Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-014-2147-2

ARTICLE

Optimization of HPV DNA detection in urine by improving collection, storage, and extraction A. Vorsters & J. Van den Bergh & I. Micalessi & S. Biesmans & J. Bogers & A. Hens & I. De Coster & M. Ieven & P. Van Damme

Received: 22 January 2014 / Accepted: 28 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported. In this paper, we examined the impact of a DNAconservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided firstvoid and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.

The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample. In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.

A. Vorsters (*) : S. Biesmans : A. Hens : I. De Coster : P. Van Damme Centre for the Evaluation of Vaccination, Vaccine & Infectious Disease Institute, University of Antwerp, Universiteitsplein 1, 2610 Antwerpen, Belgium e-mail: [email protected]

I. Micalessi : J. Bogers Applied Molecular Biology Research Group, Laboratory of Cell Biology & Histology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium

S. Biesmans e-mail: [email protected]

Introduction A bivalent vaccine that targets HPV 16/18 and a quadrivalent vaccine that targets HPV 6/11/16/18 show excellent efficacy

I. Micalessi e-mail: [email protected] J. Bogers e-mail: [email protected]

A. Hens e-mail: [email protected] I. De Coster e-mail: [email protected] P. Van Damme e-mail: [email protected] J. Van den Bergh Laboratory of Experimental Hematology, Tumor Immunology Group, Vaccine & Infectious Disease Institute, Antwerp University Hospital, University of Antwerp, Wilrijkstraat 10, 2650 Edegem, Belgium e-mail: [email protected]

M. Ieven Laboratory for Microbiology, Vaccine & Infectious Disease Institute, Antwerp University Hospital, University of Antwerp, Wilrijkstraat 10, 2650 Edegem, Belgium e-mail: [email protected]

Eur J Clin Microbiol Infect Dis

and safety [1]. HPV vaccination induces a high concentration of antibodies that lead to a sterilizing protection against infection [2]. Recent data confirm that the vaccines not only protect the vaccinated individual against high-grade lesions but also reduce HPV infection in a non-vaccinated subpopulation, indicating decreased circulation of HPV [3, 4]. To control a disease at the population level, good surveillance, including sensitive and affordable testing and practical sampling, is required to monitor the infection. Detection of HPV DNA in urine, a specimen that can easily be obtained by non-invasive self-sampling and (if necessary) mailed to a laboratory, has been suggested for surveillance and impact studies [5, 6]. Indeed, several countries are beginning to use urine samples collected in chlamydia surveillance programs to determine baseline HPV epidemiological status and monitor the impact of HPV vaccination programs. However, the detection of HPV DNA in urine is not as straightforward as many authors assume [5]. Methods used for HPV detection in cervical smears or for chlamydia detection in urine may be less sensitive when applied to HPV DNA detection in urine. The impact of the assay used to detect HPV DNA is well known and has recently been reconfirmed [7]. A full understanding of how and in what form HPV DNA enters the urine remains lacking. The most realistic scenario is that secreted mucus containing debris of desquamated infected cells from the outer layers of the mucosa (in women, this material includes debris of desquamated cells from the cervix) contaminates the first-void urine. This proposed scenario implies that depending on the life cycle of the virus and the stage of the infection, HPV particles, free HPV DNA, free HPV episomal DNA, and/or cell-associated HPV DNA may be present in the urine. In addition, more HPV DNA would be present in the first fraction of a void than in the remaining void. Spiking experiments with HPV plasmid DNA in urine have shown major reductions in detection rates compared to spiking in water or in phosphate-buffered saline (PBS) [5, 8]. Research has reported that the detection of HPV DNA from HPV DNA-containing cell lines such as HeLa is less hampered by DNA degradation [5, 9, 10]. According to research on the detection of free DNA in urine, the rapid disappearance of detectable free DNA is attributed to nuclease activity [11–13]. To our knowledge, only one study has examined the presence of cell-free HPV DNA in urine [14]. Objectives of the study The objectives of this study are to evaluate the effects of storage, sample preparation, extraction, and sampling on the detection of HPV DNA in the urine of HPV-positive women by (a) comparing five extraction methods and analyzing the impact of a urine-conservation medium (UCM1) that was developed in-house based on quantitation of HPV DNA and human DNA, (b) comparing the effect of untreated urine

stored at room temperature for 7 days, urine mixed with an in-house conservation medium, and urine mixed with a commercially available preservation buffer, and (c) investigating the impact of testing first-void versus midstream urine on the detection of HPV DNA and human DNA.

Materials and methods Urine samples Women who were recruited for a phase-one HPV therapeutic vaccine trial were enrolled in the present study. In line with the inclusion criteria of the vaccine trial, the participants had a cervical sample with normal cytology that tested positive for HPV 16 or 18 DNA. The study was approved by the Ethical Committee of the University Hospital in Antwerp, and all women provided informed consent to participate in the study. Three separate experiments were designed. In the first two experiments, during consultation, the study doctor asked the participants (25 women in the first experiment and 13 women in the second experiment) to deliver a first-void urine sample of approximately 50 ml in a standard receptacle for urine collection (100 ml). The urine sample was processed immediately after collection, as depicted in Fig. 1a and b. In brief, in the first experiment comparing the different extraction methods, one fraction was stored untreated and one fraction was mixed (two parts urine and one part UCM1). UCM1 is based on a phosphate-buffered saline (PBS) solution with the addition of a chelating agent, a microbicide, a fungicide, and bovine serum albumin. Samples were stored at room temperature for 72 ± 4 h. Subsequently, the samples were mixed and aliquots of 1.4 ml and 1 ml were subtracted. All material was then frozen for 7–90 days at −20 °C until further processing. Figure 1b shows the sample flow for the second experiment, in which the in-house buffer was compared with no treatment and a commercial preservation medium (BD ProbeTec™, BD Benelux N.V. Erembodegem). The samples were aliquoted into three vials, as follows: one vial without conservation medium (UCM0), one with an in-house medium (UCM1), and one containing a commercial preservation medium (BD ProbeTec™). Urine samples were stored at room temperature for 7 days and frozen at −20 °C until further processing. In the third experiment, ten participants were asked to provide an approximately 50-ml first-void urine sample and the subsequent fraction in two separate containers. The urine was treated with UCM1 as described for the above experiments. Samples were immediately frozen at −20 °C. In total 48 urine samples originating from 44 different women were collected over the three experiments.

Eur J Clin Microbiol Infect Dis

a

b

Fig. 1 a Sample flow to examine the impact of a urine-conservation medium and five extraction methods. b Sample flow to study the effect of storage for 7 days at room temperature without preservation buffer, an in-house buffer, and a commercial conservation medium

DNA extractions Figure 2 shows the five extraction methods and extracted volumes for the first experiment. The QIAamp DNA mini kit (Qiagen GmbH, Germany) blood and body-fluid spin protocol and reagents were used for all four manual DNA extractions, as follows: (a) 200 μl of urine was used directly in the QIAamp DNA mini kit protocol and (b) a modified protocol was used for the Amicon Ultra-4 50 K centrifugal filter devices (Merck Millipore, Belgium). After a 20-min centrifugation at 4000g, the filter was rinsed with 180 μl PBS to maximize recovery. Subsequently, 20 μl of proteinase K and 200 μl of lysis buffer were added and the solution was mixed thoroughly. After a 10-min incubation at 56 °C, the lysate was transferred to a 1.5-ml microcentrifuge tube, and the standard protocol was followed.One ml was centrifuged for 1 h at 23000g; (c) 200 μl of the supernatant (SN) was

directly extracted using the Qiagen DNA mini kit; (d) after removal of the remaining supernatant, the pellet was suspended in 180 μl PBS before proteinase K and lysis buffer were added. Extraction proceeded using the Qiagen DNA mini kit and (e) 1 ml was extracted using the Nuclisens easyMAG™ automate (BioMérieux, Benelux), following the standard protocol with off-board lysis. The elution volumes for the manual extractions and the easyMAG extraction were 50 μl and 55 μl, respectively. All extractions from a given sample were completed within 48 h of each other. In the second experiment, 4 ml of urine (UCM0 arm), 4 ml of urine/ UCM1 mixture (UCM1 arm), and 3.2 ml of urine (BD arm) were concentrated using the Amicon filters, as described above. Following filtration, the filter was rinsed with 2 ml of NucliSENS Lysis Buffer (BioMérieux, Benelux) and incubated for 10 min. Subsequently, DNA extraction was performed using the generic easyMAG off-board lysis protocol. The

Eur J Clin Microbiol Infect Dis Fig. 2 Overview of the sample flow and DNA extraction methods performed in the first experiment

elution volume was 100 μl. This adapted lysis on the filter allows to combine EasyMag extraction and Amicion filtration. In the third experiment, the urine was treated with UCM1 and extraction was performed as described for the second experiment.

concentration (ng/μl) were determined based on the standard curves. The amount of genomic DNA (ng) present in each sample was divided by the weight of one genome equivalent (6.6 pg/cell) to obtain the human DNA copy number.

Statistics Real-time PCR: quantification of HPV 16, HPV 18, and human DNA The quantitative PCRs (qPCRs) for HPV 16, HPV 18, and human DNA (hDNA) were based on Taqman technology and performed with the LightCycler® 480 (Roche Applied Science) in 20-μl volumes containing 1× LightCycler® 480 Probes Master (Roche Applied Science), 0.5 μM of each primer, 0.1 μM of the probe, and 5 μl of template DNA. The amplification conditions were 10 min at 95 °C for FastStart Taq DNA Polymerase activation, followed by 45 cycles of 10 s at 95 °C and 15 s at 60 °C. Table 1 provides an overview of the sequences of the primers and probes [15–17]. To perform absolute quantification of the amount of HPV and human DNA present in an unknown sample, standard curves and calibrators were made to correlate DNA quantity and Ct value. External standard curves were generated for each HPV type based on 10-fold serial dilutions of DNA plasmids containing full-length HPV 16 and HPV 18 (Clonit, Milan, Italy; range: 5x106 to 50 copies, three reactions per concentration). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) standard curve was obtained through the amplification of 10-fold serial dilutions of female human DNA (261 μg hDNA/mL, Promega, Fitchburg, WI, USA; range 1.31x105 to 1.31 ng, with three reactions per concentration). The HPV copy number (copies/μl) and DNA

For the statistical analysis, we used IBM SPSS Statistics Version 20 software. Based on the quantitative data, significant differences between the different treatments and extraction methods were examined using a Friedman test for multiple extractions or a related-samples Wilcoxon signed-rank test for two-by-two comparisons. Differences in the second and third experiments were analyzed using a related-samples Wilcoxon signed-rank test. Statistical significance was defined as p