Stability, enrichment, and quantification of total and HPV16-specific IgG present in first-void urine.

First-void urine (FVU) samples, containing human papillomavirus (HPV)-specific IgG from female genital tract secretions, provide a non-invasive option for disease monitoring and vaccine impact assessment. This study explores the utility of FVU for IgG quantification, exploring stability and compatibility with DNA preservation methods, alongside various IgG enrichment methods. Healthy female volunteers provided FVU and serum samples. FVU was collected with or without urine conservation medium (UCM) and stored under different conditions before freezing at -80 °C. Four IgG enrichment methods were tested on FVU samples. All samples were analyzed using three total human IgG quantification assays and an in-house HPV16-specific IgG assay. Samples stored with UCM buffer had higher total and HPV16-specific IgG concentrations (p ≤ 0.01) and IgG remained stable for at least 14 days at room temperature. Among IgG enrichment methods, Amicon filtration (AM) and AM combined with Melon Gel purification (AM-MG) provided similar HPV16-IgG concentrations, correlating strongly with serum levels. Protein G magnetic beads methods were incompatible with time-resolved fluorescence-based assays. This study highlights FVU as a reliable and convenient sample for IgG quantification, demonstrating stability for at least 14 days at room temperature and compatibility with UCM DNA preservation. It emphasizes the need to select appropriate IgG enrichment methods and confirms the suitability of both AM and AM-MG methods, with a slightly better performance for AM-MG.

Concentration strategies for spiked and naturally present biomarkers in non-invasively collected first-void urine.

BACKGROUND: First-void urine (FVU) provides a non-invasive method for collecting a wide range of biomarkers found in genital tract secretions. To optimize biomarker collection in FVU, this study investigated the impact of naturally present and supplemented precipitating agents: uromodulin (UMOD) and polyethylene glycol (PEG), on the concentration of human papillomavirus (HPV) pseudovirions (PsV), cell-free DNA (cfDNA), and cellular genomic DNA (gDNA) through centrifugation. METHODS: FVU samples from ten healthy female volunteers, along with a control sample, were spiked with seal herpesvirus 1 (PhHV-1) DNA, HPV16 plasmid DNA, and HPV16 PsV with an enhanced green fluorescent protein (EGFP) reporter. The samples were subjected to various concentration protocols involving PEG precipitation, low-speed centrifugation (5 min at 1000×g), and medium-speed centrifugation (1 h at 3000×g). Subsequently, quantitative PCR (qPCR) was used to assess cellular and cell-free glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, cell-free PhHV-1 and HPV16 DNA, and PsV (EGFP) DNA. In addition, UMOD levels were measured. RESULTS: The findings revealed that PEG significantly increased the concentration of cfDNA and gDNA in the pellet after centrifugation, with the most pronounced effect observed for cfDNA. Moreover, low-speed centrifugation without PEG effectively depleted cellular gDNA while preserving cfDNA in the supernatants. Pseudovirions were consistently pelleted, even with low-speed centrifugation, and a positive but not significant effect of PEG on PsV (EGFP) DNA yield in the pellet was observed. Additionally, a significant correlation was observed between UMOD and GAPDH, HPV16, and PsV (EGFP) DNA quantities in the pellet. Furthermore, large variations among the FVU samples were observed. CONCLUSIONS: With this study, we provide novel insights into how various biomarker precipitation protocols, including both the properties of FVU and the use of PEG as a precipitating agent, influence the concentration of cfDNA, cellular gDNA, and pseudovirions.

Follow-up of humoral immune response after HPV vaccination using first-void urine: A longitudinal cohort study.

Assessment of humoral immune responses following human papillomavirus (HPV) vaccination currently relies on invasive blood sampling. This longitudinal cohort study explores the usability of first-void urine as a noninvasive alternative sample for antibody detection. In this study, 58 women receiving three doses of the 9vHPV vaccine within a Gardasil9 (9vHPV) Phase III randomized controlled trial were included. Participants provided paired first-void urine and blood samples before vaccination (M0), 1 month after the third dose (M7), and ~3 years after the third dose (M43). Type-specific antibody responses to the 9vHPV types were analyzed in 174 first-void urine and 172 serum samples using a virus-like particle-based IgG multiplex enzyme-linked immunosorbent assay. Additionally, total human IgG concentrations were determined using the BioPlex assay. At M7, 1 month after complete 9vHPV vaccination, 95%-100% of first-void urine and 100% of serum samples had detectable concentrations, varying by HPV type. At M43, 84%-100% of first-void urine and 98%-100% of serum samples had HPV-specific antibody concentrations. Results show significant Spearman rank correlations between type-specific HPV-antibody concentrations for paired first-void urine and serum at all time points. This study confirms the potential feasibility of utilizing first-void urine as a noninvasive immunological sample within HPV vaccine trials.

Triage of human papillomavirus infected women by methylation analysis in first-void urine.

Host cell DNA methylation analysis in urine provides promising triage markers for women diagnosed with a high-risk (HR) human papillomavirus (HPV) infection. In this study, we have investigated a panel of six host cell methylation markers (GHSR, SST, ZIC1, ASCL1, LHX8, ST6GALNAC5) in cervicovaginal secretions collected within the first part of the urine void (FVU) from a referral population. Cytology, histology, and HPV DNA genotyping results on paired FVU and cervical samples were available. Urinary median methylation levels from HR-HPV (n = 93) positive women were found to increase for all markers with severity of underlying disease. Significantly elevated levels were observed for GHSR and LHX8 in relation to high-grade cervical intraepithelial neoplasia (CIN2 +; n = 33), with area under de curve values of 0.80 (95% Confidence Interval (CI) 0.59-0.92) and 0.76 (95% CI 0.58-0.89), respectively. These findings are the first to support the assertion that methylation analysis of host cell genes is feasible in FVU and holds promise as molecular, triage strategy to discern low- from high-grade cervical disease in HR-HPV positive women. Molecular testing on FVU may serve to increase cervical cancer screening attendance in hard-to-reach populations whilst reducing loss to follow-up and await further optimization and validation studies.

Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine.

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.