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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Phylogenetic trees of (A) UL54, (B) UL97, and (C) UL27 nucleotide sequences from the patients with multiple drug-resistance mutations (B and I) and 11 laboratory and wild-type HCMV strains. Maximum likelihood analysis was performed with MEGA6, generating a tree with 1000 replicates. Bootstrap values of 55 or above are shown. (A) UL54 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (B) UL97 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (C) UL27 phylogenetic tree. Samples from patients B do not cluster monophyletically. No previously-reported resistance mutations were detected within patient B sequences (the only patient to receive maribavir).
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Figure 3: Phylogenetic trees of (A) UL54, (B) UL97, and (C) UL27 nucleotide sequences from the patients with multiple drug-resistance mutations (B and I) and 11 laboratory and wild-type HCMV strains. Maximum likelihood analysis was performed with MEGA6, generating a tree with 1000 replicates. Bootstrap values of 55 or above are shown. (A) UL54 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (B) UL97 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (C) UL27 phylogenetic tree. Samples from patients B do not cluster monophyletically. No previously-reported resistance mutations were detected within patient B sequences (the only patient to receive maribavir).

Mentions: To examine further the complex drug resistance patterns seen in patients B and I, we constructed a phylogenetic tree for each of the three target regions, including all samples from these patients and 11 publically available HCMV genomes from GenBank (Figures 3A–C). For patient B, UL27 consensus sequences clustered in different parts of the tree in a time dependent manner (Figure 3C). The consensus sequences of genes UL54 and UL97 show change over time in patients B and I that is compatible with sequence evolution due to anti-viral drug pressure (Garrigue et al., 2016). In Patient B the changes in phylogenetic clustering for UL27 occurred after the start of MBV on day 133, and may reflect recombination or re-infection with a second strain of HCMV in this patient.


Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus
Phylogenetic trees of (A) UL54, (B) UL97, and (C) UL27 nucleotide sequences from the patients with multiple drug-resistance mutations (B and I) and 11 laboratory and wild-type HCMV strains. Maximum likelihood analysis was performed with MEGA6, generating a tree with 1000 replicates. Bootstrap values of 55 or above are shown. (A) UL54 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (B) UL97 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (C) UL27 phylogenetic tree. Samples from patients B do not cluster monophyletically. No previously-reported resistance mutations were detected within patient B sequences (the only patient to receive maribavir).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016526&req=5

Figure 3: Phylogenetic trees of (A) UL54, (B) UL97, and (C) UL27 nucleotide sequences from the patients with multiple drug-resistance mutations (B and I) and 11 laboratory and wild-type HCMV strains. Maximum likelihood analysis was performed with MEGA6, generating a tree with 1000 replicates. Bootstrap values of 55 or above are shown. (A) UL54 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (B) UL97 phylogenetic tree, showing evolution of patient sequences. Changes in the consensus sequences of patients B and I due to high-frequency drug resistance mutations emerging during treatment drive the evolution of their phylogenetic position. (C) UL27 phylogenetic tree. Samples from patients B do not cluster monophyletically. No previously-reported resistance mutations were detected within patient B sequences (the only patient to receive maribavir).
Mentions: To examine further the complex drug resistance patterns seen in patients B and I, we constructed a phylogenetic tree for each of the three target regions, including all samples from these patients and 11 publically available HCMV genomes from GenBank (Figures 3A–C). For patient B, UL27 consensus sequences clustered in different parts of the tree in a time dependent manner (Figure 3C). The consensus sequences of genes UL54 and UL97 show change over time in patients B and I that is compatible with sequence evolution due to anti-viral drug pressure (Garrigue et al., 2016). In Patient B the changes in phylogenetic clustering for UL27 occurred after the start of MBV on day 133, and may reflect recombination or re-infection with a second strain of HCMV in this patient.

View Article: PubMed Central - PubMed

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.

No MeSH data available.