Nathalie Dekeersmaeker

Subgroup Prevalence and Genotype Circulation Patterns of Human Respiratory Syncytial Virus in Belgium during Ten Successive Epidemic Seasons

ABSTRACT Human respiratory syncytial virus (HRSV) is the leading viral cause of severe respiratory illness for infants and young children worldwide. Two major antigenic groups (A and B) of HRSV exist, and viruses from both subgroups can cocirculate during epidemics; however, their frequencies might differ between seasons. The subgroup prevalence and genotype distribution patterns of HRSV strains were investigated in a community in Belgium during 10 successive epidemic seasons (1996 to 2006). A regular 3-year cyclic pattern of subgroup dominance was observed, consisting of two predominant HRSV-A seasons, followed by a single HRSV-B-dominant year. HRSV infections with both subgroups were more prevalent among children younger than 6 months and had a peak incidence in December. The most frequently detected genotypes were GA5 and GB13, the latter including strains with the 60-nucleotide duplication in the G gene. Furthermore, GA5 remained the dominant HRSV genotype in two consecutive epidemic seasons twice during the study period. Additional variability was detected among the GB13 isolates, due to the usage of a novel termination codon in the G gene. Dual infections with both HRSV subgroups were detected for 9 patients, and subsequent infections with the heterologous HRSV subgroup were documented for 15 patients. Among five patients with homologous reinfections, only one was caused by HRSV-B. Our results support the hypothesis that the overall prevalence of HRSV-A over HRSV-B could be due to a more-transient subgroup A-specific immune protection.

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.

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.

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.

The rare HIV-1 gp41 mutations 43T and 50V elevate enfuvirtide resistance levels of common enfuvirtide resistance mutations that did not impact susceptibility to sifuvirtide

Mutations that are selected at low frequency and/or reside outside the enfuvirtide target region, amino acid 36-45 of gp41, might still be important determinants for drug resistance. This study aimed to investigate the phenotypic impact against enfuvirtide and sifuvirtide of uncharacterized gp41 mutations 42G, 43T and 50V, selected in patients failing enfuvirtide-containing regimens. As single mutations, neither 42G, 43T nor 50V conferred resistance to enfuvirtide. However, 50V increased slightly resistance levels for 36D, 38M, 43D or 43T as did 43T for 38M. All mutants displayed a reduced replication capacity, except 42S, 50V and 36D+/-50V. None of the mutants displayed resistance to the next-generation fusion inhibitor sifuvirtide. This study highlights the necessity to confirm the in vitro effect of infrequently selected mutations as 42G was not associated with enfuvirtide resistance whereas 43T and 50V should be considered as secondary enfuvirtide resistance mutations.

Evolution of genotypic resistance to enfuvirtide in HIV-1 isolates from different group M subtypes.

BACKGROUND Enfuvirtide is active against isolates from different HIV-1 subtypes. In vitro and in vivo studies reveal that resistance mutations are primarily found within the region spanning amino acid 36-45 of gp41. However, most studies include only subtype B strains, while it is known that especially the env region is very divergent among subtypes. OBJECTIVES To analyze the gp41 HR1 genetic evolution during failure of enfuvirtide-containing salvage regimens in 19 HIV-1 patients infected with strains from different group M subtypes. STUDY DESIGN The gp41 sequence was determined at baseline and upon failure in 19 patients. For a subset of 7 patients, samples were available after discontinuation of enfuvirtide. RESULTS Our results confirmed the conserved nature of the HR1 region. Escape mutants during chronic treatment with enfuvirtide were mainly observed within region 36-45. One novel mutation was identified, i.e. S42G in a subtype A1 strain. CONCLUSIONS Different subtypes escape enfuvirtide selective pressure through similar mutational patterns, however a new S42G variant was observed. The in vivo selection of S42G suggests that it might play a role in enfuvirtide resistance. Therefore, it could be considered as a candidate mutation to be included within drug resistance interpretation systems.

Horizontal gene transfer from human host to HIV-1 reverse transcriptase confers drug resistance and partly compensates for replication deficits

We investigated the origin and the effect of insertion D67D-THGERDLGPA within HIV-1 RT from a patient failing antiviral therapy. The insertion developed within the context of pre-existing NRTI and NNRTI mutations (M41L, L210W, T215Y and N348I). Concurrently, the NRTI mutations T69I and V118I and the NNRTI mutations K103N and Y181C were detected for the first time. High-level drug resistance (fold-changes≥50) and a good replication capacity (87% of wild-type) were observed, significantly higher than for the previous virus without insertion. The insertion was very similar to a region within human chromosome 17 (31/34 nucleotide identity), and had already been detected independently in a Japanese HIV-1 isolate. These results suggest that a particular sequence within human chromosome 17 is prone to horizontal gene transfer into the HIV-1 RT finger subdomain. This insertion confers selective advantage to HIV-1 by its contribution to multi-drug resistance and restoration of impaired replication capacity.

Characterization of amino acids Arg, Ser and Thr at position 70 within HIV-1 reverse transcriptase

Abstract Objectives: The amino acid position 70 in HIV-1 reverse transcriptase (RT) plays an important role in nucleoside RT inhibitor (NRTI) resistance. K70R is part of the thymidine analog mutations, but also other amino acid changes have been associated with NRTI resistance, such as K70E and K70G. In this study, we investigated the in vivo selection of the HIV-1 RT mutations K70S and K70T and their in vitro effect on drug resistance and replication capacity. Methods: Recombinant viruses with RT mutations were generated to measure the in vitro drug susceptibility and replication capacity. Bayesian network analysis and three-dimensional modeling were performed to understand the selection and impact of the RT70 mutations. Results: K70S and K70T were found at a low frequency in RTI-experienced HIV-1 patients (0.10% and 0·20%). Baeyesian network learning identified no direct association with the in vivo exposure to any specific RTI. However, direct associations of K70S with mutations within the Q151M-complex and of K70T with K65R were observed. In vitro phenotypic testing revealed only minor effects of K70R/S/T as single mutations, associated with Q151M and within the context of the Q151M-complex. Discussion: These results suggest that the selection of K70S/T and their phenotypic impact are influenced by the presence of other mutations in RT. However, the low impact on in vitro phenotype here observed, alongside with the low in vivo prevalence, the exclusive direct association with known major RTI mutations and the unknown correlation with in vivo response, do not yet necessitate the inclusion of K70S/T in drug resistance interpretation systems.