Rapid development of ganciclovir-resistant cytomegalovirus infection in children after allogeneic stem cell transplantation in the early phase of immune cell recovery.

As recently reported, children having T cell-depleted peripheral blood stem cell transplantation (PBSCT) might be at increased risk for the development of drug resistance. To investigate if delayed immune recovery was a potential risk factor, the recovery of the CD3(+), CD4(+), CD8(+) and CD19(+) cells was related retrospectively to genotypic detected resistance development in three pediatric patients with ganciclovir (GCV)-resistant human cytomegalovirus (HCMV)-infection out of 79 receiving allogeneic PBSCT. Selected control groups consisted of HCMV-seronegative patients without any infection (A, n = 8), asymptomatic infected patients with viral leuko- and plasmaDNAemia (B, n = 4) and patients with HCMV-disease (pneumonia) (C, n = 3). Patient No. 1 with very early resistance development exhibited a rapid immune recovery with higher T cell counts than in group A. Immune recovery of patient No. 2 was delayed, as also observed in groups B and C. Patient No. 3 showed an immune recovery comparable to group A. Resistance developed before (No. 2) or during (Nos 1 and 3) the recovery of the relevant CD3(+), CD4(+), CD8(+) lymphocytes. GCV-resistance development did not necessarily coincide with delayed immune recovery, but appeared in all three cases in the early phase of immune recovery (range: day +44 to day +95). Therefore, children seem to be at special risk for resistance development in the early phase after transplantation before immune cells have recovered. These results suggest that GCV treatment of an HCMV infection in the early posttransplant phase of children after T cell-depleted PBSCT/BMT should promote more stringent resistance screening.

Drug-resistant human cytomegalovirus infection in children after allogeneic stem cell transplantation may have different clinical outcomes.

Three seropositive pediatric recipients of allogeneic stem cell transplantation out of a group of 42 patients receiving T-cell-depleted, unrelated transplants and 37 patients receiving T-cell-depleted, haploidentical transplants were monitored longitudinally for human cytomegalovirus (HCMV) infection and the emergence of antiviral drug resistance. Early in the posttransplant course, all 3 patients developed HCMV mutations conferring drug resistance to ganciclovir. One child additionally developed multidrug resistance to foscarnet and cidofovir, with mutations in the viral phosphotransferase gene (UL97) and the DNA-polymerase gene (UL54) being found. These data show that resistant HCMV infection does not necessarily correlate with a severe clinical outcome. The early detection of genotypic resistance up to 129 days before the emergence of phenotypic resistance and the dissociation of resistance patterns among different body sites emphasize the importance of genotypic analyses of different DNA specimens for an efficient antiviral therapy. T-cell-depleted children having transplantation might be at an increased risk for the development of drug resistance.

Comprehensive restriction analysis of the UL97 region allows early detection of ganciclovir-resistant human cytomegalovirus in an immunocompromised child.

Children with innate immunodeficiencies may be at high risk for early development of ganciclovir-resistant human cytomegalovirus (HCMV) infection after bone marrow transplantation (BMT). For early and frequent monitoring of the occurrence of ganciclovir resistance-associated mutations in codons of the UL97 gene, a panel of previously described restriction assays was expanded for use on codons 591, 592, and 603. This technique enabled detection of suddenly emerging ganciclovir-resistant HCMV after BMT in a 7-year-old child with a T cell defect. Resistance emerged among the isolation of a ganciclovir-sensitive HCMV strain 32 days after transplantation, the first detection of genotypical resistance at day 44, and the isolation of resistant HCMV (ID50>12 microM) at day 54. Simple and yet comprehensive methods for therapy surveillance may be important in this patient group, in which the restriction assays proved useful.

Evaluation of restriction fragment length polymorphism analysis of the UL10-UL13 genomic region for rapid identification of human cytomegalovirus strains.

A sensitive semi-nested polymerase chain reaction (PCR) was established which allows rapid identification of human cytomegalovirus strains directly on clinical specimens, thereby permitting virus isolation and propagation on cell cultures to be avoided. The assay is based on restriction analysis of PCR products derived from the polymorphic UL10-UL13 region of the human cytomegalovirus genome. The method was evaluated using clinical samples from 23 subjects comprising 16 breast-feeding mothers and seven bone marrow transplantation recipients. For eight mothers, postnatal virus transmission to their offspring via breast milk was studied. Interestingly, for one mother-infant pair, a double infection with two distinct human cytomegalovirus strains could be demonstrated. Stepwise digestion with different restriction enzymes raised the possibility of detecting different strains almost twofold compared to analysis with only one enzyme. This assay is a practical tool for monitoring human cytomegalovirus transmission in various clinical settings.

A simplified assay for screening of drug resistance of cell-associated cytomegalovirus strains.

BACKGROUND: Conventional phenotypic drug resistance determination of cell-free clinical human cytomegalovirus (HCMV) isolates is usually very laborious and may take 8-12 weeks, since serially passages of slowly growing viral isolates in tissue cultures are required to obtain a sufficient viral titer for an appropriate inoculum. Rapid screening of a large number of samples would therefore only be possible if simplified, less work-intensive methods are employed. OBJECTIVE: The aim of this work was to develop an assay which speeds up the whole procedure of phenotypic drug resistance determination. Steps of the classical plaque reduction assay should be simplified or omitted, but on the other hand, the assay should be reliable and reproducible. STUDY DESIGN: Twenty-six clinical HCMV isolates from 20 immunocompromised patients (ten pre-treatment and 16 post-treatment with ganciclovir) were tested for drug susceptibility with the simplified plaque reduction assay. Most isolates were tested at least twice in independent assays. Virus titration could be avoided by using four different doses of cell-associated virus from the secondary culture for coculture susceptibility testing. Drug susceptibility values were determined by plaque titration and Probit analysis. RESULTS: All clinical HCMV isolates tested showed a mean ganciclovir ID50 value of 1.98 microM, (range 0.2-12.2; median 0.95) and a mean foscarnet ID50 value of 92.4 microM (range 35.7-181; median 81). All except one isolate were classified ganciclovir sensitive when compared to ID50 values of two ganciclovir resistant control stains (53.7 +/- 6.4 and 12.7 +/- 0.9 microM) and the sensitive laboratory strain Towne (2.1 +/- 0.8 microM). Repeated tests of individual isolates were reproducible, although the infectivity of the inoculum has not been determined prior of the assay. The mean time which elapsed between receipt of the clinical specimen and read-out of the assay was circa 4 weeks. CONCLUSIONS: Phenotypic resistance testing of HCMV isolates following to this protocol drastically reduces expenditure of time and work. The assay allows reliably the discrimination of HCMV isolates as drug resistant or sensitive according to the recent classification criteria of the AIDS Clinical Trials Group (ACTG). The simple handling and uncomplicated calibration of this assay facilitates the screening of large specimen numbers and renders drug susceptibility determination of HCMV more accessible to diagnostic routine use.