Detection of Herpesviridae in Whole Blood by Multiplex PCR DNA-Based Microarray Analysis after Hematopoietic Stem Cell Transplantation
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France Debaugnies, Laurent Busson, Alina Ferster, Philippe Lewalle, Nadira Azzi, Mickael Aoun, Godelieve Verhaegen, Bhavna Mahadeb, Jérôme de Marchin, Olivier Vandenberg and Marie Hallin J. Clin. Microbiol. 2014, 52(7):2552. DOI: 10.1128/JCM.00061-14. Published Ahead of Print 14 May 2014.
Detection of Herpesviridae in Whole Blood by Multiplex PCR DNABased Microarray Analysis after Hematopoietic Stem Cell Transplantation Center for Molecular Diagnostic, iris-Lab, Iris-Brussels Public Hospital Network, Brussels, Belgiuma; Department of Microbiology, iris-Lab, Iris-Brussels Public Hospital Network, Brussels, Belgiumb; Department of Haematology and Oncology, Hôpital Universitaire Des Enfants Reine Fabiola, Brussels, Belgiumc; Department of Haematology, Jules Bordet Institute, Brussels, Belgiumd; Division of Infectious Diseases, Jules Bordet Institute, Brussels, Belgiume
Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests ⴝ 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test.
E
ither directly by causing end-organ damage or indirectly by facilitating bacterial or fungal infections, viral agents contribute to increased morbidity and mortality in hematopoietic stem cell transplantation (HSCT) (1–3). It is during the postengraftment period from day 30 until day 100, marked by cell-mediated and humoral immune deficiencies, that HSCT recipients are prone to devastating diseases generated by chronic or latent viruses of the Herpesviridae group. A close virological monitoring is therefore recommended as a preemptive strategy for early detection and treatment of patients experiencing viral primary infection and reactivation, as they are at risk of developing overt disease, especially from cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (4–6). This strategy is based on quantitative determination of viral load in blood once per week. In our institution, the cutoffs usually accepted for initiation of preemptive treatment are 10,000 copies for CMV and 1,000 copies for EBV in accordance with previously published data (7–9). As far as human herpesvirus 6 (HHV-6) infections are concerned, reactivations are associated with delayed platelet engraftment and early posttransplantation mortality. Furthermore, monitoring of HHV-6 viral load in blood could allow a differential diagnosis of graft-versus-host disease (GvHD)-like symptoms (10). Data to guide preemptive monitoring to prevent potential HHV-6-associated disease are lacking, and further studies are needed to reach more robust conclusions. Real-time quantitative PCR techniques have gradually replaced conventional methods of virus detection for the monitoring of transplanted patients. These molecular techniques allow an early detection and a reliable quantification of the virus and are
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more automatable and standardized. However, given the number of tests recommended in the guidelines, they represent mounting costs for the laboratories. New molecular techniques such as multiplex PCR amplification followed by microarray analysis are now being developed. They allow the simultaneous detection and identification of several viruses in one run. Microarrays offer an interesting alternative diagnostic tool, their potential benefits being the decrease in turnaround time and costs of analysis. Moreover, a larger panel of viruses can be tested simultaneously. Microarray techniques have already been evaluated in routine settings for the diagnosis of central nervous system and respiratory tract viral infections (11–16). To the best of our knowledge, microarrays have not yet been evaluated prospectively for the detection of Herpesviridae in a cohort of patients undergoing HSCT. In this study, we assessed the application of a commercial multiplex PCR DNA microarray, the Clart Entherpex kit, on a routine basis in the virological surveillance of transplanted patients. This kit allows the simultaneous qualitative detection and identifica-
Received 10 January 2014 Returned for modification 5 February 2014 Accepted 30 April 2014 Published ahead of print 14 May 2014 Editor: Y.-W. Tang Address correspondence to France Debaugnies, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.00061-14
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France Debaugnies,a Laurent Busson,b Alina Ferster,c Philippe Lewalle,d Nadira Azzi,c Mickael Aoun,e Godelieve Verhaegen,a Bhavna Mahadeb,b Jérôme de Marchin,a Olivier Vandenberg,b Marie Hallina
Detection of Herpesviridae by PCR DNA Microarray
tion of the Herpesviridae CMV, EBV, HHV-6, herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), HHV-7 and HHV-8. The aim of the study was to evaluate its analytical performances for the simultaneous qualitative detection of CMV, EBV, and HHV-6 in a cohort of patients undergoing HSCT compared to the PCR assays routinely used in our laboratory. MATERIALS AND METHODS
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RESULTS
Two hundred fifty-five blood samples were analyzed for the virological follow-up of 16 patients (8 males and 8 females) who had undergone allogeneic (n ⫽ 15) and autologous (n ⫽ 1) HSCT. For each patient, one sample was collected before transplantation and the other samples were collected posttransplantation. Nine patients were children (aged 16 years or less), and seven patients were adults. One patient died before transplantation. The monitoring period varied from 12 days for the last patient enrolled to 169 days for the first one enrolled. Among the 255 samples, 5 were completely excluded (4 samples with inhibited PCR for the 3 viruses and 1 sample with invalid microarray result). Diagnosis and management of viral infections by quantitative real-time PCR. Among the 250 remaining samples, 48% (n ⫽ 120) were completely negative and 52% (n ⫽ 130) were positive. Out of these positive samples, 82% (n ⫽ 106) were positive for a single virus and 18% (n ⫽ 24) were positive for several viruses. CMV was detected in 27% of the samples (66 out of 249; one more PCR was excluded from the analysis as it was inhibited even after sample dilution). EBV was detected in 19% of the samples (47 out of 244; 6 PCRs were excluded as 5 were inhibited and one sample was lacking). HHV-6 was detected in 14% (35 out of 246; 4 PCRs were excluded as they were inhibited). Fourteen episodes of active viral replication were observed (CMV, n ⫽ 6; EBV, n ⫽ 5; HHV-6, n ⫽ 3) in 9 patients: CMV for 3 of the patients, CMV and EBV for 3 other patients, EBV and HHV-6 for 2 of the patients, and HHV-6 for 1 of the patients. These episodes resulted in an adaptation of the antiviral therapy for 8 of the 9 patients. Given that the viral load was low and symptoms were absent in the last patient, his antiviral therapy was not modified. Performances of the microarray, the Clart Entherpex assay. Forty-eight percent of samples were completely negative (n ⫽ 120), and 52% (n ⫽ 130) were positive. Out of these positive samples, 65% (n ⫽ 84) were positive for a single virus and 35% (n ⫽ 46) were positive for several viruses. CMV was detected in 26% of the samples (64 out of 250). EBV was detected in 20% of the samples (50 out of 250). HHV-6 was detected in 17% (42 out of 250). Additionally, HHV-7 was detected in 48 samples from 9 patients. One sample was also positive for HSV-1 and confirmed by routine PCR assay; the other targeted viruses included in the range of the array were not found in any of the analyzed samples. The comparison of qualitative PCR DNA microarray and quantitative real-time PCR results is shown in Fig. 1. The sensitiv-
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Patients and samples. From November 2011 to May 2012, all new patients undergoing HSCT from the Jules Bordet Institute (adults) and the Queen Fabiola Children’s University Hospital (pediatrics), Brussels, Belgium, were prospectively enrolled in the study. At least one EDTA-anticoagulated whole-blood sample was collected per week for each patient, and these samples were tested with both real-time quantitative PCRs routinely used in the laboratory and the new multiplex PCR DNA microarray. Clinical data were prospectively collected during the evaluation in the daily report of hospitalization and during weekly clinical meetings. Laboratory data, including neutrophil and platelet counts, C-reactive protein levels, and positive microbiological results, were also collected. The study was approved by the institutional Ethical Committees of the two hospitals. DNA extraction. Total nucleic acid extraction was performed using the MagNA Pure LC instrument (Roche Diagnostics, Mannheim, Germany) with the total nucleic acid high-performance isolation kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Total nucleic acid was extracted from 200 l of EDTA-whole blood and recovered in 100 l of elution buffer. Aliquots were stored at ⫺80°C before use. Routine CMV, EBV, and HHV-6 viral load assessment by quantitative real-time PCR. CMV and EBV DNA viral loads were determined using the Dia-CMVQ-050 and Dia-EBVQ-050 kits (Diagenode Diagnostics, Liège, Belgium). HHV-6 DNA viral load was determined using an in-house real-time quantitative PCR, as described previously by Locatelli et al. (17). PCRs were performed on the ABI 7500 Fast PCR instrument (Applied Biosystems, Ghent, Belgium). All samples were tested in duplicate. Results were given in copy number/milliliter of whole blood. The lower limit of quantification is 800 copies/ml for CMV and EBV and 1,500 copies/ml for HHV-6. The lower limit of detection observed in our laboratory is 500 copies/ml of whole blood for CMV and EBV and 800 copies/ml for HHV-6. Simultaneous qualitative detection of CMV, EBV, and HHV-6 by multiplex PCR DNA microarray. The Clart Entherpex assay (Genomica, Coslada, Spain) was performed according to the manufacturer’s instructions. Briefly, in a first step, 5 l of total nucleic acid extract of each sample is amplified in two tubes containing different reaction mixtures. An internal control is added in each amplification tube to control amplification efficiency. The second step consists of hybridization of the biotinylated PCR products with target-specific binding probes. A microarray reader piloted by specific software performs the detection by colorimetric reaction and the interpretation of the results. The lower limit of reproducible detection, as determined by Lévêque et al. (11), is 250 copies/ml for CMV, 2,000 copies/ml for EBV, and 500 copies/ml for HHV-6. Statistical analyses. To evaluate the analytical performances of the microarray in the detection of CMV, EBV, and HHV-6 in blood samples, the sensitivity, specificity, and negative and positive predictive values were calculated considering the routine PCR assays as the reference method. The kappa statistics were calculated to evaluate the agreement between microarray and routine PCR assays. For each virus, the viral load levels measured by PCR in samples positive by microarray were compared using a Mann-Whitney U test to the viral load levels measured by PCR in samples negative by microarray. A P value of less than 0.05 was considered significant. Data were analyzed using GraphPad Prism 5 (GraphPad Software, Inc., USA). Definitions. “Herpesvirus infection” was defined as detection of nucleic acid of herpesvirus in blood sample. An “active viral replication” is confirmed by an increased viral load on the following sample(s) resulting
from a primary infection, a reinfection, or a reactivation of a latent virus. We used the term “intermittent viremia” in cases of detection of virus nucleic acid in the blood by PCR and/or microarray sporadically, at a low viral load, without an increased viral load on the next sample, which corresponded to a latent stage of infection. Patients with chromosomally integrated HHV-6 (ciHHV-6) were defined as persistent HHV-6 viral load over time greater than 5.5 log10 copies/ml of whole blood (18). Discrepant results between microarrays and routine PCR assays were interpreted as follows: positive PCR results, negative by microarray, were deemed “false negative”; negative PCR results, positive by microarray, were deemed “likely true positive” if either the previous or the next sample obtained from the same patient was positive by PCR, “possibly true positive” if the result was consistent with the serological status of the patient, and “false positive” in the other cases.
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(number of copies/ml of whole blood) are plotted on the y axis. The x axis indicates the positive (⫹) and negative (⫺) microarray results. The dashed lines represent the lower limits of quantification of PCR assays.
ity and specificity of the microarray were 83 and 95%, 85 and 95%, and 57 and 90% for CMV, EBV, and HHV-6, respectively. The kappa coefficients between the microarray and routine PCR assays were equal to 0.8 for CMV and EBV and 0.4 for HHV-6. Thirtythree false-negative results were observed in a total of 29 samples (Table 1). Regarding CMV (n ⫽ 11) and EBV (n ⫽ 7), all but one false-negative case were observed for viral loads below or around
the limit of quantification of the PCR. Only one sample had a viral load (CMV) above 1,000 copies/ml of whole blood with 2 positive replicates by PCR. CMV and EBV PCR-positive samples that were not detected by microarray had median viral loads significantly lower than those positive by PCR and microarray (852 copies/ml [500 to 3,439] versus 2,329 copies/ml [500 to 560,338], P ⬍ 0.05 for CMV; 500 copies/ml [500 to 575] versus 1,001 copies/ml [500
TABLE 1 Summary of the blood sample results obtained with the microarray assay compared to our routine PCR assaysa Result by microarray No. of samples Category by routine PCR
Virus positive
Virus negative
CMV Positive Negative
55 9
11 174
EBV Positive Negative
40 10
7 187
HHV-6 Positive Negative
20 22
15 189
a b
Kappa test (95% CI)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
No. of evaluable samplesb
0.79 (0.71–0.88)
83
95
86
94
249
0.78 (0.68–0.88)
85
95
80
96
244
0.43 (0.28–0.58)
57
90
48
93
246
Abbreviations: CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value. Samples with inhibited PCR or an invalid microarray or that were unavailable were excluded.
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FIG 1 Comparison of qualitative PCR DNA microarray and quantitative real-time PCR results (CMV, n ⫽ 249; EBV, n ⫽ 244; HHV-6, n ⫽ 246). The viral loads
Detection of Herpesviridae by PCR DNA Microarray
copies/ml of blood. (A) Undiluted sample. (B and C) Sample diluted 1:100 (B) and 1:1,000 (C) before the first multiplex PCR step of the microarray. (D and E) Sample diluted 1:100 (D) and 1:1,000 (E) before the hybridization step of the microarray.
to 49,972], P ⬍ 0.01 for EBV). Fifteen false-negative results were observed for HHV-6. Surprisingly, nine out of these 15 samples had, by PCR, extremely high levels of HHV-6 (above 6 log10 copies/ml of whole blood). HHV-6 PCR-positive samples that were not detected by microarray had median viral loads that were not statistically different from those of samples positive by both PCR and microarray (1.5 ⫻ 106 copies/ml [2,920 to 1.5 ⫻ 106] versus 47,666 [4,839 to 1.5 ⫻ 106]). To investigate the cause of these false-HHV-6-negative results, we tested if a hybridization could be observed when diluting one of these false-negative samples before or after performing the amplification step (dilution of the DNA extract versus dilution of the PCR product). Since the signal was recovered only when the sample was diluted before amplification (Fig. 2), we inferred that the amplification step was affected by a large amount of target. This “false-negative” phenomenon was observed in 9 out of the 12 HHV-6 viral loads above 6 log10 copies/ml collected during the study but was not observed with samples having similarly high CMV or EBV viral loads. Forty-one microarray-positive results were not confirmed by PCR (Table 1). Fifteen of them, corresponding to an early detection of reactivation or collected after the initiation of antiviral therapy, were classified as likely true positive (6 CMV and 9 HHV6). Thirteen results were possibly true positive (3 CMV and 10 EBV), consistent with the serological status of the patient. Thirteen results, all HHV-6 positive, were classified as false positive, serology not being available for these cases. DISCUSSION
Technology improvements in molecular diagnostic tests (possibility of automation, real-time PCR amplification, and availability of commercial kits) have promoted their implementation in the routine diagnostic virology laboratory. These more sensitive and specific as well as time- and labor-saving tests have mostly replaced conventional methods of virus detection (culture and serology), especially in the virological surveillance of transplanted patients for whom the timeliness of the results is a critical component. The main obstacle to the use of these techniques is their cost. In this context, the use of the multiplex PCR DNA microarray
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FIG 2 Microarray results for a sample with an HHV-6 viral load above 6 log10
Clart Entherpex kit for simultaneous qualitative detection of CMV, EBV, and HHV-6 in one run of testing could allow potential turnaround time reduction and cost savings. The aim of the study was to assess the performance of this microarray method in a routine clinical laboratory setting. In our cohort of patients, the microarray assay showed good performances for CMV and EBV detection compared to routine quantitative PCR. All clinically relevant episodes were associated with a positive microarray result. The few discrepant results were observed in cases presenting with low viral load (such as latent stage of infection, intermittent viremia, or early onset of an active replication) and thus had no major clinical consequences, i.e., no change in the antiviral therapy. The agreement was good for CMV and EBV but weaker for HHV-6 detection. Actually, 9 samples tested negative with the microarray had a very high viral load (⬎6 log10 copies/ml) by PCR. These results were observed for one patient with ciHHV-6 as well as for one patient with active reactivation. We believe that a very high HHV-6 viral load interferes with the amplification step of the microarray. This analytical glitch was even more problematic because, in these cases, the internal amplification controls were not inhibited as they should have been. We also suspect that coinfections, especially when quantities of the different viruses differ greatly, could affect the analytical sensitivity of the microarray and give false-negative results (data not shown). The multiplex PCR, the hybridization of PCR products, and the reading and interpretation of the microarray were achieved within 8 h. As the same extraction step was used for the microarray analysis and quantitative PCR analysis, the microarray could in theory easily be used as a weekly screening assay for the detection of Herpesviridae in the blood of transplanted patients. Each positive result can subsequently be followed by selective viral load measurement by a quantitative PCR. One of the main advantages of proceeding as such would be a reduction in the cost of analysis of around 22% (estimated on a retrospective series of 3,832 samples collected over 5 years for CMV, EBV, and HHV-6 detection [data not shown]). Another advantage of implementing this test would be that the wide range of viruses tested could help assess the pathogenicity of neglected viruses such as HHV-7, which was the most commonly identified virus in our cohort after CMV, EBV, and HHV-6. Moreover, the delay of response incurred by the implementation of one supplementary first-line test would not have a critical impact, seeing that samples would be analyzed only once or twice weekly in batches. However, the drawbacks of this algorithm would be that it would require a controlled workflow and that adding a screening test could increase the workload even if it reduces the total number of samples on which PCRs would be performed. However, as this test has analytical limitations that for the moment prevent its implementation in a routine setting, the gain over workload remains difficult to ascertain. To ensure the place of this microarray in routine laboratories, improvements should be made by the manufacturers regarding internal quality controls. Moreover, to make the most of its potential benefits for the follow-up of HSCT patients, the additional detection of adenovirus, parvovirus B19, and JC and BK polyomaviruses would be an interesting development which could alleviate the further workload generated. Conclusion. The evaluated multiplex PCR DNA microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV reactivations in the blood. How-
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9.
10.
ACKNOWLEDGMENTS This work was supported by a grant from the Iris-Recherche fund. We thank Marie-Jeanne Stouten for her skilled technical assistance.
11.
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ever, positive samples must be subsequently confirmed and the viral load must be quantified by PCR assays to obtain a more precise diagnosis. Several samples with high viral loads of HHV-6 testing negative with the microarray can be problematic both for the pretransplantation detection of chromosomally integrated HHV-6 and for follow-up of patients with active reactivation. Although it is promising, some characteristics of this diagnostic tool should be improved, particularly for quality control and a targeted virus panel, such that it could then be adapted to be used in a routine setting.
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France Debaugnies, Laurent Busson, Alina Ferster, Philippe Lewalle, Nadira Azzi, Mickael Aoun, Godelieve Verhaegen, Bhavna Mahadeb, Jérôme de Marchin, Olivier Vandenberg and Marie Hallin J. Clin. Microbiol. 2014, 52(7):2552. DOI: 10.1128/JCM.00061-14. Published Ahead of Print 14 May 2014.
Detection of Herpesviridae in Whole Blood by Multiplex PCR DNABased Microarray Analysis after Hematopoietic Stem Cell Transplantation Center for Molecular Diagnostic, iris-Lab, Iris-Brussels Public Hospital Network, Brussels, Belgiuma; Department of Microbiology, iris-Lab, Iris-Brussels Public Hospital Network, Brussels, Belgiumb; Department of Haematology and Oncology, Hôpital Universitaire Des Enfants Reine Fabiola, Brussels, Belgiumc; Department of Haematology, Jules Bordet Institute, Brussels, Belgiumd; Division of Infectious Diseases, Jules Bordet Institute, Brussels, Belgiume
Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests ⴝ 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test.
E
ither directly by causing end-organ damage or indirectly by facilitating bacterial or fungal infections, viral agents contribute to increased morbidity and mortality in hematopoietic stem cell transplantation (HSCT) (1–3). It is during the postengraftment period from day 30 until day 100, marked by cell-mediated and humoral immune deficiencies, that HSCT recipients are prone to devastating diseases generated by chronic or latent viruses of the Herpesviridae group. A close virological monitoring is therefore recommended as a preemptive strategy for early detection and treatment of patients experiencing viral primary infection and reactivation, as they are at risk of developing overt disease, especially from cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (4–6). This strategy is based on quantitative determination of viral load in blood once per week. In our institution, the cutoffs usually accepted for initiation of preemptive treatment are 10,000 copies for CMV and 1,000 copies for EBV in accordance with previously published data (7–9). As far as human herpesvirus 6 (HHV-6) infections are concerned, reactivations are associated with delayed platelet engraftment and early posttransplantation mortality. Furthermore, monitoring of HHV-6 viral load in blood could allow a differential diagnosis of graft-versus-host disease (GvHD)-like symptoms (10). Data to guide preemptive monitoring to prevent potential HHV-6-associated disease are lacking, and further studies are needed to reach more robust conclusions. Real-time quantitative PCR techniques have gradually replaced conventional methods of virus detection for the monitoring of transplanted patients. These molecular techniques allow an early detection and a reliable quantification of the virus and are
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more automatable and standardized. However, given the number of tests recommended in the guidelines, they represent mounting costs for the laboratories. New molecular techniques such as multiplex PCR amplification followed by microarray analysis are now being developed. They allow the simultaneous detection and identification of several viruses in one run. Microarrays offer an interesting alternative diagnostic tool, their potential benefits being the decrease in turnaround time and costs of analysis. Moreover, a larger panel of viruses can be tested simultaneously. Microarray techniques have already been evaluated in routine settings for the diagnosis of central nervous system and respiratory tract viral infections (11–16). To the best of our knowledge, microarrays have not yet been evaluated prospectively for the detection of Herpesviridae in a cohort of patients undergoing HSCT. In this study, we assessed the application of a commercial multiplex PCR DNA microarray, the Clart Entherpex kit, on a routine basis in the virological surveillance of transplanted patients. This kit allows the simultaneous qualitative detection and identifica-
Received 10 January 2014 Returned for modification 5 February 2014 Accepted 30 April 2014 Published ahead of print 14 May 2014 Editor: Y.-W. Tang Address correspondence to France Debaugnies, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.00061-14
p. 2552–2556
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France Debaugnies,a Laurent Busson,b Alina Ferster,c Philippe Lewalle,d Nadira Azzi,c Mickael Aoun,e Godelieve Verhaegen,a Bhavna Mahadeb,b Jérôme de Marchin,a Olivier Vandenberg,b Marie Hallina
Detection of Herpesviridae by PCR DNA Microarray
tion of the Herpesviridae CMV, EBV, HHV-6, herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), HHV-7 and HHV-8. The aim of the study was to evaluate its analytical performances for the simultaneous qualitative detection of CMV, EBV, and HHV-6 in a cohort of patients undergoing HSCT compared to the PCR assays routinely used in our laboratory. MATERIALS AND METHODS
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RESULTS
Two hundred fifty-five blood samples were analyzed for the virological follow-up of 16 patients (8 males and 8 females) who had undergone allogeneic (n ⫽ 15) and autologous (n ⫽ 1) HSCT. For each patient, one sample was collected before transplantation and the other samples were collected posttransplantation. Nine patients were children (aged 16 years or less), and seven patients were adults. One patient died before transplantation. The monitoring period varied from 12 days for the last patient enrolled to 169 days for the first one enrolled. Among the 255 samples, 5 were completely excluded (4 samples with inhibited PCR for the 3 viruses and 1 sample with invalid microarray result). Diagnosis and management of viral infections by quantitative real-time PCR. Among the 250 remaining samples, 48% (n ⫽ 120) were completely negative and 52% (n ⫽ 130) were positive. Out of these positive samples, 82% (n ⫽ 106) were positive for a single virus and 18% (n ⫽ 24) were positive for several viruses. CMV was detected in 27% of the samples (66 out of 249; one more PCR was excluded from the analysis as it was inhibited even after sample dilution). EBV was detected in 19% of the samples (47 out of 244; 6 PCRs were excluded as 5 were inhibited and one sample was lacking). HHV-6 was detected in 14% (35 out of 246; 4 PCRs were excluded as they were inhibited). Fourteen episodes of active viral replication were observed (CMV, n ⫽ 6; EBV, n ⫽ 5; HHV-6, n ⫽ 3) in 9 patients: CMV for 3 of the patients, CMV and EBV for 3 other patients, EBV and HHV-6 for 2 of the patients, and HHV-6 for 1 of the patients. These episodes resulted in an adaptation of the antiviral therapy for 8 of the 9 patients. Given that the viral load was low and symptoms were absent in the last patient, his antiviral therapy was not modified. Performances of the microarray, the Clart Entherpex assay. Forty-eight percent of samples were completely negative (n ⫽ 120), and 52% (n ⫽ 130) were positive. Out of these positive samples, 65% (n ⫽ 84) were positive for a single virus and 35% (n ⫽ 46) were positive for several viruses. CMV was detected in 26% of the samples (64 out of 250). EBV was detected in 20% of the samples (50 out of 250). HHV-6 was detected in 17% (42 out of 250). Additionally, HHV-7 was detected in 48 samples from 9 patients. One sample was also positive for HSV-1 and confirmed by routine PCR assay; the other targeted viruses included in the range of the array were not found in any of the analyzed samples. The comparison of qualitative PCR DNA microarray and quantitative real-time PCR results is shown in Fig. 1. The sensitiv-
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Patients and samples. From November 2011 to May 2012, all new patients undergoing HSCT from the Jules Bordet Institute (adults) and the Queen Fabiola Children’s University Hospital (pediatrics), Brussels, Belgium, were prospectively enrolled in the study. At least one EDTA-anticoagulated whole-blood sample was collected per week for each patient, and these samples were tested with both real-time quantitative PCRs routinely used in the laboratory and the new multiplex PCR DNA microarray. Clinical data were prospectively collected during the evaluation in the daily report of hospitalization and during weekly clinical meetings. Laboratory data, including neutrophil and platelet counts, C-reactive protein levels, and positive microbiological results, were also collected. The study was approved by the institutional Ethical Committees of the two hospitals. DNA extraction. Total nucleic acid extraction was performed using the MagNA Pure LC instrument (Roche Diagnostics, Mannheim, Germany) with the total nucleic acid high-performance isolation kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Total nucleic acid was extracted from 200 l of EDTA-whole blood and recovered in 100 l of elution buffer. Aliquots were stored at ⫺80°C before use. Routine CMV, EBV, and HHV-6 viral load assessment by quantitative real-time PCR. CMV and EBV DNA viral loads were determined using the Dia-CMVQ-050 and Dia-EBVQ-050 kits (Diagenode Diagnostics, Liège, Belgium). HHV-6 DNA viral load was determined using an in-house real-time quantitative PCR, as described previously by Locatelli et al. (17). PCRs were performed on the ABI 7500 Fast PCR instrument (Applied Biosystems, Ghent, Belgium). All samples were tested in duplicate. Results were given in copy number/milliliter of whole blood. The lower limit of quantification is 800 copies/ml for CMV and EBV and 1,500 copies/ml for HHV-6. The lower limit of detection observed in our laboratory is 500 copies/ml of whole blood for CMV and EBV and 800 copies/ml for HHV-6. Simultaneous qualitative detection of CMV, EBV, and HHV-6 by multiplex PCR DNA microarray. The Clart Entherpex assay (Genomica, Coslada, Spain) was performed according to the manufacturer’s instructions. Briefly, in a first step, 5 l of total nucleic acid extract of each sample is amplified in two tubes containing different reaction mixtures. An internal control is added in each amplification tube to control amplification efficiency. The second step consists of hybridization of the biotinylated PCR products with target-specific binding probes. A microarray reader piloted by specific software performs the detection by colorimetric reaction and the interpretation of the results. The lower limit of reproducible detection, as determined by Lévêque et al. (11), is 250 copies/ml for CMV, 2,000 copies/ml for EBV, and 500 copies/ml for HHV-6. Statistical analyses. To evaluate the analytical performances of the microarray in the detection of CMV, EBV, and HHV-6 in blood samples, the sensitivity, specificity, and negative and positive predictive values were calculated considering the routine PCR assays as the reference method. The kappa statistics were calculated to evaluate the agreement between microarray and routine PCR assays. For each virus, the viral load levels measured by PCR in samples positive by microarray were compared using a Mann-Whitney U test to the viral load levels measured by PCR in samples negative by microarray. A P value of less than 0.05 was considered significant. Data were analyzed using GraphPad Prism 5 (GraphPad Software, Inc., USA). Definitions. “Herpesvirus infection” was defined as detection of nucleic acid of herpesvirus in blood sample. An “active viral replication” is confirmed by an increased viral load on the following sample(s) resulting
from a primary infection, a reinfection, or a reactivation of a latent virus. We used the term “intermittent viremia” in cases of detection of virus nucleic acid in the blood by PCR and/or microarray sporadically, at a low viral load, without an increased viral load on the next sample, which corresponded to a latent stage of infection. Patients with chromosomally integrated HHV-6 (ciHHV-6) were defined as persistent HHV-6 viral load over time greater than 5.5 log10 copies/ml of whole blood (18). Discrepant results between microarrays and routine PCR assays were interpreted as follows: positive PCR results, negative by microarray, were deemed “false negative”; negative PCR results, positive by microarray, were deemed “likely true positive” if either the previous or the next sample obtained from the same patient was positive by PCR, “possibly true positive” if the result was consistent with the serological status of the patient, and “false positive” in the other cases.
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(number of copies/ml of whole blood) are plotted on the y axis. The x axis indicates the positive (⫹) and negative (⫺) microarray results. The dashed lines represent the lower limits of quantification of PCR assays.
ity and specificity of the microarray were 83 and 95%, 85 and 95%, and 57 and 90% for CMV, EBV, and HHV-6, respectively. The kappa coefficients between the microarray and routine PCR assays were equal to 0.8 for CMV and EBV and 0.4 for HHV-6. Thirtythree false-negative results were observed in a total of 29 samples (Table 1). Regarding CMV (n ⫽ 11) and EBV (n ⫽ 7), all but one false-negative case were observed for viral loads below or around
the limit of quantification of the PCR. Only one sample had a viral load (CMV) above 1,000 copies/ml of whole blood with 2 positive replicates by PCR. CMV and EBV PCR-positive samples that were not detected by microarray had median viral loads significantly lower than those positive by PCR and microarray (852 copies/ml [500 to 3,439] versus 2,329 copies/ml [500 to 560,338], P ⬍ 0.05 for CMV; 500 copies/ml [500 to 575] versus 1,001 copies/ml [500
TABLE 1 Summary of the blood sample results obtained with the microarray assay compared to our routine PCR assaysa Result by microarray No. of samples Category by routine PCR
Virus positive
Virus negative
CMV Positive Negative
55 9
11 174
EBV Positive Negative
40 10
7 187
HHV-6 Positive Negative
20 22
15 189
a b
Kappa test (95% CI)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
No. of evaluable samplesb
0.79 (0.71–0.88)
83
95
86
94
249
0.78 (0.68–0.88)
85
95
80
96
244
0.43 (0.28–0.58)
57
90
48
93
246
Abbreviations: CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value. Samples with inhibited PCR or an invalid microarray or that were unavailable were excluded.
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FIG 1 Comparison of qualitative PCR DNA microarray and quantitative real-time PCR results (CMV, n ⫽ 249; EBV, n ⫽ 244; HHV-6, n ⫽ 246). The viral loads
Detection of Herpesviridae by PCR DNA Microarray
copies/ml of blood. (A) Undiluted sample. (B and C) Sample diluted 1:100 (B) and 1:1,000 (C) before the first multiplex PCR step of the microarray. (D and E) Sample diluted 1:100 (D) and 1:1,000 (E) before the hybridization step of the microarray.
to 49,972], P ⬍ 0.01 for EBV). Fifteen false-negative results were observed for HHV-6. Surprisingly, nine out of these 15 samples had, by PCR, extremely high levels of HHV-6 (above 6 log10 copies/ml of whole blood). HHV-6 PCR-positive samples that were not detected by microarray had median viral loads that were not statistically different from those of samples positive by both PCR and microarray (1.5 ⫻ 106 copies/ml [2,920 to 1.5 ⫻ 106] versus 47,666 [4,839 to 1.5 ⫻ 106]). To investigate the cause of these false-HHV-6-negative results, we tested if a hybridization could be observed when diluting one of these false-negative samples before or after performing the amplification step (dilution of the DNA extract versus dilution of the PCR product). Since the signal was recovered only when the sample was diluted before amplification (Fig. 2), we inferred that the amplification step was affected by a large amount of target. This “false-negative” phenomenon was observed in 9 out of the 12 HHV-6 viral loads above 6 log10 copies/ml collected during the study but was not observed with samples having similarly high CMV or EBV viral loads. Forty-one microarray-positive results were not confirmed by PCR (Table 1). Fifteen of them, corresponding to an early detection of reactivation or collected after the initiation of antiviral therapy, were classified as likely true positive (6 CMV and 9 HHV6). Thirteen results were possibly true positive (3 CMV and 10 EBV), consistent with the serological status of the patient. Thirteen results, all HHV-6 positive, were classified as false positive, serology not being available for these cases. DISCUSSION
Technology improvements in molecular diagnostic tests (possibility of automation, real-time PCR amplification, and availability of commercial kits) have promoted their implementation in the routine diagnostic virology laboratory. These more sensitive and specific as well as time- and labor-saving tests have mostly replaced conventional methods of virus detection (culture and serology), especially in the virological surveillance of transplanted patients for whom the timeliness of the results is a critical component. The main obstacle to the use of these techniques is their cost. In this context, the use of the multiplex PCR DNA microarray
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FIG 2 Microarray results for a sample with an HHV-6 viral load above 6 log10
Clart Entherpex kit for simultaneous qualitative detection of CMV, EBV, and HHV-6 in one run of testing could allow potential turnaround time reduction and cost savings. The aim of the study was to assess the performance of this microarray method in a routine clinical laboratory setting. In our cohort of patients, the microarray assay showed good performances for CMV and EBV detection compared to routine quantitative PCR. All clinically relevant episodes were associated with a positive microarray result. The few discrepant results were observed in cases presenting with low viral load (such as latent stage of infection, intermittent viremia, or early onset of an active replication) and thus had no major clinical consequences, i.e., no change in the antiviral therapy. The agreement was good for CMV and EBV but weaker for HHV-6 detection. Actually, 9 samples tested negative with the microarray had a very high viral load (⬎6 log10 copies/ml) by PCR. These results were observed for one patient with ciHHV-6 as well as for one patient with active reactivation. We believe that a very high HHV-6 viral load interferes with the amplification step of the microarray. This analytical glitch was even more problematic because, in these cases, the internal amplification controls were not inhibited as they should have been. We also suspect that coinfections, especially when quantities of the different viruses differ greatly, could affect the analytical sensitivity of the microarray and give false-negative results (data not shown). The multiplex PCR, the hybridization of PCR products, and the reading and interpretation of the microarray were achieved within 8 h. As the same extraction step was used for the microarray analysis and quantitative PCR analysis, the microarray could in theory easily be used as a weekly screening assay for the detection of Herpesviridae in the blood of transplanted patients. Each positive result can subsequently be followed by selective viral load measurement by a quantitative PCR. One of the main advantages of proceeding as such would be a reduction in the cost of analysis of around 22% (estimated on a retrospective series of 3,832 samples collected over 5 years for CMV, EBV, and HHV-6 detection [data not shown]). Another advantage of implementing this test would be that the wide range of viruses tested could help assess the pathogenicity of neglected viruses such as HHV-7, which was the most commonly identified virus in our cohort after CMV, EBV, and HHV-6. Moreover, the delay of response incurred by the implementation of one supplementary first-line test would not have a critical impact, seeing that samples would be analyzed only once or twice weekly in batches. However, the drawbacks of this algorithm would be that it would require a controlled workflow and that adding a screening test could increase the workload even if it reduces the total number of samples on which PCRs would be performed. However, as this test has analytical limitations that for the moment prevent its implementation in a routine setting, the gain over workload remains difficult to ascertain. To ensure the place of this microarray in routine laboratories, improvements should be made by the manufacturers regarding internal quality controls. Moreover, to make the most of its potential benefits for the follow-up of HSCT patients, the additional detection of adenovirus, parvovirus B19, and JC and BK polyomaviruses would be an interesting development which could alleviate the further workload generated. Conclusion. The evaluated multiplex PCR DNA microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV reactivations in the blood. How-
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9.
10.
ACKNOWLEDGMENTS This work was supported by a grant from the Iris-Recherche fund. We thank Marie-Jeanne Stouten for her skilled technical assistance.
11.
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ever, positive samples must be subsequently confirmed and the viral load must be quantified by PCR assays to obtain a more precise diagnosis. Several samples with high viral loads of HHV-6 testing negative with the microarray can be problematic both for the pretransplantation detection of chromosomally integrated HHV-6 and for follow-up of patients with active reactivation. Although it is promising, some characteristics of this diagnostic tool should be improved, particularly for quality control and a targeted virus panel, such that it could then be adapted to be used in a routine setting.
