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Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.
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Heces , Metagenómica , Microbiota , ARN Ribosómico 16S , Humanos , Metagenómica/métodos , Metagenómica/normas , ARN Ribosómico 16S/genética , Heces/microbiología , Microbiota/genética , Sesgo , Metagenoma , Microbioma Gastrointestinal/genética , Análisis de Secuencia de ADN/métodos , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
BACKGROUND: Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. RESULTS: Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. CONCLUSIONS: Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy.
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Perfilación de la Expresión Génica , Vacunas , Humanos , Animales , Ratones , Conejos , Flujo de Trabajo , Transcriptoma , ARN , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Next-generation sequencing workflows, using either metabarcoding or metagenomic approaches, have massively contributed to expanding knowledge of the human gut microbiota, but methodological bias compromises reproducibility across studies. Where these biases have been quantified within several comparative analyses on their own, none have measured inter-laboratory reproducibility using similar DNA material. Here, we designed a multicenter study involving seven participating laboratories dedicated to partial- (P1 to P5), full-length (P6) metabarcoding, or metagenomic profiling (MGP) using DNA from a mock microbial community or extracted from 10 fecal samples collected at two time points from five donors. Fecal material was collected, and the DNA was extracted according to the IHMS protocols. The mock and isolated DNA were then provided to the participating laboratories for sequencing. Following sequencing analysis according to the laboratories' routine pipelines, relative taxonomic-count tables defined at the genus level were provided and analyzed. Large variations in alpha-diversity between laboratories, uncorrelated with sequencing depth, were detected among the profiles. Half of the genera identified by P1 were unique to this partner and two-thirds of the genera identified by MGP were not detected by P3. Analysis of beta-diversity revealed lower inter-individual variance than inter-laboratory variances. The taxonomic profiles of P5 and P6 were more similar to those of MGP than those obtained by P1, P2, P3, and P4. Reanalysis of the raw sequences obtained by partial-length metabarcoding profiling, using a single bioinformatic pipeline, harmonized the description of the bacterial profiles, which were more similar to each other, except for P3, and closer to the profiles obtained by MGP. This study highlights the major impact of the bioinformatics pipeline, and primarily the database used for taxonomic annotation. Laboratories need to benchmark and optimize their bioinformatic pipelines using standards to monitor their effectiveness in accurately detecting taxa present in gut microbiota.
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Microbioma Gastrointestinal , Microbiota , Humanos , Microbioma Gastrointestinal/genética , Reproducibilidad de los Resultados , Microbiota/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ADN Bacteriano/genética , ADN Bacteriano/análisis , ARN Ribosómico 16S/genéticaRESUMEN
Background and aims: Hepatitis B virus (HBV) infection affects 300 million individuals worldwide, representing a major factor for the development of hepatic complications. Although existing antivirals are effective in suppressing replication, eradication of HBV is not achieved. Therefore, a multi-faceted approach involving antivirals and immunomodulatory agents is required. Non-human primates are widely used in pre-clinical studies due to their close evolutionary relationship to humans. Nonetheless, it is fundamental to identify the differences in immune response between humans and these models. Thus, we performed a transcriptomic characterization and interspecies comparison of the early immune responses to HBV in human and cynomolgus macaques. Methods: We characterized early transcriptomic changes in human and cynomolgus B cells, T cells, myeloid and plasmacytoid dendritic cells (pDC) exposed to HBV ex vivo for 2 hours. Differentially-expressed genes were further compared to the profiles of HBV-infected patients using publicly-available single-cell data. Results: HBV induced a wide variety of transcriptional changes in all cell types, with common genes between species representing only a small proportion. In particular, interferon gamma signaling was repressed in human pDCs. At the gene level, interferon gamma inducible protein 16 (IFI16) was upregulated in macaque pDCs, while downregulated in humans. Moreover, IFI16 expression in pDCs from chronic HBV-infected patients anti-paralleled serum HBsAg levels. Conclusion: Our characterization of early transcriptomic changes induced by HBV in humans and cynomolgus macaques represents a useful resource for the identification of shared and divergent host responses, as well as potential immune targets against HBV.
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Hepatitis B , Transcriptoma , Animales , Humanos , Virus de la Hepatitis B/genética , Interferón gamma , Antivirales , Macaca fascicularis , Hepatitis B/genéticaRESUMEN
BACKGROUND: Compared to their healthy counterparts, patients with type 2 diabetes (T2D) can exhibit an altered gut microbiota composition, correlated with detrimental outcomes, including reduced insulin sensitivity, dyslipidemia, and increased markers of inflammation. However, a typical T2D microbiota profile is not established. The aim of this pilot study was to explore the gut microbiota and bacteria associated with prediabetes (pre-T2D) patients, and treatment naïve T2D patients, compared to healthy subjects. METHODS: Fecal samples were collected from patients and healthy subjects (from Norway). The bacterial genomic DNA was extracted, and the microbiota analyzed utilizing the bacterial 16S rRNA gene. To secure a broad coverage of potential T2D associated bacteria, two technologies were used: The GA-map® 131-plex, utilizing 131 DNA probes complementary to pre-selected bacterial targets (covering the 16S regions V3-V9), and the LUMI-Seq™ platform, a full-length 16S sequencing technology (V1-V9). Variations in the gut microbiota between groups were explored using multivariate methods, differential bacterial abundance was estimated, and microbiota signatures discriminating the groups were assessed using classification models. RESULTS: In total, 24 pre-T2D patients, 18 T2D patients, and 52 healthy subjects were recruited. From the LUMI-Seq™ analysis, 10 and 9 bacterial taxa were differentially abundant between pre-T2D and healthy, and T2D and healthy, respectively. From the GA-map® 131-plex analysis, 10 bacterial markers were differentially abundant when comparing pre-T2D and healthy. Several of the bacteria were short-chain fatty acid (SCFA) producers or typical opportunistic bacteria. Bacteria with similar function or associated properties also contributed to the separation of pre-T2D and T2D from healthy as found by classification models. However, limited overlap was found for specific bacterial genera and species. CONCLUSIONS: This pilot study revealed that differences in the abundance of SCFA producing bacteria, and an increase in typical opportunistic bacteria, may contribute to the variations in the microbiota separating the pre-T2D and T2D patients from healthy subjects. However, further efforts in investigating the relationship between gut microbiota, diabetes, and associated factors such as BMI, are needed for developing specific diabetes microbiota signatures.
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Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Estado Prediabético , Humanos , Microbioma Gastrointestinal/genética , Proyectos Piloto , ARN Ribosómico 16SRESUMEN
The gut microbiome is widely analyzed using high-throughput sequencing, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing (SMS). DNA extraction is known to have a large impact on the metagenomic analyses. The aim of this study was to compare DNA extraction protocols for 16S sequencing. In that context, four commonly used DNA extraction methods were compared for the analysis of the gut microbiota. Commercial versions were evaluated against modified protocols using a stool preprocessing device (SPD, bioMérieux) upstream DNA extraction. Stool samples from nine healthy volunteers and nine patients with a Clostridium difficile infection were extracted with all protocols and 16S sequenced. Protocols were ranked using wet- and dry-lab criteria, including quality controls of the extracted genomic DNA, alpha-diversity, accuracy using a mock community of known composition and repeatability across technical replicates. SPD improved overall efficiency of three of the four tested protocols compared with their commercial version, in terms of DNA extraction yield, sample alpha-diversity, and recovery of Gram-positive bacteria. The best overall performance was obtained for the S-DQ protocol, SPD combined with the DNeasy PowerLyser PowerSoil protocol from QIAGEN. Based on this evaluation, we strongly believe that the use of such stool preprocessing device improves both the standardization and the quality of the DNA extraction in the human gut microbiome studies.
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Microbioma Gastrointestinal , Microbiota , Humanos , Microbioma Gastrointestinal/genética , ARN Ribosómico 16S/genética , Genes de ARNr , ADN , Microbiota/genéticaRESUMEN
Mus musculus is the classic mammalian model for biomedical research. Despite global efforts to standardize breeding and experimental procedures, the undefined composition and interindividual diversity of the microbiota of laboratory mice remains a limitation. In an attempt to standardize the gut microbiome in preclinical mouse studies, here we report the development of a simplified mouse microbiota composed of 15 strains from 7 of the 20 most prevalent bacterial families representative of the fecal microbiota of C57BL/6J Specific (and Opportunistic) Pathogen-Free (SPF/SOPF) animals and the derivation of a standardized gnotobiotic mouse model called GM15. GM15 recapitulates extensively the functionalities found in the C57BL/6J SOPF microbiota metagenome, and GM15 animals are phenotypically similar to SOPF or SPF animals in two different facilities. They are also less sensitive to the deleterious effects of post-weaning malnutrition. In this work, we show that the GM15 model provides increased reproducibility and robustness of preclinical studies by limiting the confounding effect of fluctuation in microbiota composition, and offers opportunities for research focused on how the microbiota shapes host physiology in health and disease.
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Heces/microbiología , Microbioma Gastrointestinal/fisiología , Vida Libre de Gérmenes , Organismos Libres de Patógenos Específicos , Secuenciación Completa del Genoma/métodos , Animales , Bacterias/clasificación , Bacterias/genética , Peso Corporal/genética , Peso Corporal/fisiología , Femenino , Microbioma Gastrointestinal/genética , Masculino , Metagenómica/métodos , Ratones Endogámicos C57BL , Fenotipo , Especificidad de la EspecieRESUMEN
The GM15 community is a bacterial consortium used to generate a novel standardized mouse model with a simplified controlled intestinal microbiota recapitulating the specific opportunistic pathogen-free (SOPF) mouse phenotype and the potential to ensure an increased reproducibility and robustness of preclinical studies by limiting the confounding effect of microbiota composition fluctuation.
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The management of patients with colorectal cancer (CRC) and potentially resectable liver metastases (LM) requires quick assessment of mutational status and of response to pre-operative systemic therapy. In a prospective phase II trial (NCT01442935), we investigated the clinical validity of circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) detection. CRC patients with potentially resectable LM were treated with first-line triplet or doublet chemotherapy combined with targeted therapy. CTC (Cellsearch®) and Kirsten RAt Sarcoma (KRAS) ctDNA (droplet digital polymerase chain reaction (PCR)) levels were assessed at inclusion, after 4 weeks of therapy and before LM surgery. 153 patients were enrolled. The proportion of patients with high CTC counts (≥3 CTC/7.5mL) decreased during therapy: 19% (25/132) at baseline, 3% (3/108) at week 4 and 0/57 before surgery. ctDNA detection sensitivity at baseline was 91% (N=42/46) and also decreased during treatment. Interestingly, persistently detectable KRAS ctDNA (p=0.01) at 4 weeks was associated with a lower R0/R1 LM resection rate. Among patients who had a R0/R1 LM resection, those with detectable ctDNA levels before liver surgery had a shorter overall survival (p<0.001). In CRC patients with limited metastatic spread, ctDNA could be used as liquid biopsy tool. Therefore, ctDNA detection could help to select patients eligible for LM resection.
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ADN Tumoral Circulante/sangre , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/patología , Células Neoplásicas Circulantes/patología , Adulto , Anciano , Neoplasias Colorrectales/cirugía , Humanos , Estimación de Kaplan-Meier , Biopsia Líquida , Persona de Mediana Edad , Mutación/genética , Metástasis de la Neoplasia , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética , Resultado del TratamientoRESUMEN
BACKGROUND: Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS: We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS: The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS: This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.
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Receptores ErbB/genética , Genes ras , Mutación , Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodos , Biopsia , ADN Tumoral Circulante/sangre , Humanos , Límite de Detección , Biopsia Líquida , Sondas Moleculares , Neoplasias/sangre , Neoplasias/patologíaRESUMEN
BACKGROUND: In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. METHODS: Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 (TP53) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). RESULTS: Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS (r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index (P = 0.003), tumor grade (P = 0.003), and stage (P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free (P < 0.001) and overall (P = 0.006) survival. CONCLUSIONS: Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival.
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ADN de Neoplasias/sangre , Terapia Neoadyuvante , Neoplasias de la Mama Triple Negativas/sangre , Neoplasias de la Mama Triple Negativas/terapia , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Neoplasias de la Mama Triple Negativas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Triple-negative breast cancer (TNBC) is a highly aggressive disease characterized by a high number of relapses and poor overall survival. The heterogeneity of the disease and the limited treatment options compared to other breast cancer subtypes mainly explain these clinical outcomes. New biomarkers are urgently needed to improve the management of TNBC. Circulating tumor DNA, identified by tumor-related molecular alterations, could be used in the context of non-invasive "liquid biopsy" and help in TNBC diagnosis and treatment decisions. In this review, we discuss the key issues related to the potential of circulating tumor DNA to improve the management of this disease and the future steps to overcome before its implementation into clinical routine within the next 5 years.
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Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , Patología Molecular , Neoplasias de la Mama Triple Negativas/genética , Biomarcadores de Tumor/sangre , ADN de Neoplasias/sangre , Femenino , Humanos , Células Neoplásicas Circulantes/patología , Pronóstico , Neoplasias de la Mama Triple Negativas/sangre , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.
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Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Fragmentos de Péptidos/genética , Secuencia de Aminoácidos , Ciclohexanos/farmacología , Genotipo , Proteína gp120 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , VIH-1/fisiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Maraviroc , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Fenotipo , Mutación Puntual , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Reproducibilidad de los Resultados , Triazoles/farmacología , Tropismo Viral/efectos de los fármacos , Tropismo Viral/genética , Tropismo Viral/fisiologíaRESUMEN
Cell-free tumor DNA (ctDNA) has the potential to enable non-invasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The histology-independent phase II SHIVA trial matches patients with targeted therapeutics based on previous screening of multiple somatic mutations using metastatic biopsies. To evaluate the utility of ctDNA in this trial, as an ancillary study we performed de novo detection of somatic mutations using plasma DNA compared to metastasis biopsies in 34 patients covering 18 different tumor types, scanning 46 genes and more than 6800 COSMIC mutations with a multiplexed next-generation sequencing panel. In 27 patients, 28 of 29 mutations identified in metastasis biopsies (97%) were detected in matched ctDNA. Among these 27 patients, one additional mutation was found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. These results suggest that ctDNA analysis is a potential alternative and/or replacement to analyses using costly, harmful and lengthy tissue biopsies of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patient's tumor genetic content.
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ADN de Neoplasias/sangre , Técnicas de Genotipaje , Neoplasias/genética , Neoplasias/patología , Células Neoplásicas Circulantes/metabolismo , Medicina de Precisión , Anciano , Anciano de 80 o más Años , Biopsia , ADN de Neoplasias/metabolismo , Femenino , Frecuencia de los Genes/genética , Humanos , Persona de Mediana Edad , Mutación/genética , Metástasis de la Neoplasia , Neoplasias/metabolismo , Células Neoplásicas Circulantes/patologíaRESUMEN
OBJECTIVES: To track changes in the V3 env region of HIV-1 quasispecies and determine virus coreceptor use in cell reservoirs of patients on long-term suppressive antiretroviral therapy (ART). PATIENTS AND METHODS: Ten patients whose plasma viraemia had been suppressed for a median of 5.5 years were followed for 5 years. The V3 env regions of viruses in peripheral blood mononuclear cells were analysed by ultra-deep sequencing (UDS). HIV-1 tropism was predicted using the geno2pheno 5.75 algorithm and a phenotypic assay. RESULTS: The UDS and phenotypic assay data were concordant for predicting HIV-1 tropism. CXCR4-using viruses detected by UDS accounted for 14.7%-76.5% of the virus populations in samples from five patients at enrolment. Five patients harboured pure R5 virus populations and no X4 viruses emerged during the 5 years. The selection pressures estimated by the dN/dS ratio were acting on the V3 region to produce diversification of the quasispecies in CXCR4-infected patients and purification of the quasispecies in R5-infected patients on effective ART. CONCLUSIONS: UDS showed that the virus quasispecies in cell reservoirs of patients on long-term suppressive ART continued to evolve. CXCR4-using variants became more diversified. Analysis of the selection pressures on the virus quasispecies could provide a clearer picture of virus persistence in patients on effective ART.
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Antirretrovirales/uso terapéutico , Variación Genética , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/fisiología , Receptores del VIH/análisis , Tropismo Viral , Adulto , ADN Viral/genética , Femenino , Genotipo , Técnicas de Genotipaje , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , VIH-1/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Fenotipo , Cultivo de Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
OBJECTIVES: R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections (PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%-6.4% of subjects also harbour CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using viruses among HIV-1 quasispecies. METHODS: We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population. We then used UDS of the V3 env region with the geno2pheno algorithm (false positive rate = 5.75) to identify the HIV-1 quasispecies. RESULTS: CXCR4-using viruses were detected in all but 1 of the 11 patients by UDS, and accounted for 0.2%-100% of the virus populations. The frequency of CXCR4-using viruses was <20% in six subjects and 100% in four subjects. Virus populations containing 100% CXCR4-using variants during PHI persisted for at least 1-2 years after the acute phase. The CCR5 Δ32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5 (15%) viruses. CONCLUSIONS: UDS and the phenotype were concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis.
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Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Viral/genética , Receptores CXCR4/metabolismo , Tropismo Viral , Adulto , Femenino , Variación Genética , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/clasificación , Humanos , Masculino , Receptores del VIH/metabolismo , Adulto JovenRESUMEN
OBJECTIVES: Resistance of HIV-1 to CCR5 antagonists can occur without coreceptor switching by mutations in envelope glycoproteins that enable virus entry using the inhibitor-bound form of CCR5. We investigated whether mutations in the V3 region of HIV-1 from subjects naive to maraviroc could be associated with primary resistance to this drug. METHODS: The frequency of CCR5-tropic HIV-1 subtype B isolates harbouring putative V3 maraviroc resistance mutations was assessed among the HIV tropism database of Toulouse University Hospital, France. Phenotypic assessment of maraviroc susceptibility was performed for 14 isolates representative of the main mutation patterns and 14 controls. V3 mutations were reversed or introduced by site-directed mutagenesis. RESULTS: Ninety-three of 951 (9.8%) isolates harboured V3 mutations assumed to be associated with maraviroc resistance. Maraviroc completely blocked virus entry for all but 1 of the 14 isolates harbouring V3 mutations [IC50 8.6 nM; 95% CI (6.6-47.4)], as in the 14 control isolates [IC50 13.4 nM; 95% CI (7.7-50.3)] (P = 0.24). Primary resistance to maraviroc, with a plateau in entry inhibition, was found in one isolate (harbouring a 20F/21I genotype). Site-directed mutagenesis showed that V3 mutations are necessary but not sufficient to induce maraviroc resistance. CONCLUSIONS: The impact of V3 mutations depended on the env context in which they occurred. Simple assessment of the V3 genotype thus cannot accurately predict maraviroc resistance. Rather, phenotypic assessment of virus particles expressing the envelope glycoprotein as a whole is required. This approach revealed that primary resistance of CCR5-tropic HIV-1 subtype B isolates to maraviroc seems uncommon.
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Fármacos Anti-VIH/farmacología , Ciclohexanos/farmacología , Farmacorresistencia Viral , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Triazoles/farmacología , Francia , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Maraviroc , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación Missense , Análisis de Secuencia de ADNRESUMEN
Success to antiretroviral HIV treatment is reduced by the presence of resistant variants. These variants can be present at very low level in the viral population and not detected by conventional assay. High throughput sequencing technologies allow the detection of minority variants present at less than 20 % and their quantification, easily and rapidly. However, the influence of minority variants on the treatment response must be determined. The detection of minority variants resistant to non-nucleoside reverse transcriptase inhibitors (NNRTI) is associated to an increased risk of virological failure in patients with an NNRTI containing treatment. The detection of X4 using variants is also associated to an increased risk of virological failure in patients treated with a CCR5 antagonist. This association was not observed with other antiretroviral classes. Although, these data need to be confirmed in prospective studies, detection of minority variants could help in optimizing NNRTI and CCR5 antagonist containing treatment.
RESUMEN
UNLABELLED: The impact of minor drug-resistant variants of the type 1 immunodeficiency virus (HIV-1) on the failure of antiretroviral therapy remains unclear. We have evaluated the importance of detecting minor populations of viruses resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTI) during intermittent antiretroviral therapy, a high-risk context for the emergence of drug-resistant HIV-1. We carried out a longitudinal study on plasma samples taken from 21 patients given efavirenz and enrolled in the intermittent arm of the ANRS 106 trial. Allele-specific real-time PCR was used to detect and quantify minor K103N mutants during off-therapy periods. The concordance with ultra-deep pyrosequencing was assessed for 11 patients. The pharmacokinetics of efavirenz was assayed to determine whether its variability could influence the emergence of K103N mutants. Allele-specific real-time PCR detected K103N mutants in 15 of the 19 analyzable patients at the end of an off-therapy period while direct sequencing detected mutants in only 6 patients. The frequency of K103N mutants was <0.1% in 7 patients by allele-specific real-time PCR without further selection, and >0.1% in 8. It was 0.1%-10% in 6 of these 8 patients. The mutated virus populations of 4 of these 6 patients underwent further selection and treatment failed for 2 of them. The K103N mutant frequency was >10% in the remaining 2, treatment failed for one. The copy numbers of K103N variants quantified by allele-specific real-time PCR and ultra-deep pyrosequencing agreed closely (ρâ=â0.89 P<0.0001). The half-life of efavirenz was higher (50.5 hours) in the 8 patients in whom K103N emerged (>0.1%) than in the 11 patients in whom it did not (32 hours) (Pâ=â0.04). Thus ultrasensitive methods could prove more useful than direct sequencing for predicting treatment failure in some patients. However the presence of minor NNRTI-resistant viruses need not always result in virological escape. TRIAL REGISTRATION: ClinicalTrials.gov NCT00122551.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Benzoxazinas/uso terapéutico , Farmacorresistencia Viral/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Adulto , Alquinos , Ciclopropanos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto Joven , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
We used ultra-deep pyrosequencing and the Toulouse Tropism Test phenotypic assay to determine the prevalence of CXCR4-using viruses in 21 patients with primary HIV-1 infections. We found X4-containing virus populations in 9% of patients by ultra-deep pyrosequencing using position-specific scoring matrices (PSSM(X4/R5)) or geno2pheno(5.75) and in 14% using the combined 11/25 and net charge rule. The phenotypic assay identified 9% of CXCR4-using viruses. This confirms that R5 viruses are predominant in primary HIV-1 infections.