Drug Resistance Mutations Research Articles

SATCAS: A CRISPR/Cas13a-based simultaneous amplification and testing platform for one-pot RNA detection and SNPs distinguish in clinical diagnosis

The clinical diagnosis of pathogen infectious diseases increasingly requires sensitive and rapid RNA detection technologies. The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system has shown immense potential in molecular diagnostics due to its trans-cleavage activity. However, most Cas13a-based detection methods require an amplicon transcription step, and the multi-step open-tube operations are prone to contamination, limiting their widespread application. Here, we propose an ultrasensitive (single-copy range, ∼aM) and rapid (within 40 min) isothermal one-pot RNA detection platform, termed SATCAS (Simultaneous Amplification and Testing platform based on Cas13a). This method effectively distinguishes viable bacteria (0%–100%) under constant total bacterial conditions, demonstrating its robustness and universality. SATCAS excels in identifying single nucleotide polymorphisms (SNPs), particularly detecting 0.5% drug-resistant mutations. We validated SATCAS by detecting infections in biological samples from 68 HBV, 23 EBV, and 48 SARS-CoV-2 patients, achieving 100% sensitivity, 92.86% specificity, and 97.06% accuracy in HBV infection testing. We anticipate that SATCAS has broad application potential in the early diagnosis, subtyping, drug resistance detection, and point-of-care monitoring of pathogen infectious diseases.

HIV-1 subtype-specific drug resistance on dolutegravir-based antiretroviral therapy: protocol for a multicentre study (DTG RESIST)

IntroductionHIV drug resistance poses a challenge to the United Nation’s goal of ending the HIV/AIDS epidemic. The integrase strand transfer inhibitor (InSTI) dolutegravir, which has a higher resistance barrier, was...

Application of molecular epidemiology in revealing HIV-1 transmission network and recombination patterns in Tianjin, China.

Using a comprehensive molecular epidemiological approach, we characterized the transmission dynamics of HIV-1 among the MSM population in Tianjin, China. Our findings revealed that 38.56% (386/1001) of individuals clustered across 109 molecular transmission clusters (TCs), with MSM aged 50 and below being the group most commonly transmitting HIV-1. Among the identified TCs, CRF01_AE predominated, followed by CRF07_BC. Notably, CRF07_BC demonstrated a higher propensity for forming large clusters compared to CRF01_AE. Birth-death skyline analyses of the two largest clusters indicated that the HIV/AIDS transmission may be at a critical point, nearly all had Re approximately 1 by now. A retrospective analysis revealed that the rapid expansion of these large clusters was primarily driven by the introduction of viruses in 2021, highlighting the crucial importance of continuous molecular surveillance in identifying newly emerging high-risk transmission chains and adapting measures to address evolving epidemic dynamics. Furthermore, we detected the transmission of drug-resistant mutations (DRMs) within the TCs, particularly in the CRF07_BC clusters (K103N, Y181C, and K101E) and CRF01_AE clusters (P225H and K219R), emphasizing the importance of monitoring to support the continued efficacy of first-line therapies and pre-exposure prophylaxis (PrEP). Recombination analyses indicated that complex recombinant patterns, associated with increased amino acid variability, could confer adaptive traits to the viruses, potentially providing a competitive advantage in certain host populations or regions. Our study highlights the potential of integrating molecular epidemiological and phylodynamic approaches to inform targeted interventions.

A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations

Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs).In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for Multi-AS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 102 aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing.In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.

Exploring disparities in HIV-1 pretreatment and acquired drug resistance in China from 2003 to 2022.

To investigate the epidemic patterns of pretreatment drug resistance (PDR) and acquired drug resistance (ADR) in HIV-1 sequences from China. HIV-1 pol sequences and associated epidemiological data were collected from the Los Alamos HIV Sequence Database, NCBI, HIV Gene Sequence Database and PubMed. Genotypic resistance and subtypes were identified using the Stanford HIV Drug Resistance Database. A total of 36 263 sequences from ART-naïve individuals and 1548 sequences from ART-experienced individuals with virological failure were evaluated. PDR prevalence was 6.64%, initially decreasing and then increasing to 7.84% (2018-22) due to NNRTI. Pooled ADR prevalence (44.96%) increased, with NNRTI and NRTI aligning with the overall trend. The percentage of multidrug resistance was more than that of single-drug resistance in PDR and especially ADR annually. PDR was most prevalent in Central China followed by Southwest and North. ADR prevalence was highest in North China followed by Northwest and Southwest. In ADR sequences, high-level resistance was more common, especially in NRTI. PDR sequences exhibited low-level or intermediate resistance, especially PI. Drug resistance mutations revealed distinct patterns in PDR and ADR. CRF01_AE, the predominant subtype in China, exhibited the highest proportions among most ART drugs and drug resistance mutations, with a few exceptions where CRF07_BC (prominent in the Northwest), CRF55_01B and CRF08_BC (prominent in the Southwest) showed the highest proportions. HIV-1 PDR and ADR prevalence in China exhibited diverse epidemiological characteristics, underscoring the importance of ongoing national monitoring of PDR, ADR and subtype; patient education on adherence; and personalized regimens.

Calculating Protein-Ligand Residence Times through State Predictive Information Bottleneck Based Enhanced Sampling.

Understanding drug residence times in target proteins is key to improving drug efficacy and understanding target recognition in biochemistry. While drug residence time is just as important as binding affinity, atomic-level understanding of drug residence times through molecular dynamics (MD) simulations has been difficult primarily due to the extremely long time scales. Recent advances in rare event sampling have allowed us to reach these time scales, yet predicting protein-ligand residence times remains a significant challenge. Here we present a semi-automated protocol to calculate the ligand residence times across 12 orders of magnitude of time scales. In our proposed framework, we integrate a deep learning-based method, the state predictive information bottleneck (SPIB), to learn an approximate reaction coordinate (RC) and use it to guide the enhanced sampling method metadynamics. We demonstrate the performance of our algorithm by applying it to six different protein-ligand complexes with available benchmark residence times, including the dissociation of the widely studied anticancer drug Imatinib (Gleevec) from both wild-type Abl kinase and drug-resistant mutants. We show how our protocol can recover quantitatively accurate residence times, potentially opening avenues for deeper insights into drug development possibilities and ligand recognition mechanisms.

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.

Zanamivir and baloxavir combination to cure persistent influenza and coronavirus infections after hematopoietic stem cell transplant

ObjectivesImmunocompromised patients may experience prolonged shedding of influenza virus potentially leading to severe infections. Alternatives to monotherapy with neuraminidase inhibitors should be evaluated to entirely suppress viral replication and prevent drug-resistant mutations. MethodsWe investigated the clinical and virological evolution in a case of persistent influenza A and human coronavirus OC43 (HCoV-OC43) coinfection in a hematopoietic stem cell transplant recipient after different therapeutic strategies. ResultsSuccessive oseltamivir and zanamivir monotherapies failed to control both infections, with positive results persisting for over 110 days each. This led to the emergence of highly resistant oseltamivir strains due to neuraminidase mutations (E119V and R292K) followed by a deletion (del245-248), while maintaining sensitivity to zanamivir. The intra-host viral diversity data showed that the treatments impacted viral diversity of influenza virus, but not of HCoV-OC43. Considering the patient's underlying condition and the impact of prolonged viral shedding on pulmonary function, eradicating the influenza virus was necessary. A 10-day regimen combining zanamivir and baloxavir-marboxil effectively controlled influenza virus replication and was associated with the clearance of HCoV-OC43, finally resulting in comprehensive respiratory recovery. ConclusionThese observations underscore the importance of further investigating combination treatments as the primary approach to achieve influenza eradication in immunocompromised patients.

Resistance to protease inhibitors among persons living with HIV in Ghana: a case for viral load and drug resistance monitoring

ObjectiveRoutine viral load and drug resistance testing are well supported in most resource-rich settings and provide valuable benefits in the clinical care of PLWH in these communities. Undoubtedly, there exist financial and political constraints for the scale-up of viral load and drug resistance testing in Sub-Saharan Africa. To achieve the global UNAIDS 95/95/95 targets, there is the need to bridge this inequity in patient care and allow for a universal approach that leaves no community behind.MethodsVenous blood from 96 PLWH on second-line ART from Korle-Bu Teaching Hospital were collected and processed into plasma for CD4+ T- cell and viral load assessments. Ribonucleic acid (RNA) was extracted from stored plasma and the protease gene amplified, sequenced and analyzed for subtype and drug resistance mutations using the Stanford HIV drug resistance database.ResultsOut of the 96 PLWH, 37 experienced virological failure with 8 patients’ samples successfully sequenced. The predominant HIV-1 subtype identified was CRF02_AG (6/8, 75.0%) with 12.5% (1/8) each of CFR06_cpx infection and one case unable to subtype. The major PI resistance mutations identified were; M46I, I54V, V82A, I47V, I84V and L90M.ConclusionsPersons living with HIV who had experienced virologic failure in this study harboured drug resistance mutations to PI, thus compromise the effectiveness of the drugs in the second line. Resistance testing is strongly recommended prior to switching to a new regimen. This will help to inform the choice of drug and to achieve optimum therapeutic outcome among PLWH in Ghana.

Sequence Notes: Near Full-Length Genome Analysis of the First-Reported HIV-1 Circulating Recombinant Form (CRF)_10CD in Uganda.

HIV-1 is characterized by remarkable genetic diversity resulting from its high replication rate, error-prone reverse transcriptase enzyme and recombination events. In Uganda, HIV-1 subtype diversity is mostly dominated by subtypes A, D, and A1/D Unique Recombinant Forms (URFs). In this study, deep sequences of HIV from patients with known antiretroviral therapy (ART) status were analyzed to determine the subtypes and to identify drug-resistance mutations circulating in the study population. Of the 187 participant samples processed for next-generation sequencing (NGS), 137 (73%) were successfully classified. The majority of HIV-1 strains were classified as subtype A (75, 55%), D (43, 31%), with other subtypes including C (3, 2%), A1/D (9, 7%) and CRF10_CD (1, <1%). Recombinant analysis of nine complete A1/D HIV genomes identified novel recombination patterns described herein. Furthermore, we report for the first time in Uganda, an HIV-1 CRF10_CD strain from a fisherfolk in a Lake Victoria Island fishing community.

Use of next-generation sequencing on HIV-1 DNA to assess archived resistance in highly treatment-experienced people with multidrug-resistant HIV under virological control: data from the PRESTIGIO Registry.

To clarify whether next-generation sequencing (NGS) can be useful for resistance assessment in virologically suppressed highly treatment-experienced (HTE) individuals with MDR HIV. Ninety-one participants from the PRESTIGIO Registry were included. NGS was performed on HIV-DNA at 1%, 5% and 20% cut-offs; major drug resistance mutations (DRMs) were evaluated and compared with those detected in historical plasma genotypic resistance testing (h-GRT). APOBEC editing was also characterized. Participants had a complex and long treatment history [median 23 (IQR 21-25) years of ART exposure) and had been virologically suppressed since a median of 3 (IQR 2-5) years. Among all major DRMs detected by HIV-DNA NGS and/or h-GRT, 30% were exclusively found through NGS. The highest detection rate of historical major DRMs was reached with NGS set at 1%, but unusual substitutions and extensive APOBEC hypermutations suggest technical issues and poor clinical relevance in the 1%-5% interval. At NGS set at 5%, 67.2% of historical major DRMs were detected. The number of major DRMs detected exclusively by DNA-NGS as minority variants (frequency 5%-20%) was significantly higher in individuals who later experienced virological rebound compared with those who maintained virological control [median 2 (IQR 1-3) versus 1 (0-2), P = 0.030] and positively correlated with viraemia levels at rebound (rho = 0.474, P = 0.030). In non-viraemic people with an MDR virus, HIV-1 DNA NGS set at 5% is an acceptable technical cut-off that might help to reveal mutations with a potential clinical relevance. Moreover, the number of minority resistance mutations additionally detected by NGS might be associated with loss of virological control.

Retrospective study of the prevalence of acquired drug resistance after failed antiretroviral therapy in Libya.

The increasing rate of drug-resistant HIV mutations in Libya and other African countries threatens the efficacy of antiretroviral therapy (ART), thus necessitating urgent regional strategies to combat resistance and improve treatment outcomes. To characterize the frequency and pattern of acquired HIV drug resistance mutations in patients showing ART failure while using non-nucleoside reverse transcriptase inhibitors (NNRTI) prescribed by the Department of Infectious Diseases at Tripoli University Hospital, Libya. We collated retrospective data on 128 people living with HIV, aged 18 years or above, who experienced firstline treatment failure at Tripoli University Hospital, Libya, from 2014 to 2017. We analysed the extant HIV amino acid sequences to identify resistance mutations using algorithms from the Stanford University HIV drug resistance database. All included cases had at least one resistance mutation (n = 128; 100%) and 119 (93%) had both nucleoside reverse transcriptase inhibitors (NRTI) and NNRTI mutations. M184V/I was the most common NRTI mutation (n = 119; 93%) while K103N was the most common NNRTI mutation (n = 100; 78%). Thymidine analog mutations were detected in 51 cases (40%); featuring T215Y, D67N and M41L. Predicted full susceptibility was highest for Tenofovir (66.4%), followed by Etravirine (60%). We detected high level drug resistance and associated mutations among people living with HIV. Implementing drug regimens with high genetic barriers to resistance, coupled with rigorous monitoring and surveillance of HIV resistance development, could help mitigate such resistance in the future.

Long-term stability of acquired drug resistance and resistance associated mutations in the fungal pathogen Nakaseomyces glabratus (Candida glabrata).

The limited number of available antifungal drugs and the increasing number of fungal isolates that show drug or multidrug resistance pose a serious medical threat. Several yeast pathogens, such as Nakaseomyces glabratus (Candida glabrata), show a remarkable ability to develop drug resistance during treatment through the acquisition of genetic mutations. However, how stable this resistance and the underlying mutations are in non-selective conditions remains poorly characterized. The stability of acquired drug resistance has fundamental implications for our understanding of the appearance and spread of drug-resistant outbreaks and for defining efficient strategies to combat them. Here, we used an in vitro evolution approach to assess the stability under optimal growth conditions of resistance phenotypes and resistance-associated mutations that were previously acquired under exposure to antifungals. Our results reveal a remarkable stability of the resistant phenotype and the underlying mutations in a significant number of evolved populations, which conserved their phenotype for at least two months in the absence of drug-selective pressure. We observed a higher stability of anidulafungin resistance over fluconazole resistance, and of resistance-conferring point mutations as compared with aneuploidies. In addition, we detected accumulation of novel mutations in previously altered resistance-associated genes in non-selective conditions, which suggest a possible compensatory role. We conclude that acquired resistance, particularly to anidulafungin, is a long-lasting phenotype, which has important implications for the persistence and propagation of drug-resistant clinical outbreaks.

Genetic diversity accelerates canine distemper virus adaptation to ferrets.

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called "quasispecies." Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance.IMPORTANCEWhen viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines.

Two decades of molecular surveillance in Senegal reveal rapid changes in known drug resistance mutations over time

BackgroundDrug resistance in Plasmodium falciparum is a major threat to malaria control efforts. Pathogen genomic surveillance could be invaluable for monitoring current and emerging parasite drug resistance.MethodsData from two decades (2000–2020) of continuous molecular surveillance of P. falciparum parasites from Senegal were retrospectively examined to assess historical changes in malaria drug resistance mutations. Several known drug resistance markers and their surrounding haplotypes were profiled using a combination of single nucleotide polymorphism (SNP) molecular surveillance and whole genome sequence based population genomics.ResultsThis dataset was used to track temporal changes in drug resistance markers whose timing correspond to historically significant events such as the withdrawal of chloroquine (CQ) and the introduction of sulfadoxine-pyrimethamine (SP) in 2003. Changes in the mutation frequency at Pfcrt K76T and Pfdhps A437G coinciding with the 2014 introduction of seasonal malaria chemoprevention (SMC) in Senegal were observed. In 2014, the frequency of Pfcrt K76T increased while the frequency of Pfdhps A437G declined. Haplotype-based analyses of Pfcrt K76T showed that this rapid increase was due to a recent selective sweep that started after 2014.Discussion (Conclusion)The rapid increase in Pfcrt K76T is troubling and could be a sign of emerging amodiaquine (AQ) resistance in Senegal. Emerging AQ resistance may threaten the future clinical efficacy of artesunate-amodiaquine (ASAQ) and AQ-dependent SMC chemoprevention. These results highlight the potential of molecular surveillance for detecting rapid changes in parasite populations and stress the need to monitor the effectiveness of AQ as a partner drug for artemisinin-based combination therapy (ACT) and for chemoprevention.

Deviated binding of anti-HBV nucleoside analog E-CFCP-TP to the reverse transcriptase active site attenuates the effect of drug-resistant mutations

While certain human hepatitis B virus-targeting nucleoside analogs (NAs) serve as crucial anti-HBV drugs, HBV yet remains to be a major global health threat. E-CFCP is a 4′-modified and fluoromethylenated NA that exhibits potent antiviral activity against both wild-type and drug-resistant HBVs but less potent against human immunodeficiency virus type-1 (HIV-1). Here, we show that HIV-1 with HBV-associated amino acid substitutions introduced into the RT’s dNTP-binding site (N-site) is highly susceptible to E-CFCP. We determined the X-ray structures of HBV-associated HIV-1 RT mutants complexed with DNA:E-CFCP-triphosphate (E-CFCP-TP). The structures revealed that exocyclic fluoromethylene pushes the Met184 sidechain backward, and the resultant enlarged hydrophobic pocket accommodates both the fluoromethylene and 4′-cyano moiety of E-CFCP. Structural comparison with the DNA:dGTP/entecavir-triphosphate complex also indicated that the cyclopentene moiety of the bound E-CFCP-TP is slightly skewed and deviated. This positioning partly corresponds to that of the bound dNTP observed in the HIV-1 RT mutant with drug-resistant mutations F160M/M184V, resulting in the attenuation of the structural effects of F160M/M184V substitutions. These results expand our knowledge of the interactions between NAs and the RT N-site and should help further design antiviral NAs against both HIV-1 and HBV.

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.

Antiviral activity of luteolin against porcine epidemic diarrhea virus in silico and in vitro

BackgroundPorcine epidemic diarrhea virus (PEDV) mainly causes acute and severe porcine epidemic diarrhea (PED), and is highly fatal in neonatal piglets. No reliable therapeutics against the infection exist, which poses a major global health issue for piglets. Luteolin is a flavonoid with anti-viral activity toward several viruses.ResultsWe evaluated anti-viral effects of luteolin in PEDV-infected Vero and IPEC-J2 cells, and identified IC50 values of 23.87 µM and 68.5 µM, respectively. And found PEDV internalization, replication and release were significantly reduced upon luteolin treatment. As luteolin could bind to human ACE2 and SARS-CoV-2 main protease (Mpro) to contribute viral entry, we first identified that luteolin shares the same core binding site on pACE2 with PEDV-S by molecular docking and exhibited positive pACE2 binding with an affinity constant of 71.6 µM at dose-dependent increases by surface plasmon resonance (SPR) assay. However, pACE2 was incapable of binding to PEDV-S1. Therefore, luteolin inhibited PEDV internalization independent of PEDV-S binding to pACE2. Moreover, luteolin was firmly embedded in the groove of active pocket of Mpro in a three-dimensional docking model, and fluorescence resonance energy transfer (FRET) assays confirmed that luteolin inhibited PEDV Mpro activity. In addition, we also observed PEDV-induced pro-inflammatory cytokine inhibition and Nrf2-induced HO-1 expression. Finally, a drug resistant mutant was isolated after 10 cell culture passages concomitant with increasing luteolin concentrations, with reduced PEDV susceptibility to luteolin identified at passage 10.ConclusionsOur results push forward that anti-PEDV mechanisms and resistant-PEDV properties for luteolin, which may be used to combat PED.

Genotyping and Antiretroviral Drug Resistance Mutations among HIV Patients in Southern Santa Catarina, Brazil.

Therapeutic measures have been successful in increasing survival rates and quality of life of HIV/AIDS-infected people. However, some people fail to respond to antiretroviral therapy (HAART) because of viral resistance-associated mutations. To identify virus genotype and the presence of mutations that alter the susceptibility to HAART, and factors associated with the occurrence of these mutations. A cross-sectional study was conducted on adults living with HIV attending a specialized outpatient clinic in southern Santa Catarina, Brazil. The participants were interviewed and had blood samples collected for analysis. Those with detectable viral load were genotyped. Out of the 629 patients recruited, 127 subjects were included due to having a detectable viral load. The most common mutations were M184V and K103N. HIV-1 subtype C was the most prevalent strain. Resistance to HAART was associated with modification in the treatment regimen (p <0.001). This study concluded that the circulating subtype virus was subtype C and that the mutations K103N and M184V were the most prevalent strains in southern Santa Catarina, Brazil.

HIV-DRIVES: HIV drug resistance identification, variant evaluation, and surveillance pipeline.

The global prevalence of resistance to antiviral drugs combined with antiretroviral therapy (cART) emphasizes the need for continuous monitoring to better understand the dynamics of drug-resistant mutations to guide treatment optimization and patient management as well as check the spread of resistant viral strains. We have recently integrated next-generation sequencing (NGS) into routine HIV drug resistance (HIVDR) monitoring, with key challenges in the bioinformatic analysis and interpretation of the complex data generated, while ensuring data security and privacy for patient information. To address these challenges, here we present HIV-DRIVES (HIV Drug Resistance Identification, Variant Evaluation, and Surveillance), an NGS-HIVDR bioinformatics pipeline that has been developed and validated using Illumina short reads, FASTA, and Sanger ab1.seq files.