Antiviral Resistance in Human Cytomegalovirus Due to UL54 Mutations Without UL97 Mutations

Kuenyoul Park1 , Kyu-Hwa Hur2 , Heungsup Sung1 , Sang-Ho Choi3 , Mi-Na Kim1

1Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul
2Department of Laboratory Medicine, Uijeongbu Eulji Medical Center, Eulji University, Uijeongbu
3Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

Corresponding author : sung@amc.seoul.kr

ABSTRACT

Background: The concurrent detection of human cytomegalovirus (CMV) with UL97 and UL54 mutations is crucial for prescribing adequate antiviral treatment when drug-resistant CMV infection is suspected. We investigated the frequency of resistance-conferring mutations among patients with persistent or recurrent CMV infection and further reviewed the subgroup with UL54 mutations without UL97 mutations.
Methods: Patients with persistent or recurrent CMV infection after 4 weeks of treatment with ganciclovir or foscarnet were consecutively enrolled between November 2012 and May 2019. The direct sequencing of UL97 and UL54 was performed to detect resistance mutations in CMV.
Results: A total of 101 sequencing datasets were obtained from 65 enrolled patients. CMV UL97 and UL54 mutations were detected in 15.4% (10/65) and 9% (6/65) of patients, respectively. The CMV retrieved from two patients (3%) had mutations in both genes. Four patients with CMV UL54 mutations alone had a history of haploidentical peripheral blood stem cell transplantation, and foscarnet was administered for over 4 weeks to these patients; 21.5% of patients had suspected resistant CMV infection with either UL97 or UL54 mutations.
Conclusion: In this study, CMV UL54 mutations but not UL97 mutations were found in patients subjected to prolonged foscarnet administration for CMV disease.

Keywords

Antiviral resistance, Cytomegalovirus, UL54, UL97

INTRODUCTION

Human cytomegalovirus (CMV) disease is commonly treated with ganciclovir (GCV) or its oral prodrug, valganciclovir. Second-line treatment drugs for CMV disease, including foscarnet (FOS) and cidofovir (CDV), target UL54 DNA polymerase. Prolonged antiviral therapy can lead to mutations in the UL97 and/or UL54 genes [1]. CMV resistance to these drugs is uncommon, ranging from 0.4% to 11.9% in solid organ transplant patients and 1% to 5% in patients who have undergone hematopoietic stem cell transplant [2,3]. However, drug-resistant CMV infection is associated with inferior prognosis in these two patient groups [2,4].

UL97 mutations are well-known as the most prevalent causes for CMV resistance [5,6]. A previous study [6] showed that approximately 30% of 570 samples submitted to a commercial laboratory in the US were positive for UL97 or UL54 gene mutations, and 62.5% of 64 samples with UL54 mutations also possessed UL97 mutations. Therefore, UL97 mutations are primarily tested in patients suspected to be infected with resistant strains of CMV, and UL54 mutations are tested concurrently or only in UL97 mutation-negative cases. UL54 mutations, which usually appear after UL97 mutations, are associated with resistance to not only the .rst-line drug GCV, but also to the other second-line agents such as CDV and FOS [7]. Therefore, UL54 as well as UL97 mutations should be investigated in CMV patients suspected with antiviral resistance. Here, we describe the frequency of CMV resistance-related gene mutations among patients with persistent or recurrent CMV infection, and further reviewed the subgroup possessing UL54 mutations without UL97 mutations.

MATERIALS AND METHODS

Patients suspected to be infected with resistant CMV strains following persistent or recurrent CMV detection after 4 weeks of antiviral therapy using GCV or FOS were subjected to UL97 and UL54 mutation testing in a consultation-based setting between November 2012 and May 2019. Clinical and laboratory data were obtained from the hospital’s electric medical records and laboratory information system. CMV polymerase chain reaction (PCR) was performed with Artus CMV PCR Test (QIAGEN Gaithersburg, Inc., Gaithersburg, MD, USA) using a Rotor-Gene Q (Qiagen Inc., Hilden, Germany), and the threshold of CMV detection was set as 2.42 log copies/mL. In September 2018, Artus CMV PCR Test was replaced with Abbott RealTime CMV Assay (Abbott Molecular Inc., Des Plaines, IL, USA) with the limit of detection value set at 1.49 log IU/mL. Direct sequencing of UL97 and UL54 genes was performed for detecting the CMV resistance mutations as described previously [8,9]. Variants were detected by comparing with the reference strain AD169 (GenBank accession number BK000394). Detected variants were analyzed along with the previously reported ones and synonymous variants were not analyzed [10,11]. Categorical data were analyzed with chi-square test using MedCalc (version 20.011; MedCalc Software, Ostend, Belgium). The Institutional Review Board (IRB) of the Asan Medical Center (No. 2012-0401) approved this study.

RESULTS

A total of 101 CMV DNA samples from 65 patients were subjected to CMV resistance testing. The median age of study patients was 19 years and 62% of them were male. 75% of study patients had underlying disease of hematologic malignancy. All mutations in UL97 or UL54 genes were identi.ed in 14 patients (21.5%). 10 (15.4%) and 6 (9.2%) patients exhibited mutations in the UL97 and UL54 genes, respectively. Observed mutations from both genes are presented in Table 1. Between patients with and without any mutation in UL97 and UL54 genes, there was no signi.cant difference of underlying disease of hematologic malignancy (P=0.31) and sex (P=0.32). Changes in the test numbers of samples and % sample harboring any mutation were not signi.cant during the study period; the test numbers of samples (% sample harboring any mutation) were 5 (0%) in 2012, 4 (25.0%) in 2013, 12 (8.3%) in 2014, 18 (16.7%) in 2015, 15 (13.3%) in 2016, 17 (23.5%) in 2017, 26 (26.9%) in 2018, and 4 (25.0%) in 2019.

 

Table 1. Human cytomegalovirus UL97/UL54 gene mutations observed in this study

Case No.

Year

UL97 mutations (specimen)

UL54 mutations (specimen)

Viral loads

3

2019

A594P (blood)

4.12 log IU/mL

6

2018

F412L (blood)

3.03 log IU/mL

7

2018

L595F (blood)

N408D (blood)

4.40 log copies/mL

11

2017

V715M (blood)

3.34 log copies/mL (blood)

14

2018

L501I (CSF)

4.73 log copies/mL (CSF)

19

2017

L802M (CSF)

5.92 log copies/mL

22

2017

505-506 in frame deletion (anterior chamber fluid)

V787L (blood)

4.72 log copies/mL

29

2017

C603W (blood)

N/A

32

2016

C603W (blood)

N408D/P522S (blood)

3.57 log copies/mL

40

2016

M460V (blood)

5.69 log copies/mL

46

2015

A594V (blood)

4.12 log copies/mL

55

2015

597-600 in frame deletion (blood)

2.42 log copies/mL

60

2014

L595S (blood)

2.42 log copies/mL

65

2013

M460I (blood)

4.59 log copies/mL

40

2019

M460V (blood)

3.99 log IU/mL

46

2015

A594V (blood)

2.42 log copies/mL

55

2014

597-600 in frame deletion (blood)

2.42 log copies/mL

60

2013

L595S (blood)

4.59 log copies/mL

65

2019

M460I (blood)

3.99 log IU/mL

Abbreviations: CSF, cerebrospinal fluid; N/A, not available.

 

Four cases had UL54 mutations alone. Patient characteristics and CMV kinetics of these cases are described in Table 2 and Fig. 1, respectively. They all had a history of haploidentical peripheral blood stem cell transplantation (HaploPBSCT) and FOS was administered to these four patients before the detection of variants.

 

Table 2. Patient characteristics of four cases with UL54 mutations alone

Patient No.

Sex

Age (yr)

Underlying disease

CMV disease

Outcome

UL54 mutations

6

Female

15

MDS, HaploPBSCT

Pneumonia

Retinitis

Encephalitis

Expired

F412L

11

Female

18

AA, HaploPBSCT

Pneumonia

Encephalitis

Expired

V715M/L501I

14

Female

18

AA, HaploPBSCT

Encephalitis

Improved

L802M

19

Female

19

AA, HaploPBSCT

Retinitis

Improved

V787L

Abbreviations: CMV, cytomegalovirus; MDS, myelodysplastic syndrome; HaploPBSCT, haploidentical peripheral blood stem cell transplantation; AA, aplastic anemia.

 

Fig. 1. CMV kinetics of four cases with UL54 mutations alone (Patients #6, #11, #14, #19). Bar graph indicates the duration of antiviral drug administration. Blue arrows indicate the specimen dates of CMV viruses with specified UL54 mutations. CMV, cytomegalovirus; PBSCT, peripheral blood stem cell transplantation; CSF, cerebrospinal fluid

DISCUSSION

Approximately, one-.fth of the patients with persistent or recurrent CMV exhibited at least one mutation in UL97 and UL54. High prevalence of CMV mutations was mainly due to the inclusion criteria of patients with persistent or recurrent CMV after 4 weeks of antiviral treatment using GCV or FOS. Resistance emergence varied along with organ type among solid organ transplantation (SOT) patients [3]; thus, the composition of the included SOT patients could affect the mutation prevalence in this study.

All except two mutations were well-known mutations [12-14]. The two unknown mutations are both in-frame deletions. In-frame deletion of codon 597-600 is within the hot-spots for GCV resistance (codons 460, 520, and 590-607) [12]. In-frame deletion of the UL97 gene, especially at codons 591 to 603 is less-common but has been reported continuously, and some reported these mutations to confer 4- to 10-fold resistance [13]. The other one was an in-frame deletion of codons 505-506 located on the kinase domain, which is not a well-known mutation or polymorphism [14], but a variant of unknown signi.cance.

All cases with UL54 mutations alone were related to HaploPBSCT and exhibited a prolonged treatment history with FOS. A previous study reported three patients with UL54 mutations alone, who were hematopoietic stem cell transplantation recipients, and all were treated with both GCV and FOS [15]. In line with this, prolonged use of FOS is possibly related to CMV resistance conferred by UL54 mutations without UL97 mutations. This .nding highlights the importance of testing UL54 mutations especially in patients treated with FOS.

Our study has several limitations due to its observational nature. Furthermore, our study population was limited to patients who were clinically suspected to have resistant CMV infection, and therefore, some patients subjected to more than 4 weeks of antiviral therapy for CMV treatment were probably excluded. Additionally, the number of patients infected with drug-resistant CMV in one institution was relatively small.

Conclusively, 21.5% of patients suspected with resistant CMV infection possessed one of the two UL97 and UL54 mutations. In addition, UL54 mutations without UL97 mutations was found in patients subjected to prolonged administration of FOS for CMV disease.

요약

배경: 약제 내성 human cytomegalovirus (CMV) 감염이 의심될 때, UL97UL54 변이를 모두 확인하는 것이 적절한 항바이러스제 치료를 하는데 중요하다. 지속성 혹은 재발성 CMV 감염이 의심되는 환자에서 CMV 내성유발 변이들의 빈도를 조사하고, UL97 변이 음성이면서 UL54 변이를 가진 하위 그룹을 추가로 검토하였다.
방법: 2012년 11월부터 2019년 5월까지, 4주 동안 ganciclovir 또는 foscarnet 치료 후 지속적 또는 반복적 CMV 감염 환자를 연구에 포함시켰다. CMV 내성유발 변이를 검출하기 위해 UL97UL54를 염기서열 분석하였다.
결과: 65명의 등록된 환자로부터 총 101개의 염기서열 자료를 얻었다. 환자의 15.4% (10/65)와 9% (6/65)에서 각각 CMV UL97UL54 변이가 검출되었다. 환자 2명(3%)에서 CMV는 두 유전자에 모두 변이가 있었다. UL54 변이를 가진 4건의 경우 반일치 말초혈액줄기세포 이식을 받았고, foscarnet을 이 환자들에 4주 이상 투여한 기록이 있었다. 약제 내성 CMV 감염이 의심되는 환자의 21.5%는 UL97 또는 UL54 돌연변이를 보유하고 있었다.
결론: 본 연구에서 UL97 돌연변이가 없는 UL54 변이는 모두 foscarnet을 장기투여한 환자군에서 발견되었다.

CONFLICTS OF INTEREST

No potential conflicts of interest relevant to this article were reported.

FUNDING

This study was supported by a grant from the Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare, Republic of Korea (Grant No. HI18C2383).

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