Search

Background: European guidelines recommend treatment of chronic hepatitis B virus infection (CHB) with the nucleos(t)ide analogs (NAs) entecavir or tenofovir. However, many European CHB patients have been exposed to other NAs, which are associated with therapy failure and resistance. The CAPRE study was performed to gain insight in prevalence and characteristics of NA resistance in Europe., Methods: A survey was performed on genotypic resistance testing results acquired during routine monitoring of CHB patients with detectable serum hepatitis B virus DNA in European tertiary referral centers., Results: Data from 1568 patients were included. The majority (73.8%) were exposed to lamivudine monotherapy. Drug-resistant strains were detected in 52.7%. The most frequently encountered primary mutation was M204V/I (48.7%), followed by A181T/V (3.8%) and N236T (2.6%). In patients exposed to entecavir (n = 102), full resistance was present in 35.3%. Independent risk factors for resistance were age, viral load, and lamivudine exposure (P < .001)., Conclusions: These findings support resistance testing in cases of apparent NA therapy failure. This survey highlights the impact of exposure to lamivudine and adefovir on development of drug resistance and cross-resistance. Continued use of these NAs needs to be reconsidered at a pan-European level., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Dual therapy refers to combinations of two antiretroviral drugs applied in different clinical settings; they are considered and studied due to possibly reduced drug toxicities. In antiretroviral-naive patients, dual combinations have lower virologic efficacy than standard therapy; the sole efficacious regimen is lamivudine plus lopinavir/ritonavir. Due to a higher possibility of virologic failure, these regimens are generally not allowed in this clinical setting. In antiretroviral-experienced patients, dual regimens are examined in studies with a small sample size, centered on clinical practice, and should be ritonavir-boosted protease inhibitor-based. These combinations have a good virological efficacy; combinations with the integrase inhibitor raltegravir have small sample size and demonstrated efficacy only with etravirine. Virological aspects involving dual therapy should always consider genetic barriers, particularly in simplification strategies, and ritonavir-boosted protease inhibitors are mandatory. As far as immunological aspects are concerned, nucleoside reverse transcriptase inhibitor-sparing regimens have some encouraging data, probably due to the bone marrow toxicity of this class. Combinations with maraviroc were effective in reducing inflammation, but data about immunological recovery are conflicting. The choice of regimen should focus on specific class toxicity since dual regimens are studied in particular for improving safety and tolerability. This review will analyze different dual regimens in the clinical setting, with a peculiar focus on ameliorating toxicities and improving quality of life.

Objectives: To evaluate the replication capacity and phenotypic susceptibility to dolutegravir and raltegravir of wild-type and raltegravir-resistant HIV-1 strains in several cellular systems., Methods: The antiviral activities of dolutegravir and raltegravir were evaluated in human primary monocyte-derived macrophages (MDMs), peripheral blood mononuclear cells (PBMCs) and C8166 T lymphocytic cells. The following raltegravir resistance mutations were analysed: N155H, Y143C, N155H + Y143C and G140S+Q148H., Results: In the absence of drug, the replication capacity of raltegravir-resistant viruses was strongly reduced compared with wild-type in all cellular models analysed. In MDMs and PBMCs, a dramatic decrease in viral replication was observed for the double mutants N155H + Y143C and G140S + Q148H (ranging from 0.1% to 2.5% compared with wild-type). In MDMs, dolutegravir exhibited high potency, with EC50 and EC90 values of 1.1 ± 0.9 and 5.5 ± 3.4 nM, respectively (comparable to raltegravir). These values (particularly for EC90) were significantly lower than those observed in PBMCs (EC50: 2.7 ± 1.5 nM; EC90: 14.8 ± 0.9 nM) and C8166 cells (EC50: 5.5 ± 0.8 nM; EC90: 64.8 ± 5.8 nM). In all cellular models analysed, dolutegravir showed full efficacy against N155H and Y143C mutants (dolutegravir fold-change resistance ranging from 0.1 to 1.4; raltegravir fold-change resistance ranging from 0.1 to 10.3). In C8166 (the only cell model in which replication capacity was sufficient to perform the test) dolutegravir showed full efficacy against mutations N155H + Y143C (dolutegravir fold-change resistance: 0.6) and a slightly lower activity against G140S+Q148H (dolutegravir fold-change resistance: 2.1)., Conclusions: Dolutegravir is effective in different HIV cellular targets and against raltegravir-resistant mutants. The high efficacy of dolutegravir in MDMs (cells with limited metabolism) has relevant clinical implications in light of the role of MDMs in the transmission of HIV infection and dissemination in different body compartments., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

While the selection of complex HBV drug-resistance patterns on therapeutic failure can compromise the efficacy of anti-HBV therapies, recent data show that patients failing treatment without drug-resistance have a rate of virological success close to drug-naive patients. The goal of this study is defining, in clinical practice, the burden of drug-resistance mutations in a cohort of patients treated with anti-HBV drugs. Prevalence and patterns of drug-resistance were analyzed by RT-sequencing in 204 patients infected chronically: 148 experiencing virological rebound (defined as an increase in serum HBV-DNA > 20 IU/ml after achieving virological success [HBV-DNA < 20 IU/ml]), and 56 null/partial responders (always detectable serum HBV-DNA [>20 IU/ml] within 48 weeks of therapy). The highest rate of drug-resistance was observed in patients experiencing virological rebound (prevalence, 79.1%). Conversely, almost half (46.4%) null/partial responders have no evidence of drug-resistance. The rate of drug-resistance was higher in patients treated with lamivudine (76.8% [109/142]) and telbivudine (83.3% [5/6]), followed by adefovir (62.5% [15/24]), and entecavir (52.2% [12/23]). Complex mutational patterns characterized by the co-presence of rtM204V/I-rtA181T/V (impairing the efficacy of all anti-HBV drugs) were detected in four patients (2.7%) with virological rebound. Drug-resistance is the main cause of failure to therapy in patients experiencing virological rebound, supporting the need of rapid switch to anti-HBV drugs with higher genetic barrier and potency (entecavir/tenofovir). Conversely, nearly half of null/partial responders shows no evidence of drug-resistance mutations, maintaining high chance of achieving therapeutic success with the same class of drug. In this setting, genotypic resistance may help in selecting patients still carrying wild-type viruses, that may take major benefits from antiviral treatment., (Copyright © 2013 Wiley Periodicals, Inc.)

Objective: To describe the prevalence of the L76V protease inhibitors resistance-associated mutation (PI-RAM) in relation with patients' characteristics and protease genotypic background in HIV-1 B- and "non-B"-infected patients., Methods: Frequency of the L76V mutation between 1998 and 2010 was surveyed in the laboratory database of 3 clinical centers. Major PI-RAMs were identified according to the IAS-USA list. Fisher's and Wilcoxon tests were used to compare variables., Results: Among the overall 29,643 sequences analyzed, the prevalence of L76V was 1.50%, while was 5.42% in PI-resistant viruses. Since 2008 the prevalence of L76V was higher in "non-B"-infected than in B-infected patients each year. Median time since diagnosis of HIV-1 infection and median time under antiretroviral-based regimen were both shorter in "non-B"- than in B-infected patients (8 vs 11 years, P<0.0001; and 7 vs 8 years, P = 0.004). In addition, "non-B"-infected patients had been pre-exposed to a lower number of PI (2 vs 3, P = 0.016). The L76V was also associated with a lower number of major PI-RAMs in "non-B" vs B samples (3 vs 4, P = 0.0001), and thus it was more frequent found as single major PI-RAM in "non-B" vs B subtype (10% vs 2%, P = 0.014)., Conclusions: We showed an impact of viral subtype on the selection of the L76V major PI-RAM with a higher prevalence in "non-B" subtypes observed since 2008. In addition, in "non-B"-infected patients this mutation appeared more rapidly and was associated with less PI-RAM.

Background: Resistance to the fusion inhibitor enfuvirtide (ENF) is achieved by changes in the gp41 subunit of the HIV envelope glycoprotein (Env). Specific ENF-associated mutational pathways correlate with immunological recovery, even after virological failure, suggesting that the acquisition of ENF resistance alters gp41 pathogenicity. To test this hypothesis, we have characterized the expression, fusion capability, induction of CD4+ T cell loss and single CD4+ T cell death of 48 gp41 proteins derived from three patients displaying different amino acids (N, T or I) at position 140 that developed a V38A mutation after ENF-based treatment., Results: In all cases, intra-patient comparison of Env isolated pre- or post-treatment showed comparable values of expression and fusogenic capacity. Furthermore, Env with either N or T at position 140 induced comparable losses of CD4+ T-cells, irrespective of the residue present at position 38. Conversely, Env acquiring the V38A mutation in a 140I background induced a significantly reduced loss of CD4+ T cells and lower single-cell death than did their baseline controls. No altered ability to induce single-cell death was observed in the other clones., Conclusions: Overall, primary gp41 proteins with both V38A and N140I changes showed a reduced ability to induce single cell death and deplete CD4+ T cells, despite maintaining fusion activity. The specificity of this phenotype highlights the relevance of the genetic context to the cytopathic capacity of Env and the role of ENF-resistance mutations in modulating viral pathogenicity in vivo, further supporting the hypothesis that gp41 is a critical mediator of HIV pathogenesis.

Background: For the expression of late viral genes, HIV-1 efficiently exploits the nuclear export by using Rev viral protein, which specifically binds the RNA Rev Responsive Element (RRE). This region is contained within the gp120-gp41 encoding sequence. Enfuvirtide is the first approved HIV-1 fusion-inhibitor, and gp41 codons associated with primary enfuvirtide-resistance (amino-acids 36-45) are localized within the RRE structure. We previously found the co-presence of V38A+T18A resistance mutations in patients failing enfuvirtide., Methods: Collecting 476 and 135 HIV-1 B-subtype gp41 sequences from enfuvirtide-naïve and enfuvirtide-treated patients, respectively, two mutations previously found associated with enfuvirtide treatment, T18A and V38A, were analyzed. Moreover, the RNA secondary structure was displayed by CONTRAfold-software and the gp41 evolutionary pathways by a mutagenetic tree., Results: By modeling the RRE structure, we show that the T18 and V38 codons are base pairing within the RRE-stem-IIA, an important domain involved in Rev binding. While a structural RRE impairment in the presence of V38A alone was found, a restoration of the original RRE structure occurred in co-presence of V38A+T18A. By mutagenetic tree analysis, a compensatory evolution confirming our hypothesis on the structural modification mechanism was observed., Conclusion: We show that enfuvirtide pressure may also affect specific RRE domains involved in Rev binding, thus requiring a compensatory evolution able to preserve the secondary structure of the RRE., (Copyright © 2011 S. Karger AG, Basel.)

Background: Impact of hepatitis B virus genetic barrier, defined as the number and type of nucleotide substitutions required to overcome drug/immune selective pressure, on drug-resistance/immune-escape development is unknown., Methods: Genetic barrier was calculated according to Van de Vijver (2006) in 3482 hepatitis B virus-reverse transcriptase/HBV surface antigen sequences from 555 drug-naïve patients and 2927 antiviral-treated patients infected with hepatitis B virus genotypes A-G., Results: Despite high natural variability, genetic barrier for drug-resistance development is identical amongst hepatitis B virus genotypes, but varies according to drug-resistance mutation type. Highest genetic barrier is found for secondary/compensatory mutations (e.g. rtL80I/V-rtL180M-rtV173L), whilst most primary mutations (including rtM204V-rtA181T/V-rtI169T-rtA194T) are associated with low genetic barrier. An exception is rtM204I, which can derive from a transition or a transversion. Genotypes A and G are more prone to develop immune/diagnostic-escape mutations sT114R and sG130N. Vaccine-escape associated sT131N-mutation is a natural polymorphism in both A and G genotypes., Conclusion: Genetic barrier and reverse transcriptase/HBV surface antigen overlapping can synergistically influence hepatitis B virus drug-resistance/immune-escape development. The different immune-escape potential of specific hepatitis B virus genotypes could have important clinical consequences in terms of disease progression, vaccine strategies and correct HBV surface antigen detection., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Presence of drug-resistance mutations in drug-naïve hepatitis B virus (HBV) infected patients can seriously compromise response to antiviral treatment. Therefore, our study was aimed at defining the prevalence of HBV drug-resistance in a population of 140 patients, all infected with HBV-D-genotype (the most common HBV-genotype in Eastern Europe, Mediterranean countries and Middle East) and naïve to antiviral therapy. HBV reverse-transcriptase (RT) region was sequenced and analyzed for 20 mutations, confirmed by in vitro studies as associated with resistance to nucleos(t)ide HBV-RT inhibitors (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C/G/I-rtM204V/I-rtN236T-rtM250V). Amino acid changes at other six RT positions, potentially associated with resistance, were also analyzed (rtV84M-rtV191I-rtV207L-rtV214A-rtQ215S-rtI233V). Overall, only 2/140 (1.4%) patients carried primary drug-resistance mutations [rtA181V (0.7%), and rtA194T (0.7%)], while 3/140 (2.1%) patients harbored the secondary mutations rtV173L (1.4%) and rtL180M (0.7%). Additionally, five polymorphic mutations, with a suggested role in drug resistance, were detected [rtQ215S (12.8%), rtI233V (4.3%), rtV214A (3.6%), rtV191I (0.7%), rtV207L (0.7%)]. Notably, no YMDD mutations, namely rtM204V/I, were found. Taken together, the rate of important drug resistance mutations in naïve HBV D-genotype infected patients is today very low, and suggests the potential full efficacy of new-generation antiviral drugs used in first line therapy. Whether such low rate can be extrapolated to non HBV-D subtypes, requires a detailed investigation to be performed in a different cohort of patients., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In recent years relevant progress has been made in the treatment of HIV-1 with a consequent decrease in mortality. The availability of potent antiretroviral drugs and the ability of viral load assays that accurately evaluate the true level of viral replication, have led to a better understanding of pathogenesis of the disease and how to obtain improved therapeutic profiles. The highly active antiretroviral therapy (HAART), based on a combination of three or more antiretroviral drugs, has radically changed the clinical outcome of HIV. In particular, reverse transcriptase non-nucleoside inhibitors (NNRTIs) play an essential role in most protocols and are often used in first line treatment. The high specificity of these inhibitors towards HIV-1 has increased the number of structural and molecular modeling studies of enzyme complexes and that have led to chemical syntheses of more selective second and third-generation NNRTIs. However, a considerable percentage of new HIV-1 infections are caused by the emergence of drug-resistant mutant viruses that complicate treatment strategies. In this review we discuss relevant clinical and structural aspects for the management of antiretroviral drug resistance, with detailed explanations of mechanisms and mutation patterns useful to better understand the relation between drug resistance and therapy failure., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: To investigate lamivudine (LAM)-resistance profiles of hepatitis B virus (HBV) at the early stages of virological breakthrough (serum HBV-DNA 12-345IU/ml) or when HBV-DNA is undetectable., Methods: Sixty-four HBV-mono-infected patients were enrolled: 25 had virological breakthrough with serum HBV-DNA ranging from 12 to 345IU/ml during first-line LAM-monotherapy; 24 were on LAM-monotherapy, and 15 were on LAM+adefovir dipivoxil (ADV) with undetectable serum HBV-DNA (<12IU/ml)., Results: HBV-reverse transcriptase was successfully sequenced in 22 (88.0%) LAM-treated patients with HBV-DNA between 12 and 345IU/ml, and in 12 (30.8%) patients receiving LAM (±ADV) with HBV-DNA<12IU/ml. Drug-resistance mutations were observed in 17 (77.2%) LAM-treated patients with virological breakthrough: 8 M204V, 7 M204I, 1 M204I/V, and 1 A181T. One or ≥2 compensatory mutations were found in 10 (58.8%) and in 4 (23.5%) patients. Drug-resistance mutations were present also in patients with undetectable serum HBV-DNA: M204I was detected in 2 patients receiving LAM-monotherapy, and V84M in 1 patient receiving LAM+ADV., Conclusion: Overall findings support the existence of drug-resistance mutations even at very low levels of viral replication. The persistence of low-level HBV replication and consequent drug-resistance emergence should be considered when choosing therapeutic strategies., (Copyright © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: To investigate genotypic resistance profiles to emtricitabine + tenofovir (FTC + TDF) in-vivo and in-vitro, and compare them with lamivudine + tenofovir (3TC + TDF)., Methods: Three hundred fifty-two HIV-1 B-subtype pol sequences from 42 FTC + TDF-treated patients, 40 3TC + TDF-treated patients, and 270 patients treated with 3TC plus another nucleoside reverse transcriptase inhibitor (but not TDF). All patients never received FTC, 3TC, and TDF in their previous therapeutic regimen. 3TC/FTC ± TDF resistance was investigated using in vitro selection experiments and docking simulations., Results: The M184V mutation is less prevalent in FTC + TDF-treated patients than in 3TC + TDF-treated, and 3TC-treated/TDF-naive patients (14.3% versus 40.0%, P = 0.01 and 55.6%, P < 0.001). Multivariable analysis shows that factors correlated with a lower probability of M184V emergence at failure were the use of FTC compared with 3TC [odds ratio (OR): 0.32 (95% confidence interval (CI): 0.10 to 0.99), P = 0.04], the use of boosted protease inhibitor, and the use of TDF [OR: 0.20 (95% CI: 0.11 to 0.37), P < 0.001, and OR: 0.47 (95%CI: 0.22 to 1.01), P = 0.05, respectively]. In vitro selection experiments and docking analysis show that other reverse transcriptase (RT) mutations, even localized in RT connection domain, can be selected by 3TC + TDF or FTC + TDF in M184V absence and can affect RT affinity for 3TC/FTC and/or TDF., Conclusions: Our study shows lower rates of M184V development in FTC + TDF regimens versus 3TC + TDF and suggests a potential role of boosted protease inhibitors and TDF in delaying the M184V emergence. Novel RT mutational patterns, more complex than currently known, can contribute to 3TC, FTC, and TDF resistance.

Background: The transmission of HIV-1 drug-resistant strains in drug naive patients may seriously compromise the efficacy of a first-line antiretroviral treatment. To better define this problem, a study in a cohort of newly diagnosed HIV-1 infected individuals has been conducted. This study is aimed to assess the prevalence and the patterns of the mutations recently associated with transmitted drug resistance in the reverse transcriptase (RT) and in protease (PR) of HIV-1., Methods: Prevalence of transmitted drug resistant strains is determined in 255 newly diagnosed HIV-1 infected patients enrolled in different counselling and testing (CT) centres in Central Italy; the Avidity Index (AI) on the first available serum sample is also used to estimate time since infection. Logistic regression models are used to determine factors associated with infection by drug resistant HIV-1 strains., Results: The prevalence of HIV-1 strains with at least one major drug resistance mutation is 5.9% (15/255); moreover, 3.9% (10/255) of patients is infected with HIV nucleoside reverse transcriptase inhibitor (NRTI)-resistant viruses, 3.5% (9/255) with HIV non-NRTI-resistant viruses and 0.4% (1/255) with HIV protease inhibitor (PI)-resistant viruses. Most importantly, almost half (60.0%) of patients carries HIV-1 resistant strains with more than one major drug resistance mutation. In addition, patients who had acquired HIV through homosexual intercourses are more likely to harbour a virus with at least one primary resistance mutation (OR 7.7; 95% CI: 1.7-35.0, P = 0.008)., Conclusion: The prevalence of drug resistant HIV-1 strains among newly diagnosed individuals in Central Italy is consistent with the data from other European countries. Nevertheless, the presence of drug-resistance HIV-1 mutations in complex patterns highlights an additional potential risk for public health and strongly supports the extension of wide genotyping to newly diagnosed HIV-1 infected patients.

The gp41-encoding sequence of the env gene contains in two separate regions the Rev-responsive elements (RRE) and the alternative open reading frame of the second exon of the regulatory protein Rev. The binding of Rev to the RRE allows the transport of unspliced/singly spliced viral mRNAs out of the nucleus, an essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1). In this study, we have investigated whether the fusion-inhibitor enfuvirtide (ENF) can induce mutations in Rev and if these mutations correlate with the classical ENF resistance gp41 mutations and with viremia and CD4 cell count. Specific Rev mutations were positively associated with ENF treatment and significantly correlated with classical ENF resistance gp41 mutations. In particular, a cluster was observed for the Rev mutations E57A (E57A(rev)) and N86S(rev) with the ENF resistance gp41 mutations Q40H (Q40H(gp41)) and L45M(gp41). In addition, the presence at week 48 of the E57A(rev) correlates with a significant viremia increase from baseline to week 48 and with a CD4 cell count loss from baseline to week 48. By modeling the RRE structure, we found that the Q40(gp41) and L45(gp41) codons form complementary base pairs in a region of the RRE involved in Rev binding. The conformation of this Rev-binding site is disrupted when Q40H(gp41) and L45M(gp41) occur alone while it is restored when both mutations are present. In conclusion, our study shows that ENF pressure may also affect both Rev and RRE structures and can provide an excellent example of compensatory evolution. This highlights the multiple roles of ENF (and perhaps other entry inhibitors) in modulating the correct interplay between the different HIV-1 genes and proteins during the HIV-1 life cycle.

Abstract To date, very little information is available regarding the evolution of drug resistance mutations during treatment interruption (TI). Using a survival analysis approach, we investigated the dynamics of mutations associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTIs) during TI. Analyzing 132 patients having at least two consecutive genotypes, one at last NRTI-containing regimen failure, and at least one during TI, we observed that the NRTI resistance mutations disappear at different rates during TI and are lost independently of each other in the majority of patients. The disappearance of the K65R and M184I/V mutations occurred in the majority of patients, was rapid, and was associated with the reemergence of wild-type virus, thus showing their negative impact on viral fitness. Overall, it seems that the loss of NRTI drug resistance mutations during TI is not an ordered process, and in the majority of patients occurs without specific interaction among mutations.

Background: Human immunodeficiency virus type 1 (HIV-1) gp41 is a crucial determinant for HIV-1 pathogenicity. We investigated the correlation of enfuvirtide (ENF)-associated gp41 mutational clusters with viroimmunological parameters, as well as the potential underlying mechanisms., Methods: A total of 172 gp41 sequences and clinical follow-up data from 73 ENF-treated patients were analyzed monthly, from baseline to week 48., Results: There were 7 novel gp41 mutations positively associated with ENF treatment and correlated with classic ENF mutations. The ENF-associated clusters [V38A + N140I ] and [V 38A +T18A ] significantly correlated with an increase in CD4 cell count at week 48 ( an increase from baseline of 112 and 209 cells/microL, respectively), whereas [Q40H + L45M + 268A] significantly correlated with a decrease in CD4 cell count (-53 cells/microL), without a change in the level of viremia. Residues 38 and 18 are located complementarily to each other in the Rev-responsive element, whereas analysis of molecular dynamics showed that the copresence of [V38A + N140 I] abolishes the interaction between residue 38 and 145 important for stabilization of the 6-helix bundle. In contrast, T268A localizes in the gp41 calmodulin-binding domain responsible for gp41-induced CD4(+) T lymphocyte apoptosis., Conclusion: Specific gp41 mutational clusters associated with ENF treatment significantly correlate with increases in CD4(+) cell count. Structural analysis suggests that this immunological gain is associated with mechanisms that act at both the protein level and the RNA level (even under conditions of virological failure). This result may help in the selection of patients who can benefit most from ENF treatment and represents a driving force for the design of the next generation of entry inhibitors.

Resistance to antivirals is a complex and dynamic phenomenon that involves more mutations than are currently known. Here, we characterize 10 additional mutations (L74V, K101Q, I135M/T, V179I, H221Y, K223E/Q, and L228H/R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase which are involved in the regulation of resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs). These mutations are strongly associated with NNRTI failure and strongly correlate with the classical NNRTI resistance mutations in a data set of 1,904 HIV-1 B-subtype pol sequences from 758 drug-naïve patients, 592 nucleoside reverse transcriptase inhibitor (NRTI)-treated but NNRTI-naïve patients, and 554 patients treated with both NRTIs and NNRTIs. In particular, L74V and H221Y, positively correlated with Y181C, were associated with an increase in Y181C-mediated resistance to nevirapine, while I135M/T mutations, positively correlated with K103N, were associated with an increase in K103N-mediated resistance to efavirenz. In addition, the presence of the I135T polymorphism in NNRTI-naïve patients significantly correlated with the appearance of K103N in cases of NNRTI failure, suggesting that I135T may represent a crucial determinant of NNRTI resistance evolution. Molecular dynamics simulations show that I135T can contribute to the stabilization of the K103N-induced closure of the NNRTI binding pocket by reducing the distance and increasing the number of hydrogen bonds between 103N and 188Y. H221Y also showed negative correlations with type 2 thymidine analogue mutations (TAM2s); its copresence with the TAM2s was associated with a higher level of zidovudine susceptibility. Our study reinforces the complexity of NNRTI resistance and the significant interplay between NRTI- and NNRTI-selected mutations. Mutations beyond those currently known to confer resistance should be considered for a better prediction of clinical response to reverse transcriptase inhibitors and for the development of more efficient new-generation NNRTIs.

We characterized 16 additional mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) whose role in drug resistance is still unknown by analyzing 1,906 plasma-derived HIV-1 subtype B pol sequences from 551 drug-naïve patients and 1,355 nucleoside RT inhibitor (NRTI)-treated patients. Twelve mutations positively associated with NRTI treatment strongly correlated both in pairs and in clusters with known NRTI resistance mutations on divergent evolutionary pathways. In particular, T39A, K43E/Q, K122E, E203K, and H208Y clustered with the nucleoside analogue mutation 1 cluster (NAM1; M41L+L210W+T215Y). Their copresence in this cluster was associated with an increase in thymidine analogue resistance. Moreover, treatment failure in the presence of K43E, K122E, or H208Y was significantly associated with higher viremia and lower CD4 cell count. Differently, D218E clustered with the NAM2 pathway (D67N+K70R+K219Q+T215F), and its presence in this cluster determined an increase in zidovudine resistance. In contrast, three mutations (V35I, I50V, and R83K) negatively associated with NRTI treatment showed negative correlations with NRTI resistance mutations and were associated with increased susceptibility to specific NRTIs. In particular, I50V negatively correlated with the lamivudine-selected mutation M184V and was associated with a decrease in M184V/lamivudine resistance, whereas R83K negatively correlated with both NAM1 and NAM2 clusters and was associated with a decrease in thymidine analogue resistance. Finally, the association pattern of the F214L polymorphism revealed its propensity for the NAM2 pathway and its strong negative association with the NAM1 pathway. Our study provides evidence of novel RT mutational patterns that regulate positively and/or negatively NRTI resistance and strongly suggests that other mutations beyond those currently known to confer resistance should be considered for improved prediction of clinical response to antiretroviral drugs.

Phylogenetic analysis and evaluation of drug-resistance were carried out upon 59 plasma samples from 58 treatment-naïve HIV-1 infected patients from Mozambique, enrolled in a free antiviral-therapy protocol in the frame of Drug-Resource-Enhancement against AIDS and Malnutrition (DREAM) programme. Sequencing of the first 1,300 bases of the pol-gene shows that all virus strains cluster within clade C, with the exception of a single patient carrying a G-subtype virus. Relevant mutations in the reverse transcriptase (RT) are rare: 118A/I/L/G (four patients), 179E/D/I (three patients), 333E/D (two patients), 101R, and 210F (one patient each). In Protease (PR), V82I (10.3%) is the only relevant mutation, while natural polymorphisms/secondary mutations are found, some at very high frequency: 20R (25.9%), 36I (91.4%), 36L (8.6%), 60E (31.0%), 63P (29.3%), and 93L (96.6%). Among them, mutations with a frequency >25% were further investigated to assess their covariation pattern with PI resistance associated mutations. The pattern of covariation observed for K20R and D60E (but not L63P and M36I) was different between C and B subtype isolates from PR-inhibitor-treated patients. The sequences were also analyzed to calculate the ratio of non-synonymous to synonymous substitution. The ratio for PR and RT was 0.116 and 0.093, respectively, suggesting a greater conservation in RT than PR in both subtypes B and C HIV strains. Taken together, the results demonstrate a consistent clade-homogeneity of viral strains circulating in Mozambique, and the very limited presence, in drug-naïve patients, of mutations associated with resistance to RT-inhibitors. The high frequency of secondary mutations/polymorphisms in HIV-PR deserves further studies to evaluate its relevance in clinical settings., ((c) 2005 Wiley-Liss, Inc.)

The evaluation of resistance test perception by clinicians over the years 1999--2003 was assessed in an Italian cohort. The results on 2233 samples from 1416 HIV-1 infected patients show an increase in HIV-1 drug resistance test requests over time, with a plateau reached in the last three years. CD4-cell count at the time of genotype request progressively increased. In particular, the median CD4 cell count of drug-treated patients increased from 221x10(6) cells/l [interquartile range (IQR): 109-368] in 1999 to 296x10(6) cells/l (IQR: 166-478) in 2003 (p<0.0001). At the same time, plasma HIV-RNA level progressively decreased from a median of 103,500 copies/ml (IQR: 37,250-260,000) in 1999 to 9,444 copies/ml (IQR: 2,086-41,281) in 2003 (p<0.0001). Overall, data suggest that the genotype test is increasingly considered, and requested also for patients at earlier stages of drug history and/or at less severe disease stage.