Dataset Information

Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

ABSTRACT:

Background

Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples.

Methods

Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system.

Results

The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits.

Conclusion

The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

SUBMITTER: Zhu X

PROVIDER: S-EPMC9020426 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Publications

Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

Zhu Xunhua X Liu Pengcheng P Lu Lijuan L Zhong Huaqing H Xu Menghua M Jia Ran R Su Liyun L Cao Lingfeng L Sun Yameng Y Guo Meijun M Sun Jianyue J Xu Jin J

Virology journal 20220420 1

<h4>Background</h4>Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples.<h4>Methods</h4>Targeting four c ...[more]

PMID: 35443688

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Project description:In the transmission control of chronic and untreatable livestock diseases such as bovine leukemia virus (BLV) infection, the removal of viral superspreaders is a fundamental approach. On the other hand, selective breeding of cattle with BLV-resistant capacity is also critical for reducing the viral damage to productivity by keeping infected cattle. To provide a way of measuring BLV proviral load (PVL) and identifying susceptible/resistant cattle simply and rapidly, we developed a fourplex droplet digital PCR method targeting the BLV pol gene, BLV-susceptible bovine major histocompatibility complex (BoLA)-DRB3*016:01 allele, resistant DRB3*009:02 allele, and housekeeping RPP30 gene (IPATS-BLV). IPATS-BLV successfully measured the percentage of BLV-infected cells and determined allele types precisely. Furthermore, it discriminated homozygous from heterozygous carriers. Using this method to determine the impact of carrying these alleles on the BLV PVL, we found DRB3*009:02-carrying cattle could suppress the PVL to a low or undetectable level, even with the presence of a susceptible heterozygous allele. Although the population of DRB3*016:01-carrying cattle showed significantly higher PVLs compared with cattle carrying other alleles, their individual PVLs were highly variable. Because of the simplicity and speed of this single-well assay, our method has the potential of being a suitable platform for the combined diagnosis of pathogen level and host biomarkers in other infectious diseases satisfying the two following characteristics of disease outcomes: (i) pathogen level acts as a critical maker of disease progression; and (ii) impactful disease-related host genetic biomarkers are already identified. IMPORTANCE While pathogen-level quantification is an important diagnostic of disease severity and transmissibility, disease-related host biomarkers are also useful in predicting outcomes in infectious diseases. In this study, we demonstrate that combined proviral load (PVL) and host biomarker diagnostics can be used to detect bovine leukemia virus (BLV) infection, which has a negative economic impact on the cattle industry. We developed a fourplex droplet digital PCR assay for PVL of BLV and susceptible and resistant host genes named IPATS-BLV. IPATS-BLV has inherent merits in measuring PVL and identifying susceptible and resistant cattle with superior simplicity and speed because of a single-well assay. Our new laboratory technique contributes to strengthening risk-based herd management used to control within-herd BLV transmission. Furthermore, this assay design potentially improves the diagnostics of other infectious diseases by combining the pathogen level and disease-related host genetic biomarker to predict disease outcomes.

Project description:BackgroundThough droplet digital PCR (ddPCR) has emerged as a promising tool for early pathogen detection in bloodstream infections (BSIs), more studies are needed to support its clinical application widely due to different ddPCR platforms with discrepant diagnostic performance. Additionally, there is still a lack of clinical data to reveal the association between pathogen loads detected by ddPCR and corresponding BSIs.MethodsIn this prospective study, 173 patients with suspected BSIs were enrolled. A multiplex ddPCR assay was used to detect 18 pathogens. The results of ddPCR testing were evaluated in comparison with blood cultures (BCs) and clinical diagnosis. Taking BC as the gold standard, receiver operating characteristic curve and Cohen's kappa agreement were used to investigate whether the pathogen load could predict a corresponding culture-proven BSI for the top five microorganisms detected by ddPCR.ResultsOf the 173 blood samples collected, BC and ddPCR were positive in 48 (27.7%) and 92 (53.2%) cases, respectively. Compared to BC, the aggregate sensitivity and specificity for ddPCR were 81.3% and 63.2%, respectively. After clinical adjudication, the sensitivity and specificity of ddPCR increased to 88.8% and 86.0%, respectively. There were 143 microorganisms detected by ddPCR. The DNA loads of these microorganisms ranged from 30.0 to 3.2×105 copies/mL (median level: 158.0 copies/mL), 72.7% (104/143) of which were below 1,000 copies/mL. Further, statistical analysis showed the DNA loads of Escherichia coli (AUC: 0.954, 95% CI: 0.898-1.000, κ=0.731, cut-off values: 93.0 copies/mL) and Klebsiella pneumoniae (AUC: 0.994, 95% CI: 0.986-1.000, κ=0.834, cut-off values: 196.5 copies/mL) were excellent predictors for the corresponding BSIs. The DNA loads of Pseudomonas aeruginosa (AUC: 0.816, 95% CI: 0.560-1.000, κ=0.167), Acinetobacter baumannii (AUC: 0.728, 95% CI: 0.195-1.000), and Enterococcus spp. (AUC: 0.282, 95% CI: 0.000-0.778) had little predictive value for the corresponding culture-proven BSIs.ConclusionOur results indicate that the multiplex ddPCR is a promising platform as a complementary add-on to conventional BC. The DNA loads of E. coli and K. pneumoniae present excellent predictive value for the corresponding BSIs. Further research is needed to explore the predictive potential of ddPCR for other microorganisms.

Dataset Information

Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

Background

Methods

Results

Conclusion

Publications

Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

Similar Datasets

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