Yoeri Schrooten

Rising prevalence of HIV-1 non-B subtypes in Belgium: 1983-2001.

This study documented the HIV-1 subtype distribution in 2 Belgian hospitals and determined predictive demographics for non-B subtypes. Overall, subtype B was the most prevalent subtype in this population, followed by subtypes A and C. Several recombinants were detected, circulating recombinants as well as new ones. We found a rise in non-B subtypes from 0% in 1983 to 57% in 2001. The Cochran-Armitage trend test (P < 0.001) as well as the correlation analysis (R = 0.71, P = 0.0006) was highly significant. Recombinants were also increasing in this patient population from 0% in 1983 to 10% in 2001, with good support from the statistical analyses (trend test P < 0.001; correlation analysis R = 0.67, P = 0.0016). Heterosexual route of infection, black African race, African origin of the virus, and year of diagnosis were predictors for infection with non-B subtypes in multivariate analysis. This analysis indicates that the prevalence of non-B subtypes and recombinants in this patient population is high and increasing. Gathering demographic and sequence information from newly diagnosed patients could be useful to further follow the spread of non-B subtypes in Belgium and Europe, but subtyping based on sequence information still remains the most reliable method.

Prevalence and epidemiology of HIV type 1 drug resistance among newly diagnosed therapy-naive patients in Belgium from 2003 to 2006.

This study is the first prospective study to assess the prevalence, epidemiology, and risk factors of HIV-1 drug resistance in newly diagnosed HIV-infected patients in Belgium. In January 2003 it was initiated as part of the pan-European SPREAD program, and continued thereafter for four inclusion rounds until December 2006. Epidemiological, clinical, and behavioral data were collected using a standardized questionnaire and genotypic resistance testing was done on a sample taken within 6 months of diagnosis. Two hundred and eighty-five patients were included. The overall prevalence of transmitted HIV-1 drug resistance in Belgium was 9.5% (27/285, 95% CI: 6.6-13.4). Being infected in Belgium, which largely coincided with harboring a subtype B virus, was found to be significantly associated with transmission of drug resistance. The relatively high rate of baseline resistance might jeopardize the success of first line treatment as more than 1 out of 10 (30/285, 10.5%) viruses did not score as fully susceptible to one of the recommended first-line regimens, i.e., zidovudine, lamivudine, and efavirenz. Our results support the implementation of genotypic resistance testing as a standard of care in all treatment-naive patients in Belgium.

Molecular testing of multiple HIV-1 transmissions in a criminal case.

Objective:To test the a priori hypothesis of HIV-1 transmission from one suspect to six recipients in a criminal case. Methods:Partial pol and/or env sequences were obtained for at least two samples of the suspect and the victims. Appropriate local controls were sampled based on epidemiological and subtype criteria. Phylogenetic testing was performed using different reconstruction methods. Results:Phylogenetic analyses consistently inferred a monophyletic cluster for the suspect and victim samples in both genome regions. This was highly supported by parametric and non-parametric bootstrapping techniques. Moreover, the controls most closely related to the suspect–victim cluster had a similar geographical origin to the suspect. Conclusions:Taking into account the limitations on the conclusions that can be drawn from molecular investigations we could infer that our molecular data is consistent with a scenario of multiple HIV transmission between suspect and victims.

A genotypic assay for the amplification and sequencing of integrase from diverse HIV-1 group M subtypes

Recently, the Food and Drug Administration (FDA) of the USA approved the first integrase inhibitor for inclusion in treatment regimens of HIV-1 patients failing their current regimens with multi-drug resistant strains. However, treatment failure has been observed during integrase inhibitor-containing therapy. Several mutational pathways have been described with signature mutations at integrase positions 66, 92, 148 and 155. Therefore, a genotypic assay for the amplification and sequencing of HIV-1 integrase was developed. The assay displayed a detection limit of 10 HIV-1 III(B) RNA copies/ml plasma. As the HIV-1 pandemic is characterised by a large genetic diversity, the new assay was evaluated on a panel of 74 genetically divergent samples belonging to the following genetic forms A, B, C, D, F, G, J, CRF01-AE, CRF02-AG, CRFF03-AB, CRF12-BF and CRF13-cpx. Their viral load ranged from 178 until >500,000 RNA copies/ml. The amplification and sequencing was successful for 70 samples (a success rate of 95%). The four failures were most probably due to low viral load or poor quality of RNA and not to subtype issues. Some of the sequences obtained from integrase inhibitor-naïve patients displayed polymorphisms at integrase positions associated with resistance: 74IV, 138D, 151I, 157Q and 163AE. The relevance of these polymorphisms in the absence of the signature mutations remains unclear.

Current levels of drug resistance among therapy-naive HIV-infected patients have significant impact on treatment response

Resistant HIV can be transmitted from one patient t o another. 1 Recent large-scale European research found onetenth of viruses from untreated patients showing at least 1 resistance-related mutation. 2 Such investigations are mainly driven by the concern that resistant virus c ould at some point hamper optimal treatment response. However, the impact of baseline drug resistance on treatment response is not well studied. Various gen otypic interpretation methods are used to assess resistanc e in drug-naive patients. When transmitted resistan ce is the topic of interest, irrespective of treatment, prima ry mutations are considered most indicative. Second ary mutations could also be the result of natural varia tion. However, when interpreting resistance in view of the response to the installed treatment, all positions possibly contributing to the selective advantage of the virus in the presence of drug should be taken into account.

Antiretroviral drug resistance in HIV-1 therapy-naive patients in Cuba

In Cuba, antiretroviral therapy rollout started in 2001 and antiretroviral therapy coverage has reached almost 40% since then. The objectives of this study were therefore to analyze subtype distribution, and level and patterns of drug resistance in therapy-naive HIV-1 patients. Four hundred and one plasma samples were collected from HIV-1 therapy-naive patients in 2003 and in 2007-2011. HIV-1 drug resistance genotyping was performed in the pol gene and drug resistance was interpreted according to the WHO surveillance drug-resistance mutations list, version 2009. Potential impact on first-line therapy response was estimated using genotypic drug resistance interpretation systems HIVdb version 6.2.0 and Rega version 8.0.2. Phylogenetic analysis was performed using Neighbor-Joining. The majority of patients were male (84.5%), men who have sex with men (78.1%) and from Havana City (73.6%). Subtype B was the most prevalent subtype (39.3%), followed by CRF20-23-24_BG (19.5%), CRF19_cpx (18.0%) and CRF18_cpx (10.3%). Overall, 29 patients (7.2%) had evidence of drug resistance, with 4.0% (CI 1.6%-4.8%) in 2003 versus 12.5% (CI 7.2%-14.5%) in 2007-2011. A significant increase in drug resistance was observed in recently HIV-1 diagnosed patients, i.e. 14.8% (CI 8.0%-17.0%) in 2007-2011 versus 3.8% (CI 0.9%-4.7%) in 2003 (OR 3.9, CI 1.5-17.0, p=0.02). The majority of drug resistance was restricted to a single drug class (75.8%), with 55.2% patients displaying nucleoside reverse transcriptase inhibitor (NRTI), 10.3% non-NRTI (NNRTI) and 10.3% protease inhibitor (PI) resistance mutations. Respectively, 20.7% and 3.4% patients carried viruses containing drug resistance mutations against NRTI+NNRTI and NRTI+NNRTI+PI. The first cases of resistance towards other drug classes than NRTI were only detected from 2008 onwards. The most frequent resistance mutations were T215Y/rev (44.8%), M41L (31.0%), M184V (17.2%) and K103N (13.8%). The median genotypic susceptibility score for the commonly prescribed first-line therapies was 2.5. This analysis emphasizes the need to perform additional surveillance studies to accurately assess the level of transmitted drug resistance in Cuba, as the extent of drug resistance might jeopardize effectiveness of first-line regimens prescribed in Cuba and might necessitate the implementation of baseline drug resistance testing.

Trends and Predictors of Transmitted Drug Resistance (TDR) and Clusters with TDR in a Local Belgian HIV-1 Epidemic

We aimed to study epidemic trends and predictors for transmitted drug resistance (TDR) in our region, its clinical impact and its association with transmission clusters. We included 778 patients from the AIDS Reference Center in Leuven (Belgium) diagnosed from 1998 to 2012. Resistance testing was performed using population-based sequencing and TDR was estimated using the WHO-2009 surveillance list. Phylogenetic analysis was performed using maximum likelihood and Bayesian techniques. The cohort was predominantly Belgian (58.4%), men who have sex with men (MSM) (42.8%), and chronically infected (86.5%). The overall TDR prevalence was 9.6% (95% confidence interval (CI): 7.7–11.9), 6.5% (CI: 5.0–8.5) for nucleoside reverse transcriptase inhibitors (NRTI), 2.2% (CI: 1.4–3.5) for non-NRTI (NNRTI), and 2.2% (CI: 1.4–3.5) for protease inhibitors. A significant parabolic trend of NNRTI-TDR was found (p = 0.019). Factors significantly associated with TDR in univariate analysis were male gender, Belgian origin, MSM, recent infection, transmission clusters and subtype B, while multivariate and Bayesian network analysis singled out subtype B as the most predictive factor of TDR. Subtype B was related with transmission clusters with TDR that included 42.6% of the TDR patients. Thanks to resistance testing, 83% of the patients with TDR who started therapy had undetectable viral load whereas half of the patients would likely have received a suboptimal therapy without this test. In conclusion, TDR remained stable and a NNRTI up-and-down trend was observed. While the presence of clusters with TDR is worrying, we could not identify an independent, non-sequence based predictor for TDR or transmission clusters with TDR that could help with guidelines or public health measures.

A Combination of Poor Adherence and a Low Baseline Susceptibility Score is Highly Predictive for HAART Failure

The relationship between adherence, virological response to highly active antiretroviral therapy (HAART) and the presence and development of genotypic resistance was assessed in 41 HIV-infected patients on HAART. Four adherence parameters (drug taking adherence, dosing adherence, timing adherence and drug holidays) were scored prospectively using electronic event monitoring. Genotypic resistance at baseline and after therapy failure was scored retrospectively and a genotype-based susceptibility score was calculated. Overall median adherence rates were high. All adherence parameters were better in virological responders (n=31) compared to non-responders (n=10), drug taking adherence and number of drug holidays being significantly different. Responders had a significantly higher susceptibility score. Stepwise logistic regression showed that the number of drug holidays and a low susceptibility score were highly predictive for therapy failure. Despite the presence of a limited number of baseline resistance mutations, perfectly adherent patients can control virus replication for a prolonged period.

Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs

ABSTRACT Combination therapy can successfully suppress human immunodeficiency virus (HIV) replication in patients but selects for drug resistance, requiring subsequent resistance-guided therapeutic changes. This report describes the development and validation of a novel assay that offers a uniform method to measure susceptibility to all clinically approved HIV type 1 (HIV-1) drugs targeting reverse transcriptase (RT), protease (PR), integrase (IN), and viral entry. It is an assay in which the antiviral effect on infection within a single replication cycle is measured in triply transfected U87.CD4.CXCR4.CCR5 cells, based on homologous recombination between patient-derived amplicons and molecular proviral clones tagged with the enhanced green fluorescent protein (EGFP) reporter gene and from which certain viral genomic regions are removed. The deletions stretch from p17 codon 7 to PR codon 98 in pNL4.3-ΔgagPR-EGFP, from PR codons 1 to 99 in pNL4.3-ΔPR-EGFP, from RT codons 1 to 560 in pNL4.3-ΔRT-EGFP, from IN codons 1 to 288 in pNL4.3-ΔIN-EGFP, and from gp120 codon 34 to gp41 codon 237 in pNL4.3-Δenv-EGFP. The optimized experimental conditions enable the investigation of patient samples regardless of viral subtype or coreceptor use. The extraction and amplification success rate for a set of clinical samples belonging to a broad range of HIV-1 group M genetic forms (A-J, CRF01-03, CRF05, and CRF12-13) and displaying a viral load range of 200 to >500,000 RNA copies/ml was 97%. The drug susceptibility measurements, based on discrimination between infected and noninfected cells on a single-cell level by flow cytometry, were reproducible, with coefficients of variation for resistance ranging from 7% to 31%, and were consistent with scientific literature in terms of magnitude and specificity.

High frequency of antiviral drug resistance and non-B subtypes in HIV-1 patients failing antiviral therapy in Cuba

BACKGROUND Emergence of HIV-1 drug resistance may limit the sustained benefits of antiretroviral therapy (ART) in settings with limited laboratory monitoring and drug options. OBJECTIVES Surveillance of drug resistance and subtypes in HIV-1 patients failing ART in Cuba. STUDY DESIGN This study compiled data of ART-experienced HIV-1 patients attending a clinical center in Havana in 2003 and 2009-2011. The first period included results of a cross-sectional study, whereas in the second period genotyping was performed as part of routine care. Drug resistance mutations and levels were determined using HIVdb version 6.0.9. RESULTS Seventy-six percent received solely ART containing at least 3 drugs, of which 79.1% ever receiving unboosted protease inhibitors (PI). Patients from 2009 to 2011 were longer treated and exposed to more ART regimens. Subtype B (39%) and CRF19_cpx (18%) were the most prevalent genetic forms. Subtype distribution did not change significantly between both periods, except for BG recombinants that increased from 6% to 14%. Nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside RTI (NNRTI) and PI mutations were present in 69.5%, 54.8% and 44.4%. Full-class resistance (FCR) to NRTI, NNRTI, PI and multidrug resistance (MDR) were detected in 31.8%, 37.9%, 18.5% and 15.4%. FCR to NRTI, NNRTI, PI and MDR were present in 9.8%, 14.1%, 0%, 0% after first-line failure and in 19.8%, 20.8%, 2.9% and 2.9% after second-line failure. CONCLUSIONS Our study found a high prevalence of drug resistance and supports the need for appropriate laboratory monitoring in clinical practice and access to drug options in case of virological failure.