Stability of BK polyomavirus IgG seroreactivity and its correlation with preceding viremia.

BACKGROUND: Recently we showed that the level of BK polyomavirus (BKPyV) IgG seroreactivity in kidney donors predicted viremia and BKPyV-associated nephropathy in kidney transplant recipients (KTRs). This observation could be explained by assuming a direct association between BKPyV seroreactivity and the amount of persistent infectious virus in the renal allograft. OBJECTIVES: Since the renal BKPyV reservoir is probably sowed by viremia during primary BKPyV infection, we systematically analysed the dynamics of BKPyV IgG seroreactivity in relation to preceding BKPyV viremia in KTRs and healthy individuals. STUDY DESIGN: A cohort of 85 KTRs consisting of BKPyV viremic and nonviremic subjects was analysed for BKPyV IgG seroreactivity at five fixed time points until one year after transplantation. A cohort of 87 healthy blood donors (HBDs) was used as controls. RESULTS: Baseline BKPyV seropositivity was high in both KTRs and HBDs, and the baseline mean BKPyV IgG level comparable. BKPyV IgG levels in nonviremic KTRs and HBDs remained stable during follow-up, while a considerable increase was observed in viremic KTRs (p=0.015). The increase of BKPyV seroreactivity in viremic KTRs was associated with the duration and peak level of BKPyV viremia. CONCLUSIONS: BKPyV IgG seroreactivity was stable over time in immunocompetent subjects, which enables the use of this potential pretransplantation biomarker in kidney donors. The observed dose-dependent relationship of BKPyV IgG seroreactivity with preceding BKPyV replication is in agreement with the assumption that BKPyV seroreactivity reflects past BKPyV activity and correlates with the amount of latent BKPyV residing within a kidney allograft.

Primary Polyomavirus Infection, Not Reactivation, as the Cause of Trichodysplasia Spinulosa in Immunocompromised Patients.

Classic human polyomaviruses (JC and BK viruses) become pathogenic when reactivating from latency. For the rare skin disease trichodysplasia spinulosa, we show that manifestations of the causative polyomavirus (TSPyV) occur during primary infection of the immunosuppressed host. High TSPyV loads in blood and cerebrospinal fluid, sometimes coinciding with cerebral lesions and neuroendocrine symptoms, marked the acute phase of trichodysplasia spinulosa, whereas initiation and maturation of TSPyV seroresponses occurred in the convalescent phase. TSPyV genomes lacked the rearrangements typical for reactivating polyomaviruses. These findings demonstrate the clinical importance of primary infection with this rapidly expanding group of human viruses and explain the rarity of some novel polyomavirus-associated diseases.

Human polyomavirus 9 infection in kidney transplant patients.

Several human polyomaviruses of unknown prevalence and pathogenicity have been identified, including human polyomavirus 9 (HPyV9). To determine rates of HPyV9 infection among immunosuppressed patients, we screened serum samples from 101 kidney transplant patients in the Netherlands for HPyV9 DNA and seroreactivity. A total of 21 patients had positive results for HPyV9 DNA; positivity rates peaked at 3 months after transplantation, but the highest viral loads were measured just after transplantation. During 18 months of follow-up, HPyV9 seroprevalence increased from 33% to 46% among transplant patients; seroprevalence remained stable at ≈30% in a control group of healthy blood donors in whom no HPyV9 DNA was detected. Further analysis revealed an association between detection of HPyV9 and detection of BK polyomavirus but not of cytomegalovirus. Our data indicate that HPyV9 infection is frequent in kidney transplant patients, but the nature of infection-endogenous or donor-derived-and pathogenic potential of this virus remain unknown.

Persistence and antiviral resistance of varicella zoster virus in hematological patients.

BACKGROUND: Varicella zoster virus (VZV) infections are a relevant cause of morbidity and mortality in hematological patients and especially in hematopoietic stem cell transplant (HSCT) recipients. The present study aimed to investigate the prevalence and clinical significance of viral persistence and antiviral resistance by systematically analyzing all episodes of VZV diagnosed in our laboratory in pediatric and adult hematological patients between 2007 and 2010. METHODS: Patient charts were reviewed to document patient and disease characteristics. VZV loads were determined in all available clinical samples from the day of diagnosis and thereafter. Persistent VZV infection was defined as a VZV infection that lasted at least 7 days. Analysis of resistance was performed in all patients with persistent VZV infection by sequence analysis of viral thymidine kinase and DNA polymerase genes. RESULTS: In total, 89 episodes occurred in 87 patients, of whom 65 were recipients of an allogeneic HSCT. Follow-up samples were available in 54 episodes. Persistent VZV was demonstrated in 32 of these episodes (59%). Complications occurred in 16 of the persistent episodes (50%) vs 2 of 22 nonpersistent episodes (9%). Mutations possibly associated with resistance were found in 27% of patients with persistent VZV, including patients with treatment-unresponsive dermatomal zoster that progressed to severe retinal or cerebral infection. CONCLUSIONS: In hematological patients, VZV-related complications occur frequently, especially in persistent infections. Antiviral resistance is a relevant factor in persistent infections and needs to be investigated in various affected body sites, especially when clinical suspicion of treatment failure arises.

Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR.

BACKGROUND: Susceptibility testing of herpes simplex virus type 1 (HSV-1) is traditionally performed by a plaque reduction assay (PRA), but this is labor intensive, time consuming and has a manual read out. OBJECTIVES: The goal of this study was to develop an internally controlled real time PCR-based phenotypical susceptibility test for HSV-1 that is suitable for use in a clinical diagnostic setting. STUDY DESIGN: A DNA reduction assay (DRA) was developed and validated on a test panel of 26 well-characterized isolates of varying susceptibility to aciclovir or foscarnet, including low-level resistant isolates. The DRA consisted of pre-culture of a clinical sample for 48 h and subsequent culture in the presence of antivirals for 24 h. Viral DNA concentration in the culture lysates was measured by an internally controlled quantitative real-time HSV-1 PCR and corrected for cell count and lysis by beta-globin PCR. DRA results were compared to results from PRA and sequence analysis. RESULTS: DRA results were in accordance with PRA results for both aciclovir and foscarnet susceptibility and appeared to have good discriminative value for low-level resistance due to UL30 gene mutations. Although the direct application of DRA in clinical samples appeared not possible, short pre-culture of 48 h was sufficient and ensured results within a clinically relevant time frame of 5 days. CONCLUSIONS: DRA is an accurate, rapid and easy to perform phenotypical susceptibility test for HSV-1.

Failure of pre-emptive treatment of cytomegalovirus infections and antiviral resistance in stem cell transplant recipients.

BACKGROUND: Treatment of cytomegalovirus (CMV) infections after stem cell transplantation (SCT) does not always lead to a rapid viral response. The causes of treatment failure may be either viral resistance or immunological failure to control viral replication. This study investigated the response to pre-emptive treatment in CMV infections in order to define risk factors for treatment failure, including the role of antiviral resistance. METHODS: Adult recipients of allogeneic T-cell depleted SCT were studied retrospectively (n=92). CMV infections were treated with (val)ganciclovir according to a CMV DNA-load-based pre-emptive strategy. Treatment failure was defined as a CMV DNA load of 1,000 copies/ml or more after at least 2 weeks of treatment. Resistance was analysed by nucleotide sequence analysis of the UL97 and UL54 genes in the first CMV DNA-positive sample and in samples during treatment failure. RESULTS: Treatment failure occurred in 26 of the 47 pre-emptively treated patients (55%) and in 39 of 86 (45%) treatment episodes. The risk of treatment failure was increased during first treatment episodes (P=0.01) and during the use of immunosuppressive medication (P=0.02). Antiviral resistance was found in only 1 patient (4%) with treatment failure. CONCLUSIONS: A slow response to pre-emptive antiviral treatment occurred frequently in CMV infections in SCT recipients. Antiviral resistance was observed but played a minor role in treatment failure.

Mass spectrometry-based comparative sequencing to detect ganciclovir resistance in the UL97 gene of human cytomegalovirus.

BACKGROUND: Persistent infections with herpesviruses such as human cytomegalovirus (HCMV) frequently occur after solid organ or stem cell transplantation, and are due to either failure of the host to immunologically control the virus or emerging resistance of the virus to the antiviral drug(s) used. Antiviral therapy can be guided by viral drug susceptibility testing based on screening for known resistance-inducing mutations in the viral genome. Mass spectrometry-based comparative sequence analysis (MSCSA) might be advantageous for this purpose because of its suitability for semi-automation. OBJECTIVES: The applicability of MSCSA to detect sequence polymorphisms and drug resistance-inducing mutations in the HCMV genome was investigated. STUDY DESIGN: We analyzed the 3' part of the HCMV UL97 gene, which encodes the kinase that is activated by the commonly used anti-HCMV drug ganciclovir. Sequences obtained by MSCSA of material from HCMV-infected patients (43 samples) and the HCMV type strain were compared to conventional cycle sequencing results. RESULTS: In 94.1% of all samples the results obtained by MSCSA of the UL97 gene were identical to those from conventional cycle sequencing. The threshold to detect mutant sequences in a mixture with wild-type material was 20% using either technique. Furthermore, MSCSA was successfully applied to study the development of drug resistance in a patient who developed encephalitis due to ganciclovir-resistant HCMV. CONCLUSIONS: MSCSA was found to be equally accurate compared to conventional cycle sequencing in the analysis of UL97 of HCMV.

Preemptive versus sequential prophylactic-preemptive treatment regimens for cytomegalovirus in renal transplantation: comparison of treatment failure and antiviral resistance.

BACKGROUND: Cytomegalovirus (CMV) infections after transplantation are commonly treated using a prophylactic or preemptive regimen with (val)ganciclovir. It remains unclear, which approach is most effective in preventing CMV disease in D+R- patients. The aim of this retrospective study was to compare the treatment response and antiviral resistance in CMV infections between two treatment regimens in D+R- renal transplant recipients. METHODS: Before 2006, a preemptive treatment regimen with valganciclovir was applied (42 patients). From 2006 onwards, patients first received prophylaxis with valganciclovir for 90 days, followed by a preemptive regimen (29 patients). CMV infections were monitored by regular determination of the CMV DNA load in plasma. Patient charts were reviewed for antiviral treatment data, and resistance was analyzed by nucleotide sequence analysis of the UL97 and UL54 genes in CMV DNA-positive samples. RESULTS: Treatment failure, defined as a CMV DNA load more than or equal to 1000 copies/mL after at least 2 weeks of treatment, occurred less frequently in the prophylaxis cohort than in the preemptive cohort (14% vs. 71%, P<0.001). No CMV end-organ disease occurred in either cohort. Resistant viral isolates were found during treatment in one patient in the prophylaxis cohort versus in three patients in the preemptive group. All CMV infections with resistant virus were cleared without switch of (val)ganciclovir treatment. CONCLUSIONS: Treatment failure of CMV infections occurred less frequently in D+R- renal transplant patients on a sequential prophylaxis-preemptive regimen than in patients on a purely preemptive regimen. Antiviral resistance was observed infrequently and apparently played a minor role in treatment failure.