Mayte Pérez-Olmeda

Understanding HIV-1 latency provides clues for the eradication of long-term reservoirs

HIV-1 can infect both activated and resting, non-dividing cells, following which the viral genome can be permanently integrated into a host cell chromosome. Latent HIV-1 reservoirs are established early during primary infection and constitute a major barrier to eradication, even in the presence of highly active antiretroviral therapy. This Review analyses the molecular mechanisms that are necessary for the establishment of HIV-1 latency and their relationships with different cellular and anatomical reservoirs, and discusses the current treatment strategies for targeting viral persistence in reservoirs, their main limitations and future perspectives.

Prevalence of genotypic resistance to nucleoside analogues in antiretroviral-naive and antiretroviral-experienced HIV-infected patients in Spain.

Objective: To determine the prevalence of genotypic resistance to nucleoside analogues (NA) in a large group of HIV‐infected individuals in Spain, some of whom had no previous treatment with antiretroviral drugs (antiretroviral‐naive) and some of whom had such experience (antiretroviral‐experienced). Setting: Cross‐sectional study in outpatient clinics in three reference hospitals for HIV/AIDS located in Barcelona, Madrid and Seville, Spain. Patients and methods: Primary mutant genotypes were examined in plasma HIV RNA collected from 150 antiretroviral‐naive subjects, half in 1993 and the other half in 1997. Furthermore, drug resistance mutations were analysed in plasma collected from another 150 antiretroviral‐experienced patients who had received 2 NA for longer than 1 year, either in sequence as monotherapy or as combination therapy. A line probe assay was used for recognizing mutations conferring resistance to zidovudine (ZDV), didanosine (ddI), zalcitabine (ddC), and lamivudine (3TC). A point‐mutation nested‐PCR assay was used for examining a codon 151 mutation associated with multiple drug resistance. Results: One or more mutations associated with primary resistance to NA were seen in 10 antiretroviral‐naive (13.3%) patients in 1993 and in nine (12%) in 1997. In all but two cases, they were associated with ZDV resistance. In contrast, all but six (96%) of the antiretroviral‐experienced subjects harboured drug‐resistant mutant viruses. The codon 184 mutation (associated with resistance to 3TC) was detected in 92% of patients treated with 3TC, but also in 18% of those treated with only ddI or ddC. The codon 215 mutation was found in 67.3% of patients who had been exposed to ZDV; the codon 69 mutation was found in 15% of patients treated with ddC; and the codon 74 mutation was found in only 7.2% of patients treated with ddI. Finally, the codon 151 multidrug resistant mutation was found in four (2.7%) of 150 patients with a longterm exposure to NA. Conclusions: Overall, the prevalence of drug‐resistant HIV‐1 genotypes was 12.7% in antiretroviral‐naive patients, most of whom had ZDV‐resistant mutants. There is no evidence of an increase during the last 5 years. However, multidrug‐resistant HIV genotypes are currently circulating in Spain.

Activity of tenofovir on hepatitis B virus replication in HIV-co-infected patients failing or partially responding to lamivudine

Treatment of hepatitis B virus (HBV) with lamivudine may not completely suppress viral replication and often fails as a result of lamivudine resistance. Tenofovir is a new HIV inhibitor with further activity against HBV, which was explored in 12 HBV/HIV-co-infected patients with detectable levels of serum HBV-DNA, despite receiving a lamivudine-containing antiretroviral regimen. Lamivudine-resistance mutations were found in HBV from seven patients. HBV-DNA levels dropped a median of 3.78 logs from baseline to 24 weeks. Tenofovir was very effective at reducing HBV-DNA levels in HIV/HBV-co-infected patients carrying either wild-type or lamivudine-resistant viruses.

Primary genotypic and phenotypic HIV-1 drug resistance in recent seroconverters in Madrid.

Objective: Transmission of drug‐resistant HIV‐1 strains is increasing with widespread use of antiretroviral drugs in developed countries. This study examined the prevalence of resistant viruses in recent seroconverters in Madrid, Spain. Design: HIV isolates from 30 consecutive participants with positive or indeterminate HIV antibody test results and a negative test result at a mean of 6.6 months earlier were examined for HIV drug resistance. All study subjects admitted to having very recently engaged in high‐risk practices. All were therapeutically naive and were recruited between 1997 and 1999 in a referring health care facility for sexually transmitted diseases. Methods: Population‐based sequencing of the viral reverse transcriptase (RT) and protease (PR) regions derived from plasma viral RNA was performed. Phenotypic resistance was assessed by a recombinant virus assay. Results: Overall prevalence of genotypes associated with reduced susceptibility was 26.7% (8 of 30 participants). Resistance mutations were seen against nucleoside analogues in 7 (23.3%), nonnucleoside reverse transcriptase inhibitors in 1 (3.3%), and protease inhibitors in 2 (6.7%). Zidovudine‐resistance mutations M41L and/or T215Y were the commonest, found in 20% (6 of 30 participants). Resistance mutations to at least two antiretroviral families (multidrug‐resistance) were detected in 2 (6.7%) study subjects. A median infectious dose (IC50) increase of fourfold for any drug was found in 7 patients, and in 2 was > tenfold for zidovudine (genotype M41L + T215Y) and lamivudine (genotype M184V), respectively. Conclusions: Drug‐resistant HIV variants were present in over one quarter of individuals recently diagnosed as infected in Madrid, Spain. Therefore, resistance testing at baseline should be considered for the optimal design of first‐line antiretroviral combinations.

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain.

ObjectiveTo examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain. MethodsA cross-sectional study was conducted including 601 HIV-infected patients who attended 20 Spanish hospitals in June 1998. Drug resistant mutations were examined using hybridization line probe assays (LiPA). The 6 bp insert at position 69 and the codon 75 mutant were examined by sequencing analysis in specimens lacking reactivity to 69/70 and 74 bands on LiPA, respectively. ResultsPrimary resistance to NRTI was recognized in nine out of 52 (17%) naïve individuals, whereas primary resistance to PI was found in seven out of 126 (6%) PI-naïve patients. The codons most frequently involved in NRTI resistance were at positions 70 (66%), 184 (44%), 215 (33%), and 41 (11%), whereas the most common PI resistance mutation was at codon 82 (6/7 subjects). In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI. The most frequent NRTI mutations occurred at codons 184 (38.5%), 215 (30.1%), and 41 (22.5%), whereas the most frequent PI mutations in pre-treated subjects were found at positions 82 (15.8%) and 84 (11.4%). Overall, patients who began triple combinations as initial therapy showed a lower number of key resistance mutations than those who began highly active antiretroviral therapy (HAART) after being exposed to NRTI for a period of time (mean number of mutations, 0.1 versus 1.8, P  < 0.05). Codon 75 mutant was found in three out of 387 patients (0.7%), whereas no insertions at codon 69 were recognized. ConclusionThe prevalence of primary genotypic resistance to NRTI and PI in Spain was 17% and 6%, respectively. Zidovudine, lamivudine, indinavir and ritonavir were the drugs most frequently affected. These data support the use of resistance testing prior to the introduction of first-line antiretroviral therapies in Spain. Among pre-treated subjects, drug resistance genotypes were less prevalent in those who began HAART as initial therapy.

Role of hepatitis C virus genotype in the development of severe transaminase elevation after the introduction of antiretroviral therapy.

Objective: To assess the role of different hepatitis C virus (HCV) genotypes in the development of transaminase elevation after treatment with highly active antiretroviral therapy (HAART). Design: Retrospective cohort study at one referral HIV outpatient clinic. Methods: HCV genotype was determined in plasma samples from all consecutive HCV‐HIV coinfected patients initiating HAART between March 1998 and January 2000. Clinical and laboratory data were recorded during the following 9 months. Severe transaminase elevation was defined as ≥ fivefold increase over upper normal limits (AIDS Clinical Trials Group grades 3 or 4) when baseline alanine transaminase (ALT) and aspartate transaminase (AST) values were normal, and as ≥ 3.5‐fold increase above baseline ALT and AST values if they were abnormal. Results: Twelve of 70 subjects (17%) developed severe transaminase elevation. Their HCV genotypes were distributed as follows: type 1, 5/39 (13%); type 2, 0/3 (0%); type 3, 7/21 (33%); and type 4, 0/7 (0%). The incidence of severe transaminase elevation was significantly higher among subjects with HCV genotype 3 (HCV‐3) compared with those with non‐type 3 (OR, 4.4 [95%CI, 1.2‐16.1]; P = .02). In the multivariate analysis, HCV‐3 remained associated with severe transaminase elevation when adjusted for baseline HCV viral load and degree of immune recovery seen during follow‐up evaluation. Conclusions: HCV‐3 is an independent risk factor for developing severe transaminase elevation after HAART. HCV genotyping before initiating antiretroviral therapy may be useful for assessing the risk of hepatotoxicity and for choosing the most appropriate drugs to prescribe for HIV‐HCV coinfected patients. Given that the best response to interferon plus ribavirin occurs in patients with HCV‐3, treatment should be specially encouraged in coinfected persons carrying HCV‐3.

Hepatitis C virus (HCV) relapses after anti-HCV therapy are more frequent in HIV-infected patients.

The response to standard or pegylated interferon (IFN) plus ribavirin (RBV) seems to be lower in hepatitis C virus (HCV)/HIV-coinfected subjects than in HCV-monoinfected patients. Thus, the principles guiding anti-HCV therapy in HIV-negative patients may not apply in the setting of HIV infection. We examined the rate of HCV relapse in 58 HCV/HIV-coinfected subjects who showed undetectable HCV-RNA (<600 IU/ml) at the end of anti-HCV combination therapy. Overall, 19 (32.8%) patients relapsed after discontinuing treatment, a rate significantly higher than that seen in HIV negatives, which is in the range of 15-20%. There were no significant differences between HCV genotypes (33.3% for HCV genotypes 2-3 versus 31.8% for HCV genotypes 1-4) and/or the use of either standard or pegylated IFN (37% versus 29%, respectively). Thus, extended periods of anti-HCV therapy might reduce HCV relapses in HIV-coinfected patients initially responding to therapy.

Distribution of hepatitis B virus genotypes in HIV-infected patients with chronic hepatitis B: Therapeutic implications

Hepatitis B virus (HBV) genotypes were examined in HIV-infected patients with chronic HBV infection seen in our clinic during 2002. A total of 28 of 1100 individuals (prevalence 2.5%) were found to be HBsAg and HBV-DNA positive. HBV genotypes could be determined in 23 of them. HBV-A was the most common (57%), followed by HBV-D (39%). HBV-A predominated among homosexual men (67%) while HBV-D predominated among intravenous drug users (67%). The presence of serum HBeAg was significantly associated with the HBV-A genotype.

A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism

OBJECTIVES To develop a sensitive phenotypic assay based on recombinant viruses (RVs) for characterizing HIV-1 tropism. METHODS Viral tropism was assessed in 159 plasma samples. The env gene was amplified and ligated into pNL-lacZ/env-Ren, which carries a luciferase reporter gene. Resulting constructs were transfected into HEK293T cells to generate RVs. To assess co-receptor tropism, U87.CD4.CXCR4/CCR5 cells were infected and luciferase activity was measured. RESULTS RVs containing env from different HIV-1 subtypes were replication competent. Minor variants were detectable in 1% of the viral population. Tropism was determined in 65% of samples with a viral load of <1000 copies/mL. The phenotypic assay described here was validated with the Trofile™ and Trofile™ES assays. Considering the Trofile™ assay as a reference, the sensitivity for R5 and R5X4/X4 detection was 90% and 77%, and the specificity was 77% and 90%, respectively. Our assay was 86% concordant with Trofile™ (90% for R5 and 77% for R5X4/X4). When our system was compared with Trofile™ES, the concordance was 89% (86% for R5 and 92% for R5X4/X4), the sensitivity for R5 was 86% and for R5X4/X4 was 92%, and the specificity for R5 was 92% and for R5X4/X4 was 86%. The phenotypic results were compared with those obtained using the following V3 genotypic prediction tools: position-specific scoring matrix; geno2pheno[coreceptor]; C4.5; C4.5 using positions 8 and 12; PART; support vector machines; and the charge rule. CONCLUSIONS We describe a system to assess co-receptor tropism based on the generation of chimeric replication-competent viruses with high sensitivity in the detection of minor populations. A good correlation of our results with Trofile™ assays was found.

Liver injury after beginning antiretroviral therapy in Hiv/hepatitis C virus co-infected patients is not related to immune reconstitution

Transaminase elevations occur more frequently after beginning antiretroviral therapy in HIV-positive patients co-infected with hepatitis C virus (HCV). The mechanism of liver injury in these individuals is unknown, although immune reconstitution phenomena have been postulated. In 42 HIV/HCV co-infected individuals followed after beginning potent antiretroviral therapy, the development of liver injury was not associated with significant changes in serum HCV-RNA levels nor with greater CD4 cell increases. Underlying chronic hepatitis may thus increase the risk of liver toxicity by other mechanisms.