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Objectives Results of earlier external quality assessment (EQA) rounds suggested remarkable differences in the sensitivity of SARS-CoV PCR assays. Although the test systems are intended to detect SARS-CoV-2 in individual samples, screening is often applied to sample pools to increase efficiency and decrease costs. However, it is unknown to what extent these tests actually meet the manufacturer’s specifications for sensitivity and how they perform when testing sample pools. Methods The sensitivity of assays in routine use was evaluated with a panel of positive samples in a round of a SARS-CoV-2 virus genome detection EQA scheme. The panel consisted of samples at or near the lower limit of detection (“weakly positive”). Laboratories that routinely test sample pools were asked to also analyze the pooled EQA samples according to their usual pool size and dilution method. Results All participants could detect a highly positive patient-derived sample (>106 copies/mL). Most (96%) of the test systems could detect at least 1,000 copies/mL, meeting the minimum acceptable benchmark, and many (94%) detected the vRNA in a sample with lower concentration (500 copies/mL). The false negative ratio increased to 16 and 26% for samples with 100 and 50 copies/mL, respectively. Conclusions The performance of most assays met or exceeded their specification on sensitivity. If assays are to be used to analyze sample pools, the sensitivity of the assay and the number of pooled samples must be balanced.

Potent immunosuppressive drugs have been introduced into clinical care for solid organ transplant recipients. It is now time to guide these drugs on an individual level to optimize their efficacy. An ideal tool simultaneously detects overimmunosuppression and underimmunosuppression, is highly standardized, and is straightforward to implement into routine. Randomized controlled interventional trials are crucial to demonstrate clinical value. To date, proposed assays have mainly focused on the prediction of rejection and were based on the assessment of few immune compartments. Recently, novel tools have been introduced based on a more integrated approach to characterize the immune function and cover a broader spectrum of the immune system. In this respect, the quantification of the plasma load of a highly prevalent and apathogenic virus that might reflect the immune function of its host has been proposed: the torque teno virus (TTV). Although TTV control is driven by T cells, other major immune compartments might contribute to the hosts' response. A standardized in-house polymerase chain reaction and a conformité européenne-certified commercially available polymerase chain reaction are available for TTV quantification. TTV load is associated with rejection and infection in solid organ transplant recipients, and cutoff values for risk stratification of such events have been proposed for lung and kidney transplantation. Test performance of TTV load does not allow for the diagnosis of rejection and infection but is able to define at-risk patients. Hitherto TTV load has not been used in interventional settings, but two interventional randomized controlled trials are currently testing the safety and efficacy of TTV-guided immunosuppression.

Background The accuracy of individual test results affects the reliability of epidemiological indicators during pandemics. Therefore, the performance of test facilities and test systems must be continuously monitored. In this respect, external quality assessment (EQA) schemes are unique data sources, and their providers may serve as valuable contact for non-EQA inquiries on technical-analytical topics. However, the epidemiological importance of EQA during pandemics has not yet been evaluated. Methods Seventeen publications on SARS-CoV-2 virus genome detection EQA schemes were evaluated for their content of information which was aggregated and classified as “relevant”, “conditionally relevant” or “imperceptibly relevant” for the quality of public health microbiology. The effort required to report on any missing information was estimated. Extra-EQA services of EQA providers were also assessed for their epidemiological relevance. Findings Publications contained six to nine of the ten information criteria classified as relevant for public health microbiology. The effort required to report missing information appeared low. Information classified as epidemiologically conditionally relevant was collected only from dedicated EQA rounds. Extra-EQA services from EQA providers seemed particularly important in times of a pandemic. Interpretation Professional operation of non-EQA services and competent design of EQA schemes by their providers ensure that test facilities receive the best possible support during pandemics and that relevant information on the performance of infection diagnostics is made available. The importance of EQA for public health emergency response is therefore evident. Funding None.

Objectives External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. Methods Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. Results A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (C t ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of C t values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. Conclusions Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.

BACKGROUND AND AIMS The apathogenic and torque teno virus (TTV) is associated with the state of immunosuppression in solid organ transplant recipients. After kidney transplantation, quantification of TTV viral load may serve as a risk stratification tool for allograft rejection and infectious events. Besides a robust and independent association between recipient age, sex and TTV load, respectively, limited data exists on other potential determinants of TTV load. This trial was designed to analyse the association between TTV and detailed recipient and donor baseline characteristics and clinical follow-up parameters. METHOD This retrospective analysis included all 386 consecutive adult recipients of a kidney allograft transplanted between 1 January 2016 and 30 June 2018 at the Medical University Vienna from the prospective observational TTV-POET trial (institutional review board approval number: 1785/2016; German Clinical Trials Registry number: DRKS00012335). For the present analysis, we included TTV measures before transplantation (d0), at month 1 (m1), at month 3 (m3) and at month 12 (m12) after transplantation. TTV DNA was extracted from patient plasma and assessed by real-time PCR with laboratory-developed primers. TTV load was associated with recipient and donor baseline characteristics and follow-up data. RESULTS The median recipient and donor age at transplantation was 55 years. A total of 135 (35%) kidney allograft recipients were female; 74 patients (19%) had a history of prior kidney transplantation; and 318 patients (82%) received a deceased donor kidney transplant. A total of 34 patients (9%) had pre-formed donor-specific antibodies (DSA); 19 patients (5%) received an AB0-incompatible transplant. For the whole cohort, 1-year patient survival was 95.6% and 1-year death censored graft survival was 94.8%. Baseline TTV load (d0) was lower in female recipients (P = 0.003) and higher in older recipients {β 0.033, [95% confidence interval (95% CI) 0.002–0.066]; P = 0.039}. Baseline TTV load was not associated with recipient body mass index and a history of immunological causes of end-stage renal disease and diabetes mellitus, respectively. TTV load within the first year after kidney transplantation did not show an association with the prevalence of preformed DSA, induction treatment, AB0-incompatibility and HLA-mismatch, respectively. Our trial detected an association between donor age and TTV load. Recipients from older donors showed higher TTV loads (m1, β 0.037, 95% CI 0.008–0.065, P = .01; m3, β 0.04, 95% CI 0.012–0.068, P = .005; m12, β 0.065, 95% CI 0.028–0.01, P = .001). There was no association between TTV load during the first year after transplantation and donor sex. When investigating the association between TTV load and clinical follow-up data, TTV levels showed a negative correlation with peripheral blood leukocyte and lymphocyte counts during the first year after transplantation [lymphocytes: m1, β –0.867, 95% CI –1.367–0.364, P = .01; m3, β –0.985, 95% CI –1.709–0.260, P = .008; m12, β –1.304, 95% CI –2.156–0.452, P = .003; data on leukocytes not shown). These findings might reflect the role of lymphocytes for TTV control. Additionally, TTV viral load negatively correlated with the estimated glomerular filtration rate (MDRD) within the first year after kidney transplantation (m1, β –0.043, 95% CI –0.066–0.02, P < .001; m3, β –0.024, 95% CI –0.049–0.001, P = .06; m12, β –0.078, 95% CI –0.111–0.045, P < .001). One might speculate that a higher eGFR might be a proxy for a healthier donor being more capable of controlling TTV infection. TTV viral load was associated with tacrolimus trough levels in month 12 after transplantation (β 1.262, 95% CI 0.0221–0.250; P = .019), but not with the amount of mycophenolic acid dosage. CONCLUSION This study revealed an association between TTV viral load and distinct baseline donor and recipient characteristics and clinical follow-up data, including kidney function. Further analysis, especially longitudinal assessment of TTV levels, may help to further dissect the interplay between TTV viral load and the state of immunosuppression in patients undergoing kidney transplantation.

BACKGROUND AND AIMS The nonpathogenic and ubiquitous Torque Teno virus (TTV) plasma load is associated with immunosuppression in solid organ transplant recipients. An optimal TTV range has been defined for risk stratification of graft rejection and infection in the first year post kidney transplant applying an in-house PCR. Recently, a commercial PCR—the TTV R-GENE® kit—has been CE certified for clinical use. The present study was designed to validate and refine the optimal TTV range applying the TTV R-GENE® kit. METHOD Patients and events were selected from the prospective TTV-POET trial, including all 628 consecutive adult recipients of a kidney allograft transplanted at the Medical University of Vienna, between January 2016 and July 2020. Patients were followed for 12 months post-transplant or until drop-out. TTV was quantified longitudinally by the TTV R-GENE® kit. The primary outcome was biopsy proven graft rejection and the secondary end-point was infection. RESULTS A total of 78 patients with 85 graft biopsies (rejection, n = 18) and 274 patients with 80 infectious events following TTV quantification after reaching steady state in month 3 post-transplantation were selected. TTV was quantified at a median of 17 days before rejection was diagnosed and 63 days before onset of infection. The risk for rejection decreased by 25% with every log level increase in TTV load [RR 0.75, 95% confidence interval (95% CI) 0.67–0.85; 7log10 c/mL a high specificity of 91% for infection was calculated. Multivariate modelling revealed an independent associated between TTV and rejection and infection, respectively. CONCLUSION These data support the value of TTV quantified by TTV R-GENE® for risk stratification of graft rejection and infection in the first year post kidney transplantation. The optimal TTV range defined within this study will be applied in an interventional randomized controlled trial to assess the safety of TTV-guided immunosuppression: the TTVguideIT trial.

Background: The detection of SARS-CoV-2 vRNA in clinical samples has relied almost exclusively on RT-qPCR as the gold standard test. Published results from various external quality assessments (“ring trials”) worldwide have shown that there is still a large variability in results reported for the same samples. As reference standards of SARS-CoV-2 RNA are available, we tested whether using standard curves to convert Ct values into copies/mL (cp/mL) improved harmonization. Methods: Nine laboratories using 23 test systems (15 of which were unique) prepared standard dilution curves to convert Ct values of 13 SARS-CoV-2 positive samples to cp/mL (hereafter IU/mL). The samples were provided in three rounds of a virus genome detection external quality assessment (EQA) scheme. We tested the precision and accuracy of results reported in IU/mL, and attempted to identify the sources of variability. Results: Reporting results as IU/mL improved the precision of the estimated concentrations of all samples compared to reporting Ct values, although some inaccuracy remained. Variance analysis showed that nearly all variability in data was explained by individual test systems within individual laboratories. When controlling for this effect, there was no significant difference between all other factors tested (test systems, EQA rounds, sample material). Conclusions: Converting results to copies/mL improved precision across laboratory test systems. However, it seems the results are still very specific to test systems within laboratories. Further efforts could be made to improve accuracy and achieve full harmonization across diagnostic laboratories.

The nonpathogenic and ubiquitous torque teno virus (TTV) is associated with immunosuppression in solid organ transplant recipients. Studies in kidney transplant patients proposed TTV quantification for risk stratification of graft rejection and infection. In this prospective trial (DRKS00012335) 386 consecutive kidney transplant recipients were subjected to longitudinal per‐protocol monitoring of plasma TTV load by polymerase chain reaction for 12 months posttransplant. TTV load peaked at the end of month 3 posttransplant and reached steady state thereafter. TTV load after the end of month 3 was analyzed in the context of subsequent rejection diagnosed by indication biopsy and infection within the first year posttransplant, respectively. Each log increase in TTV load decreased the odds for rejection by 22% (odds ratio [OR] 0.78, 95% confidence interval [CI] 0.62‐0.97; P = .027) and increased the odds for infection by 11% (OR 1.11, 95% CI 1.06‐1.15; P, This trial defines an optimal plasma level range of the nonpathogenic and ubiquitous torque teno virus, which reflects the immunosuppression of its host, to minimize the risk for rejection and infection in kidney allograft recipients in the first year after transplantation. Fernández‐Ruiz comments on page 1963.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe clinical disease in immunosuppressed patients and congenitally infected newborn infants. Viral envelope glycoproteins represent attractive targets for vaccination or passive immunotherapy. To extend the knowledge of mechanisms of virus neutralization, monoclonal antibodies (MAbs) were generated following immunization of mice with HCMV virions. Hybridoma supernatants were screened for in vitro neutralization activity, yielding three potent MAbs, 6E3, 3C11, and 2B10. MAbs 6E3 and 3C11 blocked infection of all viral strains that were tested, while MAb 2B10 neutralized only 50% of the HCMV strains analyzed. Characterization of the MAbs using indirect immunofluorescence analyses demonstrated their reactivity with recombinantly derived gH. While MAbs 6E3 and 3C11 reacted with gH when expressed alone, 2B10 detected gH only when it was coexpressed with gB and gL. Recognition of gH by 3C11 was dependent on the expression of the entire ectodomain of gH, whereas 6E3 required residues 1 to 629 of gH. The strain-specific determinant for neutralization by Mab 2B10 was identified as a single Met→Ile amino acid polymorphism within gH, located within the central part of the protein. The polymorphism is evenly distributed among described HCMV strains. The 2B10 epitope thus represents a novel strain-specific antibody target site on gH of HCMV. The dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. IMPORTANCE HCMV infections are life threatening to people with compromised or immature immune systems. Understanding the antiviral antibody repertoire induced during HCMV infection is a necessary prerequisite to define protective antibody responses. Here, we report three novel anti-gH MAbs that potently neutralized HCMV infectivity. One of these MAbs (2B10) targets a novel strain-specific conformational epitope on gH that only becomes accessible upon coexpression of the minimal fusion machinery gB/gH/gL. Strain specificity is dependent on a single amino acid polymorphism within gH. Our data highlight the importance of strain-specific neutralizing antibody responses against HCMV. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. In addition, the dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex.

Objectives Mutation-specific PCR assays have quickly found their way into laboratory diagnostics due to their capacity to be a fast, easy to implement and high-throughput method for the detection of known SARS-CoV-2 variants of concern (VoCs). However, little is known about the performance of such assays in routine laboratory analysis. Methods The results reported in a recent round of an external quality assessment (EQA) scheme for SARS-CoV-2 mutation-specific PCR were retrospectively analyzed. For the determination of individual variant-specific sequences as well as for the interpretation results for certain virus variants, correct, incorrect, and unreported results were evaluated, and their possible causes were investigated. Results A total of 34 laboratories participated in this study. For five samples containing the VoC Alpha + E484K, Beta, Gamma, Delta, or B.1.1.318 (as a variant of interest), 848 results for SARS-2-CoV mutation detection were reported, 824 (97.2%, range per sample 88–100%) of which were correct. Melting curve assays gave 99% correct results, real-time RT-qPCR 94%, microarray-based assays 100%, and MALDI-TOF MS 96%. A total of 122/167 (73%) reported results for SARS-CoV-2 variant determination were correct. Of the 45 inconclusive or incorrect results, 33 (73%) were due to inadequate selection of targets that did not allow identification of contemporary VoC, 11 (24%) were due to incorrect results, and one (3%) was due to correct results of mutation-specific PCR. Conclusions Careful and up-to-date selection of the targets used in mutation-specific PCR is essential for successful detection of current SARS-CoV-2 variants.

Supplemental Digital Content is available in the text., Background. Nonpathogenic torque teno viruses (TTVs) are highly prevalent in transplant recipients and associated with immunosuppression. Studies in kidney transplant patients have proposed assessment of TTV load for risk stratification of clinically overt graft rejection. The value of TTV quantification in the context of subclinical rejection has not been evaluated. Methods. In this prospective trial, 307 consecutive kidney transplant recipients were subjected to per-protocol monitoring of plasma TTV. TTV was analyzed in the context of protocol biopsies (n = 82), scheduled 1 year posttransplantation. Results. TTV load at the time of biopsy was lower in recipients with rejection (n = 19; according to Banff, including borderline changes suspicious for acute T cell-mediated rejection) than those without rejection (n = 63) whereby each log increase in TTV copies/mL decreased the risk for rejection by 9% (risk ratio 0.91, 95% confidence interval, 0.85-0.97; P = 0.004). Development of chronic lesions (cg, cv, ci, ct, ah, ptcml) was associated with the number of days with a TTV load

Background Human pegivirus 1 (HPgV1) may cause persistent infections in immunocompetent and immunosuppressed individuals. Its clinical relevance, however, has not been determined. Previous studies have described a higher prevalence of HPgV1 infection in organ transplant recipients compared to healthy controls, but its occurrence in lung transplant recipients (LTRs) and its association with immunosuppressive therapy has not been assessed. Objectives The aim of this study was to evaluate the prevalence and clinical significance of HPgV1 infection in LTRs, and to compare HPgV1 loads and kinetics to Torque Teno Virus (TTV) kinetics, which reflects the level of immunosuppression. Study design From each of 110 LTRs, five consecutive plasma samples were collected within the first year after transplantation and tested for HPgV1 RNA and TTV DNA loads by quantitative PCR. Data were related to demographic data and clinical parameters followed up for 3 years post transplantation. Results HPgV1 prevalence in LTRs was 18,2%. HPgV1 detection was significantly associated with younger age, but not with graft rejections or other microbial infections. The viral replication level remained unaffected by immunosuppressive therapy. This was in contrast to TTV loads which increased after initiation of immunosuppressive therapy, independent of the patients’ HPgV1 infection status. Conclusions In contrast to TTV, HPgV1 kinetics do not reflect the level of immunosuppression after lung transplantation, and there is no correlation between the replication of both persistent viruses in the post transplantation follow up. Thus the individual virus host interactions seem to differ substantially and require further investigation.

Background Drug-induced immunosuppression in kidney transplant recipients is crucial to prevent allograft rejection, but increases risk for infectious disease. Immunologic monitoring to tailor immunosuppressive drugs might prevent alloreactivity and adverse effects simultaneously. The apathogenic torque teno virus (TTV) reflects the immunocompetence of its host and might act as a potential candidate for a holistic monitoring. Methods We screened all 1010 consecutive patients from the prospective Vienna Kidney Transplant Cohort Study for availability of allograft biopsies and adequately stored sera for TTV quantification by polymerase chain reaction. Results Patients with acute biopsy-proven alloreactivity according to the Banff classification (n = 33) showed lower levels of TTV in the peripheral blood compared to patients without rejection (n = 80) at a median of 43 days before the biopsy. The risk for alloreactivity decreased by 10% per log level of TTV copies/mL (risk ratio, .90 [95% confidence interval, .84–.97]; P = .005). TTV levels >1 × 106 copies/mL exclude rejection with a sensitivity of 94%. Multivariable generalized linear modeling suggests an independent association between TTV level and alloreactivity. Conclusions TTV is a prospective biomarker for risk stratification of acute biopsy-proven alloreactivity in kidney transplant recipients and might be a potential tool to tailor immunosuppressive drug therapy., Torque teno virus level in peripheral blood is associated with the immunocompetence of its host. Low levels of torque teno virus after kidney transplantation might mirror insufficient immunosuppression and thus indicate deleterious alloreactivity in patients with acute graft dysfunction.

Objectives The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. Methods Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. Results A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. Conclusions In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.

Highlights • Implementation of SARS-CoV-2 genome testing by numerous laboratories in Austria • Broad range of combinations of nucleic acid extraction and RT-PCR methods • Variable analytical performance of different protocols • Good performance of some analytical setups • Potential for optimisation of pre-RT-PCR dilution steps, Background Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHOrecommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARSCoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. Methods For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. Results and Conclusion A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60%) detected all positive samples correctly as positive, but 25/67 tests (37%) did not detect the weakest positive sample (S3), and 3% reported S2 and S3 as falsenegative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.

Background High viral loads are observed in Torque Teno Virus (TTV) infection after hematopoietic stem cell transplantation (HSCT). We aimed to analyze the kinetics of plasma TTV-DNA load in pediatric patients who received immunosuppressive therapy and developed infection complications in the first 100 days after HSCT. Methods As a patient group; 113 plasma samples taken from 33 pediatric HSCT recipients at a time interval after transplantation and as a control group; 38 plasma samples from 38 children without known chronic disease were included in the study. Viral nucleic acid isolation was performed by using the NucliSENS easyMAG (bioMerieux, France) system. A laboratory designed quantitative polymerase chain reaction process was performed on 7300 Real-Time PCR system (Applied Biosystems, CA, USA) with the amplification mixture containing primer and probe sequences for the UTR gene region. Results TTV-DNA was detected in all patient's samples and the median viral load was calculated as 7.67 Log10 copies/mL (range: 2.84–9.59). In the control group, the TTV-DNA median viral load was calculated as 5.51 Log10 copies/mL (range: 2.50–7.04), except for one negative sample. A significant difference was observed between the control group and the patient group in terms of TTV viral load levels. In nine patients, a median 2.15 Log10 copies/mL viral load increase was observed at 31–60 days post-transplant compared to the pre-transplant period. Conclusion TTV-DNA levels should be closely monitored to understand the immune status of the first 100 days after transplantation and the effects of treatment regimens on patients with HSCT.

Background Human cytomegalovirus (HCMV) may cause severe infections in lung transplant recipients (LTRs). The impact of the host antibody (AB)-dependent cytotoxicity (ADCC) on HCMV is still unclear. Therefore, we analyzed the AB-response against HCMV glycoprotein B (gB) and the pentameric complex (PC) and the ADCC response in HCMV-seropositive (R+) LTRs and in seronegative recipients of positive organs (D+/R−). Methods Plasma samples were collected from 35 R+ and 28 D+/R− LTRs for 1 (R+) or 2 (D+/R−) years posttransplantation and from 114 healthy control persons. The PC- and gB-specific ABs were assessed by enzyme-linked immunosorbent assay. The ADCC was analyzed by focal expansion assay and CD107 cytotoxicity assay. Results In R+ LTRs, significantly higher gB-specific AB levels developed within 1 year posttransplantation than in controls (immunoglobulin [Ig]G1, P Conclusions We show that a strong ADCC is elicited after transplantation and is especially based on gB-specific ABs.

Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO trimer and gH/gL/UL128-131 pentamer, are important for cell-free HCMV entry. While soluble NRP2-Fc (sNRP2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble platelet-derived growth factor receptor α-Fc (sPDGFRα-Fc) interacts with gO, thereby inhibiting infection of all cell types. Since gO is the most variable subunit, we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, and GT5 sequences in the bacterial artificial chromosome (BAC) TB40-BAC4-luc background (where luc is luciferase). All mutants were tested for fibroblast and epithelial cell infectivity, for virion content of gB, gH, and gO, and for infection inhibition by sPDGFRα-Fc and sNRP2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in gB and gH levels in mutant virions. All gO GT mutants except recombinant gO GT1c/3 displayed a nearly complete inhibition at 1.25 μg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%), and all experienced complete inhibition on fibroblasts (≥99%). While gO GT replacement did not influence sNRP2-Fc inhibition at 1.25 μg/ml on epithelial cells (97% to 99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblast inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope of >1.0), sNRP2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0, suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2 in a cell type-dependent manner. IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism, as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the amounts of trimer in virions and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information for the future development of anti-HCMV therapeutic compounds based on sPDGFRα.

Human cytomegalovirus (HCMV) causes severe infections in transplant recipients. The significance of the HCMV-specific antibody (Ab) response in limiting HCMV replication is not clear. Therefore, we analyzed the HCMV-specific subclass Ab profile in lung transplant recipients (LTRs) and its association with the genomic immunoglobulin G (IgG) heavy-chain variants GM3/17 and HCMV DNAemia.We determined HCMV-specific total IgG, IgG1 and IgG3 Ab levels by enzyme-linked immunoassay and HCMV-DNAemia by quantitative polymerase chain reaction during post-transplant follow-up in 57 LTRs and, in 44 of these recipients, the genetic allotype marker 359a/g variants (reflecting GM3/17 allotypes) by genotyping.In seropositive LTRs there was a significant Ab response to HCMV viremia (p = 0.0005), especially when low HCMV DNA levels were detected (1,000 copies/ml: p = 0.0012; DNAemia1,000 copies/ml: p = 0.0516). In particular, IgG3 but not IgG1, increased with viremia (IgG3: p = 0.0004). IgG1 levels were significantly lower in patients with 359 g/g (GM3/3) than in those with 359 a/g (GM3/17) variant (p0.0001). Of note, the IgG3 increase with viremia occurred particularly in patients carrying the IgG1 low-level 359 g/g variant (p0.0002).These data suggest that the HCMV-specific Ab response, and especially the IgG3 subclass response, correlate significantly with HCMV replication after transplantation. The patients' GM 3/17 variant is significantly associated with their HCMV IgG subclass profile.

Background Different human cytomegalovirus (HCMV) strains may persistently coexist in the human host. In immunosuppressed patients infection with mixed HCMV populations was associated with a more severe course of infection. Congenital HCMV infection may lead to severe fetal disease and possibly mixed HCMV strain infections might have also impact on the clinical consequences for the newborn. Mixed HCMV strain populations were so far detected in saliva but only rarely in urine of congenitally infected newborns. Objectives We have therefore analyzed the extent of mixed HCMV genotype populations in urine of congenitally infected newborns using a highly sensitive deep sequencing method. Study design Twenty urine samples (17 initial and 3 follow-up samples) from 17 congenitally infected newborns with a median HCMV DNA load of 7.5 log 10 copies/ml were included. Deep sequencing was applied for gO (UL74) genotyping and quantitative real-time PCR assays were used for gB (UL55) and gH (UL75) genotyping. Results In none of the urine samples a gO genotype mixture was detected, although a mean of 10.000 sequence reads per amplicon was analyzed, which allows to explore gO genotypes down to less than 1% of the total gO sequences. Also only one gB genotype was detected in the patients' initial samples, while a gH genotype mixture was detected in one case using real time PCR with a sensitivity of 5% for minor populations. Conclusion Mixed HCMV genotype populations are only rarely found in urine of congenitally infected newborns even when highly sensitive HCMV genotyping methods are applied.

Purpose New biomarkers reflecting the degree of immunosuppression in transplant recipients are needed to provide an optimal balance between risk for rejection and risk of severe infections. Methods We prospectively measured plasma TTV-DNA dynamics in 365 lung transplant recipients (LuTR) transplanted between 1/2013 and 12/2020. Severe infections, acute cellular and humoral rejection, incidence of CLAD and malignancies were recorded and correlated with dynamic changes in TTV levels. Results Patient demographics are summarized in Table 1. 95% of patients had detectable TTV levels pre-transplant with a mean log value of 4.0±1.6. Post-transplant, all LuTR became TTV positive with a peak value of log9.6±0.6 (range 5.9-10.9). TTV peaked at a mean of 143±109 days after transplantation. Mean TTV PCR values after 3, 4 and 5 years were log6.9±1,4, log6,4±1,4 and log6.0±1.2, respectively. TTV loads were significantly lower in LuTR who had episodes of acute cellular rejection (TTV PCR Conclusion TTV PCR measurement is a potent tool to predict acute cellular rejection episodes, infectious complications and CLAD. It represents a promising tool to monitor individualized immunosuppressive therapy in near future.

Purpose Human Cytomegalovirus (HCMV) is an important viral pathogen in lung-transplant recipients (LTRs), which may cause generalized infection and tissue-invasive disease. The risk for HCMV-replication depends on the donor (D) and recipient (R) serostatus as well as on HCMV-specific immune responses such as HCMV-specific antibodies (AB) and NK-cells, which may limit viral spread after lung-transplantation. In this study we analyzed the glycoprotein B (gB) and pentameric complex (PC)-specific AB-responses as well as the antibodies’ neutralizing and NK-cell activating effector functions following lung transplantation. Methods 35 R+, 26 D+/R- LTRs, who received a lung transplant between 2013 and 2016 at the Medical University of Vienna and 134 non-transplanted control individuals were included in the study. From each transplanted patient, follow-up plasma samples were collected in a three-month interval in a one (R+) or two (D+/R-) year period post-transplantation. PC-specific as well as gB-specific IgG titers were determined by ELISA. 50% neutralizing titers were determined with epithelial cells, fibroblasts and the laboratory strain TB40E-BAC4. The NK-cell response was determined using NK92-CD16a cells in a serum-dependent focal expansion and a CD107 cytotoxicity assay. Results The R+ LTRs reached a higher level of gB- but not of PC-specific IgG-titers in the first year post-transplantation compared to healthy seropositive individuals (IgG1 one year post-transplantation gB: p Conclusion From the present data it appears that R+ patients with pre-existing HCMV-specific ABs show a high level of NK-cell activation by gB-specific ABs.

Torque teno viruses (TTV) are small DNA-viruses, of the genus Alphatorquevirus, whose replication is linked to immune status. TTV load may be an indicator for efficacy of IS in lung transplant recipients (LTRs). In a prospective single-center-study 143 LTRs were followed up and tested by quantitative TTV-DNA PCR. Using multivariate Cox-regression contribution of TTV-load to the occurrence of severe infections, chronic lung allograft dysfunction (CLAD), acute cellular rejection (ACR), and death was assessed. During follow-up 28 (20%) patients developed infections with a rate of 7.7 per 100 patient-years (PY). The hazard-ratio (HR) associated with a one-log10 increase of TTV-load before the event was 5.05. CLAD occurred with a rate of 6.0%-PY. HR for a 1 log10 increase of the lowest TTV level before the event was 0.71 (CI: 0.54-0.93). TTV-load predicts clinical events and may be useful to optimize IS during the first years of follow-up of LTRs.

Background Drug-induced immunosuppression following kidney transplantation is crucial to prevent allograft rejection, but increases risk for infectious disease. Tailoring of drug dosing to prevent both rejection and infection is greatly desirable. The apathogenic and ubiquitous torque teno virus (TTV) reflects immunocompetence of the host and might be a potential candidate for immunologic monitoring. Methods To assess TTV as an infection biomarker, virus load was prospectively quantified in peripheral blood of 169 consecutive renal allograft recipients at the Medical University Vienna. Results Patients with infection showed higher TTV levels compared to patients without infection (4.2 × 108 copies/mL [interquartile range, IQR, 2.7 × 107-1.9 × 109] vs 2.9 × 107 [IQR 1.0 × 106-7.2 × 108]; P = .006). Differences in TTV load became evident almost 3 months before infection (median 77 days, IQR 19-98). Each log level of TTV copies/mL increased the odds ratio for infection by 23% (95% confidence interval 1.04-1.45; P = .014). TTV >3.1 × 109 copies/mL corresponded to 90% sensitivity to predict infections. Logistic regression demonstrated independent association between TTV levels and infection. Conclusions TTV quantification predicts infection after kidney transplantation and might be a potential tool to tailor immunosuppressive drug therapy.

The human virome plays an important role for the clinical outcome of lung transplant recipients (LTRs). While pathogenic viruses may cause severe infections, non-pathogenic viruses may serve as potential markers for the level of immunosuppression. However, neither the complexity of the virome in different compartments nor the dynamics of the virus populations posttransplantation are yet understood. Therefore, in this study the virome was analyzed by metagenomic sequencing in simultaneously withdrawn bronchoalveolar lavage (BAL) and plasma samples of 15 LTRs. In seven patients, also follow-up samples were investigated for abundance and dynamics of virus populations posttransplantation. Five eukaryotic and two prokaryotic virus families were identified in BAL, and nine eukaryotic and two prokaryotic families in plasma. Anelloviruses were the most abundant in both compartments, followed by Herpes- and Coronaviruses. Virus abundance was significantly higher in LTRs than in healthy controls (Kruskal-Wallis test, p

Glycoprotein O (gO) of the human cytomegalovirus (HCMV) is the critical subunit of the envelope trimer gH/gL/gO as it interacts with platelet-derived growth factor alpha receptor upon fibroblast entry, and triggers gB-mediated fusion for fibroblast and epithelial cell infection. Eight genotypes (GT) of the highly polymorphic gO gene are described, yet it is unclear whether the distinct GTs differ in their function. Thus, we aimed to elucidate potential functional differences between two highly diverse gO GTs in an otherwise genomically identical HCMV strain. Therefore, resident gO GT1c sequence of strain TB40-BAC4-luc was entirely replaced by gO GT4 of strain Towne and both, GT1c and GT4 viruses, were investigated for their growth properties in fibroblasts and epithelial cells. In addition, two conserved gO cysteines involved in gH/gL/gO stabilization were mutated to serine either in GT1c (C218S and C343S) or GT4 (C216S and C336S) and their effects on cell-free infectivity were assessed. GT4 viruses displayed a significantly enhanced epithelial cell tropism and this resulted in higher virus release upon replication in epithelial cells when compared to GT1c viruses. Further, when the two cysteines were individually mutated in gO GT1c no impairment in cell-free infectivity was observed. This, however, was in sharp contrast to gO GT4, in which both of the corresponding cysteine mutations led to a substantial reduction in cell-free infectivity which was even more pronounced upon mutation of GT4-C336 than of GT4-C216. In conclusion, these findings provide evidence that the two highly diverse gO genotypes, GT1c and GT4, differ in their functional properties as revealed by their different infection capacities for epithelial cells and by their different responsiveness to mutation of strictly conserved cysteine residues. Thus, it is likely that the gO heterogeneity influences cell-free infectivity of HCMV also in vivo which may have important implications for virus host transmission.

Antibody-mediated rejection (AMR) represents one of the cardinal causes of late allograft loss after kidney transplantation, and there is great need for noninvasive tools improving early diagnosis of this rejection type. One promising strategy might be the quantification of peripheral blood DNA levels of the highly prevalent and apathogenic Torque Teno virus (TTV), which might mirror the overall level of immunosuppression and thus help determine the risk of alloimmune response.To assess the association between TTV load in the peripheral blood and AMR, 715 kidney transplant recipients (median, 6.3 years posttransplantation) were subjected to a systematical cross-sectional AMR screening and, in parallel, TTV quantification.Eighty-six of these recipients had donor-specific antibodies and underwent protocol biopsy, AMR-positive patients (n = 46) showed only 25% of the TTV levels measured in patients without AMR (P = 0.003). In a generalized linear model, higher TTV levels were associated with a decreased risk for AMR after adjustment for potential confounders (risk ratio 0.94 per TTV log level; 95% confidence interval 0.90-0.99; P = 0.02).Future studies will have to clarify whether longitudinal assessment of TTV load might predict AMR risk and help guide the type and intensity of immunosuppression to prevent antibody-mediated graft injury.

Human cytomegalovirus (HCMV) infection is limited by HCMV-specific antibody functions. Here the association between the genetic marker (GM) 3/17 variants in the immunoglobulin G1 (IgG1) heavy chain constant region, virus neutralization, and natural killer (NK)-cell activation was investigated. In 100 HCMV-seropositive individuals, the GM3/17 polymorphism, serum 50% HCMV antibody neutralization titer (NT50), and in vitro HCMV-specific antibody NK-cell activation were assessed. The HCMV NT50 was higher in heterozygous GM3/17 persons than in GM3/3 persons (P = .0276). Furthermore, individuals expressing GM3/17 exhibited significantly higher NK-cell activation than persons carrying GM3/3 (P < .0001) or GM17/17 (P = .0095). Thus, persons expressing GM3/17 have potentially a selective advantage in HCMV defense.

Human cytomegalovirus (HCMV) does not exist as one defined virus genotype, but as a variety of different strains. Several studies have investigated the significance of specific viral genotypes for the clinical course of HCMV infection. Upon reinfection, patients may acquire additional HCMV strains, and infections with a mixture of HCMV strains appear to be quite common. The analysis of such mixed infections has become increasingly important, not only for investigating the clinical implications of mixed-genotype infections, but also for understanding the pathogenesis of subsequent reinfections with HCMV strains, and this is also of importance for HCMV vaccine development. This article summarizes the clinical implications of infection with individual HCMV genotypes and focuses on infection with mixed populations of HCMV strains.

Human cytomegalovirus (CMV) is a major cause of disease and transplant dysfunction in lung transplant recipients. Simultaneous emergence of more than one CMV-genotype can occur, and appears to be disadvantageous for the patient. In this study, the dynamics of individual CMV-genotypes in blood and lung was assessed within mixed CMV-genotype populations emerging after lung transplantation. In 69 plasma and 76 bronchoalveolar lavage samples of 16 lung transplant recipients with mixed CMV-genotype infections within the first year posttransplantation each of the major glycoprotein B (gB) and glycoprotein H (gH) genotypes was selectively quantified by genotype-specific quantitative TaqMan assays. The data obtained revealed that individually different genotype dynamics occurred for the individual patients and that the relative levels of the genotypes to each other may change over time. The quantitative development was independent of the specific gB–gH-genotype. In 10 of the 16 lung recipients the patient's individual genotype composition was the same in blood and lung. Genotype development during the follow-up was influenced by antiviral treatment. These data show for the first time that the CMV load used as diagnostic tool after transplantation is not always a constant entity but reflects the sum of the individual CMV-genotype dynamics developing over time. J. Med. Virol. 80:1405–1414, 2008. © 2008 Wiley-Liss, Inc.

Human cytomegalovirus (CMV) contains one of the largest genomes within the herpesvirus family and includes 12 multigene families. One of these is the RL11 family, whose members encode a characteristic domain, called RL11D. In the present study, the sequence variability of RL11D within the UL1, UL4, UL6, UL7, and UL10 genes of the RL11 family was investigated. For this purpose, these genes were analyzed in 70 clinical isolates obtained from urine, bronchoalveolar lavage, and blood of different patients. Substantial genetic variability among the clinical isolates was observed in all five genes analyzed. Based on phylogenetic analysis of variations in RL11D, the clinical isolates could be classified into three genotypes for UL1, 7, and 10 and, four genotypes for UL4 and 6. Further analysis showed statistically significant linkages between the following pairs of genes: UL6/UL7, UL4/UL7, UL1/UL4, and UL4/UL6. The data show that CMV strains exhibit a high interstrain variability in the RL11D domain of various RL11 family genes. Sequence variations, however, can be clearly grouped into a limited number of distinct genotypes. The genetic linkages found probably reflect a low frequency of recombination between genes that are arranged in close proximity on the viral genome.