Improved Detection of Emerging Drug-Resistant Mutant Cytomegalovirus Subpopulations by Deep Sequencing

Abstract

In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.

Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance

“…The novel UL54 substitutions E303D/G and D413Y reported here add to the considerable number of exonuclease domain mutations associated with dual CDV and GCV resistance (5,11,12). The only other drug resistance mutation that has been mapped to CMV exonuclease domain I (codons 300 to 304) is substitution D301N, identified in a single plaque of a clinical isolate picked under GCV (13).…”

“…Instead, drug resistance as a risk factor for early treatment failure with CDV or BCV may arise mainly from prior exposure to other antivirals, especially GCV, that select for cross-resistant UL54 mutants, as illustrated by clinical case reports of CDV or BCV as salvage therapy (11,21,22).…”

Sequencing Drug-Resistant Cytomegalovirus in Pediatric Patients: Toward Personalized Medicine

“…Amplicons are sequenced to high depth using a variety of next-generation sequencing platforms. This approach detects resistance mutations sooner, at lower frequencies, than Sanger sequencing of PCR products, with studies reporting detection of resistance mutations at frequencies of 3% [9,10]. Unfortunately, as with Sanger sequencing, this approach still relies on labor-intensive PCR for the growing number of genes targeted by new antivirals and other therapies.…”

“…Published data suggest that resistance mutations seen at high frequencies at the point of clinical diagnosis of resistance can be detected weeks earlier by deep sequencing, at frequencies <10% [10]. Researchers and clinicians need data on whether, in the presence of a low-frequency drug resistance mutation, modifying an existing treatment schedule to increase the dosage or change drug delivery method to achieve better tissue penetration is a viable treatment option.…”

How I treat resistant cytomegalovirus infection in hematopoietic cell transplantation recipients

“…68 CMV resistance to ganciclovir can result from either modification of pUL97 or alteration of pUL54 (discussed in the next section) (Figure 4). Multiple UL97 mutations with amino acid substitutions have been described, 69,70 and the 7 most common "canonical" mutations, constituting .80% of the UL97 resistance mutations, confer a 5-to 15-fold increase in IC 50 (Table 1). Other deletion mutations conferring varying levels of resistance have been reported in the codon range 590 to 607.…”