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Performance of a real-time PCR–based approach and droplet digital PCR in detecting human parechovirus type 3 RNA
Accepted Manuscript Title: Performance of a Real-time PCR–based Approach and Droplet Digital PCR in Detecting Human Parechovirus Type 3 RNA Authors: Yuta Aizawa Akihide Koyama Tomohiko Ishihara Osamu Onodera Akihiko Saitoh M.D., Ph.D. PII: DOI: Reference:
S1386-6532(16)30542-X http://dx.doi.org/doi:10.1016/j.jcv.2016.09.009 JCV 3698
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
3-5-2016 3-9-2016 21-9-2016
Please cite this article as: Aizawa Yuta, Koyama Akihide, Ishihara Tomohiko, Onodera Osamu, Saitoh Akihiko.Performance of a Real-time PCR–based Approach and Droplet Digital PCR in Detecting Human Parechovirus Type 3 RNA.Journal of Clinical Virology http://dx.doi.org/10.1016/j.jcv.2016.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Performance of a Real-time PCR–based Approach and Droplet Digital PCR in Detecting Human Parechovirus Type 3 RNA
Running title: Detection of HPeV3 RNA by droplet digital PCR
Yuta Aizawa,a Akihide Koyama,b Tomohiko Ishihara,b Osamu Onodera,b Akihiko Saitoha#
Department of Pediatrics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japana; Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japanb
#Corresponding Author Akihiko Saitoh, M.D., Ph.D. Department of Pediatrics 1
Niigata University Graduate School of Medical and Dental Sciences 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan Tel: +81-25-227-2222 Fax: +81-25-227-0778 E-mail: [email protected]
Word count: Abstract 233 words, Text 1,944 words
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Highlights
Real-time RT-PCR detection limits for HPeV3 have not been fully evaluated.
RT-ddPCR is the novel technique with precise quantitation of low-copy target genes.
Two-step RT-ddPCR was less variable and more specific than one-step RT-ddPCR.
Two-step RT-ddPCR detected HPeV3 RNA in real-time RT-PCR-negative CSF samples.
Abstract Background Human parechovirus type 3 (HPeV3) is an emerging virus that causes sepsis and meningoencephalitis in neonates and young infants. Correct diagnosis of HPeV3 infection is critical in determining appropriate management and predicting patients’ clinical course. Real-time reverse transcription PCR (RT-PCR) analysis of serum and/or cerebrospinal fluid (CSF) has been used to diagnose HPeV3 infection; however, the assay detection limits have not been fully evaluated.
3
Objectives We tested the hypothesis that droplet digital RT-PCR (RT-ddPCR)—a novel technique that precisely quantitates low-copy target genes by diluting and partitioning samples into compartments—increases the detection rate of HPeV3 RNA as compared with real-time RT-PCR. Study design Using samples with predetermined HPeV3 copy numbers, we evaluated one-step and two-step RT-ddPCR. Then, we tested two-step RT-ddPCR and real-time RT-PCR, using clinical samples with low copy numbers. Finally, we used two-step RT-ddPCR to evaluate clinical samples obtained from HPeV3-infected patients with positive serum but negative CSF, as determined by real-time RT-PCR. Results Two-step RT-ddPCR was less variable and more specific than one-step RT-ddPCR. Two-step RT-ddPCR detected HPeV3 RNA in all six CSF samples; four samples (67%) were reproducibly positive and the other two samples (33%) were positive at least once in four replicates. Finally, no nonspecific droplet was positive by two-step RT-ddPCR. Conclusions Two-step RT-ddPCR may enhance the rate of HPeV3 RNA detection from samples with low viral loads, thereby improving diagnosis and management of HPeV3-infected patients. Key Words: digital PCR; human parechovirus type 3; real-time PCR; cerebrospinal fluid; neonates; infants 4
Background Human parechoviruses (HPeVs) are small, non-enveloped, single-stranded RNA viruses within the family Picornaviridae.1 Among the 17 genotypes identified, human parechovirus type 3 (HPeV3) is a major pathogen that causes sepsis and meningoencephalitis in neonates and young infants.1, 2 Researchers have not yet determined why these severe conditions are specific to young infants; however, maternal antibodies may play a critical role.3 Rapid and accurate diagnosis of HPeV3 infection is important because antimicrobials are ineffective and the disease may lead to neurological sequelae4,5 and death.6 Real-time reverse transcription PCR (RT-PCR) has been widely used for diagnosis of HPeV3 infection.1 Detection of HPeV3 in blood and/or cerebrospinal fluid (CSF) is important in diagnosis,7,8 and a serum sample collected at disease onset is more reliable than a CSF sample in the detection of HPeV3 RNA.7 However, in previous studies on the diagnosis of HPeV3 infection, CSF samples6, 9-15 were used more often than blood samples.7, 16-18 We reported previously that real-time RT-PCR using CSF samples was unsuccessful in detecting HPeV3 RNA in some HPeV3-infected patients with positive serum samples, as determined by real-time RT-PCR.7
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Digital PCR (dPCR) is a new nucleic acid amplification technique that achieves sensitive and accurate absolute quantitation.19-21 In addition to a microfluidics chip system, droplet digital PCR (ddPCR) utilizes a platform that forms droplets by water-in-oil emulsion to partition a single reaction into thousands of nanoliter-scale reactions. Poisson statistics enable the counting of the number of positive droplets, which yields an accurate absolute quantitation of the number of target nucleic acids in the original reaction. Another advantage of this technique is that it is better than real-time PCR at low viral load detection and detection of low-abundance mutants19,21 (e.g., antiviral resistance22). Because dPCR has only recently become commercially available, its diagnostic utility in the field of clinical virology remains to be determined.19,20 In clinical settings a CSF sample is often the only type of sample available for diagnosis of HPeV3 infection in neonates and young infants. Reverse transcription-ddPCR (RT-ddPCR) might be able to detect HPeV3 RNA in CSF samples that yield negative results by real-time RT-PCR. To date, only two studies have used CSF samples for dPCR detection: for human immunodeficiency virus type 1 (HIV-1)23 and human T cell lymphotropic virus 124 in CSF pellets. During a recent HPeV3 epidemic in Japan, we evaluated patients with a coincident positive serum result and negative CSF result by real-time RT-PCR, 6
which led us to question if these samples were true negatives or simply below the detection limit of real-time RT-PCR. Objectives The objective of this study was to evaluate the clinical utility of RT-ddPCR, using clinical samples from HPeV3-infected patients analyzed by real-time RT-PCR. Study design Real-time RT-PCR For detection of HPeV3 RNA, one-step real-time RT-PCR was performed and targeted the conserved 5’ untranslated region of HPeVs.25 When the results of this assay were positive, quantitative real-time PCR was performed by means of two-step real-time RT-PCR with a standard curve established by plasmids7 (more details at Supplementary data). Two-step RT-droplet digital PCR Two-step RT-ddPCR was performed using 2× ddPCR Supermix for Probes (no dUTP) (Bio-Rad Laboratories, Hercules, CA, USA) and the same primers and probe as those in real-time PCR (more details at Supplementary data). One-step RT-droplet digital PCR 7
One-step RT-ddPCR was performed using a One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad). The primers were identical to those used in real-time PCR,25 except that the probe used was a double-quencher probe. The remaining procedures were the same as those for two-step RT-ddPCR, except for the PCR thermal cycling conditions (more details at Supplementary data).
Patients and samples This study evaluated six CSF samples from HPeV3-infected patients, all of which were negative for HPeVs by real-time RT-PCR but positive by the same assay when a serum sample from the same patient was analyzed (more details at Supplementary data).
HPeV3 RNA quantification in two-step and one-step RT-ddPCR A serum sample from the HPeV3-infected patient, quantified as 10,300 copies/reaction by two-step RT-ddPCR, was diluted 10 times (high concentration), 100 times (moderate concentration), and 1,000 times (low concentration). To evaluate the variability and specificity of two-step and one-step RT-ddPCR assays, the diluted samples and Milli-Q water, as a negative control,26 were quantified simultaneously by both assays. Each assay was performed four times for each sample. 8
Lower detection limit of HPeV3 RNA in real-time RT-PCR and two-step RT-ddPCR A serum sample from the HPeV3-infected patient, quantified as 10,300 copies/reaction by two-step RT-ddPCR, was diluted 100 times (moderate concentration), 1,000 times (low concentration), and 10,000 times (very low concentration). To evaluate the lower detection limits of the real-time RT-PCR and two-step RT-ddPCR, the diluted samples were subjected to both assays. Each assay was performed four times for each sample.
Evaluation of real-time RT-PCR–negative CSF samples by two-step RT-ddPCR Two-step RT-ddPCR was performed in triplicate for negative CSF samples, using real-time RT-PCR. Another run with triplicates was performed when all the results in triplicate yielded <10 copies/reaction. HPeV3 RNA in serum samples from patients was also evaluated by two-step RT-ddPCR.
Statistical analyses
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The data analyses were performed using SPSS Statistics 23.0 (IBM SPSS, Chicago, IL, USA). The mean and standard deviation for HPeV3 RNA copy numbers were calculated for samples quantified by RT-ddPCR. The coefficient of variation was evaluated based on threshold cycles in real-time RT-PCR and HPeV3 copy numbers in RT-ddPCR. HPeV3 RNA copy numbers quantified by one-step and two-step RT-ddPCR were compared by using the Student two-tailed t test. The Spearman rank correlation coefficient was used to evaluate the correlation between serum and CSF HPeV3 RNA levels. A P-value of <0.05 was considered to indicate statistical significance. Results Two-step RT-ddPCR and one-step RT-ddPCR for HPeV3 RNA quantification HPeV3 copy numbers quantified by one-step RT-ddPCR were one-third to one-half those quantified by two-step RT-ddPCR, at all concentrations (Table 1), and the differences in HPeV3 copy numbers between the two assays were statistically significant (P<0.02). At high and moderate concentrations, two-step RT-ddPCR values were less variable than those for one-step RT-ddPCR, although variability was similar at the low concentration. One-step RT-ddPCR yielded positive results for controls (50%), which were considered nonspecific positive droplets, whereas two-step RT-ddPCR showed no positive droplets in 10
negative controls. Of note, during this study, including the preliminary experiments, 16 negative controls yielded negative results in 6 independent runs of two-step RT-ddPCR.
The detection limit for HPeV3 RNA was lower for two-step RT-ddPCR than for real-time RT-PCR Because two-step RT-ddPCR exhibited better performance than one-step RT-ddPCR, we selected two-step RT-ddPCR for comparison with real-time RT-PCR. Real-time RT-PCR yielded positive results only for the moderate concentration sample (Table 2). In contrast, two-step RT-ddPCR yielded 100% positive results for the low concentration sample and 50% positive results for the very low concentration sample, namely, 1.4 and 1.6 copies/reaction, respectively. Importantly, no nonspecific reactions were observed in negative controls in either assay.
Successful detection of HPeV3 RNA by two-step RT-ddPCR analysis of real-time RT-PCR–negative CSF samples Among six CSF samples that were negative for HPeVs by real-time RT-PCR, four CSF samples (67%; Patients 2, 3, 4, and 7; Table 3) yielded reproducibly positive results by two-step RT-ddPCR. These 11
results indicate that serum HPeV3 RNA level was not associated with CSF HPeV3 RNA level (P=0.23). A similar serum HPeV3 RNA level resulted in an approximately 70-fold difference in CSF HPeV3 RNA level (Patient 3, 3.2±0.8; Patient 4, 228.7±31.0). In contrast, identical HPeV3 RNA levels yielded a similar CSF HPeV3 RNA level (Patient 5, 0.85±0.98; Patient 6, 0.40±0.80). Importantly, one sample with approximately 200 copies/reaction by two-step RT-ddPCR was not detected by real-time RT-PCR (Patient 4). The lower detection limit for CSF samples (Patients 5 and 6) by two-step RT-ddPCR appeared to be close to approximately 1.5 copies/reaction, which is consistent with results obtained by dilution of serum samples (Table 2). The upper quantification limit of two-step RT-ddPCR was exceeded when there were more than 360,000 copies/reaction of serum samples by real-time RT-PCR, which indicates a limitation of ddPCR. Lower CSF HPeV3 RNA levels exhibited greater variability (Table 3). Discussion This study detected HPeV3 RNA in cell-free CSF by dPCR analysis of samples that yielded negative results by real-time RT-PCR. Our findings suggest that two-step RT-ddPCR could become a useful tool for detecting HPeV3 RNA in samples with low copy numbers, thus facilitating accurate diagnosis of HPeV3 infection.
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We found samples that yielded inconsistently positive results by this assay (Patients 5 and 6; Table 3). These results are difficult to interpret because the assay is usually performed only once in clinical settings. However, because two-step RT-ddPCR was consistent with the finding of no nonspecific positive droplets in negative controls, those patients might have had a small amount of HPeV3 RNA in CSF. No correlation was observed between HPeV3 RNA levels in serum and CSF quantified by two-step RT-ddPCR, which contradicts a previous report of a positive correlation of these sample types when quantified by real-time RT-PCR.7 Leakage of HPeV3 RNA from blood into CSF may explain why HPeV3 RNA was detected in CSF,27 and the small sample size of this study might have contributed to inconsistency in the correlation between viral load in serum and CSF. Previous studies compared the sensitivity of dPCR and real-time PCR for other viruses. For cytomegalovirus, real-time PCR was superior to ddPCR when the template DNA added in real-time PCR was four times that in ddPCR.28 The sensitivity of the two assays was comparable when similar amounts of DNA were added.29 For HIV-1 DNA, the sensitivity of dPCR was greater than30 or equal to31 that of real-time PCR. For cell-associated HIV-1 RNA, the detection rate was equal for two-step RT-ddPCR and semi-nested real-time RT-PCR.32 Two-step microfluidic RT-dPCR had greater sensitivity and precision as compared 13
with two-step real-time RT-PCR for the occult RNA virus GB virus type C.33 Among picornaviruses, enterovirus 71–derived cDNA plasmids were quantified accurately and consistently by both real-time PCR and ddPCR.34 In contrast, real-time PCR was more sensitive than ddPCR for low levels of hepatitis B virus DNA.35 Overall, dPCR has achieved equal or superior performance to real-time PCR for detecting viruses. This is the first study to perform one-step RT-ddPCR for CSF, which resulted in inferior performance to two-step RT-ddPCR. This was surprising, because a previous report found that one-step real-time RT-PCR was more sensitive than two-step real-time RT-PCR.36 Possible explanations for this finding are a difference in RT efficiency and/or presence of unsuitable reagents in the one-step RT-ddPCR products—it required a double-quencher probe to clearly separate positive droplets from negative droplets. dPCR is a new technology and may therefore need additional improvements for use in clinical virology.20 Few studies have evaluated the performance of the one-step RT-dPCR for human samples. For enteric viruses, viral copy numbers quantified by one-step microfluidic RT-dPCR were lower than those by one-step real-time RT-PCR.37 Future studies are needed in order to compare the utility of one-step and two-step RT-dPCR. This study has some limitations. First, the numbers of samples were limited because most HPeV3-infected patients were serum- and 14
CSF-positive by real-time RT-PCR, which made it difficult to identify serum-positive, CSF-negative patients. Second, HPeV3 RNA copy numbers were calculated using the plasmid standard curve in the real-time RT-PCR, which did not evaluate RNA extraction and RT efficiencies. Finally, to test the validity of HPeV3 two-step RT-ddPCR, we did not test CSF samples of undetermined etiology by real-time RT-PCR. In conclusion, two-step RT-ddPCR successfully detected HPeV3 RNA in CSF samples that yielded negative results by real-time RT-PCR. Two-step RT-ddPCR may enhance the rate of HPeV3 RNA detection from samples with low viral loads, thereby improving diagnosis and management of HPeV3-infected patients. This assay might also be useful in diagnosing other infections in samples with low viral loads.
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Funding information This work was supported by the Japan Society for Promotion of Science, Grants-in-Aid for Scientific Research [26461569] to AS.
Competing interests: None declared.
Ethical approval: This study was approved by the Ethics Committee of Niigata University.
Acknowledgments The authors thank Yuko Suzuki for laboratory assistance and David Kipler for editing the manuscript.
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TABLE 1. HPeV3 Sampleb (copies/reaction) High concentration (1,000)
RNA quantitation in two-step and one-step RT-ddPCR Two-step RT-ddPCR One-step RT-ddPCR Copies/reaction Mean ± SD CV (%) Copies/reaction Mean ± SD CV (%) 1,174;1,176; 1,189 ± 20.3 1.7 474; 494; 508; 504.5 ± 28.6 5.7 1,188; 1,218 542
P values <0.001a
Moderate concentration (100)
150; 152; 160; 164
156.5 ± 6.6
4.2
40; 44; 46; 68
49.5 ± 12.6
25.4
<0.001a
Low concentration (10)
14.8; 24; 26; 34
24.7 ± 7.9
31.9
4.8; 8; 9.4; 10
8.1 ± 2.3
28.9
0.007a
Negative 0; 0; 0; 0 0 N/A 0; 0; 1.6; 1.8 0.85 ± 0.98 115.9 0.18 control aStatistically significant (P <0.05) bThe serum sample was obtained from an HPeV3-infected patient and was quantified as approximately 10,000 copies/reaction by two-step RT-ddPCR. The sample was diluted 10 times (high concentration, approximately 1,000 copies/reaction), 100 times (moderate concentration, approximately 100 copies/reaction), and 1,000 times (low concentration, approximately 10 copies/reaction). Abbreviations: HPeV3, human parechovirus type 3; RT-ddPCR, reverse transcription droplet digital PCR; CV, coefficient of variation; N/A, not available
25
TABLE 2. Lower detection limit of HPeV3 RNA for one-step real-time RT-PCR and two-step RT-ddPCR Sample (copy or copies/reaction)a Negative control Very low concentration (1) Low concentration (10) Moderate concentration (100)
One-step real-time RT-PCR No. positive/Total CV (%) 0/4 N/A 0/4 N/A
Two-step RT-ddPCR No. positive/Total Copies/reaction 0/4 0; 0; 0; 0 2/4 0; 0; 1.4; 1.6
Mean ± SD 0 0.75 ± 0.87
CV (%) N/A 116.0
0/4
N/A
4/4
9; 18; 22; 22
17.8 ± 6.1
34.5
4/4
0.65
4/4
96; 112; 116; 124
112 ± 11.8
10.5
The actual copy numbers of positive results with four replications are shown in each sample. aThe HPeV3-infected patient’s serum sample, quantified as approximately 10,000 copies/reaction by two-step RT-ddPCR, was diluted 100 times (moderate concentration, 100 copies/reaction), 1,000 times (low concentration, 10 copies/reaction), and 10,000 times (very low concentration, 1 copy/reaction). Abbreviations: HPeV3, human parechovirus type 3; RT-PCR, reverse transcription PCR; RT-ddPCR, reverse transcription droplet digital PCR; CV, coefficient of variation; N/A, not available
26
TABLE 3. Detection of HPeV3 RNA by two-step RT-ddPCR analysis of real-time RT-PCR–negative CSF samples Patient Number
1a
2
3 4
5 6
7
Age (days)
22
37
47 21
65 38
7
Sex
Male
Female
Male Male
Male Female
Male
Diagnosis
Sepsis
Serum
Day 0
One-step real-time RT-PCR Copies/reaction 361,608
Sepsis
CSF Serum
Day 0 Day 0
41,379 658,793
CSF Serum CSF Serum
Day Day Day Day
0 8,547 0 6,843
Copies/reaction Mean ± SD Exceeded upper limit 21,140 Exceeded upper limit 40; 46; 54 46.7 ± 7.0 9,420 2; 3.2; 3.6; 3.8 3.2 ± 0.8 10,300
CSF
Day 0
0
198; 228; 260
Serum CSF Serum
Day 0 Day 0 Day 4
6,640 0 2,678
2,380 0; 0; 1.6; 1.8 2,380
0.85 ± 0.98
115.9
Day 3
0
0; 0; 0; 1.6
0.40 ± 0.80
200.0
Day 0 Day 0
581 0
430 7; 7; 13
9.0 ± 3.5
38.5
Sepsis Sepsis-like illness
Sepsis
Sample type
Septic shock Pulmonary CSF hemorrhage Sepsis Serum CSF
Timing of sample collectionb
0 0 0 0
27
Two-step RT-ddPCR
CV (%)
228.7 ± 31.0
13.6
15.1 25.6
Analysis of serum samples by two-step RT-ddPCR was performed once. Analysis of CSF samples by two-step RT-ddPCR was performed at least three times. If the result of all triplicates was <10 copies/reaction, the assay was repeated once (total, four times). aPatient 1 is a positive control for the CSF sample from an HPeV3-infected patient for whom both serum and CSF samples were positive by real-time RT-PCR. This sample was used to confirm the consistency of results between the two assays. bDay 0 was defined as the day of disease onset. Abbreviations: HPeV3, human parechovirus type 3; RT-PCR, reverse transcription PCR; RT-ddPCR, reverse transcription droplet digital PCR; CSF, cerebrospinal fluid; CV, coefficient of variationtable
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S1386-6532(16)30542-X http://dx.doi.org/doi:10.1016/j.jcv.2016.09.009 JCV 3698
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
3-5-2016 3-9-2016 21-9-2016
Please cite this article as: Aizawa Yuta, Koyama Akihide, Ishihara Tomohiko, Onodera Osamu, Saitoh Akihiko.Performance of a Real-time PCR–based Approach and Droplet Digital PCR in Detecting Human Parechovirus Type 3 RNA.Journal of Clinical Virology http://dx.doi.org/10.1016/j.jcv.2016.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Performance of a Real-time PCR–based Approach and Droplet Digital PCR in Detecting Human Parechovirus Type 3 RNA
Running title: Detection of HPeV3 RNA by droplet digital PCR
Yuta Aizawa,a Akihide Koyama,b Tomohiko Ishihara,b Osamu Onodera,b Akihiko Saitoha#
Department of Pediatrics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japana; Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japanb
#Corresponding Author Akihiko Saitoh, M.D., Ph.D. Department of Pediatrics 1
Niigata University Graduate School of Medical and Dental Sciences 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan Tel: +81-25-227-2222 Fax: +81-25-227-0778 E-mail: [email protected]
Word count: Abstract 233 words, Text 1,944 words
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Highlights
Real-time RT-PCR detection limits for HPeV3 have not been fully evaluated.
RT-ddPCR is the novel technique with precise quantitation of low-copy target genes.
Two-step RT-ddPCR was less variable and more specific than one-step RT-ddPCR.
Two-step RT-ddPCR detected HPeV3 RNA in real-time RT-PCR-negative CSF samples.
Abstract Background Human parechovirus type 3 (HPeV3) is an emerging virus that causes sepsis and meningoencephalitis in neonates and young infants. Correct diagnosis of HPeV3 infection is critical in determining appropriate management and predicting patients’ clinical course. Real-time reverse transcription PCR (RT-PCR) analysis of serum and/or cerebrospinal fluid (CSF) has been used to diagnose HPeV3 infection; however, the assay detection limits have not been fully evaluated.
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Objectives We tested the hypothesis that droplet digital RT-PCR (RT-ddPCR)—a novel technique that precisely quantitates low-copy target genes by diluting and partitioning samples into compartments—increases the detection rate of HPeV3 RNA as compared with real-time RT-PCR. Study design Using samples with predetermined HPeV3 copy numbers, we evaluated one-step and two-step RT-ddPCR. Then, we tested two-step RT-ddPCR and real-time RT-PCR, using clinical samples with low copy numbers. Finally, we used two-step RT-ddPCR to evaluate clinical samples obtained from HPeV3-infected patients with positive serum but negative CSF, as determined by real-time RT-PCR. Results Two-step RT-ddPCR was less variable and more specific than one-step RT-ddPCR. Two-step RT-ddPCR detected HPeV3 RNA in all six CSF samples; four samples (67%) were reproducibly positive and the other two samples (33%) were positive at least once in four replicates. Finally, no nonspecific droplet was positive by two-step RT-ddPCR. Conclusions Two-step RT-ddPCR may enhance the rate of HPeV3 RNA detection from samples with low viral loads, thereby improving diagnosis and management of HPeV3-infected patients. Key Words: digital PCR; human parechovirus type 3; real-time PCR; cerebrospinal fluid; neonates; infants 4
Background Human parechoviruses (HPeVs) are small, non-enveloped, single-stranded RNA viruses within the family Picornaviridae.1 Among the 17 genotypes identified, human parechovirus type 3 (HPeV3) is a major pathogen that causes sepsis and meningoencephalitis in neonates and young infants.1, 2 Researchers have not yet determined why these severe conditions are specific to young infants; however, maternal antibodies may play a critical role.3 Rapid and accurate diagnosis of HPeV3 infection is important because antimicrobials are ineffective and the disease may lead to neurological sequelae4,5 and death.6 Real-time reverse transcription PCR (RT-PCR) has been widely used for diagnosis of HPeV3 infection.1 Detection of HPeV3 in blood and/or cerebrospinal fluid (CSF) is important in diagnosis,7,8 and a serum sample collected at disease onset is more reliable than a CSF sample in the detection of HPeV3 RNA.7 However, in previous studies on the diagnosis of HPeV3 infection, CSF samples6, 9-15 were used more often than blood samples.7, 16-18 We reported previously that real-time RT-PCR using CSF samples was unsuccessful in detecting HPeV3 RNA in some HPeV3-infected patients with positive serum samples, as determined by real-time RT-PCR.7
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Digital PCR (dPCR) is a new nucleic acid amplification technique that achieves sensitive and accurate absolute quantitation.19-21 In addition to a microfluidics chip system, droplet digital PCR (ddPCR) utilizes a platform that forms droplets by water-in-oil emulsion to partition a single reaction into thousands of nanoliter-scale reactions. Poisson statistics enable the counting of the number of positive droplets, which yields an accurate absolute quantitation of the number of target nucleic acids in the original reaction. Another advantage of this technique is that it is better than real-time PCR at low viral load detection and detection of low-abundance mutants19,21 (e.g., antiviral resistance22). Because dPCR has only recently become commercially available, its diagnostic utility in the field of clinical virology remains to be determined.19,20 In clinical settings a CSF sample is often the only type of sample available for diagnosis of HPeV3 infection in neonates and young infants. Reverse transcription-ddPCR (RT-ddPCR) might be able to detect HPeV3 RNA in CSF samples that yield negative results by real-time RT-PCR. To date, only two studies have used CSF samples for dPCR detection: for human immunodeficiency virus type 1 (HIV-1)23 and human T cell lymphotropic virus 124 in CSF pellets. During a recent HPeV3 epidemic in Japan, we evaluated patients with a coincident positive serum result and negative CSF result by real-time RT-PCR, 6
which led us to question if these samples were true negatives or simply below the detection limit of real-time RT-PCR. Objectives The objective of this study was to evaluate the clinical utility of RT-ddPCR, using clinical samples from HPeV3-infected patients analyzed by real-time RT-PCR. Study design Real-time RT-PCR For detection of HPeV3 RNA, one-step real-time RT-PCR was performed and targeted the conserved 5’ untranslated region of HPeVs.25 When the results of this assay were positive, quantitative real-time PCR was performed by means of two-step real-time RT-PCR with a standard curve established by plasmids7 (more details at Supplementary data). Two-step RT-droplet digital PCR Two-step RT-ddPCR was performed using 2× ddPCR Supermix for Probes (no dUTP) (Bio-Rad Laboratories, Hercules, CA, USA) and the same primers and probe as those in real-time PCR (more details at Supplementary data). One-step RT-droplet digital PCR 7
One-step RT-ddPCR was performed using a One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad). The primers were identical to those used in real-time PCR,25 except that the probe used was a double-quencher probe. The remaining procedures were the same as those for two-step RT-ddPCR, except for the PCR thermal cycling conditions (more details at Supplementary data).
Patients and samples This study evaluated six CSF samples from HPeV3-infected patients, all of which were negative for HPeVs by real-time RT-PCR but positive by the same assay when a serum sample from the same patient was analyzed (more details at Supplementary data).
HPeV3 RNA quantification in two-step and one-step RT-ddPCR A serum sample from the HPeV3-infected patient, quantified as 10,300 copies/reaction by two-step RT-ddPCR, was diluted 10 times (high concentration), 100 times (moderate concentration), and 1,000 times (low concentration). To evaluate the variability and specificity of two-step and one-step RT-ddPCR assays, the diluted samples and Milli-Q water, as a negative control,26 were quantified simultaneously by both assays. Each assay was performed four times for each sample. 8
Lower detection limit of HPeV3 RNA in real-time RT-PCR and two-step RT-ddPCR A serum sample from the HPeV3-infected patient, quantified as 10,300 copies/reaction by two-step RT-ddPCR, was diluted 100 times (moderate concentration), 1,000 times (low concentration), and 10,000 times (very low concentration). To evaluate the lower detection limits of the real-time RT-PCR and two-step RT-ddPCR, the diluted samples were subjected to both assays. Each assay was performed four times for each sample.
Evaluation of real-time RT-PCR–negative CSF samples by two-step RT-ddPCR Two-step RT-ddPCR was performed in triplicate for negative CSF samples, using real-time RT-PCR. Another run with triplicates was performed when all the results in triplicate yielded <10 copies/reaction. HPeV3 RNA in serum samples from patients was also evaluated by two-step RT-ddPCR.
Statistical analyses
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The data analyses were performed using SPSS Statistics 23.0 (IBM SPSS, Chicago, IL, USA). The mean and standard deviation for HPeV3 RNA copy numbers were calculated for samples quantified by RT-ddPCR. The coefficient of variation was evaluated based on threshold cycles in real-time RT-PCR and HPeV3 copy numbers in RT-ddPCR. HPeV3 RNA copy numbers quantified by one-step and two-step RT-ddPCR were compared by using the Student two-tailed t test. The Spearman rank correlation coefficient was used to evaluate the correlation between serum and CSF HPeV3 RNA levels. A P-value of <0.05 was considered to indicate statistical significance. Results Two-step RT-ddPCR and one-step RT-ddPCR for HPeV3 RNA quantification HPeV3 copy numbers quantified by one-step RT-ddPCR were one-third to one-half those quantified by two-step RT-ddPCR, at all concentrations (Table 1), and the differences in HPeV3 copy numbers between the two assays were statistically significant (P<0.02). At high and moderate concentrations, two-step RT-ddPCR values were less variable than those for one-step RT-ddPCR, although variability was similar at the low concentration. One-step RT-ddPCR yielded positive results for controls (50%), which were considered nonspecific positive droplets, whereas two-step RT-ddPCR showed no positive droplets in 10
negative controls. Of note, during this study, including the preliminary experiments, 16 negative controls yielded negative results in 6 independent runs of two-step RT-ddPCR.
The detection limit for HPeV3 RNA was lower for two-step RT-ddPCR than for real-time RT-PCR Because two-step RT-ddPCR exhibited better performance than one-step RT-ddPCR, we selected two-step RT-ddPCR for comparison with real-time RT-PCR. Real-time RT-PCR yielded positive results only for the moderate concentration sample (Table 2). In contrast, two-step RT-ddPCR yielded 100% positive results for the low concentration sample and 50% positive results for the very low concentration sample, namely, 1.4 and 1.6 copies/reaction, respectively. Importantly, no nonspecific reactions were observed in negative controls in either assay.
Successful detection of HPeV3 RNA by two-step RT-ddPCR analysis of real-time RT-PCR–negative CSF samples Among six CSF samples that were negative for HPeVs by real-time RT-PCR, four CSF samples (67%; Patients 2, 3, 4, and 7; Table 3) yielded reproducibly positive results by two-step RT-ddPCR. These 11
results indicate that serum HPeV3 RNA level was not associated with CSF HPeV3 RNA level (P=0.23). A similar serum HPeV3 RNA level resulted in an approximately 70-fold difference in CSF HPeV3 RNA level (Patient 3, 3.2±0.8; Patient 4, 228.7±31.0). In contrast, identical HPeV3 RNA levels yielded a similar CSF HPeV3 RNA level (Patient 5, 0.85±0.98; Patient 6, 0.40±0.80). Importantly, one sample with approximately 200 copies/reaction by two-step RT-ddPCR was not detected by real-time RT-PCR (Patient 4). The lower detection limit for CSF samples (Patients 5 and 6) by two-step RT-ddPCR appeared to be close to approximately 1.5 copies/reaction, which is consistent with results obtained by dilution of serum samples (Table 2). The upper quantification limit of two-step RT-ddPCR was exceeded when there were more than 360,000 copies/reaction of serum samples by real-time RT-PCR, which indicates a limitation of ddPCR. Lower CSF HPeV3 RNA levels exhibited greater variability (Table 3). Discussion This study detected HPeV3 RNA in cell-free CSF by dPCR analysis of samples that yielded negative results by real-time RT-PCR. Our findings suggest that two-step RT-ddPCR could become a useful tool for detecting HPeV3 RNA in samples with low copy numbers, thus facilitating accurate diagnosis of HPeV3 infection.
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We found samples that yielded inconsistently positive results by this assay (Patients 5 and 6; Table 3). These results are difficult to interpret because the assay is usually performed only once in clinical settings. However, because two-step RT-ddPCR was consistent with the finding of no nonspecific positive droplets in negative controls, those patients might have had a small amount of HPeV3 RNA in CSF. No correlation was observed between HPeV3 RNA levels in serum and CSF quantified by two-step RT-ddPCR, which contradicts a previous report of a positive correlation of these sample types when quantified by real-time RT-PCR.7 Leakage of HPeV3 RNA from blood into CSF may explain why HPeV3 RNA was detected in CSF,27 and the small sample size of this study might have contributed to inconsistency in the correlation between viral load in serum and CSF. Previous studies compared the sensitivity of dPCR and real-time PCR for other viruses. For cytomegalovirus, real-time PCR was superior to ddPCR when the template DNA added in real-time PCR was four times that in ddPCR.28 The sensitivity of the two assays was comparable when similar amounts of DNA were added.29 For HIV-1 DNA, the sensitivity of dPCR was greater than30 or equal to31 that of real-time PCR. For cell-associated HIV-1 RNA, the detection rate was equal for two-step RT-ddPCR and semi-nested real-time RT-PCR.32 Two-step microfluidic RT-dPCR had greater sensitivity and precision as compared 13
with two-step real-time RT-PCR for the occult RNA virus GB virus type C.33 Among picornaviruses, enterovirus 71–derived cDNA plasmids were quantified accurately and consistently by both real-time PCR and ddPCR.34 In contrast, real-time PCR was more sensitive than ddPCR for low levels of hepatitis B virus DNA.35 Overall, dPCR has achieved equal or superior performance to real-time PCR for detecting viruses. This is the first study to perform one-step RT-ddPCR for CSF, which resulted in inferior performance to two-step RT-ddPCR. This was surprising, because a previous report found that one-step real-time RT-PCR was more sensitive than two-step real-time RT-PCR.36 Possible explanations for this finding are a difference in RT efficiency and/or presence of unsuitable reagents in the one-step RT-ddPCR products—it required a double-quencher probe to clearly separate positive droplets from negative droplets. dPCR is a new technology and may therefore need additional improvements for use in clinical virology.20 Few studies have evaluated the performance of the one-step RT-dPCR for human samples. For enteric viruses, viral copy numbers quantified by one-step microfluidic RT-dPCR were lower than those by one-step real-time RT-PCR.37 Future studies are needed in order to compare the utility of one-step and two-step RT-dPCR. This study has some limitations. First, the numbers of samples were limited because most HPeV3-infected patients were serum- and 14
CSF-positive by real-time RT-PCR, which made it difficult to identify serum-positive, CSF-negative patients. Second, HPeV3 RNA copy numbers were calculated using the plasmid standard curve in the real-time RT-PCR, which did not evaluate RNA extraction and RT efficiencies. Finally, to test the validity of HPeV3 two-step RT-ddPCR, we did not test CSF samples of undetermined etiology by real-time RT-PCR. In conclusion, two-step RT-ddPCR successfully detected HPeV3 RNA in CSF samples that yielded negative results by real-time RT-PCR. Two-step RT-ddPCR may enhance the rate of HPeV3 RNA detection from samples with low viral loads, thereby improving diagnosis and management of HPeV3-infected patients. This assay might also be useful in diagnosing other infections in samples with low viral loads.
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Funding information This work was supported by the Japan Society for Promotion of Science, Grants-in-Aid for Scientific Research [26461569] to AS.
Competing interests: None declared.
Ethical approval: This study was approved by the Ethics Committee of Niigata University.
Acknowledgments The authors thank Yuko Suzuki for laboratory assistance and David Kipler for editing the manuscript.
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TABLE 1. HPeV3 Sampleb (copies/reaction) High concentration (1,000)
RNA quantitation in two-step and one-step RT-ddPCR Two-step RT-ddPCR One-step RT-ddPCR Copies/reaction Mean ± SD CV (%) Copies/reaction Mean ± SD CV (%) 1,174;1,176; 1,189 ± 20.3 1.7 474; 494; 508; 504.5 ± 28.6 5.7 1,188; 1,218 542
P values <0.001a
Moderate concentration (100)
150; 152; 160; 164
156.5 ± 6.6
4.2
40; 44; 46; 68
49.5 ± 12.6
25.4
<0.001a
Low concentration (10)
14.8; 24; 26; 34
24.7 ± 7.9
31.9
4.8; 8; 9.4; 10
8.1 ± 2.3
28.9
0.007a
Negative 0; 0; 0; 0 0 N/A 0; 0; 1.6; 1.8 0.85 ± 0.98 115.9 0.18 control aStatistically significant (P <0.05) bThe serum sample was obtained from an HPeV3-infected patient and was quantified as approximately 10,000 copies/reaction by two-step RT-ddPCR. The sample was diluted 10 times (high concentration, approximately 1,000 copies/reaction), 100 times (moderate concentration, approximately 100 copies/reaction), and 1,000 times (low concentration, approximately 10 copies/reaction). Abbreviations: HPeV3, human parechovirus type 3; RT-ddPCR, reverse transcription droplet digital PCR; CV, coefficient of variation; N/A, not available
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TABLE 2. Lower detection limit of HPeV3 RNA for one-step real-time RT-PCR and two-step RT-ddPCR Sample (copy or copies/reaction)a Negative control Very low concentration (1) Low concentration (10) Moderate concentration (100)
One-step real-time RT-PCR No. positive/Total CV (%) 0/4 N/A 0/4 N/A
Two-step RT-ddPCR No. positive/Total Copies/reaction 0/4 0; 0; 0; 0 2/4 0; 0; 1.4; 1.6
Mean ± SD 0 0.75 ± 0.87
CV (%) N/A 116.0
0/4
N/A
4/4
9; 18; 22; 22
17.8 ± 6.1
34.5
4/4
0.65
4/4
96; 112; 116; 124
112 ± 11.8
10.5
The actual copy numbers of positive results with four replications are shown in each sample. aThe HPeV3-infected patient’s serum sample, quantified as approximately 10,000 copies/reaction by two-step RT-ddPCR, was diluted 100 times (moderate concentration, 100 copies/reaction), 1,000 times (low concentration, 10 copies/reaction), and 10,000 times (very low concentration, 1 copy/reaction). Abbreviations: HPeV3, human parechovirus type 3; RT-PCR, reverse transcription PCR; RT-ddPCR, reverse transcription droplet digital PCR; CV, coefficient of variation; N/A, not available
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TABLE 3. Detection of HPeV3 RNA by two-step RT-ddPCR analysis of real-time RT-PCR–negative CSF samples Patient Number
1a
2
3 4
5 6
7
Age (days)
22
37
47 21
65 38
7
Sex
Male
Female
Male Male
Male Female
Male
Diagnosis
Sepsis
Serum
Day 0
One-step real-time RT-PCR Copies/reaction 361,608
Sepsis
CSF Serum
Day 0 Day 0
41,379 658,793
CSF Serum CSF Serum
Day Day Day Day
0 8,547 0 6,843
Copies/reaction Mean ± SD Exceeded upper limit 21,140 Exceeded upper limit 40; 46; 54 46.7 ± 7.0 9,420 2; 3.2; 3.6; 3.8 3.2 ± 0.8 10,300
CSF
Day 0
0
198; 228; 260
Serum CSF Serum
Day 0 Day 0 Day 4
6,640 0 2,678
2,380 0; 0; 1.6; 1.8 2,380
0.85 ± 0.98
115.9
Day 3
0
0; 0; 0; 1.6
0.40 ± 0.80
200.0
Day 0 Day 0
581 0
430 7; 7; 13
9.0 ± 3.5
38.5
Sepsis Sepsis-like illness
Sepsis
Sample type
Septic shock Pulmonary CSF hemorrhage Sepsis Serum CSF
Timing of sample collectionb
0 0 0 0
27
Two-step RT-ddPCR
CV (%)
228.7 ± 31.0
13.6
15.1 25.6
Analysis of serum samples by two-step RT-ddPCR was performed once. Analysis of CSF samples by two-step RT-ddPCR was performed at least three times. If the result of all triplicates was <10 copies/reaction, the assay was repeated once (total, four times). aPatient 1 is a positive control for the CSF sample from an HPeV3-infected patient for whom both serum and CSF samples were positive by real-time RT-PCR. This sample was used to confirm the consistency of results between the two assays. bDay 0 was defined as the day of disease onset. Abbreviations: HPeV3, human parechovirus type 3; RT-PCR, reverse transcription PCR; RT-ddPCR, reverse transcription droplet digital PCR; CSF, cerebrospinal fluid; CV, coefficient of variationtable
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