Transmitted Drug Resistance Research Articles

Full-spectrum HIV drug resistance mutation detection by high-resolution complete pol gene sequencing.

Drug resistance mutation testing is a key element for HIV clinical management, informing effective treatment regimens. However, resistance screening in current clinical practice is limited in reporting linked cross-class resistance mutations and minority variants, both of which may increase the risk of virological failure. To address these limitations, we obtained 358 full-length pol gene sequences from 52 specimens of 20 HIV infected individuals by combining microdroplet amplification, unique molecular identifier (UMI) labeling, and long-read high-throughput sequencing. We conducted a rigorous assessment of the accuracy of our pipeline for precision drug resistance mutation detection, verifying that a sequencing depth of 35 high-throughput reads achieved complete, error-free pol gene sequencing. We detected 26 distinct drug resistance mutations to Protease Inhibitors (PIs), Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), and Integrase Strand Transfer Inhibitors (INSTIs). We detected linked cross-class drug resistance mutations (PI+NRTI, PI+NNRTI, and NRTI+NNRTI) that confer cross-resistance to multiple drugs in different classes. Fourteen different types of minority mutations were also detected with frequencies ranging from 3.2% to 19%, and the presence of these mutations was verified by Sanger reference sequencing. We detected a putative transmitted drug resistance mutation (TDRM) in one individual that persisted for over seven months from the first sample collected at the acute stage of infection prior to seroconversion. Our comprehensive drug resistance mutation profiling can advance clinical practice by reporting mutation linkage and minority variants to better guide antiretroviral therapy options.

Identification of integrase inhibitor-related drug resistance mutations in newly diagnosed ART-naïve HIV patients

BackgroundIn China, the recommended treatment regimens for HIV-infected individuals were tenofovir in combination with lamivudine or emtricitabine as NRTIs, efavirenz or rilpivirine as NNRTIs, lopinavir/ritonavir as protease inhibitors, and raltegravir or dolutegravir as INSTIs. The development of drug resistance increases the risk of viral rebound, opportunistic infections, and ultimately treatment failure such that the early detection of resistance is ideal. This study was developed to explore primary drug resistance characteristics and genotypic distributions in newly diagnosed antiretroviral therapy (ART)-naïve HIV-1 patients in Nanjing with the goal of establishing a basis for their individualized treatment in the clinic. MethodsSamples of serum were collected from newly diagnosed ART-naïve HIV patients from the Second Hospital of Nanjing between May 2021 and May 2022. The HIV-1 integrase (IN), protease (PR), and reverse transcriptase (RT) gene coding sequences were amplified from these samples, sequenced, and assessed for drug resistance-related mutations. ResultsMajor integrase resistance-related mutations were detected in 4/360 amplified samples, with 5 other patient samples exhibiting accessory resistance mutations. The overall prevalence of PR and RT inhibitor-related transmitted drug resistance mutations (TDRMs) in this patient population was 16.99% (61/359). The most common mutations were non-nucleoside reverse transcriptase inhibitor-related mutations (51/359; 14.21%), followed by those associated with nucleoside reverse transcriptase inhibitors (7/359; 1.95%) and protease inhibitors (7/359; 1.95%). Dual-resistant strains were also observed in a subset of patients. ConclusionsIn summary, this study is the first to have surveyed the prevalence of integrase inhibitor resistance-related mutations and other drug resistance-related mutations among newly diagnosed ART-naïve HIV-positive patients in Nanjing, China. These results highlight the need for further molecular surveillance-based monitoring of the HIV epidemic in Nanjing.

Investigation of antibiotic resistance genotypic and phenotypic characteristics of marine aquaculture fish carried in the Dalian area of China.

Due to the long-term and irrational use of antibiotics for the prevention and control of bacterial diseases in aquaculture, antibiotic resistance genes have become a new source of pollution in aquatic products. Factors such as the spread of drug-resistant strains and the horizontal transfer of drug-resistant genes have led to multi-drug resistance in fish-infecting bacteria, which seriously affects the quality and safety of aquatic products. In this study, 50 samples of horse mackerel and puffer fish sold in Dalian aquatic products market and seafood supermarket were collected, and the phenotypic characteristics of the bacteria carried by the fish for drugs such as sulfonamides, amide alcohols, quinolones, aminoglycosides and tetracyclines were tested and analyzed, and the resistance genes carried by fish samples were detected by SYBG qPCR. Our statistical analyses demonstrated that the drug resistance phenotypes and genotypes of bacteria carried by mariculture horse mackerel and puffer fish in the Dalian area of China were complex, and the multi-drug resistance rate reached 80%. Among the examined antibiotics, the resistance rates to cotrimoxazole, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, levofloxacin, kanamycin, and florfenicol exceeded 50%, whereas the resistance rates to gentamicin and tobramycin were 26 and 16%, respectively. The detection rate of the drug resistance genes tetA, sul1, sul2, qnrA, qnrS, and floR exceeded 70% and all samples carried more than three drug resistance genes. The correlation analysis of drug resistance genes and drug resistance phenotypes showed that the detection of the drug resistance genes sul1, sul2, floR, and qnrD was correlated with the detection of drug resistance phenotypes (p < 0.01). However, the correlation between the resistance genes cmlA, cfr, tetA, qnrA, qnrS, and aac(6')-Ib-cr and the corresponding resistance phenotype was not significant (p > 0.05). In general, our findings indicated that the multi-drug resistance of bacteria carried by marine horse mackerel and puffer fish in the Dalian area was serious. From the perspective of drug resistance rate and drug resistance gene detection rate, the aminoglycosides gentamicin and tobramycin are still considered effective in controlling bacterial infection in marine fish in the study area. Collectively, our findings provide a scientific basis for the management of drug use in mariculture, which can prevent the transmission of drug resistance through the food chain and minimize the associated human health risks.

Transmitted Drug Resistance Against Integrase Strand Transfer Inhibitors in Iranian HIV-Infected Naïve Patients.

Human Immunodeficiency Virus (HIV) has claimed the lives of millions of people during the past decades. While several antiretroviral drugs like Integrase Strand Transfer Inhibitors (INSTIs) have been introduced to control HIV, Transmitted Drug Resistance (TDR) in HIV genome caused failure in treatment. This study aimed to investigate TDR and natural occurring mutations (NOPs) in HIV integrase gene in Iranian HIV patients. In this cross-sectional study, blood samples of 30 HIV-positive patients who had never taken integrase inhibitors were considered for CD4 T cell count, RT real-time PCR, and, Nested PCR. The sequencing results were analyzed by CLC sequence viewer software and Stanford University HIV Drug Resistance Database. In all samples, nine NOPs with a high prevalence were found; however, we did not find any drug resistance mutations, except for a mutation in one sample, which showed a low resistance level. Subtype A1 was dominant in all samples. Based on the findings and compared to our previous study, all patients were sustainable to main integrase inhibitors, including bictegravir, raltegravir, bictegravir, elvitegravir and dolutegravir. It seems the resistant mutation pattern attributed to integrase inhibitors was not diffent among studied patients; hence, the prescription of such inhibitors helps physicians to control HIV infection in Iranian HIV-infected patients.

Modelling the impact of treatment adherence on the transmission of HIV drug resistance.

A lower adherence rate (percentage of individuals taking drugs as prescribed) to ART may increase the risk of emergence and transmission of HIV drug resistance, decrease treatment efficacy, and increase mortality rate. Exploring the impact of ART adherence on the transmission of drug resistance could provide insights in controlling the HIV epidemic. We proposed a dynamic transmission model incorporating the CD4 cell count-dependent rates of diagnosis, treatment and adherence with transmitted drug resistance (TDR) and acquired drug resistance. This model was calibrated and validated by 2008-2018 HIV/AIDS surveillance data and prevalence of TDR among newly diagnosed treatment-naive individuals from Guangxi, China, respectively. We aimed to identify the impact of adherence on drug resistance and deaths during expanding ART. In the base case (ART at 90% adherence and 79% coverage), we projected the cumulative total new infections, new drug-resistant infections, and HIV-related deaths between 2022 and 2050 would be 420 539, 34 751 and 321 671. Increasing coverage to 95% would reduce the above total new infections (deaths) by 18.85% (15.75%). Reducing adherence to below 57.08% (40.84%) would offset these benefits of increasing coverage to 95% in reducing infections (deaths). Every 10% decrease in adherence would need 5.07% (3.62%) increase in coverage to avoid an increase in infections (deaths). Increasing coverage to 95% with 90% (80%) adherence would increase the above drug-resistant infections by 11.66% (32.98%). A decrease in adherence might offset the benefits of ART expansion and exacerbate the transmission of drug resistance. Ensuring treated patients' adherence might be as important as expanding ART to untreated individuals.

Increased prevalence and stable clustering rate of HIV-1 transmitted drug resistance strains after 'treat-all' in a megacity of China.

The 'treat-all' strategy was implemented in Shenzhen, China in 2016. The effect of this extensive treatment on transmitted drug resistance (TDR) of HIV is unclear. TDR analysis was performed, based on the partial HIV-1 pol gene obtained from the newly reported HIV-1 positive cases from 2011 to 2019 in Shenzhen, China. The HIV-1 molecular transmission networks were inferred to analyse the spread of TDR. Logistic regression was used to identify the potential risk factors with TDR mutations (TDRMs) to cluster. A total of 12 320 partial pol sequences were included in this study. The prevalence of TDR was 2.95% (363/12 320), which increased from 2.57% to 3.52% after 'treat-all'. The TDR prevalence was increased in populations with the characteristics of CRF07_BC, being single, educated to junior college level and above, MSM and male. The sensitivities of viruses to six antiretroviral drugs were decreased. The clustering rate of TDRMs remained stable, and the sequences in the three drug resistance transmission clusters (DRTCs) were mainly found during 2011-16. CRF07_BC and CRF55_01B were the factors associated with TDRMs clustering in the networks. The 'treat-all' strategy might have contributed to a small increase in TDR, while most of the TDRMs were distributed sporadically, which implies that the 'treat-all' strategy is helpful for the control of TDR in high-risk populations.

Prospective study on outcome of MDR-TB using the shorter regimen during COVID-19 pandemic.

According to Indian TB report 2020, 66,225 MDR/RR-TB cases were detected in India, 56,569 (85%) were put on treatment, and 40,397 (75%) were initiated on shorter drug regimens at the time of diagnosis. In the absence of an effective vaccine, there is an urgent need for new treatment regimens, drugs, and diagnostics to slow the evolution of drug resistance and limit transmission of resistant variants, as well as to ameliorate the treatment outcome of patients infected with MDR/XDR M. tuberculosis strains. To evaluate the efficacy of a shorter drug regimen in MDR-PTB and estimate the adverse effects of drugs used in the regimen. This is an institution-based prospective study which included 135 confirmed MDR-PTB patients. Patients with extra-pulmonary MDR-TB and use of SLI for more than one month were excluded. The success rate using a shorter regimen was 65.2% which is respectable, given the COVID-19 pandemic considered during the study period. Minor adverse events such as nausea (39.3%) and vomiting (34.8%) were reported. Rare adverse effects such as hearing loss (8.9%) and hypothyroidism (0.2%) were also seen in the study population. Overall treatment success was similar when compared to other studies done previously. A shorter drug regimen was associated with minor adverse effects such as gastrointestinal adverse effects such as vomiting and hearing loss observed in elderly patients. Baseline unknown drug resistance and lower BMI were associated with unsuccessful outcomes. Measures should be taken to improve nutrition. Our results argue the need for improving baseline DST at peripheral areas in order to effectively evaluate resistance to other drugs, especially in settings with high levels of first and second-line drug resistance.

Modeling Drug Resistance Emergence and Transmission in HIV-1 in the UK.

A deeper understanding of HIV-1 transmission and drug resistance mechanisms can lead to improvements in current treatment policies. However, the rates at which HIV-1 drug resistance mutations (DRMs) are acquired and which transmitted DRMs persist are multi-factorial and vary considerably between different mutations. We develop a method for the estimation of drug resistance acquisition and transmission patterns. The method uses maximum likelihood ancestral character reconstruction informed by treatment roll-out dates and allows for the analysis of very large datasets. We apply our method to transmission trees reconstructed on the data obtained from the UK HIV Drug Resistance Database to make predictions for known DRMs. Our results show important differences between DRMs, in particular between polymorphic and non-polymorphic DRMs and between the B and C subtypes. Our estimates of reversion times, based on a very large number of sequences, are compatible but more accurate than those already available in the literature, with narrower confidence intervals. We consistently find that large resistance clusters are associated with polymorphic DRMs and DRMs with long loss times, which require special surveillance. As in other high-income countries (e.g., Switzerland), the prevalence of sequences with DRMs is decreasing, but among these, the fraction of transmitted resistance is clearly increasing compared to the fraction of acquired resistance mutations. All this indicates that efforts to monitor these mutations and the emergence of resistance clusters in the population must be maintained in the long term.

CASCADE protocol: exploring current viral and host characteristics, measuring clinical and patient-reported outcomes, and understanding the lived experiences and needs of individuals with recently acquired HIV infection through a multicentre mixed-methods observational study in Europe and Canada

IntroductionDespite the availability of pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART), 21 793 people were newly diagnosed with HIV in Europe in 2019. The Concerted action on seroconversion to AIDS...

The impact of attrition on the transmission of HIV and drug resistance: A mathematical modelling study.

Attrition due to loss to follow-up or termination of antiretroviral therapy (ART) among HIV-infected patients in care may increase the risk of emergence and transmission of drug resistance (TDR), diminish benefit of treatment, and increase morbidity and mortality. Understanding the impact of attrition on the epidemic is essential to provide interventions for improving retention in care. We developed a comprehensive HIV transmission dynamics model by considering CD4+ cell count dependent diagnosis, treatment, and attrition involving TDR and acquired drug resistance. The model was calibrated by 11 groups HIV/AIDS surveillance data during 2008-2018 from Guangxi, China, and validated by the prevalence of TDR among diagnosed treat-naive individuals. We aimed to investigate how attrition would affect the transmission of HIV and drug-resistance when expanding ART. In the base case with CD4+ cell count dependent per capita attrition rates 0.025∼0.15 and treatment rates 0.23∼0.42, we projected cumulative total new infections, new drug-resistant infections, and HIV-related deaths over 2022-2030 would be 145 391, 7637, and 51 965, respectively. Increasing treatment rates by 0.1∼0.2 can decrease the above total new infections (deaths) by 1.63∼2.93% (3.52∼6.16%). However, even 0.0114∼0.0220 (0.0352∼0.0695) increase in attrition rates would offset this benefit of decreasing infections (deaths). Increasing treatment rates (attrition rates) by 0.05∼0.1 would increase the above drug-resistant infections by 0.16∼0.30% (22.18∼41.15%). A minor increase in attrition can offset the benefit of treatment expansion and increase the transmission of HIV drug resistance. Reducing attrition rates for patients already in treatment may be as important as expanding treatment for untreated patients.

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.

Prevalence of Transmitted Drug Resistance among HIV-1 Patients in the Aegean Region: Results from the Western part of Turkey.

This study aimed to analyze the antiretroviral drug resistance in antiretroviral treatment-naïve HIV-positive patients in the Aegean Region of Turkey from 2012 to 2019. The study included 814 plasma samples from treatment-naïve HIV-positive patients. Drug resistance analysis was performed by Sanger sequencing (SS) between 2012-2017 and by next-generation sequencing sequencing (NGS) between 2018-2019. SS was used to analyze resistance mutations in the protease (PR) and reverse transcriptase (RT) gene regions using a ViroSeq HIV-1 Genotyping System. PCR products were analyzed with an ABI3500 GeneticAnalyzer (Applied Biosystems). The sequencing of the HIV genome in the PR, RT, and integrase gene regions was carried out using MiSeq NGS technology. Drug resistance mutations and subtypes were interpreted using the Stanford University HIV-1 drug resistance database. Transmitted drug resistance (TDR) mutation was detected in 34/814 ( 4.1 %) samples. Non-nucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor (NRTI), and protease inhibitor (PI) mutations were identified in 1.4 % (n =12), 2.4 % (n =20), and 0.3 % (n = 3) of samples, respectively. The most common subtypes were B (53.1 % ), A (10.9%), CRF29_BF (10.6%), and B + CRF02_AG (8,2%). The most common TDR mutations were E138A (3.4%), T215 revertants (1.7%), M41L (1.5%), and K103N (1.1%). Transmitted drug resistance rate in the Aegean Region is compatible with national and regional data. Routine surveillance of resistance mutations may guide the safe and correct selection of initial drug combinations for antiretroviral therapy. The identification of HIV-1 subtypes and recombinant forms in Turkey may contribute to international molecular epidemiological data.

Cohort profile: InfCareHIV, a prospective registry-based cohort study of people with diagnosed HIV in Sweden

PurposeThe Swedish InfCareHIV cohort was established in 2003 to ensure equal and effective care of people living with HIV (PLHIV) and enable long-term follow-up. InfCareHIV functions equally as a decision...

Subtype Distribution and Drug Resistance Patterns Among HIV-1 Strains Prevalent in Makassar, Indonesia.

Human immunodeficiency virus type 1 (HIV-1) is characterized by a large degree of genetic variability because of high rates of recombination and mutation, sizable population sizes, and rapid replication. Therefore, this study investigated HIV-1 subtype distribution and the appearance of drug resistance mutations (DRMs) in viruses that are prevalent in Makassar, South Sulawesi, Indonesia. The HIV-1 pol, env, and gag genes were amplified from 63 infected individuals and sequenced for a subtyping analysis. CRF01_AE was identified as the predominant HIV-1 circulating recombinant form (CRF) in Makassar, South Sulawesi, Indonesia. Subtype B and recombinant viruses containing CRF01_AE, CRF02_AG, and/or subtype B gene fragments were also detected. Several major DRMs against non-nucleoside reverse transcriptase inhibitors were found among antiretroviral therapy (ART)-experienced subjects, whereas ART-naive subjects did not possess any transmitted drug resistance. The prevalence of DRMs was very high among ART-experienced subjects; therefore, further surveillance is required in this region.

Prevalence of HIV-1 Natural Polymorphisms and Integrase-Resistance-Associated Mutations in African Children.

Integrase inhibitors (INIs) are a potent option for HIV treatment. Limited data exist on INI resistance in West Africa, particularly in children living with HIV/AIDS. We determined the prevalence of integrase gene polymorphisms and the frequency of naturally occurring amino acid (aa) substitutions at positions associated with INI resistance. Dried blood spot (DBS) samples were obtained from one hundred and seven (107) HIV-1-infected children aged less than 15 years old in two West African countries, Benin and Mali. All children were naïve to INI treatment, 56 were naïve to anti-retroviral therapy (ART), and 51 had received ART. Genetic sequencing of HIV integrase was successful in 75 samples. The aa changes at integrase positions associated with INI resistance were examined according to the Stanford HIV Genotypic Resistance database. The median ages were 2.6 and 10 years for ART-naïve and -treated children, respectively. The most common subtypes observed were CRF02_AG (74.7%) followed by CRF06_cpx (20%). No major INI-resistance mutations at positions 66, 92, 121, 143, 147, 148, 155, and 263 were detected. The most prevalent INI accessory resistance mutations were: L74I/M (14/75, 18.6%) followed by E157Q (8/75, 10.6%), G163E/N/T/Q (5/75, 6.6%), Q95A/H/P (2/75, 2.6%), and T97A (4/75, 5.3%). Other substitutions observed were M50I/L/P, H51E/P/S/Q, I72V, T112V, V201I, and T206S. Polymorphisms at positions which may influence the genetic barrier and/or drive the selection of specific INI-resistance pathways were detected. However, no transmitted drug resistance (TDR) to INI was detected among samples of INI-naïve patients. These findings support the use of this treatment class for children with HIV-1, particularly in West Africa.

Future of Antiretroviral Drugs and Evolution of HIV-1 Drug Resistance.

Highly active antiretroviral (ARV) therapy has been used for many years, but the use in low- and middle-income countries of antiretroviral drugs with low genetic barrier to resistance, combined with limited availability of viral load testing, has led to higher rates of acquired drug resistance, sustaining the rate of transmitted drug resistance. Here, we describe the evolution of ARV drugs with the ongoing development of injectable long-acting forms and the requirements regarding all new ARV drugs (i.e., no transmitted drug resistance, no cross-resistance and high genetic barrier to resistance). Then, we report the evolution of both transmitted and acquired resistance regarding new ARV drugs. The WHO has set very ambitious but motivating goals for HIV testing, treatment and viral suppression, aiming to achieve rates of 95% for all three by 2025. Reaching these goals requires a wide implementation and use of close virological monitoring in LMICs.

Exosome-transmitted circCABIN1 promotes temozolomide resistance in glioblastoma via sustaining ErbB downstream signaling

Although temozolomide (TMZ) provides significant clinical benefit for glioblastoma (GBM), responses are limited by the emergence of acquired resistance. Here, we demonstrate that exosomal circCABIN1 secreted from TMZ-resistant cells was packaged into exosomes and then disseminated TMZ resistance of receipt cells. CircCABIN1 could be cyclized by eukaryotic translation initiation factor 4A3 (EIF4A3) and is highly expressed in GBM tissues and glioma stem cells (GSCs). CircCABIN1 is required for the self-renewal maintenance of GSCs to initiate acquired resistance. Mechanistically, circCABIN1 regulated the expression of olfactomedin-like 3 (OLFML3) by sponging miR-637. Moreover, upregulation of OLFML3 activating the ErbB signaling pathway and ultimately contributing to stemness reprogramming and TMZ resistance. Treatment of GBM orthotopic mice xenografts with engineered exosomes targeting circCABIN1 and OLFML3 provided prominent targetability and had significantly improved antitumor activity of TMZ. In summary, our work proposed a novel mechanism for drug resistance transmission in GBM and provided evidence that engineered exosomes are a promising clinical tool for cancer prevention and therapy.Graphical

Potential role of doravirine for the treatment of HIV-1-infected persons with transmitted drug resistance

BackgroundDoravirine has a unique resistance profile but how this profile might increase its usefulness beyond first-line therapy in persons with susceptible viruses has not been well studied. We sought to determine scenarios in which doravirine would retain activity against isolates from ART-naïve persons with transmitted drug resistance (TDR) and to identify gaps in available doravirine susceptibility data.MethodsWe analyzed published in vitro doravirine susceptibility data and applied the results to 42,535 RT sequences from ART-naïve persons published between 2017 and 2021. NNRTI drug resistance mutations (DRMs) were defined as those with a Stanford HIV Drug Resistance Database doravirine penalty score either alone or in combination with other mutations.ResultsV106A, Y188L, F227C/L, M230L, and Y318F were associated with the greatest reductions in doravirine susceptibility. However, several NNRTI DRMs and DRM combinations lacking these canonical resistance mutations had > tenfold reduced susceptibility including G190E, one isolate with G190S, three isolates with L100I + K103N, one isolate with K103N + P225H, and isolates with L100I + K103N + V108I and K101E + Y181C + G190A. Of the 42,535 ART-naïve sequences, 3,374 (7.9%) contained a NNRTI DRM of which 2,788 (82.6%) contained 1 DRM (n = 33 distinct mutations), 426 (12.6%) contained 2 DRMs (79 distinct pairs of mutations), and 143 (4.2%) contained ≥ 3 DRMs (86 distinct mutation patterns). Among the 2,788 sequences with one DRM, 112 (4.0%) were associated with ≥ 3.0-fold reduced doravirine susceptibility while 2,625 (94.2%) were associated with < 3.0-fold reduced susceptibility. Data were not available for individual NNRTI DRMs in 51 sequences (1.8%). Among the 426 sequences with two NNRTI DRMs, 180 (42.3%) were associated with ≥ 3.0 fold reduced doravirine susceptibility while just 32 (7.5%) had < 3.0 fold reduced susceptibility. Data were not available for 214 (50.2%) sequences containing two NNRTI DRMs.ConclusionsFirst-line therapy containing doravirine plus two NRTIs is expected to be effective in treating most persons with TDR as more than 80% of TDR sequences had a single NNRTI DRM and as more than 90% with a single DRM were expected to be susceptible to doravirine. However, caution is required for the use of doravirine in persons with more than one NNRTI DRM even if none of the DRMs are canonical doravirine-resistance mutations.

Advantages of long- and short-reads sequencing for the hybrid investigation of the Mycobacterium tuberculosis genome.

In the fight to limit the global spread of antibiotic resistance, computational challenges associated with sequencing technology can impact the accuracy of downstream analysis, including drug resistance identification, transmission, and genome resolution. About 10% of Mycobacterium tuberculosis (MTB) genome is constituted by the PE/PPE family, a GC-rich repetitive genome region. Although sequencing using short read technology is widely used, it is well recognized its limit in the PE/PPE regions due to the unambiguously mapping process onto the reference genome. The aim of this study was to compare the performances of short-reads (SRS), long-reads (LRS) and hybrid-reads (HYBR) based analysis over different common investigative tasks: genome coverage estimation, variant calling and cluster analysis, drug resistance detection and de novo assembly. For the study 13 model MTB clinical isolates were sequenced with both SRS and LRS. HYBR were produced correcting the long reads with the short reads. The fastq from the three approaches were then processed using a customized version of MTBseq for genome coverage estimation and variant calling and using two different assemblers for de novo assembly evaluation. Estimation of genome coverage performances showed lower 8X breadth coverage for SRS respect to LRS and HYBR: considering the PE/PPE genes, SRS showed low results for the PE_PGRS family, while obtained acceptable coverage in PE and PPE genes; LRS and HYBR reached optimal coverages in PE/PPE genes. For variant calling HYBR showed the highest resolution, detecting the highest percentage of uniquely identified mutations compared to LRS and SRS. All three approaches agreed on the identification of two major clusters, with HYBR identifying an higher number of SNPs between the two clusters. Comparing the quality of the assemblies, HYBR and LRS obtained better results than SRS. In conclusion, depending on the aim of the investigation, both SRS and LRS present complementary advantages and limitations implying that for a full resolution of MTB genomes, where all the mentioned analyses and both technologies are needed, the use of the HYBR approach represents a valid option and a well-rounded strategy.

Prevalence of HIV Transmitted Drug Resistance in Nanjing from 2018 to 2021.

Transmitted drug resistance (TDR) is a major challenge in the clinical management of acquired immunodeficiency syndrome (AIDS). Therefore, this study aimed to investigate the epidemic characteristics of and risk factors for human immunodeficiency virus (HIV)-1 TDR in Nanjing from 2018 to 2021 to provide support for clinical management. The HIV-1 Pol gene was amplified by nested reverse transcription polymerase chain reaction from venous blood of 1190 HIV-infected patients who did not receive antiviral therapy, and the amplified product was sequenced using an in-house sequencing method. The sequencing result was compared with the HIV drug resistance database from Stanford University to elucidate the rates of antiviral drug resistance and distribution of drug-resistant mutation sites. Factors associated with TDR were evaluated using a logistic regression model. Detection of drug resistance at the gene level was successful in 1138 of 1190 HIV-1-infected patients (95.6%), and the overall 4-year drug resistance rate was 8.2% (93/1138). The drug resistance rate was higher for non-nucleoside reverse transcriptase inhibitors (NNRTIs; 6.7%) than for nucleoside reverse transcriptase inhibitors (NRTIs; 2.5%) or protease inhibitors (PIs; 0.1%) (χ 2 = 83.907, P<0.0001). The most common NNRTI-related mutation was V179D/E followed by K103N. M184V was the dominant NRTI-associated mutation, and M46L/I was the most prevalent PI-associated mutation. A CD4+ T cell count of <50 cells/μL was significantly associated with an increased risk of TDR (OR=3.62, 95% CI: 1.38-9.51, P=0.009). The prevalence of TDR in the city of Nanjing from 2018 to 2021 was at a moderate epidemic risk according to World Health Organization standards. Continuous monitoring of TDR can inform clinical diagnosis and treatment. Patients with advanced disease and a low CD4+ T lymphocyte count are more likely to have TDR in Nanjing.