Research for Cytomegalovirus Mutations Associated With Resistance to Antivirals in Kidney Transplant Receptors

Abstract

Cytomegalovirus (CMV) mutations associated with antiviral resistance have become a major problem related to high mortality in kidney transplant patients. The aim of the study was to investigate mutations in the CMV genes UL97 and UL54 associated with antiviral resistance. A retrospective observational cohort study was carried out at Hospital Ophir Loyola (HOL), a reference in Kidney Transplantation. A total of 81 patients who underwent kidney transplantation were followed up between 2016 and 2018 were monitored for CMV viral load by performing qPCR. Sanger sequencing was performed on 66 patients. All CMV-positive kidney transplant recipients were included. Mutations were observed in 15 samples (22.72%) from patients. Most cases involved UL97 mutations. Mutation in UL54 without mutation in UL97 was detected in only 2 cases. Resistance mutations in UL97 were identified, such as M460V, L595S, H520Q, two co-mutations D465R + Del524 and A594P + D413A and a 3 codon deletion (del598-601). The search for mutations in the CMV genes identified mutations that confer resistance to conventional antivirals, such as ganciclovir and cidofovir, used in the treatment of these patients. Confirmation of the association with increased CMV viral load in transplanted patients, due to mutation in resistance genes, requires phenotypic analysis for confirmation purposes. These were the first findings in patients in northern Brazil that we know of.

Letter

Cytomegalovirus (CMV) infection is one of the most common complications in solid organ transplant (SOT) patients. Currently, preventive treatments have reduced the incidence of disease and death from CMV ¹ . However, mutations associated with resistance to antivirals have become a major problem related to high morbidity and mortality ² .

Currently, ganciclovir (GCV) and valganciclovir (VGCV) are the treatment of choice for CMV infection in transplant patients ³ and their use has led to a decline in CMV disease and associated morbidity in SOT recipients ⁴ . However, with the advent of the widespread and prolonged use of antiviral drugs for CMV prophylaxis and the use of immunosuppressive drugs, mutants resistant to antivirals appear where they are associated with disease progression^5,6. Two CMV gene products are involved in providing resistance to anti-CMV drugs. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase and the product of the UL54 gene is the viral polymerase, which is the target of all available antivirals. Common antiviral drugs, GCV, foscarnet (FOS), and cidofovir (CDV), target DNA polymerase, so the mutation in UL54 can cause resistance to them ⁶ .

If treatment failure is suspected to be due to antiviral resistance, a CMV resistance analysis should be obtained. Phenotypic resistance assays performed on clinical isolates directly measure antiviral susceptibilities, but they are laborious and time-consuming. Therefore, genotypic resistance analysis has become the most common means of diagnosing CMV resistance ⁷ .

Thus, the aim of this study was to identify the presence of mutations in the CMV UL97 and UL54 genes associated with resistance to standard antiviral in renal transplant recipients / patients, as well as to evaluate the CMV and BKV viral load in these patients.

Eighty (81) kidney transplant recipients were included and followed up, where 60 (74%) were men and 21 (26%) women, aged between 9 and 69 years, with an average of 44.04 years.

DNA sequencing was performed in 66 (81.48%) patients. Mutations were observed in 15 samples (22.72%) of patients, where in 13 samples mutations in the UL97 gene were identified. Single mutation in the UL54 gene was observed in only two analyzed samples (Table 1).

Table 1.

Mutations in Cytomegalovirus (CMV) UL97 and UL54 Genes Associated With Antiviral Resistance.

No. of patients	UL97 mutation	UL54 mutation	Clinical response	Viral quantity copies / ml
1	M460I		Resistant	13.030
2		A614S	unknown	13.603/14.586
1	M460V		Resistant	13.040
1	S472N + C495W		unknown	3.030
1	C603W		GCV sensitive	14.080
2	L595S		Resistant	15.050/17.400
1	H520Q		Resistant	18.000
1	N461K		unknown	50.760
1		F432Y	unknown	12.789
1	D465R	Del524	Resistant-GCV and CDV	67.000
1	A594P	D413A	Resistant	73.678
1	D605E	A928V	unknown	4.713
1	Deletion 598-601		Resistant	16.500

According to the Chi-square test, the frequency of samples with detectable viral load among patients with CMV mutants did not differ significantly from that observed in samples in general (P = 0.7408). The occurrence of mutations in the CMV UL97 and UL54 genes does not affect the viral load.

A study that detected mutations in UL97 and UL54 that confer resistance to ganciclovir in renal organ transplant recipients with high CMV viral load, in which the most frequent was D605E in the UL97. This mutation is a natural replacement or polymorphism and has no noticeable role in resistance to GCV ⁸ . The frequency of the D605E mutation may be associated with geographical or racial diferences ⁹ .

The emergence of drug-resistant CMV strains, especially in immunocompromised individuals with active infection, is a life-threatening condition ¹⁰ . Approximately 95% of these strains resistant to GCV contain one or more mutations in the UL97 ¹¹ . Mutations in three specific codons (460, 594, and 595) were observed in approximately 70% of HCV strains resistant to GCV ¹² . In a previous study, a new mutation of resistance to GCV was observed in patients with kidney transplantation, which is a deletion of codon 601 from the UL97 gene ¹³ corroborating our results.

Well-characterized GCV resistance mutations in codons 460, 520, and 590-607 of UL97 impair GCV phosphorylation that is necessary for its antiviral activity, presumably by altering the substrate recognition. In contrast, Maribavir (MBV) is an UL97 kinase inhibitor and is the first new therapy for CMV to reach clinical trials at a later stage in many years. However, after the culture propagated under the drug, UL97 mutations that confer moderate to high resistance to MBV were identified in codons 353, 397, 409 and 411 ¹¹ . In our study, there is a limitation regarding these results because some codons of the UL97 gene were not sequenced, which could present some alteration and consequently resistance to this new drug.

Although the mutation in the UL97 gene far outweighs the UL54 gene, CMV isolations have shown that the mutation in UL97 alone provides low level resistance compared with simultaneous mutations in UL97 and UL54 ⁹ . The F432Y mutation in UL54, found in one of our samples is rare whose exact role in drug resistance is subject to further testing ¹¹ . Thus, the importance of using antiviral susceptibility tests in monitoring anti-CMV therapy has become more evident, serving as an indicator therapeutic strategy ¹⁴ . The viral mutation is not detected by the diagnostic kit, but there is an amplification of the virus genome. Any changes must be confirmed by the sequencing method; however, it can be inferred that the increased viral load may be related to some mutation in the viral genome, as observed in the results.

Therefore, although recombinant phenotyping is needed for confirmation, studies to identify mutations in the UL97 and UL54 genes are of paramount importance for the best management and therapy for kidney transplant patients, resulting in rapid and comprehensive results recommending a combination of phenotypic and genotypes for complete analysis of CMV antiviral resistance, as well as further definition of the clinical relationship between the new UL54 (DNApol) mutations and antiviral resistance ¹⁵ .

The search for mutations in the CMV UL97 and UL54 genes in kidney transplant recipients in Belém, Northern Brazil, identified mutations that confer resistance to standard antivirals such as ganciclovir and cidofovir used in the treatment of these patients. Interestingly, most individuals had an undetectable viral load suggesting that these changes are independent of high viral load.

Ultimately, CMV prevention will require better recognition of at-risk populations with attention to prophylactic and personalized treatment strategies that are truly effective based on individual profile.