Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed pediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1–27 weeks. Changes in consensus sequence and resistance mutations were analyzed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of 11 subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome.

Frequent pUL27 Variations in HIV-Infected Patients

“…Others were Q54E/L, D123N, V103A, L56P, L60R, P62Q, L32I, and R34S, which are reported for the first time in this study. However, N-terminal polymorphisms found in this study like Q54 and L56 are close to those found in recently published study by Houldcroft et al [18]. Polymorphisms in N-terminal hydrophobic domain may influence viral interactions.…”

“…This indicates that pUL27 may have functions potentiated under HIV-CMV co-infection. In common, with recently shown frequent pUL27 polymorphisms in immunocompromised patients [18], we found new polymorphisms in HIV-infected patients. As shown in Table 2, almost 67% of HIV-positive individuals had progressed to AIDS and are therefore considered as severely immunocompromised.…”

Clinical and biological insights from viral genome sequencing

“…Similar approaches have been used to identify resistant variants of HCV 23 , HBV 24 and influenza virus 25 . partial genomes has been used to detect drug resistance in RNA viruses, such as influenza virus 11 , and DNA viruses, such as human cytomegalovirus (HCMV) 12 . Viral genome sequencing is becoming ever more important, especially in clinical research and epidemiology.…”

“…A good example of this is HCMV, for which WGS can simultaneously capture the genes that encode targets of licensed therapies, such as UL27 (unknown function), UL54 (DNA polymerase) and UL97 (serine/ threonine protein kinase), and of newer drugs, such as letermovir, which targets UL56 (terminase complex). This enables comprehensive antiviral-resistance testing in a single test 12 . In addition, WGS can provide information on antigenic epitopes, virus evolution in a patient over time 12 , and evidence of recombination between HCMV strains 32 .…”

Ganciclovir‐Resistant Cytomegalovirus Infection in Abdominal Solid Organ Transplant Recipients: Case Series and Review of the Literature

“…As a UL97 inhibitor, maribavir is intrinsically antagonistic to the action of GCV, which requires UL97 for activation. Like GCV, however, maribavir is vulnerable to resistance mutations in UL97 , although at different locations, and resistance has been identified in vitro and in vivo . Maribavir was initially studied in the context of CMV prophylaxis and was ultimately inferior to GCV in the prevention of CMV disease after liver transplantation and HSCT, although it has been suggested this was due to suboptimal dosing (100 mg twice/day) .…”