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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.

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Related in: MedlinePlus

Maps of UL54 (A) and UL97 (B) sequence variants detected in this study associated with drug resistance. Variants in red have been previously reported as having drug resistance phenotypes or mutations with drug resistance phenotypes (G598D, Gilbert et al., 2011) when combined with concurrent UL54 mutations of unknown significance in marker transfer experiments; variants in blue are novel mutations. The highest frequency each mutation was detected at is shown in brackets. Known SNPs not shown. Plots were made using Lollipops (https://github.com/pbnjay/lollipops) and the relevant Uniprot HCMV strain Merlin protein structure.
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Figure 2: Maps of UL54 (A) and UL97 (B) sequence variants detected in this study associated with drug resistance. Variants in red have been previously reported as having drug resistance phenotypes or mutations with drug resistance phenotypes (G598D, Gilbert et al., 2011) when combined with concurrent UL54 mutations of unknown significance in marker transfer experiments; variants in blue are novel mutations. The highest frequency each mutation was detected at is shown in brackets. Known SNPs not shown. Plots were made using Lollipops (https://github.com/pbnjay/lollipops) and the relevant Uniprot HCMV strain Merlin protein structure.

Mentions: Patients B, H, I, and M developed known drug resistance mutations in UL54 and UL97 during treatment (Figure 1; Table 2). The mean time to mutation detection was 115 days (range 18–171) following the start of antiviral treatment. The mutations detected are shown in Figure 2. Patient H carried no baseline resistance mutations by deep-sequencing analysis, but Sanger sequencing detected fixed resistance mutation L501I (CDV and GCV resistance) in ORF UL54 on day 18 of treatment (day 43 post-admission). This mutation was not detected by Sanger sequencing on day 56 of treatment despite continued GCV; the patient also received FOS throughout this period. The patient developed a mutation, G598D, in UL97 on day 81 post-admission (treatment day 56) which has previously been seen in patients failing GCV therapy, detected by Sanger sequencing. However, the phenotype of this mutation without concurrent UL54 mutations has yet to be demonstrated by marker transfer (Gilbert et al., 2011). Samples taken on days 43 and 81 post-admission were not available for follow-up deep-sequencing, but neither mutation was detected by deep sequencing on days 5, 11, 14, and 18 post-admission. No previously reported UL27 anti-viral resistance mutations were detected, although a number of SNPs of unknown function were present, reported in Supplementary Table 2, and stop codons were present in UL27 sequences from patients I and L (Supplementary Figure 4A).


Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus
Maps of UL54 (A) and UL97 (B) sequence variants detected in this study associated with drug resistance. Variants in red have been previously reported as having drug resistance phenotypes or mutations with drug resistance phenotypes (G598D, Gilbert et al., 2011) when combined with concurrent UL54 mutations of unknown significance in marker transfer experiments; variants in blue are novel mutations. The highest frequency each mutation was detected at is shown in brackets. Known SNPs not shown. Plots were made using Lollipops (https://github.com/pbnjay/lollipops) and the relevant Uniprot HCMV strain Merlin protein structure.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Maps of UL54 (A) and UL97 (B) sequence variants detected in this study associated with drug resistance. Variants in red have been previously reported as having drug resistance phenotypes or mutations with drug resistance phenotypes (G598D, Gilbert et al., 2011) when combined with concurrent UL54 mutations of unknown significance in marker transfer experiments; variants in blue are novel mutations. The highest frequency each mutation was detected at is shown in brackets. Known SNPs not shown. Plots were made using Lollipops (https://github.com/pbnjay/lollipops) and the relevant Uniprot HCMV strain Merlin protein structure.
Mentions: Patients B, H, I, and M developed known drug resistance mutations in UL54 and UL97 during treatment (Figure 1; Table 2). The mean time to mutation detection was 115 days (range 18–171) following the start of antiviral treatment. The mutations detected are shown in Figure 2. Patient H carried no baseline resistance mutations by deep-sequencing analysis, but Sanger sequencing detected fixed resistance mutation L501I (CDV and GCV resistance) in ORF UL54 on day 18 of treatment (day 43 post-admission). This mutation was not detected by Sanger sequencing on day 56 of treatment despite continued GCV; the patient also received FOS throughout this period. The patient developed a mutation, G598D, in UL97 on day 81 post-admission (treatment day 56) which has previously been seen in patients failing GCV therapy, detected by Sanger sequencing. However, the phenotype of this mutation without concurrent UL54 mutations has yet to be demonstrated by marker transfer (Gilbert et al., 2011). Samples taken on days 43 and 81 post-admission were not available for follow-up deep-sequencing, but neither mutation was detected by deep sequencing on days 5, 11, 14, and 18 post-admission. No previously reported UL27 anti-viral resistance mutations were detected, although a number of SNPs of unknown function were present, reported in Supplementary Table 2, and stop codons were present in UL27 sequences from patients I and L (Supplementary Figure 4A).

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.


Related in: MedlinePlus