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Background <p>Human parvovirus B19 (HB19V) infection poses significant clinical challenges in resource-limited settings, particularly regarding blood safety surveillance and diagnosis of associated syndromes. Limited access to validated diagnostic assays restricts clinical management and transfusion safety protocols. The objective is to develop and validate an in-house duplex real-time PCR assay for HB19V detection and evaluate its clinical utility in a resource-limited setting.</p> Methods and Results <p>A duplex real-time PCR assay targeting the conserved NS1 gene of HB19V with RNase P as endogenous control was developed. Analytical validation assessed amplification efficiency, limit of detection (LOD), precision (intra- and inter-assay coefficients of variation), and cross-reactivity. Clinical validation compared qualitative agreement with a CE-certified commercial kit using Cohen’s kappa statistic. Positive samples underwent Sanger sequencing for genotype characterization. Clinical specimens from suspected HB19V infections were tested and results correlated with patient presentations. The assay demonstrated 98.82% amplification efficiency with LOD of 6 copies/µL. Precision metrics showed excellent reproducibility (CoV &lt; 1% intra-assay, &lt; 5% inter-assay) with no cross-reactivity. Qualitative agreement with the commercial reference was 100% (Cohen’s kappa &gt; 0.95). Clinical application identified three significant cases: post-transfusion HB19V in neonatal bilirubin encephalopathy, HB19V consideration in severe anaemia with ventriculitis, and active infection in paediatric Ewing sarcoma with chemotherapy-induced cytopenias requiring IVIG therapy. Genotyping revealed genotypes 1a and 3, confirming regional viral diversity.</p> Conclusion <p>This validated in-house duplex real-time PCR assay provides a cost-effective, locally sustainable diagnostic tool for HB19V detection. It addresses critical gaps in blood safety surveillance and expands diagnostic capacity for HB19V-associated syndromes in resource-limited settings, while highlighting the necessity of contextual clinical interpretation for optimal patient management.</p>

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Development and clinical validation of a PCR assay for human parvovirus B19 detection with applications in challenging diagnostic scenarios

  • 期刊论文

  • 发表日期：

    2026年7月3日

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  • Kashmi Sharma,
  • Rohan Shrivastava,
  • Somesh Mishra,
  • Poonam Parihar,
  • Debasis Biswas,
  • Narendra Kumar Chaudhary,
  • Poorva Gohiya,
  • Mahendra Jain,
  • Ram K. Nema,
  • Ashwin A. Raut,
  • Vandana Gupta,
  • Megha Katare Pandey

摘要

Background

Human parvovirus B19 (HB19V) infection poses significant clinical challenges in resource-limited settings, particularly regarding blood safety surveillance and diagnosis of associated syndromes. Limited access to validated diagnostic assays restricts clinical management and transfusion safety protocols. The objective is to develop and validate an in-house duplex real-time PCR assay for HB19V detection and evaluate its clinical utility in a resource-limited setting.

Methods and Results

A duplex real-time PCR assay targeting the conserved NS1 gene of HB19V with RNase P as endogenous control was developed. Analytical validation assessed amplification efficiency, limit of detection (LOD), precision (intra- and inter-assay coefficients of variation), and cross-reactivity. Clinical validation compared qualitative agreement with a CE-certified commercial kit using Cohen’s kappa statistic. Positive samples underwent Sanger sequencing for genotype characterization. Clinical specimens from suspected HB19V infections were tested and results correlated with patient presentations. The assay demonstrated 98.82% amplification efficiency with LOD of 6 copies/µL. Precision metrics showed excellent reproducibility (CoV < 1% intra-assay, < 5% inter-assay) with no cross-reactivity. Qualitative agreement with the commercial reference was 100% (Cohen’s kappa > 0.95). Clinical application identified three significant cases: post-transfusion HB19V in neonatal bilirubin encephalopathy, HB19V consideration in severe anaemia with ventriculitis, and active infection in paediatric Ewing sarcoma with chemotherapy-induced cytopenias requiring IVIG therapy. Genotyping revealed genotypes 1a and 3, confirming regional viral diversity.

Conclusion

This validated in-house duplex real-time PCR assay provides a cost-effective, locally sustainable diagnostic tool for HB19V detection. It addresses critical gaps in blood safety surveillance and expands diagnostic capacity for HB19V-associated syndromes in resource-limited settings, while highlighting the necessity of contextual clinical interpretation for optimal patient management.