Genotypic Drug Resistance Research Articles

Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms.

ABSTRACTHigh genetic diversity, including the emergence of recombinant forms (RFs), is one of the most prominent features of human immunodeficiency virus type 1 (HIV-1). Conventional detection of HIV-1 RFs requires pretreatments, i.e., cloning or single-genome amplification, to distinguish them from dual- or multiple-infection variants. However, these processes are time-consuming and labor-intensive. Here, we constructed a new nanopore sequencing-based platform that enables us to obtain distinctive genetic information for intersubtype RFs and dual-infection HIV-1 variants by using amplicons of HIV-1 near-full-length genomes or two overlapping half-length genome fragments. Repeated benchmark tests of HIV-1 proviral DNA revealed consensus sequence inference with a reduced error rate, allowing us to obtain sufficiently accurate sequence data. In addition, we applied the platform for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection according to Sanger sequencing-based genotyping tests for HIV-1 drug resistance. For each RF infection case, replicated analyses involving our nanopore sequencing-based platform consistently produced long consecutive analogous consensus sequences with mosaic genomic structures consisting of two different subtypes. In contrast, we detected multiple heterologous sequences in each dual-infection case. These results demonstrate that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Since the genetic diversity of HIV-1 continues to gradually increase, this system will help accelerate full-length genome analysis and molecular epidemiological surveillance for HIV-1.IMPORTANCE HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases. In this study, we developed a new analytical system for HIV-1 sequence data obtained by nanopore sequencing. The error rate of this method was reduced to ~0.06%. We applied this system for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection, which were extracted from 373 cases of HIV patients based on our retrospective analysis of HIV-1 drug resistance genotyping test results. We found that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Our protocol will be useful for epidemiological surveillance to examine HIV-1 transmission as well as for genotypic tests of HIV-1 drug resistance in clinical settings.

Characterization of HIV-1 molecular epidemiology and transmitted drug-resistance in newly diagnosed HIV-infected patients in Sichuan, China

BackgroundSichuan province is one of the highest AIDS epidemic provinces in China, with a large number of floating population. The annual number of cases of HIV/AIDS reported in Sichuan has been the highest province in China for several successive years. There is a lack of widespread and representative data on the distribution of HIV genotypes in Sichuan. We aim to investigate the characteristics of HIV-1 molecular epidemiology and transmitted drug-resistance in newly diagnosed HIV-infected patients in Sichuan, China.MethodArchived plasma samples (n = 1524) from HIV-1 newly-diagnosed individuals in April 2019 were selected by cross-sectional investigation from all 21 cities in Sichuan province. Phylogenetic relationship, transmission cluster, and genotypic drug resistance analyses were performed using HIV-1 polymerase (pol) gene sequences. We also analysed the association of demographic and virological factors with transmitted drug-resistance (TDR) and transmission clusters.ResultsPartial pol gene sequences were obtained from 1297 cases. HIV-1 epidemic strains in Sichuan province: the majority of genotypes were circulating recombinant form (CRF) 07_BC (675, 52.04%), CRF01_AE (343, 26.45%), CRF08_BC (115, 8.87%), CRF85_BC (67, 5.17%), subtype B (33, 2.54%), the other genotypes only accounted for 4.93%, and unique recombinant forms (URFs) (23, 1.77%) were observed in the study, and the difference of age, ethnicity, education, occupation, region and transmission pathway of different genotypes were statistically significant. According to WHO HIVDR surveillance threshold, the level of TDR has reached a medium level, with 72 of 1297 (5.55%) cases carrying drug-resistance mutation sites, TDR mutation frequency to nonnucleoside reverse transcriptase inhibitors (NNRTIs, 3.85%) was much higher than nucleoside reverse transcriptase inhibitors (NRTIs, 0.31%) and protease inhibitors (PIs, 1.70%), and CRF08_BC was a risk factor for TDR (odds ratio, 8.32; 95% CI 4.38–15.80 for CRF07_BC, P < 0.05). The most common drug resistance HIV-1 mutation pattern for NNRTI was V106 (1.31%, 17/1297) and E138 (1.16%, 15/1297), and for PI was M46 (0.69%, 9/1297). A total of 205 (15.8%) pol sequences were involved in the genetic transmission network clusters, CRF01_AE (odds ratio, 2.369; 95% CI 1.659–3.382; P < 0.05), subtype B (odds ratio, 13.723; 95% CI 6.338–29.71; P < 0.05), drug resistance (odds ratio, 0.306; 95% CI 0.106–0.881; P < 0.05) and different levels of education (P < 0.05) were significantly associated to be in clusters.ConclusionThe distribution of HIV-1 genotypes in Sichuan is more diverse and complex, and the Men who have sex with men (MSM) is underrated, arguing for behavior scaling up intervention in this specific population besides the elderly people with heterosexual transmission risk groups. The risk of TDR mutation frequency increased in newly diagnosed patients highlights the significance of genotypic drug resistance monitoring and molecular surveillance of pretreatment HIV-1 drug resistance. The regimen composed of TDF, 3TC and EFV was still currently the preferred solution used free first-line therapy.

Overview of the Analytes Applied in Genotypic HIV Drug Resistance Testing.

The close monitoring of HIV drug resistance using genotypic HIV drug resistance testing (HIVDRT) has become essential for effective HIV/AIDS management at both individual and population levels. Over the years, a broad spectrum of analytes or specimens have been applied or attempted in HIVDRT; however, the suitability and performance of these analytes in HIVDRT and the clinical relevance of the results from them may vary significantly. This article provides a focused overview of the performance, strengths, and weaknesses of various analytes while used in HIVDRT, which may inform the optimal analytes selection in different application contexts.

Accelerated cycling PCR: A novel tool for rapid, sensitive and specific detection of single-nucleotide mutation within 30 min

Rapid detection of single-nucleotide mutations (SNMs) has played a vital role for point-of-care testing. We herein first introduced accelerated thermal cycling into conventional allele-specific qPCR (AS-qPCR), named accelerated cycling PCR (AC-PCR) to achieve rapid and sensitive detection of SNM. It could simultaneously detect 10 copies of H. pylori DNA and identify its clarithromycin-resistance genotype within 30 min, and showed 100-fold enhanced specificity than AS-qPCR. Therefore, AC-PCR shows great potential in clinical diagnosis for drug-resistance mutation or genotyping analysis.

Probe Capture Enrichment Methods for HIV and HCV Genome Sequencing and Drug Resistance Genotyping.

Human immunodeficiency virus (HIV) infections remain a significant public health concern worldwide. Over the years, sophisticated sequencing technologies such as next-generation sequencing (NGS) have emerged and been utilized to monitor the spread of HIV drug resistance (HIVDR), identify HIV drug resistance mutations, and characterize transmission dynamics. Similar applications also apply to the Hepatitis C virus (HCV), another bloodborne viral pathogen with significant intra-host genetic diversity. Several advantages to using NGS over conventional Sanger sequencing include increased data throughput, scalability, cost-effectiveness when batched sample testing is performed, and sensitivity for quantitative detection of minority resistant variants. However, NGS alone may fail to detect genomes from pathogens present in low copy numbers. As with all sequencing platforms, the primary determinant in achieving quality sequencing data is the quality and quantity of the initial template input. Samples containing degraded RNA/DNA and/or low copy number have been a consistent sequencing challenge. To overcome this limitation probe capture enrichment is a method that has recently been employed to target, enrich, and sequence the genome of a pathogen present in low copies, and for compromised specimens that contain poor quality nucleic acids. It involves the hybridization of sequence-specific DNA or RNA probes to a target sequence, which is followed by an enrichment step via PCR to increase the number of copies of the targeted sequences after which the samples are subjected to NGS procedures. This method has been performed on pathogens such as bacteria, fungus, and viruses and allows for the sequencing of complete genomes, with high coverage. Post NGS, data analysis can be performed through various bioinformatics pipelines which can provide information on genetic diversity, genotype, virulence, and drug resistance. This article reviews how probe capture enrichment helps to increase the likelihood of sequencing HIV and HCV samples that contain low viral loads and/or are compromised.

Predominance of the East-Asian Beijing genotype in a Mycobacterium tuberculosis drug-resistant population in Central Malaysia

ObjectivesPrevious diversity studies on local Mycobacterium tuberculosis (MTB) isolates, with or without antibiotic resistance, showed predominance of Indo-Oceanic East African-Indian (EAI) strains. This study focused specifically on a drug-resistant MTB population from Central Malaysia and aimed to investigate the genotypes and resistance patterns involved. MethodsWhole-genome sequencing was performed on 56 local MTB isolates with known rifampicin resistance or multidrug resistance towards 13 anti-TB agents. Analysis of each genome sequence was performed using the widely recognized online MTB genotyping platforms, TBProfiler and Mykrobe, to determine lineage and genotypic drug resistance profiles. Results: Forty (71.4%) isolates were identified as East-Asian Beijing strains. Phenotypic to genotypic antibiotic resistance patterns differed in 33 isolates (58.9%), with one isolate showing extensive drug-resistance (XDR) previously not detected by conventional drug-susceptibility testing. ConclusionThis drug resistance population study demonstrated predominance of the East-Asian Beijing strains and a newly detected extensively drug-resistant MTB (XDR-TB) isolate in Malaysia. Information regarding the association between lineage and drug-resistant TB in Malaysia is scarce, and more studies are needed to determine the significance of such association, if any, in our local settings.

Phenotypic and genomic characterization for Salmonella isolates recovered from meat products in Beijing wholesale markets, 2014-2015

Objective: To analyze the phenotypic and genomic characteristics of Salmonella isolates recovered from meat products in Beijing wholesale markets. Methods: A total of 336 Salmonella strains from meat products collected from wholesale markets in Beijing were tested for antimicrobial resistance to 25 antimicrobial compounds by micro-broth dilution method; whole genome data were sequenced, followed by the serotype and ST type prediction by Seqsero2 and SISTR software, and the drug resistance genes and virulence factors were also predicted with CARD and VFDB databases of Abricate software; Salmonella serotyping assay kit and serum agglutination method were used for serotype confirmation of some isolates with different genome prediction results. Results: The resistance rates to Nalidixic acid and Ampicillin were 62.5% (210/336) and 55.1% (185/336), respectively, and all isolates were susceptible to Tigecyclin, Cefoxitin and Carbapenem antimicrobial compounds; 207 isolates (61.6%, 207/336) were multi-drug resistant, some could even be resistant to ten categories of drugs at the same time, and the most common antimicrobial resistance spectrum was NAL-AMP-SAM. A total of 24 serotypes were detected with predominant serotypes of Enteritidis (34.5%, 116/336), Derby (17.3%, 58/336) and Indiana (10.4%, 35/336). A total of 27 ST types were detected, the dominant type was ST11; ST types were in good consistency with serotypes; The detection rates of resistant genes referred to aminoglycosides, fluoroquinolones, β-lactams, sulfonamides and tetracyclines are more than 48%, and the first two reached 100%. The prediction of drug resistance genes was consistent with the results of antimicrobial resistance phenotype. A total of 122 virulence genes were predicted, 74 of which existing among all isolates. Conclusion: Salmonella in meat from the wholesale markets of Beijing has a high proportion of multiple drug resistance, a complex drug resistance spectrum, a variety of serotypes and ST types, and a high carrying rate of drug resistance gene and virulence gene; drug resistance phenotype and genotype are relatively consistent.

HIV-1 drug resistance genotyping success rates and correlates of Dried-blood spots and plasma specimen genotyping failure in a resource-limited setting

BackgroundHIV-1 drug resistance genotyping is critical to the monitoring of antiretroviral treatment. Data on HIV-1 genotyping success rates of different laboratory specimen types from multiple sources is still scarce.MethodsIn this cross-sectional study, we determined the laboratory genotyping success rates (GSR) and assessed the correlates of genotyping failure of 6837 unpaired dried blood spot (DBS) and plasma specimens. Specimens from multiple studies in a resource-constrained setting were analysed in our laboratory between 2016 and 2019.ResultsWe noted an overall GSR of 65.7% and specific overall GSR for DBS and plasma of 49.8% and 85.9% respectively. The correlates of genotyping failure were viral load (VL) < 10,000 copies/mL (aOR 0.3 95% CI: 0.24–0.38; p < 0.0001), lack of viral load testing prior to genotyping (OR 0.85 95% CI: 0.77–0.94; p = 0.002), use of DBS specimens (aOR 0.10 95% CI: 0.08–0.14; p < 0.0001) and specimens from routine clinical diagnosis (aOR 1.4 95% CI: 1.10–1.75; p = 0.005).ConclusionsWe report rapidly decreasing HIV-1 genotyping success rates between 2016 and 2019 with increased use of DBS specimens for genotyping and note decreasing median viral loads over the years. We recommend improvement in DBS handling, pre-genotyping viral load testing to screen samples to enhance genotyping success and the development of more sensitive assays with well-designed primers to genotype specimens with low or undetectable viral load, especially in this era where virological suppression rates are rising due to increased antiretroviral therapy roll-out.

Molecular Characterization of the pol Gene of Vertically Transmitted HIV-1 Strains in Children with Virological Failure.

HIV-1 pol gene sequences were analyzed from 77 HIV-1 positive children infected perinatally and exhibiting virological failure (VF). Viral subtyping, phylogenetic analysis, and genotypic drug resistance analysis were carried out on samples collected before start of anti retroviral treatment (ART) (baseline, BL), and at 12 months post-ART initiation (M12). Subtype C was found to be most predominant, seen in 75 of the 77 (97.4%) children. The level of pretreatment drug resistance (PDR) was 14% among these children. At BL, K103N (5), E138A/G (4), and M184V (3) were the most common mutations. At M12 the prevalence of any resistance-associated mutation (RAM) (acquired drug resistance/ADR) was 81.8% (63/77). Dual class resistance mutations were seen in 64% (49/77) of children. M184V/I, K103N/S, and Y181C were the most commonly occurring mutations, seen in 76%, 51%, and 36% children. RAMs to the second-generation non-nucleoside reverse transcriptase inhibitors (NNRTI), etravirine (ETR) and rilpivirine (RPV), were seen in 40.2% (31/77) and 48.05% (37/77) of the children, respectively. Our findings reveal similar prevalence rates of PDR and ADR in children with VF as reported in other studies. Occurrence of ETR and RPV resistance associated mutations (RAMs) is of concern and highlights the need for timely switch of regimens guided by genotypic resistance testing in perinatally infected children from India.

Protease and gag diversity and drug resistance mutations among treatment-naive Mexican people living with HIV

IntroductionIn Mexico, HIV genotyping is performed in people living with HIV (PLWH) failing their first-line antiretroviral (ARV) regimen; it is not routinely done for all treatment-naive PLWH before ARV initiation. The first nationally representative survey published in 2016 reported that the prevalence of pretreatment drug mutations in treatment-naive Mexican PLWH was 15.5% to any antiretroviral drug and 10.6% to non-nucleoside reverse transcriptase inhibitors (NNRTIs) using conventional Sanger sequencing. Most reports in Mexico focus on HIV pol gene and nucleoside and non-nucleoside reverse transcriptase inhibitor (NRTI and NNRTI) drug resistance mutations (DRMs) prevalence, using Sanger sequencing, next-generation sequencing (NGS) or both. To our knowledge, NGS has not be used to detect pretreatment drug resistance mutations (DRMs) in the HIV protease (PR) gene and its substrate the Gag polyprotein.MethodsTreatment-naive adult Mexican PLWH were recruited between 2016 and 2019. HIV Gag and protease sequences were obtained by NGS and DRMs were identified using the WHO surveillance drug resistance mutation (SDRM) list.ResultsOne hundred PLWH attending a public national reference hospital were included. The median age was 28 years-old, and most were male. The median HIV viral load was 4.99 [4.39–5.40] log copies/mL and median CD4 cell count was 150 [68.0–355.78] cells/mm3. As expected, most sequences clustered with HIV-1 subtype B (97.9%). Major PI resistance mutations were detected: 8 (8.3%) of 96 patients at a detection threshold of 1% and 3 (3.1%) at a detection threshold of 20%. A total of 1184 mutations in Gag were detected, of which 51 have been associated with resistance to PI, most of them were detected at a threshold of 20%. Follow-up clinical data was available for 79 PLWH at 6 months post-ART initiation, seven PLWH failed their first ART regimen; however no major PI mutations were identified in these individuals at baseline.ConclusionsThe frequency of DRM in the HIV protease was 7.3% at a detection threshold of 1% and 3.1% at a detection threshold of 20%. NGS-based HIV drug resistance genotyping provide improved detection of DRMs. Viral load was used to monitor ARV response and treatment failure was 8.9%.

Evolution and spread of a highly drug resistant strain of Mycobacterium tuberculosis in Papua New Guinea.

BackgroundMolecular mechanisms determining the transmission and prevalence of drug resistant tuberculosis (DR-TB) in Papua New Guinea (PNG) are poorly understood. We used genomic and drug susceptibility data to explore the evolutionary history, temporal acquisition of resistance and transmission dynamics of DR-TB across PNG.MethodsWe performed whole genome sequencing on isolates from Central Public Health Laboratory, PNG, collected 2017–2019. Data analysis was done on a composite dataset that also included 100 genomes previously sequenced from Daru, PNG (2012–2015).ResultsSampled isolates represented 14 of the 22 PNG provinces, the majority (66/94; 70%) came from the National Capital District (NCD). In the composite dataset, 91% of strains were Beijing 2.2.1.1, identified in 13 provinces. Phylogenetic tree of Beijing strains revealed two clades, Daru dominant clade (A) and NCD dominant clade (B). Multi-drug resistance (MDR) was repeatedly and independently acquired, with the first MDR cases in both clades noted to have emerged in the early 1990s, while fluoroquinolone resistance emerged in 2009 (95% highest posterior density 2000–2016). We identified the presence of a frameshift mutation within Rv0678 (p.Asp47fs) which has been suggested to confer resistance to bedaquiline, despite no known exposure to the drug. Overall genomic clustering was significantly associated with rpoC compensatory and inhA promoter mutations (p < 0.001), with high percentage of most genomic clusters (12/14) identified in NCD, reflecting its role as a potential national amplifier.ConclusionsThe acquisition and evolution of drug resistance among the major clades of Beijing strain threaten the success of DR-TB treatment in PNG. With continued transmission of this strain in PNG, genotypic drug resistance surveillance using whole genome sequencing is essential for improved public health response to outbreaks. With occurrence of resistance to newer drugs such as bedaquiline, knowledge of full drug resistance profiles will be important for optimal treatment selection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-022-07414-2.

Close Related Drug-Resistance Beijing Isolates of Mycobacterium tuberculosis Reveal a Different Transcriptomic Signature in a Murine Disease Progression Model.

Mycobacterium tuberculosis (MTB) lineage 2/Beijing is associated with high virulence and drug resistance worldwide. In Colombia, the Beijing genotype has circulated since 1997, predominantly on the pacific coast, with the Beijing-Like SIT-190 being more prevalent. This genotype conforms to a drug-resistant cluster and shows a fatal outcome in patients. To better understand virulence determinants, we performed a transcriptomic analysis with a Beijing-Like SIT-190 isolate (BL-323), and Beijing-Classic SIT-1 isolate (BC-391) in progressive tuberculosis (TB) murine model. Bacterial RNA was extracted from mice lungs on days 3, 14, 28, and 60. On average, 0.6% of the total reads mapped against MTB genomes and of those, 90% against coding genes. The strains were independently associated as determined by hierarchical cluster and multidimensional scaling analysis. Gene ontology showed that in strain BL-323 enriched functions were related to host immune response and hypoxia, while proteolysis and protein folding were enriched in the BC-391 strain. Altogether, our results suggested a differential bacterial transcriptional program when evaluating these two closely related strains. The data presented here could potentially impact the control of this emerging, highly virulent, and drug-resistant genotype.

Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Clinical Feature, Therapy, Antimicrobial Resistance Gene Distribution, and Outcome of Nosocomial Meningitis Induced by Multidrug-Resistant Enterobacteriaceae-A Longitudinal Cohort Study From Two Neurosurgical Centers in Northern China.

ObjectivesThis is a comparative cohort study aiming to evaluate the mortality risk factors for patients with nosocomial meningitis (NM) induced by multidrug-resistant Enterobacteriaceae (MDRE) in China. The clinical features and therapies of patients and the resistance mechanisms of MDRE pathogens were also assessed.MethodsMDRE-NM patients from two neurosurgical centers in China from 2014 to 2019 were included in this study. Clinical features were extracted from the medical record databases of the two centers. The molecular mechanisms underlying the microbiological resistance mechanisms of each MDRE pathogen were determined, Kaplan–Meier survival analysis was conducted, and multivariable analyses were performed using a Cox proportional hazard model.ResultsNinety MDRE-NM patients were included in this study. Klebsiella pneumoniae accounted for the highest proportion of causative pathogens (46/90, 51.1%), and 40 causative pathogens (44.4%) were meropenem-resistant. blaKPC (27/40, 67.5%) was the predominant carbapenem resistance gene. Multivariate Cox analysis showed that external ventricular drainage (EVD) [hazard ratio (HR) = 2.524, 95% confidence interval (CI) = 1.101–5.787, p = 0.029] and a Glasgow Coma Scale (GCS) score ≤;8 (HR = 4.033, 95% CI = 1.526–10.645, p = 0.005) were mortality risk factors for patients with MDRE-NM. A total of 90.0%, 94.4%, and 97.8% of MDRE-NM patients received antibiotic prophylaxis (AP), antibiotic empirical therapy (AET), and antibiotic definitive therapy (ADT), respectively.ConclusionsNM caused by MDRE is an important sign of the failure of neurosurgery. MDRE possesses multiple drug resistance genotypes, and EVD and a GCS score ≤;8 are independent mortality risk factors for patients with MDRE-NM, which deserve the attention of microbiologists and neurosurgical clinicians.

Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon deep sequencing (TADS) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, TADS Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterize the complexity of infection (COI) and drug-resistance genotypes, respectively. P. falciparum positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Of the 30 participants, 9 presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. The COI was comparable between ama1 and msp1, msp2 and glurp, however, overall ama1 detected more haplotypes. Based on ama1, a stable number of haplotypes were detected throughout treatment up until day 3. Additionally, a recrudescent infection was identified with an ama1 haplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 haplotypes and parasitaemia, we identified a fast (&lt;1h) and slow (&gt;5h) clearing haplotype. As expected, only two mdr1 haplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. This study highlights TADS as a sensitive tool for tracking parasite haplotypes throughout treatment and can detect variation in haplotype clearance estimates. TADS can also identify slow clearing haplotypes, a potential early sign of selection during treatment. Consequently, TADS has the capability of improving the discriminatory power to accurately distinguish recrudescences from reinfections.

Management of a human immunodeficiency virus case with discordant antiviral drug resistance profiles in cerebrospinal fluid compared with plasma: a case report

BackgroundHuman immunodeficiency virus-1-associated neurocognitive disorder is a known complication in individuals treated with antiretroviral therapy. Cerebrospinal fluid escape, which is defined as discordant higher cerebrospinal fluid viremia than plasma, may occur in antiretroviral therapy-experienced individuals. Different cerebrospinal fluid versus plasma mutation patterns have been observed in individuals with cerebrospinal fluid escape.Case presentationA 46-year-old adult African male with human immunodeficiency virus-1 infection and acquired immunodeficiency syndrome based on cerebral toxoplasmosis and a chronic hepatitis B virus infection developed cerebrospinal fluid escape. A different human immunodeficiency virus-1 genotypic drug resistance profile was observed in plasma compared with cerebrospinal fluid. Brain biopsy and cerebral magnetic resonance imaging indicated the development of human immunodeficiency virus encephalopathy. A discordant protease inhibitor mutation/wild-type T74PT in plasma but not in cerebrospinal fluid indicated poor central nervous system penetration due to the selective pressure of drug therapy. An intensified antiretroviral therapy regimen including dolutegravir with good central nervous system penetration improved conditions.ConclusionsThis case shows the importance of measuring human immunodeficiency virus drug resistance in cerebrospinal fluid, which might differ from resistance detected in plasma samples and target effective antiretroviral therapy treatment accordingly.

Spectrum of Activity of Raltegravir and Dolutegravir Against Novel Treatment-Associated Mutations in HIV-2 Integrase: A Phenotypic Analysis Using an Expanded Panel of Site-Directed Mutants.

Integrase inhibitors (INIs) are a key component of antiretroviral therapy for human immunodeficiency virus-1 (HIV-1) and HIV-2 infection. Although INI resistance pathways are well-defined for HIV-1, mutations that emerge in HIV-2 in response to INIs are incompletely characterized. We performed systematic searches of GenBank and HIV-2 drug resistance literature to identify treatment-associated mutations for phenotypic evaluation. We then constructed a library of 95 mutants of HIV-2ROD9 that contained single or multiple amino acid changes in the integrase protein. Each variant was tested for susceptibility to raltegravir and dolutegravir using a single-cycle indicator cell assay. We observed extensive cross-resistance between raltegravir and dolutegravir in HIV-2ROD9. HIV-2-specific integrase mutations Q91R, E92A, A153G, and H157Q/S, which have not been previously characterized, significantly increased the half maximum effective concentration (EC50) for raltegravir when introduced into 1 or more mutational backgrounds; mutations E92A/Q, T97A, and G140A/S conferred similar enhancements of dolutegravir resistance. HIV-2ROD9 variants encoding G118R alone, or insertions of residues SREGK or SREGR at position 231, were resistant to both INIs. Our analysis demonstrates the contributions of novel INI-associated mutations to raltegravir and dolutegravir resistance in HIV-2. These findings should help to improve algorithms for genotypic drug resistance testing in HIV-2-infected individuals.

Impact of combinations of clinically observed HIV integrase mutations on phenotypic resistance to integrase strand transfer inhibitors (INSTIs): a molecular study.

Routine HIV drug resistance genotyping identified an integrase sequence harbouring T97A, E138K, G140S and Q148H, with high predicted resistance to all integrase strand transfer inhibitors (INSTIs). To assess the impact of these substitutions alone and together on phenotypic INSTI susceptibility. We constructed recombinant NL4.3 viruses harbouring all mutation combinations in the autologous integrase sequence. Viruses were grown in GFP-reporter CD4+ T-cells in the presence of 0.01-1000 nM raltegravir, elvitegravir, dolutegravir, bictegravir, and cabotegravir. Infection was measured by imaging cytometry. Q148H-containing viruses lacking G140S failed to propagate or mutated in vitro, consistent with fitness costs. Statistically significant reductions in INSTI susceptibility were observed for several mutation combinations, as follows. T97A or G140S alone conferred 3.6- to 5.6-fold decreased susceptibility to raltegravir and elvitegravir. Two-mutation combinations conferred low-to-moderate resistance to raltegravir and elvitegravir only, except G140S/Q148H which eliminated raltegravir and elvitegravir activity and conferred 24.6-, 7.9-, and 107.5-fold reduced susceptibility to dolutegravir, bictegravir and cabotegravir. Addition of E138K to G140S/Q148H conferred 35.5, 11.6 and 208-fold reduced susceptibility to dolutegravir, bictegravir, and cabotegravir, while addition of T97A to G140S/Q148H conferred 318, 121 and >1000-fold reduced susceptibility to these drugs. T97A/E138K/G140S/Q148H in the autologous backbone conferred >300-fold reduced susceptibility to all INSTIs. Notably, bictegravir EC50 was significantly lower when T97A/E138K/G140S/Q148H was introduced into NL4.3, suggesting that other mutations in the autologous sequence enhanced resistance. High-level dolutegravir, bictegravir and cabotegravir resistance requires multiple integrase substitutions including compensatory mutations. T97A and E138K further enhance the resistance conferred by G140S/Q148H, yielding >300-fold decreased susceptibility to all INSTIs when all four mutations are present.

Comparing effectiveness of first-line antiretroviral therapy between peri-urban and rural clinics in KwaZulu-Natal, South Africa.

Viral suppression (VS) is the hallmark of successful antiretroviral therapy (ART) programmes. We sought to compare clinic retention, virological outcomes, drug resistance and mortality between peri-urban and rural settings in South Africa after first-line ART. Beginning in July 2014, 1000 (500 peri-urban and 500 rural) ART-naïve patients with HIV were enrolled and managed according to local standard of care. Clinic retention, virological suppression, virological failure (VF), genotypic drug resistance and mortality were assessed. The definition of VS was a viral load ≤1000 copies/ml. Time to event analyses were stratified by site, median age and gender. Kaplan-Meier curves were calculated and graphed with log-rank modelling to compare curves. Based on 2741 patient-years of follow-up, retention and mortality did not differ between sites. Among all 1000 participants, 47%, 84% and 91% had achieved VS by 6, 12 and 24months, respectively, which was observed earlier in the peri-urban site. At both sites, men aged <32years had the highest proportion of VF (15.5%), while women aged >32years had the lowest, at 7.1% (p=0.018). Among 55genotypes, 42 (76.4%) had at one or more resistance mutations, which did not differ by site. K103N (59%) and M184V (52%) were the most common mutations, followed by V106M and K65R (31% each). Overall, death was infrequent (<4%). No significant differences in treatment outcomes between peri-urban and rural clinics were observed. In both settings, young men were especially susceptible to clinic attrition and VF. More effective adherence support for this important demographic group is needed to achieve UNAIDS targets.

Analysis of HIV quasispecies and virological outcome of an HIV D+/R+ kidney\u2013liver transplantation

IntroductionTransplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney–liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring.MethodsThe donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts.ResultsDonor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up.ConclusionsNo evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations.