Thomas Toni

Antiretroviral Drug Resistance in Human Immunodeficiency Virus Type 2

Scientists established long ago that human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS ([4][1], [30][2], [48][3]). HIV is known to severely damage the immune system by selectively infecting T-helper (CD4+) lymphocytes. This can lead to serious infections by agents that would

Characterization of the E138K Resistance Mutation in HIV-1 Reverse Transcriptase Conferring Susceptibility to Etravirine in B and Non-B HIV-1 Subtypes

ABSTRACT We have selected for resistance to etravirine (ETR) and efavirenz (EFV) in tissue culture using three subtype B, three subtype C, and two CRF02_AG clinical isolates, grown in cord blood mononuclear cells. Genotypic analysis was performed at baseline and at various weeks of selection. Phenotypic resistance in regard to ETR, EFV, and nevirapine (NVP) was evaluated at weeks 25 to 30 for all ETR-selected viruses and in viral clones that contained specific resistance mutations that were inserted by site-directed mutagenesis into pNL-4.3 and AG plasmids. The results show that ETR selected mutations at positions V90I, K101Q, E138K, V179D/E/F, Y181C, V189I, G190E, H221H/Y, and M230L and that E138K was the first of these to emerge in most instances. The time to the emergence of resistance was longer in the case of ETR (18 weeks) compared to EFV (11 weeks), and no differences in the patterns of emergent mutations could be documented between the B and non-B subtypes. Viral clones containing E138K displayed low-level phenotypic resistance to ETR (3.8-fold) and modestly impaired replication capacity (2-fold) compared to wild-type virus. ETR-selected virus showed a high degree of cross-resistance to NVP but not to EFV. We identified K101Q, E138K, V179E, V189I, G190E, and H221Y as mutations not included among the 17 currently recognized resistance-associated mutations for ETR.

Prevalence and virological profiles of hepatitis B infection in human immunodeficiency virus patients

AIM To determine the prevalence of hepatitis B virus (HBV) in adult human immunodeficiency virus (HIV) patients with CD4+ T-cell count less than 500/mm(3) and without antiretroviral therapy; to describe different HBV-HIV coinfection virological profiles; and to search for factors associated with HBs antigen (HBsAg) presence in these HIV positive patients. METHODS During four months (June through September 2006), 491 patients were received in four HIV positive monitoring clinical centers in Abidjan. INCLUSION CRITERIA HIV-1 or HIV-1 and 2 positive patients, age ≥ 18 years, CD4+ T-cell count < 500/mL and formal and signed consent of the patient. Realized blood tests included HIV serology, CD4+ T-cell count, quantitative HIV RNA load and HBV serological markers, such as HBsAg and HBc antibody (anti-HBcAb). We performed HBeAg, anti-HBe antibody (anti-HBeAb), anti-HBc IgM and quantitative HBV DNA load in HBsAg positive patients. Anti-HBsAb had been tested in HIV patients with HBsAg negative and anti-HBcAb-positive. HBV DNA was also tested in 188 anti-HBcAb positive patients with HBsAg negative status and without anti-HBsAb. Univariate analysis (Pearson χ(2) test or Fischer exact test) and multivariate analysis (backward step-wise selection logistic regression) were performed as statistical analysis. RESULTS Mean age of 491 patients was 36 ± 8.68 years and 73.3% were female. Type-1 HIV was found in 97% and dual-type HIV (type 1 plus type 2) in 3%. World Health Organization (WHO) clinical stage was 1, 2, 3 and 4 respectively in 61 (12.4%), 233 (47.5%), 172 (35%) and 25 patients (5.1%). Median CD4+ T-cell count was 341/mm(3) (interquartile range: 221-470). One hundred and twelve patients had less than 200 CD4+ T-cell/mm(3). Plasma HIV-1 RNA load was elevated (≥ 5 log(10) copies/mL) in 221 patients (45%). HBsAg and anti-HBcAb prevalence was respectively 13.4% and 72.9%. Of the 66 HBsAg positive patients, 22 were inactive HBV carriers (33.3%), 21 had HBeAg positive hepatitis (31.8%) and 20 had HBeAg negative hepatitis (30.3%). HBeAg and anti-HBeAb were indeterminate in 3 of them. Occult B infection prevalence (HBsAg negative, anti-HBcAb positive, anti-HBsAb negative and detectable HBV DNA) was 21.3%. Three parameters were significantly associated with the presence of HBsAg: male [odds ratio (OR): 2.2; P = 0.005; 95% confidence interval (CI): 1.3-3.8]; WHO stage 4 (OR: 3.2; P = 0.01; 95% CI: 1.3-7.9); and aspartate aminotransferase (AST) level higher than the standard (OR: 1.9; P = 0.04; 95% CI: 1.02-3.8). CONCLUSION HBV infection prevalence is high in HIV-positive patients. HBeAg positive chronic hepatitis and occult HBV infection are more frequent in HIV-positive patients than in HIV negative ones. Parameters associated with HBsAg positivity were male gender, AIDS status and increased AST level.

Nucleoside and Nucleotide Analogs Select in Culture for Different Patterns of Drug Resistance in Human Immunodeficiency Virus Types 1 and 2

ABSTRACT Recent findings suggest bidirectional antagonisms between the K65R mutation and thymidine analogue mutations in human immunodeficiency virus type 1 (HIV-1)-infected, treatment-experienced patients, yet little is known about HIV-2 in this regard. This study addressed the effects of innate polymorphisms in HIV-2 on emergent resistance to nucleoside/nucleotide analogues. Emergent drug resistance profiles in HIV-2 subtypes A (n = 3) and B (n = 1) were compared to those of HIV-1 subtypes B and C. Drug resistance was evaluated with cord blood mononuclear cells (CBMCs) and MT2 cells, using selective pressure with tenofovir (TFV), zidovudine (ZDV), stavudine (d4T), didanosine (ddI), abacavir (ABC), lamivudine (3TC), emtricitabine (FTC), or various dual-drug combinations. Resistance was evaluated using conventional and ultrasensitive sequencing approaches. In agreement with our previous findings, dual-drug combinations of TFV, ddI, ABC, d4T, ZDV, and 3TC preferentially selected for K65R in HIV-1 subtype C isolates. In HIV-1 subtype B, TFV-3TC and ZDV-3TC selected for M184I and D67N, respectively. In contrast, selections with all four HIV-2 cultures favored the development of M184I in dual-drug combinations that included either 3TC or FTC. Since HIV-2 cultures did not develop K65R, an ultrasensitive allele-specific real-time PCR assay was developed to distinguish the presence of 65R from wild-type K65 after 16 cycles with a discriminatory ability of 0.1% against a population of wild-type virus. These results underscore potential differences in emergent drug resistance pathways in HIV-1 and HIV-2 and show that polymorphisms may influence the development of the resistance pathways that are likely to emerge.

Medium-term probability of success of antiretroviral treatment after early warning signs of treatment failure in West African adults.

West African adults with warning signs of failure of antiretroviral treatment (ART) at 6 months were assessed for the probability and factors associated with success at 36 months. After 6 months on ART, patients were included if they had a bad immunologic response (BIR) (month 6 CD4 count < pre-ART CD4 count + 50/mm(3)), incomplete virologic suppression (IVS) (month 6 plasma HIV-1 RNA >300 copies/ml), or both (Dual). They were followed for 30 months after inclusion. CD4 counts and HIV-1 RNA were measured every 3 months. We estimated the probability of reaching immunovirologic success (CD4 count >350/mm(3) and plasma HIV-1 RNA <300 copies/ml) and looked for determinants using Cox analysis. A total of 208 adults were included. Among patients in the IVS and Dual groups, 23% and 38% had at least one genotypic resistance mutation at month 6. The 36-month cumulative probability of immunovirologic success was 0.84 in BIR, 0.81 in IVS, and 0.67 in Dual (p = 0.02). Adjusting for CD4 count, viral load, ART regimen, and morbidity, patients who had no genotypic resistance mutations at month 6 or a medication possession ratio (MPR) >90% between month 6 and month 36 had a likelihood of success 3.8 and 3.6 higher than other patients. The 36-month probability of success was 0.56 and 0.86 in patients with an MPR <90% and >90% and 0.59 and 0.84 in patients with and without resistance. After warning signs of failure at 6 months, a large proportion of patients reaches immunovirologic success before 36 months provided there is a high rate of adherence to medication and the absence of early resistance mutations.

Near Full-Length Genomic Analysis of a Novel Subtype A1/C Recombinant HIV Type 1 Isolate from Canada

The rapid introduction of non-B HIV-1 subtypes into Quebec, mostly from persons from regions where HIV prevalence is high and where different variants circulate, means that recombination must now be an important consideration in the epidemiologic surveillance of HIV infection. No circulating recombinant form (CRF), currently involving exclusively subtypes A1 and C, exists in the Los Alamos HIV database. This study presents a near full-length genomic analysis of a novel HIV-1 recombinant involving subtypes A1 and C. Bootscanning revealed that the recombinant structure involves three breakpoints that separate the genome into four regions, alternating between subtypes A1 and C. The intersubtype recombinant breakpoint in the pol gene was at midpoint between the protease and reverse transcriptase open reading frames. This is the first report of a recombinant involving subtypes A1 and C in Canada, the epidemiologic significance of which is not yet known.

Tissue culture drug resistance analysis of a novel HIV-1 protease inhibitor termed PL-100 in non-B HIV-1 subtypes

PL-100 is a novel HIV-1 protease inhibitor (PI) that maintains activity against viruses that are resistant to other PIs. To further characterize this compound, we used it to select for drug resistance in tissue culture, using two non-B HIV-1 subtypes, viz. subtype C and a CRF01_AE recombinant virus. PL-100 selected for both minor and major PI resistance mutations along either of two distinct pathways. One of these involved the V82A and L90M resistance mutations while the other involved a mutation at position T80I, with other mutations being observed at positions M46I/L, I54M, K55R, L76F, P81S and I85V. The resistance patterns in both subtype C and CRF01_AE were similar and an accumulation of at least three mutations in the flap and active sites were required in each case for high-level resistance to occur, demonstrating that PL-100 has a high genetic barrier against the development of drug resistance.

Use of new resistance markers to predict virologic response to antiretrovirals

Results Among the reasons that more ultrasensitive assays may sometimes be needed for more accurate assessments of drug resistance is the differential effect of certain mutations on viral replicative capacity. As an example, the K65R mutation is known to adversely affect HIV replication, and this may be one of the reasons that it is found relatively infrequently among individuals who fail antiretroviral therapy. In contrast, the use of AS-PCR for K65R in subtype C viruses has shown that this method was able to detect the presence of this mutation in an additional 4 of 30 samples who had tested negative by bulk sequencing methods. Now, it also appears as though the transmission of the K65R mutation, while rare, can also be detected in higher numbers by AS-PCR than bulk sequencing, and that this is also more common among subtype C than subtype B viruses. The likely reason is that subtype C viruses are more prone to develop K65R as a consequence of treatment failure and are therefore more likely than subtype B viruses to contain this mutation at the time that transmission takes place. In the case of the M184V mutation, it has also been observed that AS-PCR methods can detect this substitution more efficiently than bulk sequencing among newly-infected individuals.