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Journal of Clinical Virology xxx (2013) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Journal of Clinical Virology

j ourna l h om epage: www.elsev ier .com/ locate / j cv

ase report

mergence of antiviral resistance during oral valganciclovir treatment of annfant with congenital cytomegalovirus (CMV) infection

. Yeon Choia,1, B. Sharona, H.H. Balfour Jr. b, K. Belanic, T.C. Pozosc, M.R. Schleissa,∗

Division of Pediatric Infectious Diseases and Immunology, Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota Medical School, 2001 6thtreet SE, Minneapolis, MN 55455, United StatesDivision of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN 55455, United StatesInfectious Diseases and Immunology, Children’s Hospitals and Clinics of Minnesota, 2525 Chicago Avenue South, Minneapolis, MN 55404, United States

a r t i c l e i n f o

rticle history:eceived 25 January 2013eceived in revised form 31 March 2013ccepted 4 April 2013

eywords:

a b s t r a c t

Congenital infection with human cytomegalovirus (CMV) is a major cause of morbidity, including sen-sorineural hearing loss (SNHL), in newborns. Antiviral therapy with ganciclovir (GCV) and its oral prodrug,valganciclovir (VAL-GCV) are increasingly being administered to infected infants, toward the goal ofimproving neurodevelopmental and auditory outcomes. In this case report, we describe a symptomaticcongenitally infected infant treated with VAL-GCV in whom GCV resistance was suspected, based on a 50-

ongenital cytomegalovirus infectionntiviral therapyanciclovir resistanceL97 mutation

fold increase in viral load after 6 weeks of oral therapy. Analyses of CMV sequences from both blood andurine demonstrated populations of viruses with M460V and L595F mutations in the UL97 phosphotrans-ferase gene. In contrast, analysis of viral DNA retrieved from the newborn dried blood spot demonstratedwild-type UL97 sequences. DNAemia resolved after the discontinuation of VAL-GCV. Long-term VAL-GCVtherapy in congenitally infected infants can select for resistant viral variants, and anticipatory virologicalmonitoring may be warranted.

© 2013 Elsevier B.V. All rights reserved.

. Why this case is important

Human cytomegalovirus (CMV) is an important cause of diseaseollowing solid organ and hematopoietic stem cell transplantationHSCT), in the setting of HIV infection, and in newborn infants withongenital infection.1–4 Most experience with treatment of CMVas been accrued with the nucleoside analog, ganciclovir (GCV),nd its oral prodrug, valganciclovir (VAL-GCV).5–11 Although useful,hese drugs cause toxicities, particularly neutropenia, and long-erm therapy can lead to the emergence of antiviral resistance.12–14

ost reports of GCV resistance have been in adult patients, andess information is available for pediatric patients, particularlynfants. However, with the increasing use of GCV for treatmentf congenital CMV infection,15–19 GCV resistance may be identi-ed more commonly in infants in the future. An ongoing studyy the Collaborative Antiviral Study Group (CASG) is examining

otential benefits of prolonged (six months) VAL-GCV therapy onhe neurodevelopmental and audiologic status of infants with con-enital CMV infection.20 If long-term VAL-GCV therapy becomes

∗ Corresponding author. Tel.: +1 612 624 1112.E-mail address: schleiss@umn.edu (M.R. Schleiss).

1 Current address: Department of Microbial & Molecular Pathogenesis, Texas And M Health Sciences Center, College Station, Texas, United States.

386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jcv.2013.04.004

increasingly utilized for the treatment of congenitally infectedinfants, anticipating and identifying the emergence of resistantCMV strains will be required as an element of the ongoing clinicalmanagement of infants and children on this antiviral suppressionregimen.

2. Case description

A 30-year-old gravida 2 para 1 woman was noted by ultra-sound at 25 weeks gestation to be carrying a fetus with intrauterinegrowth retardation, hepatomegaly, and brain calcifications. Amnio-centesis was positive for CMV by PCR. Caesarian section wasperformed at 35 weeks gestational age due to decreased fetal move-ment. At birth, the male infant weighed 1270 g, with a lengthof 40.5 cm and a head circumference of 27.5 cm (all values <3rdpercentile). The placenta was positive for CMV antigen by immuno-histochemistry and demonstrated histological findings of CMVplacentitis.

Neonatal urine and blood specimens were both positive byPCR for CMV DNA, confirming congenital infection. Renal insuf-ficiency was noted, with reduced urine output and elevated

serum creatinine. Because of documented CMV end-organ disease,including central nervous system involvement, GCV therapy wasinitially commenced at a 3 mg/kg dose (6 mg/kg/day), adjusted forprematurity and renal insufficiency. After the infant’s weight

JCV-2706; No. of Pages 5

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2 K.Y. Choi et al. / Journal of Clinical Virology xxx (2013) xxx– xxx

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ig. 1. Time course of DNAemia and evolution of GCV resistance. The initial course oeutropenia, oral VAL-GCV was commenced, with emergence approximately 8 wee

eached 1500 g, the dose was increased to 7.5 mg/kg/day. After 4eeks of IV therapy, GCV was discontinued because of neutrope-ia [Fig. 1], which resolved. At approximately 3 months of age,

ollowing hospital discharge and on an outpatient basis, VAL-GCVherapy was commenced at a dose of 20 mg/kg twice daily. A dura-ion of six additional months of treatment was planned, based onhe regimen employed in the ongoing CASG study of long-term oralAL-GCV therapy in congenitally infected infants (Clinicaltrials.gov

dentifier: NCT00466817)20.The dynamics of CMV DNAemia in this patient are summa-

ized in Fig. 1. Initially, the magnitude of DNAemia decreasedrom 14,500 to 3000 copies/ml on GCV therapy. Within 4 weeksfter GCV was discontinued, viral load had increased to 13,000opies/ml, essentially identical to the pre-treatment level. Fol-owing the initiation of VAL-GCV, viral load remained essentiallytable, at 9500 copies/ml, for the next two weeks. The parentseported adherence to the VAL-GCV regimen, and the patient hado symptoms of CMV disease. At approximately 5 months of age6 weeks into oral therapy), viral load dramatically rose to 476,000opies/ml [Fig. 1], prompting an evaluation for possible GCV resis-ance.

All studies were done with parental consent and with approvalf the University of Minnesota Institutional Review Board. To testor genotypic resistance, DNA extracted from blood at day of life50 (at the time of peak DNAemia, Fig. 1) was PCR-amplified andequenced to test for UL54 (DNA polymerase) and UL97 (ganci-lovir kinase) mutations.21 For UL97, two primer sets were usedo amplify regions covering codons 377–653. For UL54, 4 primerets were used to amplify codon regions 276-1004. PCR productsere cloned into the TA cloning vector (Invitrogen) and, following

ransformation in E. coli, 15–20 clones from each PCR amplificationere sequenced. These analyses demonstrated two UL97 mutations

n the majority of the PCR-amplification products. In total, 60% ofequenced clones (12/20) demonstrated a M460V mutation, and5% of clones (12/16) demonstrated a L595F mutation [Fig. 2]. NoL54 mutations were observed.

To confirm that these resistance mutations had emerged duringhe course of GCV therapy, the newborn viral culture isolate wasbtained, and CMV DNA was amplified by PCR. PCR was also per-ormed, following parental consent, on the archived newborn driedlood spot. All sequences were wild-type in the UL97 and UL54

enes, confirming that a GCV-susceptible genotype was present atirth (Fig. 2B).

In light of high-grade DNAemia and suspected (and later con-rmed) GCV resistance, VAL-GCV therapy was discontinued after

nteral GCV was limited to 4 weeks of therapy due to neutropenia. After resolution ofr of a resistant isolate associated with high systemic viral load (476,000 copies/ml).

8 weeks of oral therapy. Viral load declined to 300 copies/ml at 13months of age, and DNAemia was no longer detectable at 20 monthsof age. Cerebral palsy and bilateral severe-to-profound sensorineu-ral hearing loss, requiring cochlear implantation, were observed onlong-term follow-up.

3. Other similar and contrasting cases in the literature

GCV resistance has been reported as early as 1–3 weeks afterthe initiation of preemptive therapy in young children with immu-nodeficiency disorders undergoing T-cell-depleted haploidenticalstem-cell transplantation.22 Resistance to GCV, cidofovir and fos-carnet was described in an infant with severe combined immunedeficiency with severe CMV disease, probably acquired via breastmilk.21 Multiple GCV resistance mutations, detected by PCR andpyrosequencing, have been described in an infant with symp-tomatic congenital CMV infection treated sequentially with GCV,VAL-GCV, and foscarnet.23 Detection of GCV-resistant variantsusing pyrosequencing was also described in a fatal case of dissemi-nated CMV disease in a preterm infant with congenital infection.24

4. Discussion

GCV and, more recently VAL-GCV, have been increasinglyemployed in the treatment of congenital CMV infection duringthe course of the past decade, toward the goal of improv-ing neurodevelopmental and auditory outcomes in symptomaticnewborns.15–20 In this case, we describe GCV resistance in aninfant being treated sequentially with intravenous GCV and long-term oral VAL-GCV for congenital CMV infection. The emergenceof GCV-resistance most likely occurred during oral VAL-GCVtherapy, since the viral load initially decreased on intravenous ther-apy. However, it is possible that emergence of resistant strainsoccurred during the period of intravenous GCV therapy. The ini-tial dose of GCV was low, and was intentionally administeredat a reduced dose in light of prematurity and renal insuffi-ciency. There is little information available about drug dosagein premature infants with renal insufficiency, and, unfortunately,GCV drug levels were not available for this patient. The oralVAL-GCV dose prescribed for this patient, 20 mg/kg twice daily,

achieves therapeutic levels in both premature and term infants,25,26

making it unlikely that inadequate dosing during oral therapyplayed any role in promoting the documented emergence of resis-tance.

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K.Y. Choi et al. / Journal of Clinical Virology xxx (2013) xxx– xxx 3

Fig. 2. Demonstration of GCV resistance mutations. Blood PCR at approximately 5 months of age (while on oral VAL-GCV therapy) demonstrated resurgence of high systemicviral load. PCR of UL97 coding regions associated with GCV resistance followed by DNA sequence analysis demonstrated simultaneous appearance of both an M460V (leftpanel, post-GCV) and a L595F mutation (right panel, post-GCV). These mutations were not noted when PCR and subsequent sequence analyses were performed using DNAp utati

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urified from the original viral isolate obtained at birth, indicating that resistance m

In this case, clinical and genotypic evidence of resistancemerged and was detectable after approximately 6 weeks ofral VAL-GCV therapy, [Fig. 1]. There is precedent for resis-ance emerging after comparable long-term treatment courses indults. Following long-term GCV therapy for AIDS-associated CMVetinitis, the prevalence of GCV resistance was reported to be 38%n patients still shedding CMV after 3 months of therapy, andstimated to be 7.6% overall, although these studies assessed forhenotypic, not genotypic, resistance.27 In a prospective studyomparing intravenous GCV and oral VAL-GCV in transplantatients, the risk of developing a GCV resistance mutation was.3% for GCV, and 3.6% for VAL-GCV, after 7 weeks of therapy.28 In

ediatric solid organ transplant patients receiving VAL-GCV pro-hylaxis for 100 days, 2 of 46 patients (4%) demonstrated UL97utations in blood samples.29 Our case is similar to a case described

y Campanini and colleagues,23 who also described GCV resistance

ons emerged after the initiation of antiviral therapy.

in an infant with congenital CMV infection treated sequentiallywith GCV and VAL-GCV. Our study relied on sequencing of sub-cloned PCR amplification products, and demonstrated that 67% ofsequenced clones demonstrated drug-resistance mutations, usingDNA purified from blood obtained while the infant was on VAL-GCV therapy. This is similar to the 47% of positive clones notedduring GCV therapy of another infant with congenital CMV infec-tion when pyrosequencing was used to identify UL97 mutations invirus subpopulations.23 UL97 mutations continued to be detectedin our patient months after discontinuation of VAL-GCV therapy;although the M460V mutation had reverted to wild-type sequenceby 14 months of age, the L595F mutation persisted, (data not

shown). This observation was surprising in light of mathemat-ical models showing a reduced in vivo fitness of GCV-resistantUL97 mutants in the absence of the drug.30,31 A recent review sug-gested that a range of 3–8 months is required for repopulation of

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ARTICLEK.Y. Choi et al. / Journal of Cl

CV-resistant CMV by wild-type virus following discontinuation ofCV in the setting of documented resistance.32 Long-term molec-lar virological follow-up will be required to evaluate whetherur patient eventually sheds a completely GCV-susceptibletrain.

Our case differs from that described by Campanini in that ouratient was commenced on VAL-GCV on an outpatient basis, at aime that he was asymptomatic, solely toward the goal of maximiz-ng the likelihood of a favorable neurodevelopmental outcome.16,20

ndeed, the resurgence of high-grade DNAemia on VAL-GCV ther-py in this infant was not associated with symptoms. DNAemiaapidly cleared in this infant following discontinuation of antiviralherapy, presumably because of the development of an effec-ive immune response. Low-level viral replication (and low gradeNAemia) may promote development of cytotoxic T-lymphocyte

mmunity, and complete ablation of viral replication with GCVay conceivably retard the development of effective long-term

mmunity in solid organ transplant patients.33 Whether prolongedAL-GCV therapy delays the development of an effective cellu-

ar immune response in congenital CMV infection requires furthertudy.

In summary, although most available data on the emergence ofCV resistance in the setting of long-term therapy suggests that

he risk is low,28,29,34 our case demonstrates that GCV resistancean nonetheless emerge in asymptomatic infants being treated onn outpatient basis for congenital CMV infection with oral VAL-CV suppression. The potential risks of treatment, coupled with

he emergence of resistance as demonstrated in this report, provideupport for a recommendation that prolonged VAL-GCV should note administered to infants with congenital CMV infection outside ofontrolled clinical trials.20,35 Strategies to enable rapid, “real-time”etection of genotypic resistance will be needed to more preciselyefine the risk and aid in the clinical management of infants on

ong-term therapy.24,36–38

unding

Grant support from NIH HD044064 and 038416 is acknowl-dged (M.R.S.). These studies were also supported by an Amplatzhildren’s Discovery award (B.Z.).

ompeting interests

None declared.

thical approval

Genotypic analyses were performed on DNA from clinical sam-les collected in the context of standard care for the infantescribed in this report. All studies of viral DNA and viral strainsere performed with approval of the University of Minnesota Insti-

utional Review Board (study number: 0709M17161) and parentalonsent.

cknowledgements

The authors acknowledge the collaboration and assistance ofhe Minnesota Department of Health (Mark McCann and Carrie

olf). The critical review of the manuscript by Dr. Mark Robiens gratefully acknowledged.

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