Computational Estimation of Residues Involving Resistance to the SARS-CoV-2 Main Protease Inhibitor Ensitrelvir Based on Virtual Alanine Scan of the Active Site.

Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.

No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Dynamic diversity of SARS-CoV-2 genetic mutations in a lung transplantation patient with persistent COVID-19.

Numerous SARS-CoV-2 variant strains with altered characteristics have emerged since the onset of the COVID-19 pandemic. Remdesivir (RDV), a ribonucleotide analogue inhibitor of viral RNA polymerase, has become a valuable therapeutic agent. However, immunosuppressed hosts may respond inadequately to RDV and develop chronic persistent infections. A patient with respiratory failure caused by interstitial pneumonia, who had undergone transplantation of the left lung, developed COVID-19 caused by Omicron BA.5 strain with persistent chronic viral shedding, showing viral fusogenicity. Genome-wide sequencing analyses revealed the occurrence of several viral mutations after RDV treatment, followed by dynamic changes in the viral populations. The C799F mutation in nsp12 was found to play a pivotal role in conferring RDV resistance, preventing RDV-triphosphate from entering the active site of RNA-dependent RNA polymerase. The occurrence of diverse mutations is a characteristic of SARS-CoV-2, which mutates frequently. Herein, we describe the clinical case of an immunosuppressed host in whom inadequate treatment resulted in highly diverse SARS-CoV-2 mutations that threatened the patient's health due to the development of drug-resistant variants.

Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Target-based drug design strategies to overcome resistance to antiviral agents: opportunities and challenges.

Viral infections have a major impact in human health. Ongoing viral transmission and escalating selective pressure have the potential to favor the emergence of vaccine- and antiviral drug-resistant viruses. Target-based approaches for the design of antiviral drugs can play a pivotal role in combating drug-resistant challenges. Drug design computational tools facilitate the discovery of novel drugs. This review provides a comprehensive overview of current drug design strategies employed in the field of antiviral drug resistance, illustrated through the description of a series of successful applications. These strategies include technologies that enhance compound-target affinity while minimizing interactions with mutated binding pockets. Furthermore, emerging approaches such as virtual screening, targeted protein/RNA degradation, and resistance analysis during drug design have been harnessed to curtail the emergence of drug resistance. Additionally, host targeting antiviral drugs offer a promising avenue for circumventing viral mutation. The widespread adoption of these refined drug design strategies will effectively address the prevailing challenge posed by antiviral drug resistance.

FMO-guided design of darunavir analogs as HIV-1 protease inhibitors.

The prevalence of HIV-1 infection continues to pose a significant global public health issue, highlighting the need for antiretroviral drugs that target viral proteins to reduce viral replication. One such target is HIV-1 protease (PR), responsible for cleaving viral polyproteins, leading to the maturation of viral proteins. While darunavir (DRV) is a potent HIV-1 PR inhibitor, drug resistance can arise due to mutations in HIV-1 PR. To address this issue, we developed a novel approach using the fragment molecular orbital (FMO) method and structure-based drug design to create DRV analogs. Using combinatorial programming, we generated novel analogs freely accessible via an on-the-cloud mode implemented in Google Colab, Combined Analog generator Tool (CAT). The designed analogs underwent cascade screening through molecular docking with HIV-1 PR wild-type and major mutations at the active site. Molecular dynamics (MD) simulations confirmed the assess ligand binding and susceptibility of screened designed analogs. Our findings indicate that the three designed analogs guided by FMO, 19-0-14-3, 19-8-10-0, and 19-8-14-3, are superior to DRV and have the potential to serve as efficient PR inhibitors. These findings demonstrate the effectiveness of our approach and its potential to be used in further studies for developing new antiretroviral drugs.

Resistencia transmitida de VIH-1 en pacientes sin exposición a tratamiento antirretroviral: Guatemala / Transmitted HIV-1 resistance in patients without exposure to antiretroviral treatment: Guatemala / Resistência transmitida ao HIV-1 em pacientes sem exposição ao tratamento antirretroviral: Guatemala

El objetivo del estudio fue describir los niveles de resistencia transmitida de VIH-1 en adultos atendidos en Unidades de Atención Integral de Guatemala. El estudio incluyó registros de 185 pacientes adultos VIH-1 positivo, de reciente diagnóstico sin antecedente de uso de TAR, de noviembre del 2019 a noviembre del 2020. El análisis se realizó en el software DeepChek® v2.0, para la clasificación de la resistencia se siguió el algoritmo de Stanford HIVdb (v9.4 - 07/12/2022). Se encontró 18.4% (IC 95% 13.1 - 24.7%) de resistencia general a alguna familia de ARVs. Se evidenció 15.1% (IC 95% 10.3 - 21.1%) de resistencia individual a la familia de INNTR afectando principalmente a NVP y EFV; 2.2% (IC 95% 0.6 - 5.4%) de resistencia a INTR, mayormente a FTC/3TC; y 2.7% (IC 95% 0.9 - 6.2%) de resistencia intermedia y baja los IP NFV y LPV/r. Tres casos presentaron resistencia múltiple a los INTR + INNTR. Las mutaciones más frecuentemente encontradas fueron K103N (41.2%), M184V/I (8.8%) y M46I (5.9%). La elevada resistencia transmitida del VIH-1 en pacientes atendidos en distintas Unidades de Atención Integral del VIH, demuestra la importancia de analizar periódicamente la tendencia de la resistencia en personas que no han estado expuestas a ARVs, lo cual a su vez es un marcador indirecto de presencia de resistencia adquirida en el país, datos que evidencian la necesidad de acciones e intervenciones prontas y efectivas dado su impacto en la salud pública.

The objective of this study was to describe the levels of transmitted HIV-1 resistance in patients with a recent HIV diagnosis before starting ART, treated in Comprehensive Care Units in Guatemala during the years 2019 and 2020. The study included records of 185 HIV-positive adult patients, recently diagnosed with HIV without a history of ART use. The analysis was carried out in the DeepChek® v2.0 software, the Stanford HIVdb algorithm (v9.4 - 07/12/2022) was followed to classify resistance. 18.4% (95% CI 13.1 - 24.7%) of general resistance to some family of ARVs was found. There was evidence of 15.1% (95% CI 10.3 - 21.1%) of individual resistance to the NNRTI family, mainly affecting NVP and EFV; 2.2% (95% CI 0.6 - 5.4%) resistance to INTR, mostly to FTC/3TC; and 2.7% (95% CI 0.9 - 6.2%) of intermediate and low resistance IP NFV and LPV/r. Three cases presented multiple resistance to NRTIs + NNRTIs. The most frequently found mutations were K103N (41.2%), M184V/I (8.8%) and M46I (5.9%). The high transmitted resistance of HIV-1 in patients treated in different Comprehensive HIV Care Units demonstrates the importance of periodically analyzing the trend of resistance in people who have not been exposed to ARVs, which in turn is an indirect marker. of the presence of acquired resistance in the country, data that demonstrate the need for prompt and effective actions and interventions given its impact on public health.

O objetivo deste estudo foi descrever os níveis de resistência transmitida ao HIV-1 em adultos tratados em Unidades de Cuidados Integrais na Guatemala. O estudo incluiu prontuários de 185 pacientes adultos HIV-1 positivos, recentemente diagnosticados sem histórico de uso de TARV, no período de novembro de 2019 a novembro de 2020. A análise foi realizada no software DeepChek® v2.0, para classificação da resistência, O algoritmo Stanford HIVdb (v9.4 - 07/12/2022) foi seguido. Foi encontrada 18.4% (IC 95% 13.1 - 24.7%) de resistência geral a alguma família de ARVs. Houve evidência de 15.1% (IC 95% 10.3 - 21.1%) de resistência individual à família de NNRTI, afetando principalmente NVP e EFV; 2.2% (IC 95% 0.6 - 5.4%) resistência ao INTR, principalmente ao FTC/3TC; e 2.7% (IC 95% 0.9 - 6.2%) de resistência intermediária e baixa ao IP NFV e LPV/r. Três casos apresentaram resistência múltipla a NRTIs + NNRTIs. As mutações mais frequentemente encontradas foram K103N (41.2%), M184V/I (8.8%) e M46I (5.9%). A elevada resistência transmitida do HIV-1 em pacientes atendidos em diferentes Unidades de Cuidados Integrados ao HIV demonstra a importância de analisar periodicamente a tendência de resistência em pessoas que não foram expostas aos ARVs, o que por sua vez é um marcador indireto da presença de ARVs adquiridos. resistência no país, dados que demonstram a necessidade de ações e intervenções rápidas e eficazes dado o seu impacto na saúde pública.

Checklist for studies of HIV drug resistance prevalence or incidence: rationale and recommended use.

HIV drug resistance (HIVDR) is a major challenge to the effectiveness of antiretroviral therapy. Global efforts in addressing HIVDR require clear, transparent, and replicable reporting in HIVDR studies. We describe the rationale and recommended use of a checklist that should be included in reports of HIVDR incidence and prevalence. After preliminary consultations with experts on HIVDR and establishing the need for guidance on HIVDR reporting, we used a sequential, explanatory, mixed methods approach to create the checklist; together with the accompanying articles, the checklist was reviewed by the authors and validated externally. The checklist for studies on HIVDR prevalence or incidence (CEDRIC-HIV) includes 15 recommended items that would enhance transparency and facilitate interpretation, comparability, and replicability of HIVDR studies. CEDRIC-HIV will help authors of HIVDR studies prepare research reports and assist reviewers and editors in assessments of completeness of reporting. The checklist will also facilitate statistical pooling and interpretation of HIVDR data.

Resistencia genotípica del HIV a los antirretrovirales en niños de Argentina entre 2006 y 2021 / HIV genotypic drug resistance to antiretrovirals in children in Argentina between 2006 and 2021

Introducción: La resistencia del HIV a los antirretrovirales (ARVs) es una de las principales causas de fallo terapéutico en niños, niñas y adolescentes que conviven con el virus. Desde el año 2006, el Laboratorio de Biología Celular y Retrovirus del Hospital Garrahan realiza el estudio genotípico de resistencia (ER) del HIV-1 a los ARVs a fin de identificar mutaciones que disminuyen la susceptibilidad del virus a los fármacos que componen el tratamiento ARV. Objetivos: El objetivo del trabajo fue estudiar el tipo y frecuencia de resistencia del HIV a los ARVs, a través de un análisis de 371 ER realizados entre los años 2006 y 2021 en niños, niñas y adolescentes con HIV-1 adquirido por transmisión vertical y con solicitud médica de ER por presentar fallo terapéutico. Resultados: Entre los años 2006 y 2013 la proporción de casos con resistencia a al menos una clase de fármaco ARV fue mayor al 90%, sugiriendo una asociación directa entre el fallo virológico y la disminución en la susceptibilidad del HIV-1 a uno o más componentes del TARV. A partir del año 2012, se observa una disminución progresiva del nivel de resistencia de HIV-1, llegando al 50% en 2021 (p<0.0001). La frecuencia de mutaciones de resistencia fue diferente para cada una de las clases de ARVs. Mientras que la resistencia a INNTR no sufrió cambios significativos a lo largo del período de estudio, oscilando entre 27% y 75%. La proporción de mutaciones a IPs en pacientes con fallo virológico disminuyó de 87% en 2006 a 17% en 2021 y para los INTR, disminuyó de 79% en 2006 a 45% en 2021. Conclusión: El nivel de resistencia a los ARVs ha disminuido de manera sustancial a lo largo de los últimos 16 años, probablemente por el uso de nuevos fármacos ARV con alta potencia que posibilitaron la intensificación de los tratamientos ARV y la implementación de criterios de fallo terapéutico más estrictos tanto a nivel clínico como virológico (AU)

Introduction: HIV resistance to antiretroviral (ARV) drugs is one of the main causes of therapeutic failure in children and adolescents living with the virus. Since 2006, the Cell Biology and Retrovirus Laboratory of the Garrahan Hospital has been performing the genotypic study of HIV-1 resistance to ARV drugs in order to identify mutations that reduce the susceptibility of the virus to the drugs that constitute ARV treatment. Objectives: The aim of this study was to assess the type and frequency of HIV resistance to ARV drugs through an analysis of 371 genotype studies performed between 2006 and 2021 in children and adolescents with HIV-1 acquired through motherto-child transmission and with medical request for genotype study due to therapeutic failure. Results: Between 2006 and 2013, the proportion of cases with resistance to at least one ARV drug class was greater than 90%, suggesting a direct association between virologic failure and decreased susceptibility of HIV-1 to one or more components of ART. From 2012 onwards, a progressive decrease in the level of HIV-1 resistance was observed, reaching 50% in 2021 (p<0.0001). The frequency of resistant mutations was different for each of the ARV classes, while resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) did not change significantly over the study period, ranging from 27% to 75%. The proportion of drug-resistant mutations to protease inhibitors (PI) in patients with virologic failure decreased from 87% in 2006 to 17% in 2021 and for NNRTIs from 79% in 2006 to 45% in 2021. Conclusion: The level of resistance to ARV drugs has decreased substantially over the last 16 years, probably due to the use of new ARV drugs with high potency that allowed the intensification of ARV treatments and the implementation of stricter criteria for therapeutic failure both clinically and virologically (AU)

In-vivo and human evidence for potential efficacy of therapeutic polyclonal RSV neutralizing antibodies for palivizumab-resistant RSV infections.

BACKGROUND: Monoclonal antibody (palivizumab), intravenous immune globulin (IGIV), or respiratory syncytial virus (RSV)-polyclonal-hyperimmune-globulin (RSV-IG as Respigam®, RI-001, RI-002) are used with ribavirin in RSV-infected immunocompromised patients, with debated efficacy. Palivizumab-resistance (PR) can arise during treatment of persistent infections in this population. RSV-IG may confer benefit in PR-RSV infection. METHODS: RSV-IG [RI-001] was provided for an immunocompromised infant with RSV-pneumonitis refractory to ribavirin and palivizumab. RSV-neutralizing antibody, respiratory RSV load (qPCR), and F-gene-sequence-detection of PR was determined. Prophylactic RSV-IG [RI-002] or palivizumab was administered in a cotton-rat model infected with wild-type and PR-RSV. Lung RSV load and neutralizing antibody were measured. RESULTS: As protective RI-001-neutralizing antibody titers waned in the infant, a subpopulation of PR-escape mutants were detected with a fatal RSV-burden in the lungs. In PR-RSV-infected cotton rats, prophylactic RI-002 reduced RSV-load in the lungs (2.45 vs 0.28 log10 PFU/g lung-tissue reduction, respectively, p < 0.05) and provided protective RSV-neutralizing antibody. CONCLUSIONS: RSV-IG and ribavirin use in immunocompromised patients requires further study.

Acyclovir-resistant HSV-1 isolates among immunocompromised patients in southern Taiwan: Low prevalence and novel mutations.

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections.

Novel UL23 and UL30 substitutions in HSV1 and HSV2 viruses related to polymorphism or drug resistance.

Data on herpes simplex virus (HSV) polymorphism as well as acyclovir (ACV) and foscarnet (FOS) resistance mutations are not exhaustive and may hinder accurate diagnosis by next-generation sequencing (NGS). Here, we report novel UL23 and UL30 substitutions for HSV1 and HSV2 identified in immunocompromised patients treated for hematological malignancies during the last 6 years of HSV resistance surveillance at the University Hospital of Lyon. For HSV1, 35 novel UL23 substitutions and 52 novel UL30 substitutions were identified. For HSV2, 2 novel UL23 substitutions and 12 novel UL30 substitutions were identified. These results allow to complete the database of HSV1 and HSV2 substitutions, related either to polymorphism or to ACV and FOS resistance.

[Investigation of Ganciclovir Resistance in Cytomegalovirus Isolates by Phenotypic and Genotypic Methods]. / Sitomegalovirüs Izolatlarinda Gansiklovir Direncinin Fenotipik ve Genotipik Yöntemlerle Arastirilmasi.

Ganciclovir-resistant cytomegalovirus (CMV) strains are reported following long-term antiviral agent use, especially for immune-suppressive patients. In this study, it was aimed to investigate the mutations in the UL97 gene of CMV, which causes ganciclovir (GCV) resistance by genotypic and phenotypic methods in patients who developed CMV infection following hematopoietic cell (HCT) or solid organ transplantation (SOT). Thirty patients who had HCT or SOT in Mediterranean University Hospital and developed CMV infection during routine follow-up with a viral load of CMV over 1000 copies/mL were included in the study. CMV DNA was analyzed by an automated system (Cobas Ampliprep/COBAS TaqMan CMV Test, Roche Diagnostics, Germany) quantitatively. DNA sequence analysis of the regions including codons 420-664 in the UL97 gene region was done by the Sanger sequencing method to detect mutations causing antiviral resistance and compared with defined mutations. In order to investigate antiviral resistance by phenotypic methods, heparinized blood samples of the patients were collected, 'buffy coat (leukocyte layer)' was inoculated into MRC-5 cells by centrifugation method and CMV growth in these cells was controlled with monoclonal antibodies when growth was detected, virus titer was determined and plaque reduction test was applied as recommended. It was determined that 22 of the 30 patients were HCT recipients and eight were SOT (five kidney, three liver) recipients. When the CMV serology pattern of the patients was evaluated before transplantation, 29 (96.7%) patients were found to be seropositive and one (3.3%) patient was found to be seronegative. Totally, nine CMV UL97 mutations were detected in seven (23.3%) pediatric patients who had HCT, including six seropositive and one seronegative case. In addition, one mutation (D605E) not known to cause GCV resistance was detected in a seronegative recipient and three previously unidentified mutations were detected (1474T, F499S, V559A) in a seronegative recipient. Five of the mutations defined were UL97 mutations with a defined clinical resistance against GCV in each of the five recipients (C603W, C592G, H520Q, M460V, A594T). In the plaque reduction test using 3 µM, 12 µM, 48 µM and 96 µM concentrations of GCV in CMV strains, the IC50 value was determined to be ≥ 8 µM for the five CMV strains, and the phenotypic presence of GCV resistance was shown. Clinical resistance associated with CMV UL97 mutation was detected in five (22.7%) of 22 patients who had HCT. GCV resistance was also demonstrated in these patients by phenotypic methods. No UL97 mutation was detected in the patients who had SOT.

Epidemiology of cytomegalovirus antiviral resistance testing for solid organ and bone marrow transplant patients from 2011 - 2019.

BACKGROUND: CMV reactivation post-transplantation is common, with need for prompt identification of patients most at-risk for CMV antiviral drug resistance (AVDR). OBJECTIVES: This study describes CMV AVDR frequencies, antiviral prescribing practices, and AVDR risk factors in patients from 2011 to 2019 in British Columbia, Canada. STUDY DESIGN: Retrospective review of demographics, transplant type, viral loads, antiviral exposure duration, and 12-month mortality was conducted for all patients with samples submitted for CMV AVDR testing from 2011 to 2019. Genotyping of AVDR mutations occurred at the national reference laboratory. Mann-Whitney U, T-test or Fisher's exact tests examined differences between patients with and without AVDR. RESULTS: Fifty-three plasma and three tissue/fluid specimens successfully underwent CMV AVDR testing; of these samples, 27/56 (48%) had AVDR mutations detected. The commonest AVDR mutations were at UL97 loci A594 (20%), H596 (12%) and L595 (12%). Mutations occurred more frequently in requests from solid organ than hematopoietic stem cell transplant patients (58% vs. 27%, p = 0.05). Previous resistance testing was a significant risk factor for AVDR (p < 0.001). Patients with AVDR had approximately 51 more days of antiviral therapy (p = 0.007) and took 9 days longer to clear viremia (p = 0.23). The median turnaround time from sample send-out to reporting was nine days. However, empiric use of second-line antivirals occurred in most cases (39/53, 74%) before results were available. DISCUSSION: Laboratories should strive to provide timely CMV AVDR testing for transplant patients, to minimize unnecessary exposure to second-line antiviral agents. The findings of this study may help guide clinicians when selecting empiric antiviral therapy.

Cytomegalovirus drug resistance mutations in transplant recipients with suspected resistance.

Resistant CMV infections are challenging complications after SOT and HSCT. Prompt recognition of ARMs is imperative for appropriate therapy. 108 plasma samples from 96 CMV + transplant recipients with suspected resistance were analysed in CNM in a retrospective nationwide study from January 2018 to July 2022 for resistance genotyping. ARMs in UL97 and UL54 were found in 26.87% (18/67) and 10.60% (7/66) of patients, respectively. Patients' ARM distribution in UL97 was as follows: L595S n = 3; L595S/M460I n = 1; L595S/N510S n = 1; L595W n = 1; C603W n = 4; A594V n = 3; A594E n = 1; C607Y n = 1; L397R/T409M/H411L/M460I n = 1; L397I n = 1; H520Q n = 1; four patients showed ARMs in UL54 as well (F412C n = 1; T503I n = 2; P522S n = 1), whereas three patients exhibited ARMs in UL54 only (L501I/T503I/L516R/A834P n = 1; A987G n = 2). L516R in UL54 and L397R/I and H411L in UL97 have been found for the first time in a clinical sample. L595S/W was the most prevalent ARM found to lend resistance to GCV. In UL54 all ARMs lent resistance to GCV and CDV. In addition, A834P, found in one patient, also lent resistance to FOS. CMV load did not differ significantly in patients with or without ARMs, and no differences were found either between patients with ARMs in UL97 or in UL97 and UL54. Despite extensive use of classical antivirals for the treatment of CMV infection after HSCT and SOT, ARMs occurred mainly in viral UL97 kinase, which suggests that CDV and mostly FOS continue to be useful alternatives to nucleoside analogues after genotypic detection of ARMs.

Clinical next-generation sequencing assay combining full-length gene amplification and shotgun sequencing for the detection of CMV drug resistance mutations.

Cytomegalovirus (CMV) causes severe systemic and tissue-invasive disease in immunocompromised patients, particularly solid organ and hematopoietic stem cell transplant recipients. While antiviral drugs offer promising efficacy, clinical management is complicated by the high frequency of drug resistance-associated mutations. The most commonly encountered mutations occur in the genes encoding for the drug targets: UL54 (DNA polymerase), UL56 (terminase complex), and UL97 (phosphotransferase), conferring resistance to ganciclovir/cidofovir/foscarnet, letermovir, and ganciclovir/maribavir, respectively. Currently, standard practice for detecting drug resistance is sequencing-based genotypic analysis by commercial reference laboratories with strictly prescribed sample requirements and reporting parameters that can often restrict testing in a highly vulnerable population. In order to circumvent these limitations, we developed a dual-step next-generation sequencing (NGS)-based clinical assay that utilizes full-length gene amplification by long-range PCR followed by shotgun sequencing for mutation analysis. This laboratory-developed test (LDT) achieved satisfactory performance with 96.4% accuracy, 100% precision, and an analytical sensitivity of 300IU/mL with 20% allele frequency. Highlighted by two clinical cases, our NGS LDT was able to provide critical results from patient specimens with viral loads <500IU/mL and volumes <0.5 mL - conditions otherwise unacceptable by reference laboratories. Here, we describe the development and implementation of a robust NGS LDT that offers greater testing flexibility and sensitivity to accommodate a more diverse patient population.

Genetic Variants Associated with Drug Resistance of Cytomegalovirus in Hematopoietic Cell Transplantation Recipients.

Cytomegalovirus (CMV) infection is a serious complication in hematopoietic cell transplantation (HCT) recipients. Drug-resistant strains make it more challenging to treat CMV infection. This study aimed to identify variants associated with CMV drug resistance in HCT recipients and assess their clinical significance. A total of 123 patients with refractory CMV DNAemia out of 2271 HCT patients at the Catholic Hematology Hospital between April 2016 and November 2021 were analyzed, which accounted for 8.6% of the 1428 patients who received pre-emptive therapy. Real-time PCR was used to monitor CMV infection. Direct sequencing was performed to identify drug-resistant variants in UL97 and UL54. Resistance variants were found in 10 (8.1%) patients, and variants of uncertain significance (VUS) were found in 48 (39.0%) patients. Patients with resistance variants had a significantly higher peak CMV viral load than those without (p = 0.015). Patients with any variants had a higher risk of severe graft-versus-host disease and lower one-year survival rates than those without (p = 0.003 and p = 0.044, respectively). Interestingly, the presence of variants reduced the rate of CMV clearance, particularly in patients who did not modify their initial antiviral regimen. However, it had no apparent impact on individuals whose antiviral regimens were changed due to refractoriness. This study highlights the importance of identifying genetic variants associated with CMV drug resistance in HCT recipients for providing appropriate antiviral treatment and predicting patient outcomes.

A Herpes Simplex Virus 1 DNA Polymerase Multidrug Resistance Mutation Identified in a Patient With Immunodeficiency and Confirmed by Gene Editing.

BACKGROUND: Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management. METHODS: Seventeen herpes simplex virus 1 isolates were obtained from orofacial/anogenital lesions in a patient with leaky severe combined immunodeficiency over 7 years, before and after stem cell transplantation. Spatial/temporal evolution of drug resistance was characterized genotypically-with Sanger and next-generation sequencing of viral thymidine kinase (TK) and DNA polymerase (DP)-and phenotypically. CRISPR/Cas9 was used to introduce the novel DP Q727R mutation, and dual infection-competition assays were performed to assess viral fitness. RESULTS: Isolates had identical genetic backgrounds, suggesting that orofacial/anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by next-generation sequencing, undetectable by Sanger sequencing. Thirteen isolates were acyclovir resistant due to TK mutations, and the Q727R isolate additionally exhibited foscarnet/adefovir resistance. Recombinant Q727R mutant virus showed multidrug resistance and increased fitness under antiviral pressure. CONCLUSIONS: Long-term follow-up of a patient with severe combined immunodeficiency revealed virus evolution and frequent reactivation of wild-type and TK mutant strains, mostly as heterogeneous populations. The DP Q727R resistance phenotype was confirmed with CRISPR/Cas9, a useful tool to validate novel drug resistance mutations.

HIV-1 3'-Polypurine Tract Mutations Confer Dolutegravir Resistance by Switching to an Integration-Independent Replication Mechanism via 1-LTR Circles.

Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an in vivo systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles. As the replication of these virus variants is activated by the human T-lymphotropic virus 1 (HTLV-1) Tax protein, a factor that reverses epigenetic silencing of episomal HIV DNA, these data indicate that the 3'PPT-mutated viruses escape from the integrase inhibitor DTG by switching to an integration-independent replication mechanism. IMPORTANCE The integrase inhibitor DTG is a potent inhibitor of HIV replication and is currently recommended in drug regimens for people living with HIV. Whereas HIV normally escapes from antiviral drugs by the acquisition of specific mutations in the gene that encodes the targeted enzyme, mutational inactivation of the viral 3'PPT sequence, an RNA element that has a crucial role in the viral reverse transcription process, was found to allow HIV replication in the presence of DTG in cell culture experiments. While the integration of the viral DNA into the cellular genome is considered one of the hallmarks of retroviruses, including HIV, 3'PPT inactivation caused integration-independent replication, which can explain the reduced DTG sensitivity. Whether this exotic escape route can also contribute to viral escape in HIV-infected persons remains to be determined, but our results indicate that screening for 3'PPT mutations in patients that fail on DTG therapy should be considered.