Integrase Region Research Articles

Comparison of short-read and long-read next-generation sequencing technologies for determining HIV-1 drug resistance.

Accurate HIV-1 genome sequencing is necessary to identify drug resistance mutations (DRMs) in people with HIV-1 (PWH). Next-generation-sequencing (NGS) allows the detection of minor variants and is now available in many laboratories. Our study aimed to compare two NGS approaches, a "short read" sequencing protocol using DeepChek® Whole Genome HIV-1 Assay on Illumina, and a "long read" sequencing protocol of HIV-1 pol and env single-molecule real-time sequencing (SMRT) on Pacific Biosciences (PacBio). We analyzed 16 plasma samples and 13 cellular samples from PWH. HIV-1 whole genome was amplified into five amplicons using DeepChek® Whole Genome HIV-1 Assay and sequenced on an iSeq. 100. In parallel, HIV-1 pol and env genes were separately amplified and sequenced using PacBio SMRT system with the circular consensus sequencing mode on a Sequel IIe. Concordance rates for determining DRMs with both approaches varied depending on the HIV-1 region, with higher concordance in the integrase region compared to the reverse transcriptase and protease regions. DeepChek® Whole Genome HIV-1 Assay exhibited better sensitivity in HIV-1 RNA sequencing of plasmas with lower viral loads. In cell HIV-1 DNA sequencing, the DeepChek® Whole Genome HIV-1 Assay performed better in pol and env sequencing but detected more APOBEC-induced DRMs, which can represent defective proviruses. Our findings indicate that both DeepChek® Whole Genome HIV-1 Assay and PacBio SMRT sequencing exhibit good performance for subtype determination, detection, and quantification of DRMs of the HIV-1 genome. However, some discrepancies were found in cellular samples, highlighting the challenges of interpreting HIV-1 DNA DRMs.

HIV Drug Resistance in Newly Diagnosed Young Children in the Western Cape, South Africa.

Pretreatment of HIV drug resistance among children living with HIV (CLHIV) can compromise antiretroviral therapy (ART) effectiveness. Resistance may be transmitted directly from mothers or acquired following exposure to antiretrovirals consumed through breastfeeding or administered as prophylaxis. We performed resistance testing in children aged <3 years, newly diagnosed with HIV in Western Cape, South Africa (2021-2022), who either (1) acquired HIV via possible breastfeeding transmission from mothers who received ART (any regimen) during pregnancy/postpartum and/or (2) were exposed to protease inhibitors or integrase strand transfer inhibitors (INSTIs) in utero. Possible breastfeeding transmission was defined as testing HIV-polymerase chain reaction positive at age >28 days, after previously testing negative. We used surveillance drug-resistance mutation lists to define mutations. We included 135 CLHIV. Most mothers started ART prepregnancy (73%). Overall, 57% (77/135) of children had resistance mutations detected. Nonnucleoside reverse transcriptase inhibitor-associated, nucleoside reverse transcriptase inhibitor-associated, protease inhibitor-associated and INSTI-associated mutations were found in 55% (74/135), 10% (13/135), <1% (1/135) and <1% (1/122) of children tested, respectively. One child with breastfeeding transmission had high-level INSTI resistance detected at HIV diagnosis, aged 18 months (E138K and G118R mutations). Although not clinically relevant, nonnucleoside reverse transcriptase inhibitor-associated mutations were common. Dolutegravir is currently the preferred first-line treatment for adults and CLHIV age ≥4 weeks, and although very low INSTI resistance levels have been observed in adults, limited data exist on genotyping the integrase region in children. Pretreatment INSTI resistance in children is likely to be unusual, but future surveillance, including longitudinal studies with paired mother-child resistance testing, is needed.

Full-Spectrum Surveillance of Pre-Treatment HIV Drug Resistance in Southeastern China.

HIV drug resistance compromises the ability of anti-retroviral therapy (ART) to suppress viral replication, resulting in treatment failure. This study investigates the prevalence of pre-treatment drug resistance (PDR) in newly diagnosed individuals in a prosperous city (Wenzhou) in Southeastern China. A cross-sectional investigation was carried out among 473 newly diagnosed ART-naive HIV-1-infected individuals between January and December 2022. The protease-reverse transcriptase (PR-RT) region and integrase (IN) region of HIV-1 were amplified by two separately nested PCRs, followed by sequencing. Drug resistance mutations (DRMs) and drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and integrase strand transfer inhibitors (INSTIs) were analyzed. The PDR prevalence was 6.5% [95% CI: 4.4-9.1] for any anti-retroviral drug, 0.9% [95% CI: 0.3-2.3] for NRTIs, 4.1% [95% CI: 2.5-6.5] for NNRTIs, 1.8% [95% CI: 0.8-3.6] for PIs and 0.5% [95% CI: 0.1-1.8] for INSTIs. According to the subtyping results of the PR-RT region, 11 different subtypes and 31 unique recombinant forms (URFs) were found. CRF07_BC was the dominant subtype (53.7%, 233/434), followed by CRF01_AE (25.3%, 110/434). V179D (1.6%) and K103N (1.4%) were the most predominant types of NNRTI DRMs. Q58E (1.2%) and M184V (0.7%) were the most frequent PI DRMs and NRTI DRMs, respectively. The INSTI-related DRMs Y143S (causes high-level resistance to RAL) and G163K (causes low-level resistance to EVG and RAL) were found in one patient each. Given the relatively high PDR prevalence of NNRTI (4.1%), non-NNRTI-based ART may be preferred in the future. It is recommended to include genotypic resistance testing before starting ART in regions where feasible.

1601. Transmitted Drug Resistance in Treatment-Naïve People Living with HIV in Costa Rica

Abstract Background Transmitted drug resistance (TDR) occurs in people living with HIV infection (PLWH) who are not exposed to antiretroviral drugs but who are infected with a virus with mutations associated with resistance. In Costa Rica, HIV genotyping is performed in PLWH failing their first-line ART regimen, but it is not routinely done for all treatment-naïve PLWH before ART initiation, therefore, little is known about TDR prevalence. Methods The aim of this study was to determine the prevalence of TDR and the HIV mutation patterns. During 2020, 162 cases of newly diagnosed treatment-naïve PLWH from all of the country’s HIV clinics were included for a genotyping study. Viral ARN was extracted from plasma samples obtained from the HIV National Reference Laboratory and protease (PR), reverse transcriptase (RT), and integrase (IN) regions were amplified and sequenced. The HIV database Program (Stanford University) and the World Health Organization (WHO) 2009 TDR surveillance mutation list were used for interpretation of resistance-associated mutations. Results The overall prevalence of TDR was 36% (N=59/162). According to ART class, TDR was 22% in the nucleoside reverse transcriptase inhibitors (NRTI), followed by 13% in the non-nucleoside reverse transcriptase inhibitors (NNRTI); for the protease inhibitors (PI) and the integrase inhibitors (INI), TDR was 8% and 3%, respectively. (Figures 1-3). Most common mutations were: in NRTI, M41L (9.9%), and M184I (6.8%); in NNRTI, K103N (7.4%); in PI, M46I (4.3%); in INI, T97A (1.9%). (Table 1). The median age was 32 years-old (range: 16-71), and 91% were male. Median CD4 count was 420 cells/uL (range: 11-1670) and median viral load was 588337 copies/mL (range: 1220-10000000). According to CDC classification, 20% were stage C-3. Source: local database Source: local database Source: local database Conclusion This study population has a moderate level of HIV drug resistance (HIVDR) in ART-naïve PLWH and strongly implies the need to introduce HIVDR surveillance in Costa Rica. These findings confirm the TDR and highlight the need for systematic viral load monitoring and drug resistance testing at diagnosis. Continuous surveillance of newly infected individuals is required to help strategize the best ART regimen for these patients, and should prompt a change in current local recommendations for starting ART. Source: local database Disclosures All Authors: No reported disclosures

Genetic characteristics and integration specificity of Salmonella enterica temperate phages.

Temperate phages can engage in the horizontal transfer of functional genes to their bacterial hosts. Thus, their genetic material becomes an intimate part of bacterial genomes and plays essential roles in bacterial mutation and evolution. Specifically, temperate phages can naturally transmit genes by integrating their genomes into the bacterial host genomes via integrases. Our previous study showed that Salmonella enterica contains the largest number of temperate phages among all publicly available bacterial species. S. enterica is an important pathogen that can cause serious systemic infections and even fatalities. Initially, we extracted all S. enterica temperate phages from the extensively developed temperate phage database established in our previous study. Subsequently, we conducted an in-depth analysis of the genetic characteristics and integration specificity exhibited by these S. enterica temperate phages. Here we identified 8,777 S. enterica temperate phages, all of which have integrases in their genomes. We found 491 non-redundant S. enterica temperate phage integrases (integrase entries). S. enterica temperate phage integrases were classified into three types: intA, intS, and phiRv2. Correlation analysis showed that the sequence lengths of S. enterica integrase and core regions of attB and attP were strongly correlated. Further phylogenetic analysis and taxonomic classification indicated that both the S. enterica temperate phage genomes and the integrase gene sequences were of high diversities. Our work provides insight into the essential integration specificity and genetic diversity of S. enterica temperate phages. This study paves the way for a better understanding of the interactions between phages and S. enterica. By analyzing a large number of S. enterica temperate phages and their integrases, we provide valuable insights into the genetic diversity and prevalence of these elements. This knowledge has important implications for developing targeted therapeutic interventions, such as phage therapy, to combat S. enterica infections. By harnessing the lytic capabilities of temperate phages, they can be engineered or utilized in phage cocktails to specifically target and eradicate S. enterica strains, offering an alternative or complementary approach to traditional antibiotic treatments. Our study has implications for public health and holds potential significance in combating clinical infections caused by S. enterica.

No transmitted drug resistance to HIV integrase strand-transfer inhibitors after their scale-up in Estonia in 2017

In Eastern Europe HIV-1 transmitted drug resistance (TDR) data, especially in integrase (IN) region is limited. In Estonia INSTI (Integrase Strand Transfer Inhibitors) TDR has been studied only prior to the INSTI scale-up in late 2010s. Current study aimed to determine the levels of protease (PR), reverse transcriptase (RT) and IN surveillance drug resistance mutations (SDRMs) among newly diagnosed patients in Estonia in 2017. Study included 216 newly diagnosed HIV-1 individuals from January 1 until December 31, 2017 in Estonia. Demographic and clinical data were obtained from the Estonian Health Board, Estonian HIV Cohort Study (E-HIV) and clinical laboratories' databases. The PR-RT and IN regions were sequenced and analyzed for SDRMs and subtype determination. Seventy-one percent (151/213) of available HIV-positive samples were successfully sequenced. The overall level of TDR was 7.9% (12/151; 95% CI 4.4-13.8%); no dual or triple class resistance was detected. No major INSTI mutations were found. The distribution of SDRMs for NNRTI, NRTI and PI was 5.9% (9/151), 1.3% (2/151) and 0.7% (1/151), respectively. The predominant NNRTI mutation was K103N. CRF06_cpx was predominant variant (59%) in Estonian HIV-1 population followed by subtype A (9%) and subtype B (8%). Although no major INSTI mutations were found, close monitoring of INSTI SDRMs is needed considering the extensive use of the first and second-generation INSTIs. PR-RT TDR is slowly rising in Estonia indicating the need for continuous surveillance in the future. Low genetic barrier NNRTIs should be avoided in the treatment regimens.

High-level resistance to non-nucleos(t)ide reverse transcriptase inhibitor based first-line antiretroviral therapy in Ghana; A 2017 study.

Expanding access to effective antiretroviral therapy (ART) is a major tool for management of Human Immunodeficiency Virus (HIV) infection. However, rising levels of HIV drug-resistance have significantly hampered the anticipated success of ART in persons living with HIV (PLWH), particularly those from Africa. Though great strides have been made in Ghana toward achieving the UNAIDS “95-95-95” target, a substantial number of PLWH receiving ART have not attained viral suppression. This study investigated patterns of drug resistance mutations in ART naïve as well as ART-experienced PLWH receiving first-line regimen drugs from Ghana. In a cross-sectional study, blood samples were collected from HIV-1 infected adults (≥18 years) attending HIV/AIDS clinic at the Eastern Regional Hospital, Koforidua, Ghana from September to October 2017. Viral RNA isolated from plasma were subjected to genotypic drug resistance testing for Protease Inhibitors (PI), Reverse Transcriptase Inhibitors (RTI), and Integrase Strand Transfer Inhibitors (INSTI). A total of 95 (84 ART experienced, 11 ART naïve) HIV-1 infected participants were sampled in this study. Sixty percent (50/84) of the ART-experienced participants were controlling viremia (viral load < 1,000 copies/ml). Of the 95 patient samples, 32, 34, and 33 were successfully sequenced for protease, reverse-transcriptase, and integrase regions, respectively. The dominant HIV-1 subtypes detected were CRF02_AG (70%), and A3 (10%). Major drug resistance associated mutations were only detected for reverse transcriptase inhibitors. The predominant drug resistance mutations were against nucleos(t)ide reverse transcriptase inhibitors (NRTI)—M184V/I and non-nucleos(t)ide reverse transcriptase inhibitors (NNRTI)—K103N. In the ART-experienced group, M184V/I and K103N were detected in 54% (15/28) and 46% (13/28) of individuals, respectively. Both mutations were each detected in 33% (2/6) of ART naïve individuals. Multiclass resistance to NRTI and NNRTI was detected in 57% of ART-experienced individuals and two ART naïve individuals. This study reports high-level resistance to NNRTI-based antiretroviral therapy in PLWH in Ghana. However, the absence of major PI and INSTI associated-mutations is a good signal that the current WHO recommendation of Dolutegravir in combination with an NRTI backbone will yield maximum benefits as first-line regimen for PLWH in Ghana.

Evaluation of HIV-1 integrase variability by combining computational and probabilistic approaches.

This study aimed at updating previous data on HIV-1 integrase variability, by using effective bioinformatics methods combining different statistical instruments from simple entropy and mutation rate to more specific approaches such as Hellinger distance. A total of 2133 HIV-1 integrase sequences were analyzed in: i) 1460 samples from drug-naïve [DN] individuals; ii) 386 samples from drug-experienced but INI-naïve [IN] individuals; iii) 287 samples from INI-experienced [IE] individuals. Within the three groups, 76 amino acid positions were highly conserved (≤0.2% variation, Hellinger distance: <0.25%), with 35 fully invariant positions; while, 80 positions were conserved (>0.2% to <1% variation, Hellinger distance: <1%). The H12-H16-C40-C43 and D64-D116-E152 motifs were all well conserved. Some residues were affected by dramatic changes in their mutation distributions, especially between DN and IE samples (Hellinger distance ≥1%). In particular, 15 positions (D6, S24, V31, S39, L74, A91, S119, T122, T124, T125, V126, K160, N222, S230, C280) showed a significant decrease of mutation rate in IN and/or IE samples compared to DN samples. Conversely, 8 positions showed significantly higher mutation rate in samples from treated individuals (IN and/or IE) compared to DN. Some of these positions, such as E92, T97, G140, Y143, Q148 and N155, were already known to be associated with resistance to integrase inhibitors; other positions including S24, M154, V165 and D270 are not yet documented to be associated with resistance. Our study confirms the high conservation of HIV-1 integrase and identified highly invariant positions using robust and innovative methods. The role of novel mutations located in the critical region of HIV-1 integrase deserves further investigation.

Transmission and Drug Resistance Characteristics of Human Immunodeficiency Virus-1 Strain Using Medical Information Data Retrieval System.

This study was aimed at exploring the transmission and drug resistance characteristics of acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus-1 (HIV-1). The query expansion algorithm based on Candecomp Parafac (CP) decomposition was adopted to construct a data information retrieval system for semantic web and tensor decomposition. In the latent variable model based on tensor decomposition, the three elements in the triples generated feature vectors to calculate the training samples. The HIV patient data set was selected to evaluate the performance of the system, and then, the HIV gene resistance of 213 patients was retrospectively analyzed based on the electronic medical records. 43 cases showed failure of ribonucleic acid drug resistance, the ART virological failure rate was 24.43% (43/213), and one case was not reported. There was 1 case of RNA hemolysis that could not be detected. There were 50 resistant cases of nonnucleotide reverse transcriptase inhibitors (NNRTI), accounting for 29.94% (50/167), and there were 17 resistant cases of nucleotide reverse transcriptase inhibitors (NRTI), accounting for 10.18% (17/167) of all mutation cases. Among the HIV-1 strains, 19 cases failed the detection of drug resistance sites in the integrase region, and mutations in the integrase region were significantly more than those in the protease region. There were 12 types of HIV-1 strains with drug-resistant mutations. The fusion technical scheme constructed in this study showed excellent performance in medical information retrieval. In this study, the characteristics of HIV-1 of AIDS patients were analyzed from different directions, and effective treatment was performed for patients, so as to provide reference for clinical diagnosis of AIDS patients.

Pretreatment Human Immunodeficiency Virus (HIV) Drug Resistance Among Treatment-Naive Infants Newly Diagnosed With HIV in 2016 in Namibia: Results of a Nationally Representative Study.

BackgroundThe World Health Organization (WHO) recommends routine surveillance of pretreatment human immunodeficiency virus (HIV) drug resistance (HIVDR) in children <18 months of age diagnosed with HIV through early infant diagnosis (EID). In 2016, 262 children <18 months of age were diagnosed with HIV in Namibia through EID. Levels of HIVDR in this population are unknown.MethodsIn 2016, Namibia surveyed pretreatment HIVDR among children aged <18 months following WHO guidance. Reverse transcriptase, protease, and integrase regions of HIV-1 were genotyped from remnant dried blood spot specimens from all infants diagnosed with HIV in Namibia in 2016. HIVDR was predicted using the Stanford HIVdb algorithm.ResultsOf 262 specimens genotyped, 198 HIV-1 protease and reverse transcriptase sequences and 118 HIV-1 integrase sequences were successfully amplified and analyzed. The prevalence of efavirenz/nevirapine (EFV/NVP), abacavir (ABC), zidovudine, lamivudine/emtricitabine (3TC/FTC), and tenofovir (TDF) resistance was 62.6%, 17.7%, 5.6%, 15.7%, and 10.1%, respectively. No integrase inhibitor resistance was detected.ConclusionsThe high level of EFV/NVP resistance is unsurprising; however, levels of ABC and TDF resistance are among the highest observed to date in infants in sub-Saharan Africa. The absence of resistance to dolutegravir (DTG) is reassuring but underscores the need to further study the impact of ABC and 3TC/FTC resistance on pediatric protease inhibitor– and DTG-based regimens and accelerate access to other antiretroviral drugs. Results underscore the need for antiretroviral therapy optimization and prompt management of high viral loads in infants and pregnant and breastfeeding women.

Drug Resistance to HIV-1 Integrase Inhibitors Among Treatment-Naive Patients in Beijing, China.

IntroductionIntegrase strand transfer inhibitors (INSTIs) are important drugs that are currently used as the first line treatment for HIV-1 patients. The aim of this study was to characterize HIV-1 INSTI mutations among ART-naive patients in Beijing from 2019–2021.Methods865 ART-naive patients were enrolled in this study between January 2019 and June 2021 in Beijing. The amplification of the entire pol gene containing the reverse transcriptase, protease and integrase regions was performed using a validated In-house SBS method. HIV-1 subtypes and circulating recombinant forms (CRFs) were determined using the COMET online tool (http://comet.retrovirology.lu). Stanford HIV-1 drug resistance database (HIVdb version 8.9) was used to analyze the mutations.Results865 HIV-1 pol sequences were successfully amplified and sequenced. Among them, no major INSTI-related mutations were identified, but 12 polymorphic accessory mutations were found. Two patients have E138A and G163R mutations respectively and both could cause low-level resistance to RAL and EVG. Furthermore, one patient having S230R mutation resulted in low-level resistance to RAL, EVG, DTG and BIC.ConclusionThe prevalence of INSTIs mutations remains low, which demonstrated that INSTIs have good applicability currently in our city. Nevertheless, it is very important to monitor the INSTI-related mutations in Beijing.

Yoğun Bakım Ünitesinden Alınan Alt Solunum Yolu Örneklerinde Üretilen Acinetobacter baumannii İzolatlarında Antibiyotik Direnci ve İntegron Gen Kasetleri

Objective: The spread of antibiotic resistance genes among bacteria causes serious problems in the treatment of infectious diseases. Recently, it has been shown that these resistance genes are also found in integrons. This study investigated the class 1, 2 integrons and the antibiotic-resistant genes in the infection agent Acinetobacter baumannii produced in respiratory tract samples.Material and Methods: The study involved 86 A. baumannii strains isolated from lower respiratory tract samples collected between March 2014 and March 2015 in the Intensive Care Units. Identification and antibiograms of the isolates were made using conventional methods and Vitek 2 Compact systems. The presence of integrons in strains was investigated by polymerase chain reaction method using specific primer pairs for class 1and class 2 integrase regions. All samples in which integron amplification was carried out were subjected to DNA sequence analysis both by cloning and as a PCR product. The resistance genes were screened by polymerase chain reaction. Results: Isolates were obtained from specimens including tracheal aspirates (33.7%), bronchoalveolar lavage (29.1%), sputum (22.1%), bronchial washing fluid/bronchial brushing (4.7%), transbronchial biopsy (4.7%), pleural fluid (3.4%) and lung aspirates/abscess (2.3%). While resistance to imipenem, meropenem, ampicillin-sulbactam, ceftazidime, and piperacillin-tazobactam was determined in all isolates, resistance rates to ciprofloxacin, levofloxacin, gentamicin, amikacin and tigecycline were 97.7%, 93.0%, 60.5%, 53.5%and 9.3%, respectively. Colistin was the only antibiotic to which all strains were susceptible. The positivity rates of integrase I and II genes were 45.4%and 9.3%, respectively. Class 1 and 2 integron positivity rates in all isolates were 31.4%and 8.1%, respectively. blaTEM, blaSHV, blaCTX-M1, blaCTX-M2, blaOXA23, blaOXA40, blaOXA58 were 70.9%, 2.3%, 4.7%, 7.0%, 95.3%, 5.8%and 3.5%, respectively. Conclusion: Increasing association of multi drug resistant strains and integron resistant genes in A. baumannii infections will lead to the failure of antibiotic treatment strategies.

Prevalence of HIV-1 Integrase Strand Transfer Inhibitor Resistance in Treatment-Naïve Voluntary Counselling and Testing Clients by Population Sequencing and Illumina Next-Generation Sequencing in Taiwan.

PurposeIntegrase strand transfer inhibitors (INSTIs) are used as first-line therapy for HIV-1-infected patients. Next-generation sequencing (NGS) can detect low-frequency mutants; however, the clinical value of NGS to detect resistance variants is unknown. This study aimed to evaluate the prevalence of INSTI resistance in southern Taiwan and determine the clinical implications of using NGS to detect integrase region low-level resistant variants.Patients and MethodsThis retrospective cohort study included antiretroviral therapy-naïve HIV-1-infected individuals at Kaohsiung Veterans General Hospital, Taiwan, from 2013 to 2017. Drug-resistance mutations were determined, and an in-house polymerase chain reaction was used for genotyping INSTI resistance. NGS was used to assess INSTI resistance (≧1%), and the results were compared with those from population sequencing. Drug resistance-associated mutations were defined according to the 2019 IAS-USA HIV drug resistance-associated mutations list, and accessory mutations by a Stanford HIVdb score ≥10 to at least one INSTI.ResultsA total of 224 patients were included. Subtype B HIV-1 strains were found in 96% of the individuals and subtype CRF01_AE in 4%. The prevalence rates for nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and INSTI resistance were 4%, 5.8%, 0.4% and 0.9%, respectively. The most common INSTI resistance-associated mutations were G163K (0.4%) and E138A (0.4%). Of the 38 patients diagnosed in 2017 who had both NGS and population sequencing data, none had INSTI resistance-associated mutations by population sequencing; however, NGS detected four more INSTI resistance-associated mutations with low frequencies (G163R 3.25%, S153F 3.21%, S153Y 1.36% and Y143H 2.06%). Two patients with S153F and S153Y low frequencies mutations started INSTI-based highly active antiretroviral therapy, and none had virological failure by week 48.ConclusionOur findings showed a low rate of HIV drug resistance to INSTIs (0.9%) in treatment-naïve patients. NGS detected more INSTI resistance-associated mutations at a low frequency. Low-level drug resistance-associated mutations to INSTIs identified by NGS did not have an impact on the treatment response to INSTI-based first-line therapy.

HIV-1 molecular epidemiology and drug resistance-associated mutations among treatment-na\xefve blood donors in China

Surveillance of human immunodeficiency virus (HIV) molecular diversity and drug resistance-associated mutations (DRMs) among treatment-naïve blood donors is critical for monitoring viral evolution and blood safety. From 2016-2017, 199 plasma samples were collected from 24 blood centers and confirmed as HIV viral load positive or serologically reactive in National Centers for Clinical Laboratories (NCCL), of which 179 were sequenced and subtyped in the gag, protease (PR)-reverse transcriptase (RT), integrase (IN) and/or envelope (env) regions. DRMs in PR-RT and IN regions were analyzed in Stanford HIVdb Program. The majority of subtypes were circulating recombinant form (CRF) 07_BC (34.6%) and CRF01_AE (32.4%); many unique recombinant forms (URFs) (39, 21.8%) and other rare CRFs were observed in the study. Notably, CRF02_AG and CRF06_cpx strains typically found in Africa were firstly identified amongst Chinese blood donors. DRMs were common, with 28 of 179 (15.6%) specimens carrying DRMs, including the PR N88S and RT K103N mutations, which have been implicated in elevated resistance to antiretroviral drugs. Furthermore, 4 HIV-1 isolates (2.4%, 4/168) had surveillance drug-resistance mutation (SDRM), including 3 nonnucleosidereverse transcriptase inhibitors (NNRTI) SDRMs (1 K101E, 2 K103N) and 1 protease inhibitor (PI) SDRM (M46I). The HIV viral diversity among blood donors observed in this study suggest that ongoing HIV-1 recombination is becoming progressively complex in China, and lots of DRMs found in the study exacerbate the primary drug resistance landscape, which highlight the necessity of timely genotypic drug resistance monitoring and molecular surveillance of HIV-1 among blood donors.

Multidrug-resistant HIV viral rebound during early syphilis: a case report

BackgroundSyphilis has been associated with an increase in HIV RNA and a temporary decline in CD4 T cell counts in people living with HIV who are not receiving antiretroviral treatment (ART), and may be associated with a transient HIV RNA rebound in those who are receiving ART. Our case is the first to highlight the risk of a multidrug-resistant HIV viral rebound during the course of early syphilis even if antiretroviral drug concentrations are within the therapeutic range.Case presentationThis 50-year-old HIV-1-positive male patient with concomitant early syphilis presented with an HIV RNA rebound (8908 copies/mL) during a scheduled visit to our clinic. He was receiving a stable ART regimen consisting of darunavir/cobicistat plus dolutegravir, and had a 15-year history of viral suppression. Good short-term drug adherence could be inferred as liquid chromatography tandem mass spectrometry showed that his trough antiretroviral drug concentrations were within the therapeutic range: darunavir 2353 ng/mL (minimum effective concentration > 500 ng/mL) and dolutegravir 986 ng/mL (minimum effective concentration > 100 ng/mL). A plasma RNA genotype resistance test revealed wild-type virus in the integrase region and protease region (PR), but extensive resistance in the reverse transcriptase (RT) region (M41L, E44D, D67N, K70R, M184V, L210W and T215Y). Phylogenetic analysis of next-generation sequences (used to investigate the presence of minor viral variants), the PR and RT sequences from plasma HIV RNA and pro-viral DNA extracted from peripheral blood mononuclear cells during the viral rebound, and a Sanger sequence obtained during a previous virological failure suggested clonal viral expression because the previous PR resistance mutations had been lost or had not been archived in pro-viral DNA.ConclusionsThis case shows that early syphilis may cause an HIV RNA rebound in patients under stable virological control with the potential of transmitting an extensively drug-resistant virus.

Shall I trust the report? Variable performance of Sanger sequencing revealed by deep sequencing on HIV drug resistance mutation detection

BackgroundThe clinical utilisation of deep sequencing in HIV treatment has been hindered due to its unknown correlation with standard Sanger genotyping and the undetermined value of minority drug resistance mutation (DRM) detection. ObjectivesTo compare deep sequencing performance to standard Sanger genotyping with clinical samples, in an effort to delineate the correlation between the results from the two methods and to find the optimal deep sequencing threshold for clinical utilisation. MethodsWe conducted a retrospective study using stored plasma collected from August 2014 to March 2018 for HIV genotyping with the commercial Sanger genotyping kit. Samples with available Sanger genotyping reports were further deep sequenced. Drug resistance was interpreted according to the Stanford HIV drug resistance database algorithm. ResultsAt 15–25% minority detection thresholds, 9–15% cases had underestimated DRMs by Sanger sequencing. The concordance between the Sanger and deep sequencing reports was 68–82% in protease-reverse transcriptase region and 88–97% in integrase region at 5–25% thresholds. The undetected drug resistant minority variants by Sanger sequencing contributed to the lower negative predictive value of Sanger genotyping in cases harbouring DRMs. ConclusionsUse of deep sequencing improved detection of antiretroviral resistance mutations especially in cases with virological failure or previous treatment interruption. Deep sequencing with 10–15% detection thresholds may be considered a suitable substitute for Sanger sequencing on antiretroviral DRM detection.

A broad-host-range event detector: expanding and quantifying performance between Escherichia coli and Pseudomonas species

AbstractModern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications such as chemical sensing and event logging in complex environments will benefit from new host chassis. This study quantitatively compared how the same chemical event logger performed across four strains and three different microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads—Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two benchmark E. coli chassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and downstream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

HIV-1 integrase drug-resistance mutations in Iranian treatment-experienced HIV-1-infected patients.

The latest class of antiretrovirals (ARVs), including integrase strand transfer inhibitors (INSTIs), has been demonstrated to be effective for antiretroviral therapy (ART). Despite all the distinguishing characteristics of these drugs, including a high genetic barrier to resistance and lower toxicity than other ARVs, unfortunately, INSTI drug resistance mutations (DRMs) have occasionally been observed. The aim of this study was to investigate the presence of DRMs associated with INSTIs among treatment-experienced HIV-1-infected patients. From June 2012 to December 2018, a total of 655 treatment-experienced HIV-1-infected patients enrolled in this cross-sectional survey. Following amplification and sequencing of the HIV-1 integrase region of the pol gene, DRM and phylogenetic analysis were successfully carried out on the plasma samples of patients who had a viral load over 1,000IU/ml after at least 6months of ART. Out of the 655 patients evaluated, 62 (9.5%) had a viral load higher than 1,000IU/ml after at least 6months of ART. Phylogenetic analysis showed that all of the 62 HIV-1 patients experiencing treatment failure were infected with CRF35_AD, and one of these patients (1.6%) was infected with HIV-1 variants with DRMs. The DRMs that were identified belonged to the INSTI class, including E138K, G140A, S147G, and Q148R. This survey shows that DRMs belonging to the INSTI class were detected in an Iranian HIV patient who has experienced treatment failure. Therefore, regarding the presence of DRMs to INSTIs in ART-experienced patients, it seems better to perform drug resistance mutation testing in HIV patients experiencing treatment failure before changing the ART regimen and prescribing this class of medication.

Prevalence of drug resistance mutations among ART-naive and -experienced HIV-infected patients in Sierra Leone.

ObjectivesThe aim of this study was to assess the prevalence of HIV drug resistance (HIVDR) in HIV-infected ART-naive and -experienced patients in Sierra Leone.Patients and methodsWe conducted a cross-sectional study of HIV-positive adults aged ≥18 years at Connaught Hospital in Freetown, Sierra Leone in November 2017. Sequencing was performed in the reverse transcriptase, protease and integrase regions, and interpreted using the Stanford HIVDR database and WHO 2009 mutation list.ResultsTwo hundred and fifteen HIV-infected patients were included (64 ART naive and 151 ART experienced). The majority (66%) were female, the median age was 36 years and the median ART exposure was 48 months. The majority (83%) were infected with HIV-1 subtype CRF02_AG. In the ART-naive group, the pretreatment drug resistance (PDR) prevalence was 36.7% (14.2% to NRTIs and 22.4% to NNRTIs). The most prevalent PDR mutations were K103N (14.3%), M184V (8.2%) and Y181C (4.1%). In the ART-experienced group, 64.4% harboured resistance-associated mutations (RAMs) and the overall prevalence of RAMs to NRTIs and NNRTIs was 85.2% (52/61) and 96.7% (59/61), respectively. The most prevalent RAMs were K103N (40.7%), M184V (28.8%), D67N (15.3%) and T215I/F/Y (15.3%). Based on the genotypic susceptibility score estimates, 22.4% of ART-naive patients and 56% of ART-experienced patients were not susceptible to first-line ART used in Sierra Leone.ConclusionsA high prevalence of circulating NRTI- and NNRTI-resistant variants was observed in ART-naive and -experienced HIV-1-infected patients in Sierra Leone. This necessitates the implementation of HIVDR surveillance programmes to inform national ART guidelines for the treatment and monitoring of HIV-infected patients in Sierra Leone.

First evaluation of the Next-Generation Sequencing platform for the detection of HIV-1 drug resistance mutations in Belgium.

IntroductionThe WHO urges action against the threat posed by HIV drug resistance. It is well known that the sensitivity of Next-Generation Sequencing (NGS) is greater than that of Sanger Sequencing (SS). The objective of this study was to evaluate the performance of the novel NGS HIV-1 drug resistance monitoring system.Materials & methodsNGS analyses were performed on 67 plasma samples from HIV-1 infected patients using the Sentosa SQ HIV Genotyping Assay from Vela-Dx. This kit was used on a semi-automated Ion Torrent-based platform. Sequences were compared to those obtained by SS. Samples were analysed in the same and in separate runs. Quality controls (QC) were added to control sequencing processes of protease (PRO), reverse transcriptase (RT) and integrase (INT) regions.ResultsOf the 41 analysed samples, 33 (80.5%) had identical drug resistance interpretation reports. Discrepant results were observed for eight samples. Five of them were only detected by NGS and had drug resistance mutations (DRMs) with an allelic frequency below the limit of detection of the SS method (between 6.3 to 20.5%). Two DRMs were only identified using the SS method. The sequences were similar in 98.2% of cases (counting variants as mismatches) and homologous in 99.9% if missed variants. Duplicated samples in a single run were similar in 95.7% (99.9%) of cases. Duplicated samples in two different runs were 98% (100%) homologous. QC results were manually assessed with a score of 340/340 for detection of DRMs in PRO and RT and 100% for INT sequencing.ConclusionsThis is the first preliminary evaluation in Belgium employing the Sentosa SQ HIV Genotyping Assay. The NGS appears to be a promising tool for the detection of DRMs in HIV-1 patients and showed a higher sensitivity compared to SS. Large studies assessing the clinical relevance of low frequency DRMs are needed.