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The purpose of this study was to evaluate the performance of laboratories for the detection and quantification of human herpesvirus 6 (HHV-6) by an external quality assessment (EQA) evaluation. The HHV-6 EQA panel consisted of eight samples containing various concentrations of HHV-6 type A (strain GS) or type B (strain Z29), two samples containing...
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Universal screening of congenital cytomegalovirus (CMV) infection without symptoms at birth seems to be necessary to detect late-onset neurodevelopmental sequelae. Congenital CMV infection, as demonstrated by isolation of the virus within the first week of life, was diagnosed in 37 (0.31%) of 11,938 infants born between 1977 and 2002 in the city of...
The purpose of this investigation was to describe a distribution of cytomegalovirus (CMV) single and multiple genotypes among infected pregnant women, their fetuses, and newborns coming from Central Poland, as well as congenital cytomegaly outcome. The study involved 278 CMV-seropositive pregnant women, of whom 192 were tested for viral DNAemia. Hu...
Human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV) DNAs were quantified by real-time PCR assays in blood and saliva obtained from 50 patients with acute leukemia at the time of diagnosis (50 of each matrix), aplasia (65 of each matrix), remission (55 of each matrix), and relapse (20 of each matrix) to evaluate which biological matrix was...
Citations
... Available PCR tests do not always discriminate between HHV-6A and HHV-6B, which is helpful when evaluating patients if feasible [3]. Inter-assay variability has been observed, though the development of a World Health Organization (WHO) international standard for HHV-6B PCR can facilitate comparability of results between studies [75,76]. The implementation of this new standard by major diagnostic labs will be a key step towards standardization and moving the field forward. ...
HHV-6B reactivation affects approximately half of all allogeneic hematopoietic cell transplant (HCT) recipients. HHV-6B is the most frequent infectious cause of encephalitis following HCT and is associated with pleiotropic manifestations in this setting, including graft-versus-host disease, myelosuppression, pneumonitis, and CMV reactivation, although the causal link is not always clear. When the virus inserts its genome in chromosomes of germ cells, the chromosomally integrated form (ciHHV6) is inherited by offspring. The condition of ciHHV6 is characterized by the persistent detection of HHV-6 DNA, often confounding diagnosis of reactivation and disease—this has also been associated with adverse outcomes. Recent changes in clinical practice in the field of cellular therapies, including a wider use of post-HCT cyclophosphamide, the advent of letermovir for CMV prophylaxis, and the rapid expansion of novel cellular therapies require contemporary epidemiological studies to determine the pathogenic role and spectrum of disease of HHV-6B in the current era. Research into the epidemiology and clinical significance of HHV-6B in chimeric antigen receptor T cell (CAR-T cell) therapy recipients is in its infancy. No controlled trials have determined the optimal treatment for HHV-6B. Treatment is reserved for end-organ disease, and the choice of antiviral agent is influenced by expected toxicities. Virus-specific T cells may provide a novel, less toxic therapeutic modality but is more logistically challenging. Preventive strategies are hindered by the high toxicity of current antivirals. Ongoing study is needed to keep up with the evolving epidemiology and impact of HHV-6 in diverse and expanding immunocompromised patient populations.
... Quantitative real-time PCR (qPCR) may be useful for distinguishing active HHV-6 infection from these cases (23,24). Various qPCR assays for measuring HHV-6 DNA load are available, and some of them can differentiate between HHV-6A and HHV-6B DNA (28)(29)(30)(31)(32)(33)(34)(35). ...
... With the increasing number of patients undergoing HSCT or SOT, the need for quantita tive detection and differentiation of HHV-6A and HHV-6B DNA has become increasingly evident. In recent decades, several qPCR assays that can differentiate between HHV-6A and HHV-6B DNA, including the RealStar assay, have been developed and are routinely used in diagnostic laboratories (28,(31)(32)(33). In the present study, we evaluated the performance of the SMG assay, a newly developed qPCR assay, and compared the results to those of the RealStar assay. ...
The aim of this study was to compare the performance of the newly developed SMG HHV-6 Q Real-Time PCR Kit (SMG assay) with the RealStar HHV-6 PCR Kit (RealStar assay). The analytical sensitivity and specificity, linearity, and precision of the SMG assay were evaluated. The clinical performance of the SMG assay was assessed and compared with that of the RealStar assay using 207 clinical specimens (HHV-6A positive, n = 51; HHV-6B positive, n = 64; HHV-6A/B negative, n = 92). The limit of detection of the SMG assay was 2.92 log 10 copies/mL for HHV-6A DNA and 2.88 log 10 copies/mL for HHV-6B DNA. The linear range was determined to be 3.40–9.00 log 10 copies/mL for both viruses. Intra- and inter-assay variability were below 5% at concentrations ranging from 4 to 9 log 10 copies/mL. No cross-reactivity was observed with the 25 microorganisms included in the specificity panel. The clinical sensitivity and specificity of the SMG and RealStar assays compared to in-house polymerase chain reaction and sequencing were as follows: SMG assay, 98.0% and 100% for HHV-6A DNA, respectively, and 96.9% and 100% for HHV-6B DNA, respectively; RealStar assay, 98.0% and 100% for HHV-6A DNA, respectively, and 90.6% and 100% for HHV-6B DNA, respectively. The correlation coefficients between viral loads measured by the two assays were 0.948 and 0.975, with mean differences of 0.62 and 0.32 log 10 copies/mL for HHV-6A and HHV-6B DNA, respectively. These results demonstrate that the SMG assay is a sensitive and reliable tool for the quantitative detection and differentiation of HHV-6A and HHV-6B DNA.
IMPORTANCE
Quantitative real-time PCR (qPCR) that can distinguish between HHV-6A and HHV-6B DNA is recommended for diagnosis of active infection. The SMG HHV-6 Q Real-Time PCR Kit (SMG assay) is a newly developed qPCR assay that can differentiate between HHV-6A and HHV-6B DNA; however, little is known about its performance. In this study, we assessed the performance of the SMG assay and compared it with that of a commercially available qPCR assay, the RealStar HHV-6 PCR Kit (RealStar assay). The SMG assay demonstrated excellent analytical sensitivity and specificity, precision, and linearity. Furthermore, the viral loads measured by the SMG assay were highly correlated with those measured by the RealStar assay. Our results suggest that the SMG assay is a useful diagnostic tool for quantitative detection and differentiation of HHV-6A and HHV-6B DNA.
... Once a positive result is obtained, however, thresholds for the reduction of immunosuppression or initiation of antiviral therapy may vary widely based on the source of the hematopoietic stem cells (e.g., unrelated cord blood transplant), clinical context (e.g., whether the patient is symptomatic versus asymptomatic), as well as institution-specific protocols [10,[13][14][15][16][17]. The challenge in setting a clinical threshold is compounded, at least in part, due to the lack of harmonization among different HHV-6 qPCR assays [18,19]. Nevertheless, after immunosuppression has been adjusted or antiviral therapy initiated, monitoring patients for the clearance of HHV-6 DNA in blood, and if serially collected, CSF, is generally accepted [3,11,12]. ...
Background:
Quantitation of human herpesvirus-6 (HHV-6) DNA in clinical specimens is important for the diagnosis and management of HHV-6-associated infection and reactivation in immunocompromised patients, particularly transplant recipients.
Methods:
The analytical performance of the Altona RealStar ASR HHV-6 qPCR on the semi-automated AltoStar AM16 system was assessed using HHV-6 reference material in plasma and cerebral spinal fluid (CSF). Qualitative and quantitative agreement was determined using 123 clinical EDTA plasma specimens tested using a laboratory-developed HHV-6 qPCR.
Results:
The 95% Lower Limit of Detection was 20 IU/mL [95% confidence interval (CI): 10 to 29] in plasma and 78 IU/mL (95% CI: 55 to 146) in CSF. The assay was linear from 7.0 to 2.0 log10 IU/mL in both matrices. Overall agreement of the RealStar ASR HHV-6 qPCR on the AltoStar AM16 with a laboratory-developed test was 95.9% (95% CI: 90.8 to 98.7). Passing-Bablok analysis of specimens quantifiable by both methods and at levels >1000 copies/mL revealed a regression line of Y = 1.00*X-0.20, with neither systematic (95% CI Y-intercept: -0.66 to 0.26) nor proportional (95% CI slope: 0.89 to 1.10) bias compared to the reference.
Conclusions:
The RealStar ASR HHV-6 qPCR on the AltoStar AM16 provides accurate quantitation for clinical monitoring of HHV-6 in immunocompromised hosts.
... For the sake of argument, we refer to a published multi-center study, in which two bulk samples with different human herpesvirus 6 load (6000 and 200 copies/mL) were distributed and analyzed by different laboratories with their in-house diagnostic qPCR assays [47]. As should be expected, all laboratories could detect the virus in the bulk sample with a viral load of 6000 copies/mL, whereas only 80% of the laboratories could report a correct qualitative result for the 200 copies/mL bulk sample. ...
In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.
... 22,23 Not all differentiate between HHV-6A and HHV-6B, and agreement between laboratories for HHV-6 DNA levels is poor. 22,24 However, a World Health Organization standard for HHV-6B DNA is now available (http://www.nibsc.org/documents/ ifu/15-266.pdf). ...
Of the two human herpesvirus 6 (HHV-6) species, human herpesvirus 6B (HHV-6B) encephalitis is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplant. Guidelines for the management of HHV-6 infections in patients with hematologic malignancies or post-transplant were prepared a decade ago but there have been no other guidelines since then despite significant advances in the understanding of HHV-6 encephalitis, its therapy, and other aspects of HHV-6 disease in this patient population. Revised guidelines prepared at the 2017 European Conference on Infections in Leukaemia covering diagnosis, preventative strategies and management of HHV-6 disease are now presented.
... Interlaboratory variation has been previously evaluated for several different quantitative real-time PCR-based assays [10,11,17,18]. Results were variable, especially for low concentration sample, but most assays obtained reproducible and acceptable results for high concentration samples. ...
... Indeed, we observed that the one laboratory using a fully automated Roche Cobas Ampliprep assay had a good performance. Finally, the UI design of this study may also be a factor in the observed variability because, differently from other PT studies [10,11,17,18], the laboratory inspection dates were unannounced, which very likely prevented the report of unrealistic and unreliable results by laboratories due to collusive cheating behaviors, such as those found in our previous PT study [21]. ...
Sensitive and accurate hepatitis C virus (HCV) RNA quantification is essential for the management of chronic hepatitis C therapy. Currently, in Shanghai, China, many laboratories offer to quantify the HCV RNA level using various reverse transcription-polymerase chain reaction (rRT-PCR) assays, but the degree of proficiency may vary between them. The objective of this study was to realistically evaluate the performance of clinical laboratories for HCV RNA quantification by a new way of proficiency test program (unannounced inspection). Inactivated chimeric influenza viral particles (CIVP) encapsulating specific RNA sequences of HCV were prepared as positive samples. The sample panel, which consisted of two negative and eight positive samples with different concentrations of CIVP (25 IU/ml to 2.5×10⁶ IU/ml), was distributed to 40 clinical laboratories for HCV RNA quantification. The results reported by the participating laboratories were compared and scored. It was found that 75 % (30/40) of the laboratories obtained an acceptable or better performance score, while the other 10 laboratories had room for improvement. Reported results for individual samples ranged from 125 IU/ml (minimum) to 6.0×10⁴ IU/ml (maximum). The greatest variation was observed for samples with a relatively low concentration. Interlaboratory variability among replicate samples was significantly greater than intralaboratory variability (p < 0.05). Our results demonstrate that there is room for some laboratories to improve their performance in the quantification of HCV RNA in Shanghai. Additionally, this study underlines the importance of unannounced inspection for realistically monitoring the quality of diagnostic laboratories.
... A review of the PCR methods used in 46 recently published papers (2014 to 2017) revealed the use of 17 different primer sets at multiple locations throughout the genome (locations U6, U12, U13, U22, U27, U31, U32, U38, U41, U57, U65, U66, U67, U69, U90, U95, and U100), with only the U31, U65, and U66 primers used more than twice. Not surprisingly, crosslaboratory proficiency testing has shown differences in quantitation as high as 4 log units (24,25). Previous studies have identified the critical role that standardized materials play in the ability to establish clinical viral load cutoffs and assay sensitivity and to compare results between laboratories (24,(26)(27)(28)(29)(30). ...
Quantitative PCR is the diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of HHV-6A/B is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies. 11 of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1254 bp and 983 bp, respectively. Average copy number measured between 5-10X copies relative to the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and >5.5kb region from HHV-6B from U41-U43 that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-U24 region and U94/95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different labs on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.
IMPORTANCE
Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very accuracy through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well-known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6A/B biology and the standard selection process.
... All the commercial assays reported correct results, compared to 96.1% of the in-house real-time assays and conventional in-house assays, suggesting that commercial assays may have a higher sensitivity. 71 Additionally, HHV-6 PCR results are variably reported as copies/mL plasma or per one million peripheral blood lymphocytes. 72 Clinically significant HHV-6 DNA levels remain to be determined in liver transplant recipients 26,59,63 . ...
Human herpesvirus 6 (HHV-6A and HHV-6B) can cause primary infection or reactivate from latency in liver transplant recipients, which can result in a variety of clinical syndromes, including fever, hepatitis, encephalitis, and higher rates of graft dysfunction as well as indirect effects including increased risks of mortality, CMV disease, hepatitis C progression and greater fibrosis scores. Although HHV-6 infection is currently diagnosed by quantifying viral DNA in plasma or blood, biopsy to demonstrate histopathologic effects of HHV-6 remains the gold standard for diagnosis of end-organ disease. HHV-6 reactivation may be restricted to the infected organ with no evidence of active infection in the blood. HHV-6 infections in liver transplant patients are mostly asymptomatic, but clinically significant tissue-invasive infections have been treated successfully with ganciclovir, foscarnet, or cidofovir. Inherited chromosomally-integrated HHV-6 (ciHHV-6), in either the recipient or the donor organ, may create confusion about systemic HHV-6 infection. Recipients with inherited ciHHV-6 may have an increased risk of opportunistic infection and graft rejection. This article reviews the current scientific data on the clinical effects, risk factors, pathogenesis, diagnosis, and treatment of HHV-6 infections in liver transplant recipients. This article is protected by copyright. All rights reserved.
... Despite this, our findings support a quantitative association between HHV-6 viral load and the endpoints of interest. The lack of an international standard for HHV-6 DNA measurement precludes extrapolation of quantitative levels to other studies, as inter-laboratory correlation is known to be poor 27 . Lack of CSF HHV-6 testing in all patients with delirium was another limitation. ...
Human herpesvirus 6B (HHV-6B) frequently reactivates after cord blood transplantation (CBT). We previously reported an association between HHV-6B reactivation and delirium after hematopoietic cell transplantation. In this prospective study, 35 CBT recipients underwent twice-weekly plasma PCR testing for HHV-6 and thrice-weekly delirium assessment until day 84. There was a quantitative association between HHV-6B reactivation and delirium in univariable (odds ratio, 2.88; 95% confidence interval (CI), 0.97-8.59) and bivariable models. In addition, intensified prophylaxis with high-dose valacyclovir mitigated HHV-6B reactivation (adjusted hazard ratio, 0.39; 95% CI, 0.14-1.08). Larger trials are needed to explore the utility of HHV-6B prophylaxis after CBT.Bone Marrow Transplantation advance online publication, 29 June 2015; doi:10.1038/bmt.2015.154.
... he development of infections that remain a concerning cause of post - transplant morbidity and mor - tality ( Cordonnier 2008 ) . Viral infections are frequent after HSCT and may be life threatening , especially when affecting the lung , liver or central nervous system in al - logeneic HSCT and solid organ recipients ( Boutolleau et al . 2003 , de Pagter et al . 2008a , Schonberger et al . 2010 , Pollack et al . 2011 , Gotoh et al . 2014 ) . Human her - pesvirus ( HHV ) appears to play an important a role in this setting ( Jenkins et al . 2002 , Razonable & Paya 2003 , Kalpoe et al . 2006 , Ogata 2009 , Al Fawaz et al . 2014 ) . HHV - 6 was first isolated by Salahuddin et al . ( 1986 ) from peripheral blood mononuclear cells o ...
... 2014 ) , but the detection by real - time quantita - tive polymerase chain reaction ( qPCR ) ( TaqMan ® tech - nology based ) in HSCT recipients has not been systemat - ically performed in Brazilian patients . The development of diagnostics and immunology methods for HHV - 6 detection can provide better surveillance of HHV - 6 re - activation ( de Pagter et al . 2008b , 2010 , Betts et al . 2011 , Gerdemann et al . 2013 , Leibovitch et al . 2014 ) . The aim of this study is to optimise a qPCR assay for HHV - 6 de - tection and quantification in plasma samples . ...
Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.
