Emergence of Multiple Human Cytomegalovirus Ganciclovir-Resistant Mutants with Deletions and Substitutions within the UL97 Gene in a Patient with Severe Combined Immunodeficiency

Abstract

Infection with multiple ganciclovir-resistant human cytomegalovirus mutants, containing different substitutions and deletions in the UL97 gene, was found in a patient with severe combined immunodeficiency (SCID) within 3 weeks of ganciclovir therapy. A novel 11-codon deletion at positions 590 to 600 was identified. These unique findings may be related to the nature of the immunodeficiency in the SCID patient.Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised individuals, especially in transplant recipients, patients with AIDS, and children with congenital immunodeficiency disorders (1, 10). With the availability of effective antiviral drugs, prolonged prophylactic and therapeutic regimens are increasingly employed. Ganciclovir, a nucleoside homologue, is the most widely used anti-HCMV drug (11). However, prolonged ganciclovir therapy can lead to the development of ganciclovir-resistant strains. HCMV ganciclovir resistance results mainly from impaired phosphorylation of the drug, caused by mutations in the HCMV UL97 phosphotransferase (2,3,7,8,21).Until recently, ganciclovir-resistant strains have been recovered mainly from AIDS patients who received the drug for more than 3 months. With the widespread use of ganciclovir prophylaxis among transplant recipients, ganciclovir resistance is increasingly reported in the transplant setting, especially among solid-organ transplant recipients who have had prolonged exposure to ganciclovir (4,14,18). Recently, we described the early emergence of ganciclovir-resistant virus in children with primary combined immunodeficiency (22). Here, we report the unusual isolation of multiple drug-resistant variants, containing point mutations and different deletions of the UL97 gene, from one of these patients after 3 weeks of ganciclovir therapy.The patient (patient 4) was a 5-month-old girl with B Ϫ severe combined immunodeficiency (SCID) who received haploidentical T-cell-depleted bone marrow transplantation. She was HCMV seropositive before transplantation and received a bone marrow graft from a seronegative donor. She had been treated with acyclovir for 3 weeks before secondary HCMV infection with HCMV pneumonitis developed. Intravenous ganciclovir therapy (5 mg/kg of body weight twice daily) was initiated; however, the patient's condition deteriorated, and a leukocyte culture 3 weeks after initiation of therapy was positive for HCMV.Viral isolate propagation, plaque purification, and antiviral susceptibility assays of the isolate and the plaque-purified viruses were done as previously described (21). The laboratoryadapted ganciclovir-sensitive HCMV strain AD169 was included as a sensitive control with each assay. HCMV strains were considered sensitive to ganciclovir if their 50% effective doses (ED 50 ) were Յ6 M. The leukocyte isolate, the corresponding plasma specimen, and the plaque-purified viruses were subjected to direct PCR sequencing of the UL97 gene (21, 22), using primers encompassing nucleotides 1207 to 1979. The PCRs were performed under stringent c...

Sequencing of Cytomegalovirus UL97 Gene for Genotypic Antiviral Resistance Testing

“…The potential exists to further shorten the time required for the genotypic assay by extracting viral DNA directly from patient samples. Portions of the UL97 and polymerase coding sequences can be amplified from DNA extracted from blood cells or plasma (35,37,38; N. S. Lurain, unpublished data) and directly sequenced to detect resistance mutations. We are currently focusing on developing this modification of the assay.…”

“…The CMV UL97 gene is an important viral target for genotypic assays, because all documented mutations conferring GCV resistance have been found at one of three sites within the coding region for the C-terminal half of the phosphotransferase (2,7,9,11,19,26,29,38). At two of the corresponding sites in the UL97 gene there are point mutations in single codons (460 and 520), and at the third there are point mutations or short deletions within the codon range 590 to 607.…”

“…Our approach has been to define the baseline variability that occurs in the absence of drug resistance by sequencing the complete UL97 gene from a large number of clinical isolates that are phenotypically sensi-tive to GCV. These sequences from GCV-sensitive isolates combined with the discrete sites of GCV resistance mutations that have been previously identified (2,9,11,19,26,29,38) provide the basis for the assay. Our results show that amplification of the portion of the UL97 gene encoding the C-terminal half of the enzyme followed by two sequencing reactions can provide rapid identification of all presently known sites of GCV resistance attributed to this gene.…”

Cytomegalovirus UL97 mutations associated with ganciclovir resistance in immunocompromised patients from Argentina

“…Thereafter, resistant strains with mutations on different UL97 gene regions and different levels of resistance have been reported. The appearance of these mutants depends on the immunocompromised population under treatment and on the schedule of the antiviral therapy used (Limaye et al, 2000;Wolf et al, 2001). The most frequent mutations conferring GCV resistance in the UL97 gene are at codons 460, 594, 595 and deletions 595 to 607 (Chou et al, 1995a(Chou et al, , 1995bErice et al, 1989;Spector et al, 1995;Wolf et al, 2001Wolf et al, , 1995.…”

“…The appearance of these mutants depends on the immunocompromised population under treatment and on the schedule of the antiviral therapy used (Limaye et al, 2000;Wolf et al, 2001). The most frequent mutations conferring GCV resistance in the UL97 gene are at codons 460, 594, 595 and deletions 595 to 607 (Chou et al, 1995a(Chou et al, , 1995bErice et al, 1989;Spector et al, 1995;Wolf et al, 2001Wolf et al, , 1995. Ganciclovir-resistant strains can be classified into two groups: (a) those with low resistance to GCV with mutations on the UL97 gene, but wild-type UL54 gene; and (b) those with high resistance to GCV with mutations on both viral genes: the phosphotransferase and the DNA polymerase (Uwe-Vogel et al, 1997).…”