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

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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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Viraemia and anti-viral therapy in patients who developed drug resistance. In patients B and I, multiple drug-resistance mutations accrue over time at a range of frequencies, associated with a multi-drug resistance phenotype and patient death. Patients H and M developed single fixed resistance mutations and recovered from HCMV disease. ORF locations of each mutation are Table 1. Red arrows: samples deep sequenced by target enrichment. Blue arrows: samples sequenced only by reference laboratory (PCR + Sanger sequencing) leaving insufficient material for target enriched deep sequencing. Blue open circles: bone marrow or thymus transplant, or gene therapy given. Left Y axes: log10 virus copies/ml blood. Right Y axes: variant frequency. X axis: time since admission (days).
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Figure 1: Viraemia and anti-viral therapy in patients who developed drug resistance. In patients B and I, multiple drug-resistance mutations accrue over time at a range of frequencies, associated with a multi-drug resistance phenotype and patient death. Patients H and M developed single fixed resistance mutations and recovered from HCMV disease. ORF locations of each mutation are Table 1. Red arrows: samples deep sequenced by target enrichment. Blue arrows: samples sequenced only by reference laboratory (PCR + Sanger sequencing) leaving insufficient material for target enriched deep sequencing. Blue open circles: bone marrow or thymus transplant, or gene therapy given. Left Y axes: log10 virus copies/ml blood. Right Y axes: variant frequency. X axis: time since admission (days).

Mentions: From deep sequencing results we were able to stratify patients into two groups: those with no evidence of developing resistance mutations despite receiving long term antiviral treatment (A, C, D, G, J, K, L); and those patients who developed known HCMV resistance mutations: either fixed (H and M) or at low level (B and I). We plotted viral load, drugs received and mutations over time for each of these patients (Figure 1; Supplementary Figure 3).


Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus
Viraemia and anti-viral therapy in patients who developed drug resistance. In patients B and I, multiple drug-resistance mutations accrue over time at a range of frequencies, associated with a multi-drug resistance phenotype and patient death. Patients H and M developed single fixed resistance mutations and recovered from HCMV disease. ORF locations of each mutation are Table 1. Red arrows: samples deep sequenced by target enrichment. Blue arrows: samples sequenced only by reference laboratory (PCR + Sanger sequencing) leaving insufficient material for target enriched deep sequencing. Blue open circles: bone marrow or thymus transplant, or gene therapy given. Left Y axes: log10 virus copies/ml blood. Right Y axes: variant frequency. X axis: time since admission (days).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Viraemia and anti-viral therapy in patients who developed drug resistance. In patients B and I, multiple drug-resistance mutations accrue over time at a range of frequencies, associated with a multi-drug resistance phenotype and patient death. Patients H and M developed single fixed resistance mutations and recovered from HCMV disease. ORF locations of each mutation are Table 1. Red arrows: samples deep sequenced by target enrichment. Blue arrows: samples sequenced only by reference laboratory (PCR + Sanger sequencing) leaving insufficient material for target enriched deep sequencing. Blue open circles: bone marrow or thymus transplant, or gene therapy given. Left Y axes: log10 virus copies/ml blood. Right Y axes: variant frequency. X axis: time since admission (days).
Mentions: From deep sequencing results we were able to stratify patients into two groups: those with no evidence of developing resistance mutations despite receiving long term antiviral treatment (A, C, D, G, J, K, L); and those patients who developed known HCMV resistance mutations: either fixed (H and M) or at low level (B and I). We plotted viral load, drugs received and mutations over time for each of these patients (Figure 1; Supplementary Figure 3).

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