Novel C480F Mutation Found in 2 Transplant Recipients With Drug-Resistant Cytomegalovirus

The C480F mutation of the human cytomegalovirus (CMV) UL97 kinase gene was found to confer the highest level of resistance and cross-resistance to maribavir (MBV) and ganciclovir (GCV), respectively, in 2 patients who were transplant recipients with drug-resistant CMV, both of whom initially achieved CMV DNA clearance following MBV treatment, according to results of a case study published in The Journal of Infectious Diseases.

In a case study, investigators obtained clinical isolates from 1 patient who underwent hematopoietic stem cell transplantation (HSCT) and 1 who underwent kidney transplantation, both of whom had suspected resistance to anti-CMV agents. They collected plasma samples and biopsy specimens for genotypic assays, and CMV viral load measurement was performed via quantitative reverse transcription-polymerase chain reaction testing (RT-PCR). The investigators also performed genotypic testing with PCR amplification of CMV UL97 in the 2 fragments associated with GCV and MBV resistance and 4 fragments of UL54. In addition, UL27 was genotyped following Sanger sequencing.

The investigatorsperformed phenotypic testing of the UL97 C480F mutation, assessed the antiviral susceptibility of the mutation to MBV, GCV, and foscarnet (FOS), determined the 50% inhibitory concentration (IC₅₀) of the mutatation, and also estimated the effect C480F had on viral fitness.

Patient 1 was a 38-year-old woman with seropositive CMV in addition to acute myeloid leukemia who received a myeloablative allogenic HSCT from a sibling donor with seronegative CMV. On post-transplantation day 45, CMV replication was detected in the patient’s plasma and she was started on valganciclovir (VGCV). After 1 week, VGCV was discontinued due to severe cytopenia and patient 1 was then switched to FOS 60 mg/kg every 12 hours for 1 week, which resulted in CMV DNA clearance.

Two months later, patient 1 was diagnosed with gastrointestinal (GI) CMV disease and treated with GCV (5 mg/kg twice daily). After 3 weeks, GCV was discontinued due to cytopenia and she was subsequently started on MBV (400 mg twice daily) CMV viral loads had not decreased. MBV treatment continued for 6 weeks and the patient had adequate CMV clearance; however, 1 week after discontinuing MBV treatment, CMV was detected and treatment with MBV was reinitiated. After 3 weeks of treatment, the investigators noted a progressive CMV viral load increase. Resistance mutation genotyping showed the emergence of the UL97 C480F variant, which had an unknown phenotype at the time, in addition to the UL54 F669L sensitive polymorphism. Patient 1 was then switched to GCV (2.5 mg/kg twice daily) and FOS (60 mg/kg once daily) due to cytopenia. She died from multiorgan failure related to uncontrolled GI-CMV disease on day 258.

The investigators noted that all plasma samples and biopsy specimens obtained from patient 1 contained the naturally occurring polymorphism UL54 F669L, and 2 plasma samples showed previously uncharacterized UL54 mutations (Y601H, H600P) during GCV treatment on days 136 and 139. Neither of these mutations were detected in subsequent esophageal or duodenal biopsy specimens or plasma samples.

In regard to patient 1, the investigators found that the UL97 C480F variant was detected in a plasma sample 72 days after the first course of MBV treatment and 13 days after the second course of MBV treatment. The variant spread during treatment with MBV, GCV, and FOS, and was detected in every plasma sample as well as colon and gastric biopsy specimens until patient 1 died. Of note, no mutations were detected in UL27 and there were no resistance mutations to VGCV-GCV, FOS, or MBV at baseline.

Patient 2 was a 44-year-old woman who had undergone a third D⁺R⁺ kidney transplantation due to chronic kidney failure. She received primary prophylaxis, and a CMV viral load was detected with no associated symptoms 1 month after treatment with the primary prophylaxis was discontinued. After 9 days of treatment with VGCV (900 mg twice daily), patient 2 developed clinical symptoms and her CMV viral load was increased and she was started on GCV (5mg/kg twice daily). After 2 weeks of GCV treatment, viremia continued to increase and patient 2 was switched to MBV (400 mg twice daily) for 2 months until clearance.

Patient 2 developed asymptomatic viremia 16 days after MBV treatment was discontinued. UL97 C480F was detected in a resistance mutation study. There were no amino acid changes in UL54 or UL27, and patient 2 was not prescribed an anti-CMV drug due to lack of response in previous episodes. In addition, her previous immunosuppression treatment was modified to everolimus and decreased dose of mycophenolate until loss of viremia was achieved without CMV-associated clinical outcomes.

The investigators were unable to collect clinical isolates from patient 2 for retrospective genotypic studies. They identified C480F in the UL97 fragment via phenotypic testing by placing an enhanced green fluorescent protein (EGFP) gene in the standard AD169 laboratory viral strain. C480F IC₅₀ showed high resistance to MBV and GCV cross-resistance.

Growth assays showed a delay in the replication of the C480F variant compared with AD169, demonstrating deteriorated viral fitness.

“Mapping of the UL97 mutation that confers resistance to MBV and GCV may be useful for designing alternative UL97 inhibitor antivirals to avoid cross-resistance,” the investigators noted. They also recommended clinical follow-up after routine CMV diagnosis “due to the possible sudden emergence of mutants and the limited sensitivity of Sanger sequencing for detecting small populations.”

Reference

Santos Bravo M, Plault N, Sánchez Palomino S, et al. Phenotype and genotype study of novel C480F maribavir-ganciclovir cross-resistance mutation detected in hematopoietic stem cell and solid organ transplant recipients. J Infect Dis. 2021;224(6):1024-1028. doi:10.1093/infdis/jiab029