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Optimization and analytical validation of the Allplex HPV28 genotyping assay for use in first-void urine samples.

Author(s): Bell M, Baussano I, Rol M, Tenet V, Heideman DAM, Gheit T, Van Caesbroeck A, Vorsters A, Clifford G

Publication: J Clin Microbiol, 2024, Vol. , Page e0140424

PubMed ID: 39723836 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how urine processing parameters that included a centrifugation step, different extraction kits, and specimen concentration affect the detection of human papilloma virus (HPV) DNA. Specifically, HPV DNA detection was compared between (1) urine that was centrifuged or not prior to extraction with STARMag Universal Cartridge Kit and analysis by the Allplex HPV28 assay; (2) urine that was used directly for extraction with the STARMag Universal Cartridge versus the manual QIAamp Kit and analyzed with the Allplex HPV28 assay; (3) urine that was used directly for extraction with the STARMag Universal Cartridge and analyzed using the Allplex assay; and (4) urine that was concentrated using an Amicon filter, extracted using the automated NucliSENS kit and assayed using either the GP5+/6+ RLB or the E7-MPG assay. In total, 701 first void urine specimens (630 HPV positive and 71 HPV negative) were self-collected using a Colli-Pee device containing urine conditioning medium. Specimens were shipped and stored at -80°C (duration not specified). To investigate the effects of urine concentration and extraction methods, four different workflows were compared: (1) a 4 mL aliquot was concentrated using an Amicon Ultra-4 50K filter device until the volume was < 1mL and DNA was extracted with the automated NucliSENS EasyMag Kit and quantified using PCR-enzyme immunoassays for GP5+/6+ or the Luminex PCR bead-based E7-MPG assay; (2) a 2 mL aliquot was centrifuged at 4,000 × g for 20 min and 0.5 mL was used for DNA extraction with the automated STARMag Universal Cartridge Kit and quantified with the real-time PCR Allplex HPV28 assay; (3) DNA was directly extracted from 0.5 mL urine using the automated STARMag Universal Cartridge Kit and quantified with the Allplex HPV28 assay; and (4) DNA was directly extracted from 0.2 mL urine using the manual QIAamp DNA Mini Kit and quantified with the Allplex HPV28 assay.

   Summary of Findings:

   Genotype-specific positivity was very strongly correlated between urine that was centrifuged and urine that was directly extracted using the automated STARMag Universal Cartridge Kit (R² = 0.981, P < 0.001), with kappa agreements of 0.70 to 1.00 observed for each of the HPV genotypes. Similarly, genotype-specific positivity was very strongly correlated between urine that was directly extracted using the automated STARMag Universal Cartridge Kit or the manual QIAamp method (R² = 0.959, P < 0.001), with kappa agreements of 0.45 to 1.00 observed for each of the HPV genotypes.  CT values were strongly correlated with no bias observed in Bland-Altmann plots between (a) urine specimens that were centrifuged prior to extraction and those that were used directly, and (b) urine specimens extracted using the automated STARMag Universal Cartridge Kit and those extracted using the manual QIAamp method. 

   When HPV genotypes were compared between case-matched urine that was concentrated using an Amicon filter, extracted using the automated NucliSENS kit and assayed using the GP5+/6+ RLB assay and urine that was directly used for DNA extraction with the STARMag Universal Cartridge Kit and quantified with the Allplex assay, 266 specimens (38.1%) were positive with both methods, 100 (14.3%) were positive only using the Allplex assay, and 35 (5%) were positive only with the GP5+/6+ assay. The remaining specimens were either negative by both methods or were positive for a HPV genotype that was not among the 28 genotypes detectable by both assays. Direct extraction and analysis with Allplex resulted in higher positivity for all 28 shared HPV genotypes than when urine was concentrated and analyzed using the GP5+/6+ RLB assay. Kappa values between Amicon concentrated urine assayed with GP5+/6+ and non-concentrated urine assayed with Allplex ranged from 0 (genotype not detected in any specimen using the GP5+/6+ RLB assay) to 1.00. When Amicon filter-concentrated urine analyzed by the E7-MPG assay was compared to urine that was directly used for DNA extraction and analyzed with the Allplex assay, 336 specimens (48.1%) were positive with both methods, 30 (4.3%) were positive only using the Allplex assay, and 238 (34.1%) were positive only with the E7-MPG assay. Direct extraction and analysis with the E7-MPG assay resulted in higher positivity for all 21 shared HPV genotypes than when urine was concentrated and analyzed using the Allplex assay. Kappa values between Amicon-concentrated urine assayed with E7-MPG and non-concentrated urine assayed with Allplex ranged from 0.40 to 0.86.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Immunoassay
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Filtration	Concentrated with Amicon Ultra-4 50K filter device Not concentrated
   Analyte Extraction and Purification	Analyte isolation method	Automated STARMag Universal Cartridge Kit Manual QIAamp DNA Mini Kit Automated NucliSENS EasyMag Kit
   Real-time qPCR Specific	Technology platform	E7-MPG Allplex HPV28 GP5+/6+ RLB
   Biospecimen Aliquots and Components	Centrifugation	Centrifuged Not centrifuged

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