Katharina Göhring

Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

BACKGROUND Infection with human cytomegalovirus (HCMV) is the most common congenital virus infection, affecting about 0.5-2% of newborns. Using DBS on Guthrie cards, it is possible to discriminate congenital from postnatal HCMV-infection. However, a recent European trial revealed serious problems in detection of low HCMV-DNA levels from DBS-filter-cards (Barbi et al., 2008).(7) OBJECTIVES Evaluation of the most sensitive combination of sample size, DNA extraction method and PCR system for the detection of low copy numbers of HCMV-DNA from DBS-filter-cards. STUDY DESIGN We compared three different manual extraction methods for the detection of HCMV-DNA out of DBS: the QIAmp-blood-Mini-Kit, a heat-extraction-method and traditional phenol-chloroform extraction. Additionally, we tested an automated nucleic acid extraction system (NucliSense EasyMag/Biomerieux). Different punch-sizes of DBS spiked with defined HCMV AD169-DNA copy numbers were analyzed. For detection, we used a quantitative in-house-LightCycler-PCR targeting the gB-region using the hybridisation-probe-format. We compared the sensitivity of the real-time-PCR with IE1Ex4-targeted nested-PCR. RESULTS The highest sensitivity with 200 copies HCMV DNA/ml was achieved using the phenol-chloroform method in combination with the nested-PCR and 6mm, 3x3mm punches or the whole DBS. The QIAmp-blood-Mini-Kit also showed a very high sensitivity by using the whole DBS and the nested-PCR. CONCLUSION These results may have strong implications for retrospective diagnosis of congenital HCMV (cHCMV) infection, since a defined combination of the area of punch, the extraction method, and PCR method determine the probability of detection of viral DNA from DBS according to a logistic model.

Rapid Simultaneous Detection by Real-Time PCR of Cytomegalovirus UL97 Mutations in Codons 460 and 520 Conferring Ganciclovir Resistance

ABSTRACT Ganciclovir (GCV) resistance is an emerging problem for transplant recipients. A sensitive and rapid real-time PCR approach for simultaneous and semiquantitative detection of human cytomegalovirus (HCMV) UL97 mutations in codons 460/520 was established by LightCycler and confirmed by restriction fragment length polymorphism and sequencing. Results from HCMV laboratory strains were compared with results from 11 GCV-resistant clinical isolates.

Antiviral Drug- and Multidrug Resistance in Cytomegalovirus Infected SCT Patients

In pediatric and adult patients after stem cell transplantation (SCT) disseminated infections caused by human cytomegalovirus (HCMV) can cause life threatening diseases. For treatment, the three antivirals ganciclovir (GCV), foscarnet (PFA) and cidofovir (CDV) are approved and most frequently used. Resistance to all of these antiviral drugs may induce a severe problem in this patient cohort. Responsible for resistance phenomena are mutations in the HCMV phosphotransferase-gene (UL97) and the polymerase-gene (UL54). Most frequently mutations in the UL97-gene are associated with resistance to GCV. Resistance against all three drugs is associated to mutations in the UL54-gene. Monitoring of drug resistance by genotyping is mostly done by PCR-based Sanger sequencing. For phenotyping with cell culture the isolation of HCMV is a prerequisite. The development of multidrug resistance with mutation in both genes is rare, but it is often associated with a fatal outcome. The manifestation of multidrug resistance is mostly associated with combined UL97/UL54-mutations. Normally, mutations in the UL97 gene occur initially followed by UL54 mutation after therapy switch. The appearance of UL54-mutation alone without any detection of UL97-mutation is rare. Interestingly, in a number of patients the UL97 mutation could be detected in specific compartments exclusively and not in blood.

Dynamics of the Emergence of a Human Cytomegalovirus UL97 Mutant Strain Conferring Ganciclovir Resistance in a Pediatric Stem-Cell Transplant Recipient

Stem-cell transplant recipients are at risk of developing ganciclovir-resistant human cytomegalovirus (HCMV) infection caused by mutations in the viral UL97 gene. Knowledge of the relative proportions of coexisting HCMV wild-type and mutant strains may contribute to a better understanding of the dynamics of in vivo mutant strain selection under ganciclovir. Currently, genotype resistance screening for UL97 is routinely performed by restriction fragment length polymorphism detection and sequencing. We present here the longitudinal course of a pediatric recipient of an allogeneic stem-cell transplant infected with a ganciclovir-resistant HCMV strain. EDTA-treated blood samples were analyzed longitudinally. The patient acquired a primary HCMV infection shortly before transplantation and reactivated the virus following allogeneic hematopoietic stem cell transplantation, thus receiving an intensive antiviral treatment schedule. Three different methods for UL97 mutation analysis, restriction fragment length polymorphism detection, sequencing, and a new, real-time PCR approach were performed. In conclusion, for our pediatric patient, during peak viral load, the UL97 wild-type strain predominates, while during clinical deterioration with low viral load, the predominant mutant strain persists.

Dynamics of coexisting HCMV-UL97 and UL54 drug-resistance associated mutations in patients after haematopoietic cell transplantation

BACKGROUND Resistance to antiviral drugs can be a severe problem in transplant recipients. Mutations in the HCMV phosphotransferase-gene (UL97) and the polymerase-gene (UL54) are responsible for resistance against ganciclovir (GCV), cidofovir (CDV) and foscarnet (PFA). Most frequently mutations in the UL97-gene are associated with resistance to GCV. Resistance against PFA and CDV is associated to mutations in the UL54-gene. There are only few reports about multidrug-resistance with mutations in both genes in patients after allogeneic haematopoietic cell transplantation (HCT). OBJECTIVES To asses retrospectively the role of UL97/UL54-mutations for clinical deterioration. STUDY DESIGN We present here three patients after HCT developing multidrug-resistance with coexisting UL97 and UL54-mutations. Genotypical resistance screening was done with restriction-fragment-length-polymorphism (RFLP), sequencing of UL97/UL54, and LightCycler real-time PCR. Phenotyipcal testing was performed by a cell-associated plaque-reduction-assay. Plasma viral-load (VL) was determined longitudinally using Roche Cobas-Amplicor-System (Roche Diagnostics). In one case VL was also correlated to different ratios of coexisting UL97-wildtype and mutant variants. RESULTS All three patients developed multidrug resistant HCMV-infections with one or more UL97 and UL54-mutation detected by RFLP, sequencing and LightCycler-analysis. Two out of three patients showed biphasic VL kinetics with manifestation of UL97 drug-resistance prior/or at peak VL. UL54-mutations emerged also in all three patients either at increasing VL levels of ≥10(5)copies/ml or at peak VL. CONCLUSIONS The development of coexisting HCMV UL97 and UL54-mutations conferring drug-resistance after HCT is not strictly associated with fatal outcome in one of our three patients. Manifestation of drug resistant combined UL97/UL54-mutations occurred prior to a second VL peak under (V)GCV/PFA co-treatment.

Generation and characterization of a GCV resistant HCMV UL97-mutation and a drug sensitive UL54-mutation.

In transplant recipients, drug-resistant human cytomegalovirus (HCMV) infections remain a serious problem. Drug-resistance against ganciclovir (GCV), cidofovir (CDV) and foscarnet (PFA) is caused by mutations either in the phosphotransferase-gene (UL97) or in the viral polymerase (UL54). For characterization of newly emerging mutations marker transfer analysis is required. Two new HCMV-mutations, the UL54-mutation L516M and the UL97-mutation A613V, were characterized by this method.

Identification of newly detected, drug-related HCMV UL97- and UL54-mutations using a modified plaque reduction assay

BACKGROUND Drug-resistant cytomegalovirus causes major problems in immunocompromised patients and is due to mutations in the UL97-gene (phosphotransferase) and/or the UL54-gene (polymerase). OBJECTIVE Three previously unknown UL97-mutations (E596D/Y and I610T), one UL54 single point mutation (D515E) and a UL54 triple mutation (D515E+L516M+I521T) were characterized for drug-resistance by marker transfer analysis using BAC-technology. STUDY DESIGN Mutations were introduced into the bacterial artificial chromosome TB40-BACKL7-UL32EGFP. In addition, mutations M460V (UL97) and I521T (UL54) served as drug-resistant control. Phenotypic resistance testing was performed by a modified plaque reduction assay using a mixture of infected fibroblasts and uninfected ARPE-19 cells which improved formation of clearly definable plaques considerably. RESULTS Resistance testing showed ganciclovir (GCV)-resistance for UL97-mutations I610T and E596Y while mutation E596D was drug-sensitive. UL54-mutation D515E was resistant to GCV. The virus strain containing the UL54 triple mutation conferred cross-resistance to GCV and cidofovir (CDV). None of the mutations interfered with normal growth kinetics of the virus. CONCLUSIONS New mutations in the UL97- and UL54-gene of HCMV are still detected continuously. Furthermore, several mutations occurring in the same codon often show divergent phenotypes and the accumulation of multiple mutations in one virus strain may lead to increased or decreased drug-resistance. Therefore, characterization of newly detected mutations by marker transfer analysis is essential to confirm that genotypically detected mutations can produce phenotypic resistance. These results allow reliable interpretation of fast genotypic methods generally used in diagnostics.

Intrafamilial transmission of human cytomegalovirus (HCMV): Long-term dynamics of epitope-specific antibody response in context of avidity maturation

BACKGROUND The role of a special early family formation (PEKiP), which is popular in Germany, as a potential origin of HCMV-transmission to seronegative mothers is not documented. OBJECTIVES To describe the clinical courses, to identify the virological origin and to evaluate a new tool for diagnosis of a cascade of intrafamilial HCMV primary infections. STUDY DESIGN This prospectively analyzed long-term course of HCMV primary infection leading to hospitalization of two family members, included the evaluation of different IgG/IgM/IgG avidity-assays with an epitope-specific recombinant immunoblot-assay. Additionally, neutralization (NT) assays using fibroblast-and epithelial-target cells were performed to correlate NT₅₀ values to avidity maturation. HCMV gN/gO/gB-RFLP-genotyping and phylogenetic analyses were performed using urine viral isolates. RESULTS The clinical courses of the sequentially occurring intrafamilial HCMV primary infections were unusual, leading to hospitalization. Long-term-serology of the mother revealed concordant results for an unimodal IgG-course and a rapid decrease of IgM-indices from week 7 to week 21 p.i. Interestingly, the cut-off definitions for low and high avidity ranged discordantly from 15 to 25 weeks, and from 18 to 42 weeks p.i., respectively. A good correlation was found between the increase of fibroblast-adapted NT50 values and the appearance of high avidity using the epitope-specific immunoblot (>18 weeks p.i.). RFLP-genotyping and sequencing could identify the index patient as member of PEKiP-meetings. CONCLUSIONS PEKiP-meetings with naked babies may be an important source of horizontal HCMV-transmission to seronegative pregnant mothers in Germany. Using epitope-specific immunoblots, persisting HCMVp150-IgM-reactivities and good concordance between high IgG-avidity and increase of fibroblast adapted neutralization capacity were found.

Identification of resistance-associated HCMV UL97-and UL54-mutations and a UL97-polymporphism with impact on phenotypic drug-resistance

BACKGROUND Human cytomegalovirus (HCMV) drug-resistance remains of high clinical importance. While UL97-mutations can confer ganciclovir-resistance, UL54-mutations can be associated with resistance to ganciclovir, foscarnet and/or cidofovir. OBJECTIVE Three UL97-mutations (A619V, P468Q, del597-599), three UL54-mutations (V715A, A492D, L516W) and two UL97/UL54-mutation combinations (A594TUL97+V715MUL54; A591VUL97+D515EUL54, L516MUL54, I521TUL54) were characterised phenotypically. All mutations were introduced into the bacterial artificial chromosome (BAC) TB40-BACKL7-UL32EGFP. A revertant of HCMV-TB40-BACKL7-UL32EGFP/A591VUL97+D515EUL54, L516MUL54, I521TUL54 was generated. RESULTS The UL97-mutation del597-599 showed GCV-resistance while A619V and P468Q were drug-sensitive. The UL54-mutation V715A was FOS-resistant/CDV-hypersensitive and L516W was GCV/CDV cross-resistant. Mutation A594TUL97+V715MUL54 showed GCV/FOS cross-resistance. HCMV-BACKL7-UL32EGFP/A591VUL97+D515EUL54,L516MUL54, I521TUL54 was GCV/CDV cross-resistant with a remarkably increased GCV-ratio compared to a strain where only the UL54-mutations D515E+L516M+I521T were present. Since the revertant was drug-sensitive again, the increased drug-ratio is supposed to be due to the UL97-polymorphism A591V. CONCLUSION Phenotypic characterisation of newly detected mutations in UL97 and UL54 remain of high importance. Only mutations with a confirmed phenotype allow reliable interpretation of genotypic methods. Here, we provide the first description of a UL97-polymorphism that contributes to the overall drug-resistance when combined with resistance-associated UL54-mutations. The finding shows the high importance to look at mutations in the context of their genetic background.

Quantitative monitoring of HCMV DNAlactia in human milk by real time PCR assay: Implementation of internal control contributes to standardization and quality control

For cytomegalovirus screening of breastfeeding mothers of preterm infants under risk, we present a rapid, quantitative real-time PCR protocol using the hybridization format of the viral gB target region. For quantification, we used an external gB fragment cloned into a vector system. For standardization, we created an internal control-plasmid by site-directed mutagenesis with an exchange of 9 nucleotides. Spiked with internal control, patient wildtype amplicons could be discriminated from internal controls by hybridization probes using two-channel fluorescence detection. Potential bias of formerly reported false nucleotide sequence data of gB-hybridization probes was excluded. Using this approach, we could demonstrate excellent analytical performance and high reproducibility of HCMV detection during lactation. This assay shows very good correlation with a commercial quantitative HCMV DNA PCR and may help to identify rapidly HCMV shedding mothers of very low birth weight preterm infants to prevent HCMV transmission. On the other hand, negative DNA amplification results allow feeding of milk samples of seropositive mothers to their preterm infants under risk (<30 weeks of gestational age, <1000g birth weight) during the onset and late stage of HCMV shedding during lactation.