UL97 Gene Research Articles

Mutations in the cytomegalovirus UL97 gene associated with ganciclovir resistance in recipients of allogeneic hematopoietic stem cell transplants

Objective. To identify mutations in UL97 gene associated with antiviral drug resistance in recipients of allogeneic hematopoietic stem cells transplants (allo-HSCT). Materials and Methods. A total of 9 HCMV DNA samples were studied. These samples were received from the blood of 8 allo-HSCT recipients who were infected with HCMV and undergoing treatment at NMRC of Hematology (Russia) over the period of 2016 to 2017. Sanger sequencing was used to find mutations. The sequenced part of the virus DNA was analyzed using nucleotide BLAST and Genome compiler. Mutations were identified using MRA program which compared the obtained nucleotide sequence with the reference sequence of UL97 gene from Merlin strain. Results. Rate of detection of viruses with mutations that may lead to drug resistance is relatively high – 3 of 8 patients. The following mutations were identified: C592G, C607F and C603W. The obtained data shows that the presence and characteristics of mutations affect the viral load and the time when HCMV DNA is identified in blood. Conclusions. Obtained data show that presence of mutations impacts the course infection, leading to higher viral load and longer persistence of viral DNA in blood samples. Identified mutations had different resistance factor, which also impacted on the pattern of infection.

1561. Outcomes of Resistant or Refractory CMV Infection in Recipients of Allogeneic Hematopoietic Cell Transplant

BackgroundResistant or refractory CMV infections are not well defined and may be associated with high morbidity and mortality in allogeneic hematopoietic cell transplant (allo-HCT) recipients. Suspicion of resistant CMV infections is usually based on suboptimal responses to antiviral agents.MethodsWe performed a single-center retrospective chart review (January 2010 to September 2017) of Allo-HCT recipients who had CMV genotypic testing performed for suspected antiviral resistance. Based on the results, we categorized patients as either having refractory CMV (defined as CMV viremia that fails to decrease after at least 2 weeks of appropriately dosed and delivered antiviral therapy; and in the absence of known genetic mutations to the available antiviral agents) or resistant CMV infection (defined as refractory infection with identification of genetic mutations in the UL97 and/or UL54 genes correlating with in vitro antiviral resistance). Primary outcome was all-cause mortality.ResultsCMV genotypic resistance analysis was performed in 97 patients. Of those, 23 had resistant (11 had UL54 gene, 10 had UL97 gene, and two had both UL54 and 97) and 74 had refractory CMV infections. The majority of patients had AML (53%), underwent matched unrelated donor transplantation (43%), and received ATG during conditioning (64%). Patients with resistant CMV infections had a greater number of prior episodes when compared with those with refractory infections and had a longer time from transplant to suspicion of resistance (P < 0.01; each). Overall, the incidence of CMV disease was 41% (25% vs. 58% affecting the lungs and 56% vs. 17% the GI tract, in resistant vs. refractory infections, respectively). All-cause mortality was 57% (61% resistant vs. 55% refractory) and CMV-attributable mortality was 11% (9% resistant vs. 12% refractory).ConclusionOur data showed that resistant CMV infections are associated with a higher rate of CMV disease. However, both resistant and refractory CMV infections had increased all-cause mortality and similar CMV-attributable mortality. There was no difference in outcomes between allo-HCT recipients who had resistant or refractory CMV infections. New treatment strategies for resistant or refractory CMV infections are needed.Disclosures S. L. Aitken, Merck: Speaker’s Bureau, Consulting fee. E. Ariza-Heredia, Oxford Immunotec: Grant Investigator, Research grant. R. Chemaly, Merck: Consultant, Research grant; Chimerix: Consultant, Research grant; Novartis: Investigator, Research grant; Oxford Immunotec: Consultant, Research grant.

Biological relevance of Cytomegalovirus genetic variability in congenitally and postnatally infected children

BackgroundHuman cytomegalovirus (HCMV) is the leading cause of congenital infections resulting in severe morbidity and mortality among infected children. Although the virus is highly polymorphic, particularly in genes contributing to immune evasion, the mechanisms underlying its genetic variability and pathogenicity are only partially understood. ObjectivesWe aimed to characterize different HCMV clinical strains isolated from 21 congenitally- or postnatally-infected children for in vitro growth properties and genetic polymorphisms. Study designThe growth of various HCMV isolates was analyzed in different cell culture models. Genetic polymorphism was assessed by genetic and phylogenetic analysis of viral genes involved in virulence (UL144, US28, and UL18), latency (UL133-138), or drug resistance (UL54 and UL97). ResultsHere, we report a high degree of genetic and phenotypic diversity in distinct HCMV clinical isolates, as shown by their in vitro growth properties. In particular, HCMV isolates displayed the highest degree of genetic variability in the UL144 gene, where we were able to define four distinct genotypes within the cohort based on UL144 heterogeneity. Lastly, among all isolates we were able to identify 36 mutations in UL54 and 2 in UL97. ConclusionsOur findings indicate that surprisingly high levels of genetic HCMV variability correlate with a high degree of phenotypic polymorphism, which in turn might differentially influence the growth, fitness, and drug susceptibility of HCMV.

Eleusine indica Inhibits Early and Late Phases of Herpes Simplex Virus Type 1 Replication Cycle and Reduces Progeny Infectivity

The present study was aimed at determining the compounds available in Eleusine indica methanol extract and the effects on herpes simplex virus type 1 (HHV1) replication cycle and progeny infectivity. Twelve compounds mostly from the flavonoid and phenolic groups were identified by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. The effect on replication phases of HHV1 was determined by time-of-addition, time-removal and virus yield reduction assays with expression of selected genes analysed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The extract inhibited plaque formation the most during the first 2 h and at 24 h of infection. Plaque formation inhibition was also noted at all other time points but at lesser percentage. Treatment with E. indica reduced progeny infectivity when treated for 10 h and was dose-dependent. E. indica methanol extract inhibited immediate early, early and late phases of HHV1 replication cycle by modifying the expression of UL54, UL27 and UL30 genes during the infection. Immunostaining of infected cells confirmed that E. indica inhibited mainly Glycoproteins B but not Glycoprotein C and D. Thus, the methanol extract of E. indica has the ability to alter HHV1 replication cycle at almost all stages and reduce progeny infectivity.

Mo1921 - Prevalence of Ul97 Gene Mutations and Polymorphisms in Cytomegalovirus Colitis in Japan

Mo1921 - Prevalence of Ul97 Gene Mutations and Polymorphisms in Cytomegalovirus Colitis in Japan

HCMV UL97 phosphotransferase gene mutations may be associated with antiviral resistance in immunocompromised patients in Belém, PA, Northern Brazil.

Human cytomegalovirus is one of the causes of opportunist infections in immunocompromised patients, and is triggered by factors such as state of viral latency, weakened immune responses, and development of antiviral resistance to ganciclovir, the only drug offered by the public health system in Brazil to treat the infection. The goal of this study was to identify mutations that may be associated with antiviral resistance in immunocompromised patients. Molecular analysis was performed in 82 blood samples and subjected to genomic DNA extraction by a silica-based method. Three sequences of the HCMV UL97 gene, which encodes a phosphotransferase protein required for activation of ganciclovir, were amplified by polymerase chain reaction. Pyrosequencing methods were applied to one external 2096-bp segment DNA and two internal sequences between nucleotides 1087 to 1828 to detect mutations in this gene. Approximately 10% of sequences contained mutations between nucleotides 377 and 594, in conserved regions of the UL97 gene, leading to amino acid changes. Eleven coding mutations were identified, including changes leading to amino acid substitutions, E596K and S604F, which were observed in 100% of samples and are described for the first time in Brazil. In addition, one mutation (A594V) that is associated with ganciclovir resistance was detected in a kidney transplant patient. Further studies to detect mutations associated with HCMV resistance to antiviral drugs are required to demonstrate the need to increase the variety and availability of drugs used to treat viral infections in the public health care system in Brazil.

Resistant Cytomegalovirus Infection After Renal Transplantation: Literature Review

BackgroundResistant cytomegalovirus (R-CMV) is an emerging problem in the renal transplantation population. The most frequent CMVs are high-resistance mutations (UL97 gene). MethodsWe describe our experience in management of R-CMV after renal transplant at our center (2012–2016). ResultsWe encountered 3 cases of R-CMV infection after renal transplant (all primary infections). All 3 patients received induction therapy with corticosteroids, tacrolimus, and mycophenolate mofetil. The first patient (basiliximab induction, preemptive CMV) developed CMV replication on day +53, which responded poorly both to standard-dose valganciclovir (vGCV) and high-dose ganciclovir (GCV) (creatinine clearance [CrCl] >70 mL/min; vGCV 900 mg twice daily for 50 days and GCV 7.5 mg/kg twice daily for 8 days). Hematologic toxicity occurred. The R-CMV test was positive and foscarnet (FOS) was initiated (90 mg/kg twice daily for 21 days). The second patient presented CMV infection (day +30, thymoglobulin induction, CMV prophylaxis), which was not controlled with the high dose (CrCl 23 mL/min; GCV 3.5 mg/kg twice daily and vGCV 900 mg twice daily), resulting in severe neutropenia. R-CMV was detected and FOS initiated (FOS 50 mg/kg twice daily for 7 days and 50 mg/kg every 2 days for 13 days). The third patient's infection occurred on day +22 (basiliximab induction, preemptive CMV). Standard-dose vGCV was uneffective (CrCl >70 mL/min, vGCV 900 mg twice daily) and it did not respond to the high dose (GCV 7.5 mg/kg twice daily and vGCV 2700 mg/d). Moderate hematologic toxicity occurred. R-CMV was diagnosed and FOS treatment begun (FOS 70 mg/kg per day for 2 weeks). ConclusionsResistant CMV infection may be severe due to viral infection and side effects of high-dose antiviral treatment. We presented 3 cases requiring the use of FOS in the absence of response or toxic effects from the usual treatment, with an optimal sustained response (temporary in case 2) and without serious side effects.

High-dose Valganciclovir Treatment for Resistant Cytomegalovirus Colitis due to UL97 and UL54 Mutations

We report the first case of a ganciclovir-resistant cytomegalovirus (CMV) involving the gastrointestinal tract that was successfully treated with high-dose valganciclovir. A kidney transplant recipient developed drug-resistant CMV colitis which was initially treated with valganciclovir, but his CMV was found to have major resistance to ganciclovir and cidofovir due to UL97 and UL54 mutations. The patient was switched to intravenous foscarnet 40 mg/kg given every twelve hours. However, foscarnet had to be discontinued after 4 days of treatment due to acute kidney injury. Patient was restarted on valganciclovir at a higher target dose of 1800 mg twice a day based on the creatinine clearance. CMV became undetectable 2 weeks after valganciclovir treatment was completed. High-dose valganciclovir along with immune suppression reduction may be a treatment option for CMV colitis with ganciclovir resistance due to dual UL97 and UL54 gene mutations.

A Novel Ganciclovir Resistance Mutation in the UL97 Gene of the HHV-5 in an Adult Hematopoietic Stem Cell Transplant Recipient

Therapeutic management of cytomegalovirus (CMV) disease in hematopoietic stem cell transplantation patients can become a challenge because of the emergence of anti-CMV drug resistance. This case report presents a patient with clinical ganciclovir resistance due to a new mutation: histidine-to-asparagine change at residue 393 of UL97. This mutation, which is located in the nonfunctional region of the UL97 gene, is very unusual. Having more information about the mutations leading to drug resistance in CMV is important for both improved clinical management and development of new diagnostic tests and drugs.

Incidence of UL97 Resistance Mutations in Infants with Congenital Cytomegalovirus Disease Receiving 6 Months of Oral Valganciclovir Therapy

BackgroundA recently completed Phase 3 randomized, controlled, double-blind, multicenter study of infants with symptomatic congenital cytomegalovirus (CMV) disease receiving 6 months of oral valganciclovir (VGCV) therapy represents the largest such population in which to evaluate treatment-emergent antiviral resistance. The most common mechanism of CMV antiviral resistance occurs through mutations in the CMV UL97 gene that confer resistance to ganciclovir (GCV). Genotypic resistance analyses were performed on infants receiving 6 months of VGCV to assess the incidence of antiviral resistance due to UL97 sequence variants.MethodsResistance analyses were performed by conventional DNA sequencing of the UL97 gene at multiple time points. Following CMV DNA extraction from frozen whole blood specimens, the UL97 gene was amplified with a double nested polymerase chain reaction method and sequenced to identify polymorphisms and mutations that might confer GCV resistance.ResultsForty-six infants with symptomatic CMV disease who received a 6-month course of VGCV underwent resistance analysis to identify UL97 sequence variants. In addition to a range of natural polymorphisms known to have no effect on antiviral susceptibility, 2 subjects developed UL97 mutations known to confer resistance to GCV (A594V and G598S detected in one subject; E596G detected in another), yielding an incidence of 4%. Each of these resistance mutations occurred in specimens collected after at least 4 months of antiviral therapy. As evaluated in the original Phase 3 trial, neither of these infants showed an improvement in hearing outcome.ConclusionThe development of treatment-emergent UL97 resistance mutations was determined in a controlled study population of infants with congenital CMV disease receiving 6 months of VGCV. This targeted resistance analysis demonstrated an incidence approaching the total incidence of antiviral resistance for CMV disease in some immunocompromised populations, such as solid-organ transplant recipients. Further studies within this study population are warranted to elucidate the risk of emerging antiviral resistance and to assess clinical impact as well as the potential need for combination antiviral therapy.Disclosures All authors: No reported disclosures.

Human Cytomegalovirus UL54 and UL97 Mutations for Detection of Ganciclovir Resistance in Congenital Infection

Aim: Ganciclovir (GCV) is used as an antiviral drug for the treatment of human cytomegalovirus infection. The aim of this study was to demonstrate GCV-resistant human cytomegalovirus in congenitally infected neonates. Patients &amp; methods: DNA of CMV positive newborn samples was extracted and UL97 and UL54 genes were amplified by PCR and real-time PCR. Sequencing of UL97 and UL54 genes were performed and analyzed. Results: UL97 GCV resistance mutation C603W was detected in one newborn. D605E was the most common polymorphism in UL97 observed in 7/13 (53.8%) of samples. N685S, A688V, A885T and N898D were four known common UL54 polymorphisms. Conclusion: The rate of GCV resistance in congenital CMV is low. Common polymorphisms in UL97 and UL54 genes are also reported as new mutations.

Incidence of Ganciclovir Resistance in CMV-positive Renal Transplant Recipients and its Association with UL97 Gene Mutations.

Human cytomegalovirus (CMV) remains the most common infection affecting organ transplant recipients. Despite advances in the prophylaxis and acute treatment of CMV, it remains an important pathogen affecting the short- and long-term clinical outcome of solid organ transplant recipient. The emergence of CMV resistance in a patient reduces the clinical efficacy of antiviral therapy, complicates therapeutic and clinical management decisions, and in some cases results in loss of the allograft and/or death of the patient. Common mechanisms of CMV resistance to ganciclovir have been described chiefly with the UL97 mutations. Here we evaluate Incidence of ganciclovir resistance in 144 CMV-positive renal transplant recipients and its association with UL97 gene mutations. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 gene sequencing. Six mutations in six patients were detected. Three patients (2.6%) of 112 patients with history of ganciclovir (GCV) treatment had clinical resistance with single UL97 mutations at loci known to be related to resistance (including mutations at codon 594, codon 460, and codon 520). three patients who were anti-CMV drug naïve had single UL97 mutations (D605E) without clinical resistance. Our results confirm and extend our earlier findings on the specific mutations in the UL97 phosphotransferase gene in loci that have established role in ganciclovir resistance and also indicate that clinical ganciclovir resistance due to UL97 gene mutations is an issue in subjects with history of with ganciclovir treatment. D605E mutations remains a controversial issue that needs further investigations.

Epidemiology and Outcome of Ganciclovir-Resistant Cytomegalovirus Infection After Solid Organ Transplantation: A Single Transplant Center Experience in Thailand

BackgroundData on drug-resistant cytomegalovirus (CMV) infection in solid organ transplantation (SOT) are not often reported from resource-limited settings. We aimed to investigate the epidemiology and outcomes of this infection in SOT recipients at our institution. MethodsThis was a retrospective study conducted from January 2012 to May 2015. We included all SOT recipients who were suspected for drug-resistant CMV infection. Genotypic assay for UL97 gene mutation was analyzed by real-time polymerase chain reaction. Patients were reviewed for demographic data, clinical presentation, virologic data, treatment, and outcomes. ResultsThe population consisted of 18 (12 kidney, 6 liver) SOT recipients with a median age of 20 years (interquartile range [IQR], 1–49); 44% were male. Anti-CMV resistance testing was analyzed at a median time of 23 days (IQR, 14–33) after initiation of anti-CMV therapy with a median CMV load of log 3.79 copies/mL (IQR, 3.37–4.58). During a median period of 2 years (IQR, 1–3), 6 SOT recipients were identified with UL97 gene mutation in codon 460, conferring ganciclovir (GCV) resistance. Patients with UL97 gene mutation had a longer mean duration of CMV DNAemia compared with those without mutation (263 vs 107 days; P = .04). All patients received high-dose GCV. Two patients received foscarnet and cidofovir. Two patients died (non–CMV-related), and 4 patients. ConclusionsGCV-resistant CMV infection in SOT recipients is an emerging clinical problem in resource-limited country. Those with UL97 mutation CMV infection have prolonged duration of CMV DNAemia. Clinicians should be aware of this condition when caring for SOT recipients.

Genotypic resistance of cytomegalovirus to antivirals in hematopoietic stem cell transplant recipients from Portugal: A retrospective study

The aim of this study was to characterize Human Cytomegalovirus (HCMV) drug resistance mutations in UL97 and UL54 genes in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients in Portugal. We have performed a retrospective study with 22 patients from a cohort of patients with different haematological malignancies submitted to allo-HSCT between 2010 and 2014. Patients were selected according to clinical and laboratory data of HCMV infection and management. HCMV resistance mutations were characterized by sequencing of UL97 and UL54 genes. Sequence data were compared with: 1) HCMV genome reference strain AD169; and also 2) UL97 from Merlin strain (GenBank: AY446894.2), and UL54 from TB40/E strain (GenBank: ABV71585.1). Resistance mutations were identified in seven patients (32%): five harboured resistance mutations in UL97: A594V (n = 2), C592G (n = 1), L595W (n = 1), and C603W (n = 1); and two harboured resistance mutations in UL54: P522S and L957F, one in each patient. Several natural polymorphisms and unknown mutations were found in both UL97 and UL54, with the majority of the patients harbouring more than one unknown mutation in UL97 but only one in UL54. No simultaneous mutations were found. This is the first study in Portugal to characterize HCMV UL97 and UL54 sequences and to identify HCMV drug-resistance mutations in allo-HSCT patients. The UL97 resistance mutations found were amongst the most frequent resistant mutations, while UL54 L957F mutation was here reported for the first time in a clinical specimen. This information provides important information regarding HCMV strains and antiviral resistance in our population.

Assessment of the Human Cytomegalovirus UL97 Gene to identify the Resistance to Ganciclovir in Kidney Transplant Recipients in Labbafi-Nejad Hospital

Background: Cytomegalovirus (CMV) is one of the most frequently encountered opportunistic viral pathogens in renal transplantation. Approximately, in 60% of the transplant recipients CMV infection can be observed and in > 20% symptomatic diseases can be developed. However, antiviral prophylaxis and treatment have reduced the CMV morbidity and mortality at the time of development of antiviral-resistance CMV strains that can significantly contributed to the adverse clinical outcomes in transplant recipients. Mutations in the human CMV UL97 kinase gene are a major mechanism of viral resistance to the anti-CMV drug “Ganciclovir (GCV)”. GCV, as the most widely used and recognized therapy for CMV, is a substrate for the UL97 kinase. Methods: The studied patients were renal transplant recipients in Tehran Labbafinejad hospital who were positive for CMV-DNA PCR test and have been treated with Ganciclovire. Patients who have been treated for at least 3 weeks with GCV and have not shown a proper therapeutic response were candidate for UL97 gene mutations associated with GCV resistance evaluation. Results: About 60 patients with positive CMV DNA PCR were hospitalized during one year study. Eventually, after 2 times measurement of CMV viral load at the end of the third week and third month of therapy with Ganciclovire, 5 cases were candidate for antiviral resistance evaluation. Genotypic testing was performed, but no mutation neither in UL97 nor in UL 54 was detected by the laboratory. Conclusions: The increasing use of antiviral drugs in transplant patients associated with the narrow range of antiviral agents effective to treat CMV have increased our need for further understanding of the risk factor for development of CMV antiviral resistance and it’s clinical impacts. Detection of UL97 gene mutation plays a major role to determine therapeutic strategies to treat patients infected with the resistant viruses.

Assessment of the Human Cytomegalovirus UL97 Gene to identify the Resistance to Ganciclovir in Kidney Transplant Recipients in Labbafi-Nejad Hospital

Background: Cytomegalovirus (CMV) is one of the most frequently encountered opportunistic viral pathogens in renal transplantation. Approximately, in 60% of the transplant recipients CMV infection can be observed and in > 20% symptomatic diseases can be developed. However, antiviral prophylaxis and treatment have reduced the CMV morbidity and mortality at the time of development of antiviral-resistance CMV strains that can significantly contributed to the adverse clinical outcomes in transplant recipients. Mutations in the human CMV UL97 kinase gene are a major mechanism of viral resistance to the anti-CMV drug “Ganciclovir (GCV)”. GCV, as the most widely used and recognized therapy for CMV, is a substrate for the UL97 kinase. Methods: The studied patients were renal transplant recipients in Tehran Labbafinejad hospital who were positive for CMV-DNA PCR test and have been treated with Ganciclovire. Patients who have been treated for at least 3 weeks with GCV and have not shown a proper therapeutic response were candidate for UL97 gene mutations associated with GCV resistance evaluation. Results: About 60 patients with positive CMV DNA PCR were hospitalized during one year study. Eventually, after 2 times measurement of CMV viral load at the end of the third week and third month of therapy with Ganciclovire, 5 cases were candidate for antiviral resistance evaluation. Genotypic testing was performed, but no mutation neither in UL97 nor in UL 54 was detected by the laboratory. Conclusions: The increasing use of antiviral drugs in transplant patients associated with the narrow range of antiviral agents effective to treat CMV have increased our need for further understanding of the risk factor for development of CMV antiviral resistance and it’s clinical impacts. Detection of UL97 gene mutation plays a major role to determine therapeutic strategies to treat patients infected with the resistant viruses.

Assessment of the Human Cytomegalovirus UL97 Gene to identify the Resistance to Ganciclovir in Kidney Transplant Recipients in Labbafi-Nejad Hospital

Assessment of the Human Cytomegalovirus UL97 Gene to identify the Resistance to Ganciclovir in Kidney Transplant Recipients in Labbafi-Nejad Hospital

Molecular Analysis of Ganciclovir-Resistant Cytomegalovirus in Renal Transplant Recipients with High Viral Load.

Gancyclovir-resistant (GanR) cytomegalovirus (CMV) remains an issue, especially in solid organ transplant (SOT) recipients. Some mutations in UL54 and UL97 confer this resistance. Long-lasting high-dose drug exposure, high viral load, together with lack of sufficient compliance with treatment may account for these mutations. The aim of this study was to detect UL97 and UL54 putative mutations conferring ganciclovir-resistance in renal organ transplant recipients with high CMV load. In this cross-sectional study, 58 serum samples were collected from renal transplant recipients who had referred to three hospitals in Tehran from January 2014 to June 2015. Specific criteria such as CMV syndrome, presence of CMV in blood and organ dysfunction were considered. Then, they were tested for viral load in their early fourth month of intravenous ganciclovir treatment. Fifty cases revealing more than 200 copies/mL were analyzed for mutations. Two fragments of UL54 and Ul97 genes were amplified and sequenced bidirectionally. Sequence alignment and statistical analysis were performed by Mutation Surveyor software and t-test respectively. A significant difference was observed in viral load between seronegative and seropositive recipients (P = 0.036). The most frequent mutation was related to D605E in UL97 gene with the rate of 25%. Regardless of viral load, neither putative mutation nor simultaneous mutation was detected in either UL97 and UL54 regions. In spite of high viral load and persistence of symptoms, our population study did not reveal putative mutations. Hence, the direct relationship between the presence of high quantity of CMV and the occurrence of putative mutation cannot be considered. Non-putative gancyclovir resistant mutations and prolonged drug exposure may have a role in these manifestations.

Assessment of the Human Cytomegalovirus UL97 Gene for Identification of Resistance to Ganciclovir in Iranian Immunosuppressed Patients.

BackgroundHuman cytomegalovirus (HCMV) infections are a major cause of morbidity and mortality among immunocompromised patients. Prolonged antiviral therapy is a cause of mutation and drug resistance in the HCMV genome.ObjectivesThe aim of this study was to identify resistance to ganciclovir (GCV) in Iranian immunosuppressed patients at two different stages of the disease: early (before GCV is initiated) and late (after six months of GCV therapy).Patients and MethodsIn this study, 87 specimens from Iranian patients were amplified using nested PCR amplification of the UL97 gene. Sequence analyses of products were performed for identifying the mutated codons.ResultsThe present study show that the most frequent GCV-resistant mutations occurred in codons A594V (26.43%), H520Q (18.39%), and M460V (13.79%), consequently occurring at a low frequency in the L595S (2.29%), E596G (1.14%), and Del 594 (1.14%) codons, and with intermediate frequency in the C592G (10.34%), M460I (9.19%), and C603W (6.89%) codons. We describe for the first time a new GCV-resistance mutation, the deletion of codon 594, in the UL97 gene of Iranian HCMV patients after GCV therapy, following renal transplantation.ConclusionsThe findings of the present study can be utilized to detect GCV resistance patterns among Iranian immunocompromised patients and to treat HCMV infections accordingly.

Contribution of next generation sequencing to early detection of cytomegalovirus UL97 emerging mutants and viral subpopulations analysis in kidney transplant recipients

BackgroundCytomegalovirus (CMV) is the major opportunistic virus encountered after transplantation, and resistant variants challenge antiviral treatment. We studied the emergence and evolution of the canonical UL97 L595S mutation in four kidney recipients by comparing Sanger sequencing with a specific next-generation sequencing (NGS) assay, and assessed the global evolution of UL97 gene variability. Study designPlasmids harbouring wild-type and/or L595S mutated UL97 genes were used to assess the analytical performances of NGS assay. UL97 gene was retrospectively analysed in patients’ samples drawn during CMV infection follow-up, Shannon entropy (Sn) was calculated and phylogenetic analyses were performed. ResultsWild-type and L595S plasmids PCR products were mixed to obtain L595S concentrations of 0, 1, 2, 5, 10, 20 and 100%. Mean triplicate NGS results were 0, 0.71, 1.79, 5.30, 13.17, 17 and 100%, respectively, while Sanger sequencing only detected L595S when above 20%. The NGS mean error rate was 0.196±0.07%. In the four patients, emergence of L595S mutation under ganciclovir treatment was followed-up. After foscarnet rescue therapy, leading to undetectable CMV viral load, in two patients, L595S mutant re-emerged, but was only detected by NGS technology (14% and 9.6%). Using NGS data, phylogenetic trees and Sn showed a complex evolution of concomitant viral subpopulations. ConclusionsNGS technology allowed a deeper discrimination of the emergence and persistence of a drug resistance mutation, which could be pertinent to investigate when routine Sanger sequencing detects only wild-type strains. Moreover, NGS improved sensitivity helps in studying viral abundance, dynamics and diversity, less approachable with Sanger sequencing.