n=21) in HIV-1 RNA, respectively, baseline genotypic ZDV resistance was the only independent predictor of failure in a logistic regression model (P=0.003 or P=0.024, depending on whether low-level resistance was considered or not, respectively). Thus, selection of ZDV resistance mutations may impair subsequent use of different nucleoside reverse-transcriptase inhibitor compounds.

Concordance between polymerase chain reaction and antibody detection in the diagnosis of human immunodeficiency virus type 1 infection

A highly sensitive nested polymerase chain reaction (PCR) protocol was used to detect human immuno-deficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells from 271 HIV-1-seropositive patients, 240 HIV-1-seronegative subjects at increased risk for HIV-1 infection, 51 serologically indeterminate individuals, and 120 healthy blood donors. PCR was carried out in a multiplex nested configuration with pol and env region primer sets. HIV-1 DNA was detected in all of the HIV-1 seropositive patients. In contrast, HIV-1 DNA was not detected in any of the either seronegative or serologically indeterminate subjects. Only one of 37 seronegative regular sexual partners of HIV-1-infected patients who were followed longitudinally was found to seroconvert to HIV-1. However, HIV-1 DNA and antibody results were concordant in the four samples obtained from this subject prior to and after seroconversion. These results show an excellent concordance between HIV-1 DNA and antibody detection for diagnosis of HIV-1 infection and suggest that long-term HIV-1 infection in the absence of detectable antibody is likely to occur at a very low frequency.