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Journal of Clinical Microbiology, May 2009, p. 1428-1435, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02080-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Performance of a Polymer-Based DNA Chip Platform in Detection and Genotyping of Human Papillomavirus in Clinical Samples

T. Schenk,^1, T. Brandstetter,² A. zur Hausen,³ J. Alt-Mörbe,⁴ D. Huzly,¹ and J. Rühe^2*

Department of Virology, University Medical Center Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany,¹ Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Köhler-Allee 103, D-79110 Freiburg, Germany,² Institute of Pathology, University Medical Center Freiburg, Breisacher Strasse 115a, D-79106 Freiburg, Germany,³ Laboratory for DNA—Analytics, Klarastrasse 63, D-79106 Freiburg, Germany⁴

Received 28 October 2008/ Returned for modification 22 December 2008/ Accepted 2 March 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Persistent infection with human papillomavirus (HPV) is now well known to play the key role in the development of cervical carcinoma, which is the second most common malignancy in women worldwide. Certain HPV genotypes have been shown to be associated with a high risk for carcinogenesis. HPV type 16 (HPV-16) and HPV-18 are responsible for more than 70% of cervical carcinomas, whereas other high-risk genotypes (HPV-31, -33, -45, -52, -58, and others) are found less frequently (34, 38, 39).

Due to the high negative predictive value of a negative HPV test result combined with negative cytology results, it is expected that, in the future, DNA testing will complement cytology in routine gynecological screening, thus enabling the development of a more cost-efficient screening program with expanded intervals between patient visits (11). For women with low-grade squamous intraepithelial lesions or atypical squamous cells of undetermined significance, HPV testing is an important requirement for triage strategies (3, 32). Since a significant proportion of HPV infections, including high-risk genotypes HPV-16 and -18, are cleared within several months by the immune system, single detection of HPV DNA alone is not sufficient to predict cancer development, especially in younger women (5). Consequently, the ability to genotype at least the most common high-risk HPV strains, i.e., HPV-16 and -18, in order to distinguish persistent infections with the same genotype from subsequent infections with other HPV genotypes is an important feature of modern HPV assays (16, 33).

In the last 2 years, we have seen a breakthrough in the prevention of HPV infections. Much of the progress is the result of immunological studies that have paved the way for development of the first effective preventive vaccines (9, 25, 35, 37). Since the vaccines presently being marketed are considered effective only in a preventive and not in a therapeutic setting, a highly sensitive and specific diagnostic tool for HPV genotyping is required to exclude active HPV infection before vaccination (10).

Furthermore, a versatile tool for HPV genotyping will be useful for monitoring epidemiologic consequences following broad clinical use of HPV vaccination, i.e., long-term protection against genotypes included in the vaccine, cross-protection against certain other genotypes, and a potential shift in the prevalence of genotypes not covered by direct or cross-protection.

Traditional methods for HPV detection, e.g., cytological and immunological methods, show low specificity and sensitivity. Currently, molecular methods, including nucleic acid hybridization and PCR, are most frequently used for HPV detection (18, 30). However, there is an inherent danger of false-positive results due to unspecific amplification, as well as false-negative results due to variations in primer-binding sites in the target region of the virus, which decrease the amplification efficiency of some HPV genotype sequences. In addition, HPV genotyping requires either genotype-specific PCR or subsequent labor-intensive procedures, such as sequencing and hybridization. These methods exhibit difficulties in identifying all HPV genotypes present in multiple infections (36).

Recently, the first studies have been reported involving the genotyping of HPV with genotype-specific oligonucleotides and DNA microarray analysis (1, 2, 7, 12, 13, 14, 15, 19, 20, 22, 23, 24, 27, 28, 31). HPV chip technology offers advantages both in the detection of multiple infections and in sensitivity (T. Brandstetter, S. Böhmer, O. Prucker, E. Bissé, A. zur Hausen, J. Alt-Mörbe, and J. Rühe, submitted for publication).

In this study, we compared a new polymer-based DNA biochip assay (Biochip) for HPV detection and genotyping (Brandstetter et al., submitted) to a CE-marked commercially available PCR kit that includes reverse hybridization of PCR amplicons on nitrocellulose membrane strips (GenID-PCR), using clinical samples (cervical swabs, tissue biopsy samples, and others). Genetic sequencing served as a reference standard in case of divergent results from the two methods.

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Sample collection and isolation of nucleic acids. A total of 123 nonselected clinical samples sent to the Department of Virology at the University Medical Center Freiburg for routine HPV diagnosis were prospectively included in the study. All the samples were immediately stored at 4°C until further processing and underwent nucleic acid isolation procedures within 1 to 2 working days.

Cervical swabs were taken with special brushes (Duo-Brush; Laboratoire C.C.D., Paris, France), and the head of the brush was released and submerged in 2 ml of methanol containing sample fixation medium (Laboratoire C.C.D., Paris, France) for transport to the laboratory at ambient temperatures. To release attached cells prior to nucleic acid isolation, the tube containing the fixation medium and the brush head were thoroughly vortexed. Two milliliters of cell suspension was transferred to a 2-ml reaction tube and washed twice for 2 min by centrifugation at 14,000 rpm, followed by removal of supernatant and resuspension of the pellet in 1 ml of phosphate-buffered saline. After the second washing step, the pellet was resuspended in 200 µl of phosphate-buffered saline and used for DNA isolation with the Qiagen DNA blood minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions (protocol for viral DNA with elution in 60 µl of preheated buffer AE).

Tissue biopsy samples were sent to the laboratory in sterile isotonic NaCl solution. A maximum of 5 mm³ tissue was cut into small pieces with a single-use scalpel in a sterile petri dish. Fragments were transferred into a 1.5-ml reaction tube, and 180 µl of lysis buffer ATL (Qiagen, Hilden, Germany) and 20 µl proteinase K (Qiagen, Hilden, Germany) were added before the reaction mixture was incubated at 60°C overnight or until complete lysis occurred. The solution was subsequently used for DNA isolation with the Qiagen DNA blood minikit as described above, but omitting the addition of protease during the procedure.

Superficial skin swabs were transported in sterile isotonic NaCl solution. Attached cells were vigorously resuspended by vortexing prior to nucleic acid isolation. A total of 200 µl of the cell suspension was processed with the Qiagen DNA blood minikit (protocol for viral DNA) according to the manufacturer's instructions.

Purified viral and cellular DNA was immediately frozen in two aliquots of 30 µl each and stored at –20°C until PCR amplification. One of these aliquots was used for routine HPV diagnosis with the commercially available GenID-PCR.

Amplification procedure for Biochip. All the DNA samples used in the present study were prepared at the Department of Virology at the University Medical Center Freiburg. A minimum of 200 ng total DNA was used for HPV genotyping. A pair of oligonucleotide primers specific for consensus sequences within the late 1 (L1) regions of all known HPV was applied. The sequence of the sense primer was 5'-TRT TTG TTA CTG TKG TWG ATA C-3' (TAGAT 5.2, in-house primer), and that of the antisense primer was 5'-CptGptTpt CptCptH ARR GGW AYT GAT C-3' (MY09) (31), producing a PCR product of nearly 405 bp (genotype-specific length variation). The antisense primer had phosphothionate primer modifications (indicated by lowercase letters) of the first five nucleotides at the 5' end, protecting the antisense PCR strand from exonuclease digestion.

The PCR was performed in a 0.2-ml vial (Biozym, Hessisch Oldendorf, Germany) using a Primus 25 advanced system (MWG Biotech, Ebersberg, Germany), containing 1x buffer (Qiagen, Hilden, Germany), 4 mM MgCl₂ (Qiagen, Hilden, Germany), a nucleotide mix (dATP, dCTP, and dGTP, 300 µM each; Amersham, New Jersey), biotin-11-dUTP (30 µM; Fermentas, St. Leon-Rot, Germany), dTTP (80 µM; Amersham, New Jersey), the primers (1 µM), and the HotStarTaq polymerase (0.1 U/µl; Qiagen, Hilden, Germany). The total reaction volume used was 50 µl. After activating the polymerase with incubation for 15 min at 95°C, 50 cycles were performed for 1 min at 95°C, followed by annealing at 53°C for 1 min and elongation at 72°C for 1 min. A final elongation was performed for 10 min at 72°C before cooling down to 4°C.

To obtain single-stranded PCR products on the chip, PCR products were digested with a T7 gene 6 exonuclease (70025Z; USB). Therefore, the sample was mixed with 5x digestion buffer and 50 U of T7 gene 6 exonuclease (USB, Ohio), then incubated for 15 min at 37°C, and finally denatured at 95°C. After immediately cooling down the sample with –20°C precooled ethanol, it was mixed in equal parts with 200 mM NaPi buffer (a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, pH 7.0). The sample with at least 30% single-stranded antisense PCR product was then used for analysis with Biochip.

Biochip: description of assay. A total of 63 probe sequences with 5' modifications of 15-mer thymine tails (high-performance liquid chromatography purified; TIB Molbiol, Berlin, Germany) were printed in four equal subarrays (two by two) using the sciFlexarrayer S5 (Scienion AG, Berlin, Germany). The oligo probes were spotted at a concentration of 20 µM in a spotting solution consisting of 400 mM NaPi on commercially available surfaces. The spotting solution contained, in addition to buffer salt and DNA, 1 mg/ml polymer. The printing temperature used was 22°C, with a humidity of 60 to 65%. The spot volume used was about 0.8 nl per dot, resulting in a spot diameter of about 175 µm. The spot spacing was chosen as 350 µm, and the spacing of 500 µm of the subarrays was 500 µm. The printing procedure was done with a 384-well plate. The printed arrays were annealed for 1 h at 70°C, followed by photo-cross-linkage at 254 nm with 1.25 J. After preparation, the samples were stored at room temperature. The subarray design is shown below (see Fig. 3A). Three oligo probes target one HPV genotype. The HPV PCR product of about 405 bp, whose length depends on the HPV genotype, carries three heterogeneous domains in the L1 region of the HPV genome. These domains can be used for identifying the HPV genotypes. Twelve HPV genotypes were selected for analysis by this HPV chip. The probes were surrounded by methodical controls, such as a negative control containing only spotting solution, which allows us to measure the background, one negative control containing a sequence of the human leukocyte antigene gene DRB1 (NCHL), one detection control containing a 3'-biotinylated oligonucleotide probe, a dilution line of hybridization control for an HPV-positive control, which consisted of a universal antisense HPV sequence of the L1 region, a dilution line of a coupling control (oligo probe with a 3' Cy5; highest concentration, 0.25 µM), and finally, a recalibration control of the chip (a β-globin sequence). All oligonucleotide probes employed by Biochip are listed in Table 1 . The main handling steps of the method are illustrated in Fig. 1.

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FIG. 3. Representative examples of Biochip and GenID-PCR results. (A) The subarray design is illustrated in detail, with all the specific probes in the center of the subarray and the systemic and methodical controls surrounding the specific probes. (B) A typical readout of HPV-6 genotyping after an exposure time of 60 s is shown. In addition, a picture of the PCR amplicon after gel electrophoresis and a typical GenID-PCR blot strip of the sample are attached. (C) Detection and genotyping of a sample with multiple HPV infections indicated by Biochip revealed three HPV genotypes (HPV-6, -18, and -31) after an exposure time of 60 s, whereas the GenID-PCR detects only HPV-6 and -18.

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TABLE 1. List of all specific HPV probe sequences, with three probes for each genotype

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FIG. 1. Biochip handling procedure. After DNA preparation from various samples, the labeled specific PCR for the L1 region of the HPV genome takes place. To optimize hybridization conditions, the PCR amplicon is reduced to a single strand by exonuclease digestion. Hybridization of the labeled amplicon and staining of the hybridized PCR amplicon at a dilution of 1:200 with streptavidin-Cy5 in a flow cell are followed by a fluorescence readout.

Biochip handling and detection. The chips were placed in a chip reader consisting of a laser, a flow cell chamber, and a charge-coupled-device (CCD) camera (Fig. 2) (21). In the flow cell, the hybridization progress of the molecules can be observed from the liquid phase to the immobilized oligonucleotides on a transducer surface. The excitation of fluorochromes is realized with a semiconductor laser. The fluorescence emission was collected by the cooled CCD camera. The chip was mounted in such a way that a homogeneous evanescent illumination of the active area of the sensor surface was achieved. Before hybridizing the DNA on the chip with the labeled analyte DNA (labeled single-stranded PCR amplicons), the fluorescence of the chip was measured at 30, 60, and 90 s under dry conditions. Once filled into the flow cell of the reader, the sample was hybridized over the array for 20 min. Next, the sample was removed. The chip was washed with 3 ml of 100 mM NaPi, which was slowly drawn through the cell. After being dried with air/nitrogen, the chip was incubated with streptavidin-Cy5 (PA 45001, diluted 1:200 in 100 mM NaPi; GE Healthcare, Munich, Germany), detecting the biotin-labeled PCR amplicon. In the next step, the chip was washed with 3 ml NaPi buffer solution to remove unbound streptavidin-Cy5. Exposure times between 30 and 90 s were chosen for the readout. A software package designed in-house for the determination of HPV genotypes was used.

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FIG. 2. Technical details of the Biochip reader. Monochromatic light (4) is coupled into the plastic microarray substrate (1) so that total internal reflection occurs. An evanescent field travels along the surface, allowing fluorescence excitation to occur. The substrate serves as a wave guide, which allows detection of the interaction between the immobilized probes (2) and the labeled DNA in the flow cell (3). A CCD camera (7) with its filter (8) detects the fluorescence intensities. The cooling system (5), with a Peltier element (6) behind it, regulates the temperature of the hybridization solution.

HPV detection and genotyping by GenID-PCR. In brief, the HPV amplification and HPV detection kit (GenID, Strassberg, Germany) contains two biotinylated primer pairs (PN-I and PN-II), targeting a 145-bp fragment of the HPV L1 gene, for two separate conventional PCRs. In addition, a primer pair specific for the human housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is included in the reaction mixture PN-I to control for adequate content of cellular DNA. Following PCR amplification, reaction products from PN-I and PN-II are mixed and used for hybridization with HPV-specific oligonucleotide probes immobilized on a nitrocellulose strip in the HPV genotyping kit. Streptavidin-coupled alkaline phosphatase binds to the captured, biotin-labeled amplification products. After addition of BCIP-NBT (5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium) as a colorigenic substrate, the band pattern can be analyzed.

Each strip holds control bands for conjugate addition and nucleic acid isolation, as well as amplification (GAPDH). A polyspecific HPV band indicates the presence of HPV DNA from any of the covered genotypes, whereas the high-risk HPV band comprises high-risk genotypes (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -66, -68, -73, and -82), and the low-risk HPV band represents low-risk genotypes (HPV-6, -11, -40, -42, and -44). Single-specific bands are included for HPV-16 and -18 as the most frequent causes of cervical carcinoma, as well as for HPV-45, -6, and -11, the latter two of which cause genital warts (condyloma acuminata). Group-specific bands represent high-risk HPV genotypes 31, 33, 35, and 39 (3X) and high-risk HPV genotypes 51, 52, 53, 56, 58, and 59 (5X).

Sequencing. PCRs were separated on a 1.8% (wt/vol) agarose gel, and fragments were excised under UV light with 365 nm and purified with the Qiagen MinElute gel extraction kit (Qiagen, Hilden, Germany). Fragments were eluted with 12 µl H₂O, and 6 µl was subjected to sequence analysis using the fluorescein-labeled forward primer with the SequiTherm Excel II sequencing kit by Epicentre Biotechnologies (Madison, Wisconsin) on an ALF sequencer (Pharmacia, Uppsala, Sweden).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In total, 123 nonselected samples were analyzed by both methods. The majority of the samples (82.1% [101/123]) consisted of gynecological swabs (99 cervical swabs and 2 vaginal swabs). Of these, 25.7% (26/101) were taken after previous therapy (i.e., cervical conization, laser treatment, etc.) due to cytological or histological abnormalities. For 5.0% (5/101), HPV positivity had been documented in the preceding tests.

Concomitant cytological data (with the following classes: Pap II, 1 sample; Pap IIIA, 1 sample; Pap IIID, 13 samples; Pap IVA, 1 sample; Pap V, 1 sample) and/or histological data (cervical intraepithelial neoplasia grade 1 [CIN1], 3 samples; CIN2, 6 samples; CIN3, 3 samples; vulval intraepithelial neoplasia 3, 1 sample; adenocarcinoma, 1 sample) were available for 91.1% of the samples (92/101). Of these, 67 showed normal cytological and/or histological results. Of the 16 samples with Pap III cytology or higher, 14 tested HPV positive (9 with high-risk genotypes). In nine samples with histology findings of CIN2 or higher, seven were HPV positive (six with high-risk genotypes).

Of the remaining samples, 6.5% (8/123) were biopsy samples or swabs from patients with diagnosed or suspected genital warts (condyloma acuminata), 5.7% (7/123) were biopsy samples from otorhinolaryngeal sources (three were laryngeal, two palatal, one epiglottal, and one unknown), 4.1% (5/123) were dermal biopsy samples or swabs from skin warts, and 1.6% (2/123) were orolabial swabs or biopsy samples (Table 2).

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TABLE 2. Characterization of samples included prospectively into the study

Figure 3 shows representative assay results as exemplified for an HPV-6 monoinfection (Fig. 3B) and a triple infection with HPV-18, -31, and -6 (Fig. 3C). In the latter case, HPV-31 was detected only by Biochip. A total of 33.3% (41/123) of all the samples tested positive with at least one of the two methods. For 18.7% (23/123) of the samples, the results were identical for the twoassays, but 14.6% (18/123) of the positive samples yielded discrepant results. A total of 6.5% (8/123) of the samples were positive in Biochip but negative in the GenID-PCR, and in 2.4% (3/123), coinfections were detected only by Biochip. The opposite constellation was found in 4.1% (5/123) and 0.8% (1/123) of the samples, respectively. A total of 6.5% (8/123) of the samples were sequenced, six samples were in accordance with the Biochip result, and only one was in accordance with the GenID-PCR result.

Major errors, defined as failure to detect infection with high-risk HPV genotypes, were seen with equally low frequency in the two methods. In 4 of 123 cases (3.3%), a high-risk genotype infection was detected only by the GenID-PCR. Three (2.4%) infections with high-risk HPV genotypes were detected only by Biochip, and Biochip detected a coinfection of two high-risk genotypes in two (1.6%) samples, whereas only one high-risk genotype was detected by GenID-PCR (Table 3).

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TABLE 3. Samples with discrepant test results

A total of 60.1% (74/123) of the samples tested negative with both methods. A total of 5.7% (7/123) of the samples showed no GAPDH band on the GenID-PCR, leading to an invalid test result, but produced a positive β-globin signal on Biochip. Only one sample, a superficial swab taken from a genital wart, failed to produce either a GAPDH band or a β-globin signal.

For calculation of overall assay agreement, a total of 115 samples evaluable by both methods were used. Detection of an HPV genotype in one assay that was not covered by the other was not assessed as being discordant. Consequently, 87.8% (101/115) of the samples produced identical results in the two assays.

High-risk HPV genotypes were detected in 30 samples (24.3%), i.e., 29 cervical swabs and 1 skin swab. Six cases of a double infection and one case of a triple infection with different genotypes were identified, five of which involved a combination of two different high-risk genotypes. The most frequently occurring high-risk genotype was HPV-16, which was detected in 12 samples (10 with previous therapy, abnormal cytology, or pathology results), followed by HPV-45 (3 samples) and HPV-18 (2 samples). Genotypes from the HPV 3X group (genotypes 31, 33, 35, and 39) were found in seven samples, and those from the HPV 5X group (51, 52, 53, 56, 58, and 59) were found in five samples. One high-risk genotype that was not further typeable by GenID-PCR was found in a skin swab.

Low-risk genotypes were detected in 20 samples (16.2%). The most frequently detected low-risk genotype was HPV-6, and it was found in 11 samples (7 cervical swabs, 1 genital wart sample, 3 laryngeal papillomatosis samples). Other low-risk genotypes with genotype identification were HPV-11 (genital wart sample), HPV-43 (cervical swab, Pap IIID; coinfection with HPV 53), HPV-57 (skin wart sample), HPV-62 (cervical swab, Pap IIID), HPV-84 (Pap IIID/CIN3 after LASER treatment), and HPV-90 (cervical swab, Pap IIID). Three low-risk HPV infections were detected by the GenID-PCR without specific genotype identification (Table 4).

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TABLE 4. Overview of HPV genotypes detected in different sample materials

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In modern laboratories, it is essential that assay techniques are compatible with routine work flow. Procedures employed for isolation of viral DNA were identical in the two methods under investigation here. Furthermore, the time and effort needed to pipette for a conventional PCR, for amplification of the target DNA, and for the hands-on genotyping reaction are comparable. In the case of Biochip, the HPV genotyping procedure requires 60 min after amplification, including single-stranded digestion, hybridization, washing, and staining steps. Except exonuclease digestion, all handling steps and incubations take place in the Biochip reader, which consists of a hybridization flow cell (Fig. 2). A specially developed HPV software module adapted to ATR software package version 2.1 was programmed to render it instrumental for automatic identification of HPV genotypes by Biochip. Since sample hybridization takes place at room temperature, other samples can be handled consecutively by using seal frames for sample hybridization. One biochip can be processed within 15 min; hence, 12 samples can be analyzed within 3 h. The GenID-PCR uses reverse hybridization of the biotin-labeled amplification products to genotype-specific DNA probes immobilized on a nitrocellulose strip, followed by detection of the captured amplicons by a chromogenic reaction mediated by streptavidin-coupled alkaline phosphatase. This procedure requires manual pipetting steps, incubation periods in a heater/shaker water bath, and several washing steps on a shaker. In total, about 3 h are also needed to process 12 samples. However, automation of the genotyping reaction for the GenID-PCR is possible by use of a programmable blot robot, e.g., the Tecan ProfiBlot (Tecan, Mainz, Germany). Since the hybridization temperature must be kept strictly at 47°C, with a maximum deviation of only 0.5°C according to the manufacturer's instructions, a heatable unit with accurate temperature control is necessary.

In this study, we compared the results obtained by the newly developed multiplex Biochip to those obtained by GenID-PCR, a commercially available CE-marked test kit for the detection and genotyping of HPV. A total of 123 prospectively collected samples were available for comparison, representing the unselected sample population sent to our routine laboratory.

In summary, the two methods showed good interassay agreement, with 87.8% identical results. Major errors, defined by failure to detect a high-risk HPV genotype, occurred with equally low frequency in the two methods, without a clear preference for certain genotypes or source materials. Considering the slightly different panels of low-risk genotypes covered, the overall low number of discrepant results indicates a comparable analytical sensitivity for the two assays (Table 3). However, genetic sequencing, used as a gold standard for genotype determination, revealed a higher analytical specificity for Biochip.

Likewise, the two methods showed equal performance in detecting double infections (Table 3). We found one case of an assumed triple infection, which was detected only by Biochip. Thus, it can be speculated that due to the technically different nature of chip detection, Biochip might have an advantage in the identification of multiple infections involving more than two different genotypes. Interestingly, in a previous study comparing the GenID-PCR to the INNO-LiPA (line probe assay) kit (Siemens Medical Solutions Diagnostics, Leverkusen, Germany), which is also based on the reverse hybridization principle, the INNO-LiPA kit detected more high-risk infections than the GenID-PCR assay (17, 29).

Biochip is coated with sets of probes specific for a number of single HPV genotypes. In contrast, due to limited space on the hybridization strips, GenID-PCR combines multiple different genotypes in group-specific bands for the detection of high-risk HPV groups 3X and 5X. This strategy might cause problems in subsequent transient infections with high-risk genotypes of the same group, since an HPV-31 infection, for instance, which has cleared and is followed by HPV-33, would be interpreted as persisting by the GenID-PCR. It is well known that only persistent infections with the same high-risk genotype are at risk for cancer development (16). In this respect, Biochip offers a technical advantage, due to fewer restrictions for the absolute number of spots for individual genotype identification.

In agreement with published data, in our study, HPV-16 was the high-risk genotype identified most frequently, followed by HPV-45 and HPV-18. HPV-18 is commonly referred to as the second most common genotype in high-grade cervical lesions (34). The relatively low prevalence of HPV-18 in our study might be explained not only by the limited number of samples but also by an additional sampling bias. In Germany, HPV testing with cervical swabs is mostly used for the triage of atypical squamous cell of undetermined significance or low-grade squamous intraepithelial lesion findings (4), whereas high-grade lesions are surgically excised by cervical conization, and subsequent HPV testing is then performed with the excised tissue in a pathology laboratory. Hence, in our study cohort, recent high-grade lesions might be underrepresented.

The specificity of Biochip was achieved using 35- to 38-bp synthesized oligonucleotides specific for the 12 HPV genotypes studied (Brandstetter et al., submitted). To improve the PCR process carried out before Biochip analysis, we designed a new sense primer called TAGAT 5.2, which detected all 12 HPV genotypes without any loss in specificity or sensitivity compared to MY11 (data not shown). To achieve the best differentiation ability among these 12 HPV genotypes, all the oligonucleotides selected contained more than four mismatches within the corresponding heterogeneous gene loci. The PCR product of the L1 region captured three of these heterogeneous gene loci, with one oligonucleotide for each HPV genotype. In multiple infections, the amounts of type-specific PCR products can be lower due to competing amplification reactions. Together with the differences in binding affinity of detection probes, this occasionally results in fluorescence patterns with only two of three genotype-specific spots showing positive signals (HPV-6, -18, and -31) (Fig. 3C). Statistically, a minimum of two specific probes are necessary for distinct genotype identification in order to avoid false-positive results. Hence, in the case of Biochip, successful HPV genotyping is ensured, even in the presence of multiple infections with closely related HPV genotypes. In addition, high yields of HPV PCR amplification products from clinical samples, which potentially result in unspecific bindings, do not induce cross-hybridization in Biochip (data not shown).

According to a study on geographic variation in the prevalence of HPV genotypes in cervical cancer, HPV genotypes 16 and 18 are causative in 71% of cervical cancers worldwide (8, 26). The 15 most common genotypes for CIN and/or cervical carcinoma, in descending order of frequency, are 16, 18, 45, 31, 33, 52, 58, 35, 59, 56, 39, 51, 73, 68, and 66 (4). Based on this prevalence study, Biochip can detect approximately 85% of high-risk infections worldwide. According to a study by Castellsagué et al. (6), the probes printed onto Biochip cover the sequences of more than 95% of all cancer-relevant infections worldwide.

In conclusion, Biochip proved to have good usability in a routine diagnostic laboratory. Consequently, this HPV chip could become a suitable tool for routine diagnostic laboratories. Furthermore, it showed advantages in specific genotyping, especially in the case of certain genotypes for which only group-specific detection is achieved in GenID-PCR (HPV 3X and HPV 5X), as well as for multiple infections with more than one high-risk genotype. Further investigations, especially with paraffin-embedded samples, must be done to extend the capability of this biochip platform.

 
We thank Dieter Neumann-Haefelin for critical review of the manuscript, Otto Haller for support, and Franziska Rabold, Renate Mielke, and Susanne Riesterer for excellent technical support.

This research was funded by the German Research Association (DFG) under grant DFG RU 489/15-1.

 
* Corresponding author. Mailing address: Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Köhler-Allee 103, D-79110 Freiburg, Germany. Phone: 49 761 203 7160. Fax: 49 761 203 7162. E-mail: ruehe{at}imtek.de

Published ahead of print on 11 March 2009.

Current address: LADR GmbH, MVZ Baden-Baden, Lange Str. 65, D-76530 Baden-Baden, Germany.

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1
1. An, H. J., K. R. Kim, I. S. Kim, D. W. Kim, M. H. Park, I. A. Park, K. S. Suh, E. J. Seo, S. H. Sung, J. H. Sohn, H. K. Yoon, E. D. Chang, H. I. Cho, J. Y. Han, S. R. Hong, and G. H. Ahn. 2005. Prevalence of human papillomavirus DNA in various histological subtypes of cervical adenocarcinoma: a population-based study. Mod. Pathol. 18:528-534.[CrossRef][Medline]
2
2. An, H. J., N. H. Cho, S. Y. Lee, I. H. Kim, C. Lee, S. J. Kim, M. S. Mun, S. H. Kim, and J. K. Jeong. 2003. Correlation of cervical carcinoma and precancerous lesions with human papillomavirus (HPV) genotypes detected with the HPV DNA chip microarray method. Cancer 97:1672-1680.[CrossRef][Medline]
3
3. Bjerre, P., L. Silfverdal, L. Dillner, B. Hagmar, H. Edvardsson, J. Dillner, and A. Andersson-Ellström. 2008. A randomized trial of basing treatment on human papillomavirus and/or cytology results in low-grade cervical lesion triage. Am. J. Obstet. Gynecol. 199:24.e1-24.e7.[CrossRef][Medline]
4
4. Bollmann, R., A. D. Varnai, A. Bankfalvi, and M. Bollmann. 2007. Risk-adapted multimodal laboratory cervical screening—Pap test of the future? Pathologe 28:334-338.[CrossRef][Medline]
5
5. Bulkmans, N. W. J., J. Berkhof, S. Bulk, M. C. G. Bleeker, F. J. van Kemenade, L. Rozendaal, P. J. F. Snijders, and C. J. L. M. Meijer. 2007. High-risk HPV type-specific clearance rates in cervical screening. Br. J. Cancer 96:1419-1424.[Medline]
6
6. Castellsagué, X., M. Díaz, S. de Sanjosé, N. Muñoz, R. Herrero, S. Franceschi, R. W. Peeling, R. Ashley, J. S. Smith, P. J. F. Snijders, C. J. L. M. Meijer, and F. X. Bosch. 2006. Worldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactors: implications for screening and prevention. J. Natl. Cancer Inst. 98:303-315.[Abstract/Free Full Text]
7
7. Choi, Y. L., E. Y. Cho, J. H. Kim, S. J. Nam, Y. L. Oh, S. Y. Song, J. H. Yang, and D. S. Kim. 2007. Detection of human papillomavirus DNA by DNA chip in breast carcinomas of Korean women. Tumour Biol. 28:327-332.[CrossRef][Medline]
8
8. Cutts, F. T., S. Franceschi, S. Goldie, X. Castellsague, S. de Sanjose, G. Garnett, W. J. Edmunds, P. Claeys, K. L. Goldenthal, D. M. Harper, and L. Markowitz. 2007. Human papillomavirus and HPV vaccines: a review. Bull. W. H. O. 85:649-732.
9
9. Harper, D. M., E. L. Franco, C. M. Wheeler, A. B. Moscicki, B. Romanowski, C. M. Roteli-Martins, D. Jenkins, A. Schuind, S. A. Costa Clemens, and G. Dubin on behalf of the HPV Vaccine Study Group. 2006. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet 367:1247-1255.[CrossRef][Medline]
10
10. Hildesheim, A., R. Herrero, S. Wacholder, A. C. Rodriguez, D. Solomon, M. C. Bratti, J. T. Schiller, P. Gonzalez, G. Dubin, C. Porras, S. E. Jimenez, and D. R. Lowy for the Costa Rican HPV Vaccine Trial Group. 2007. Effect of human papillomavirus 16/18 L1 viruslike particle vaccine among young women with preexisting infection: a randomized trial. JAMA 298:743-753.[Abstract/Free Full Text]
11
11. Hoyer, H., C. Scheungraber, R. Kuehne-Heid, K. Teller, C. Greinke, S. Leistritz, B. Ludwig, M. Dürst, and A. Schneider. 2005. Cumulative 5-year diagnoses of CIN2, CIN3 or cervical cancer after concurrent high-risk HPV and cytology testing in a primary screening setting. Int. J. Cancer 116:136-143.[CrossRef][Medline]
12
12. Hwang, T. S., J. K. Jeong, M. Park, H. S. Han, H. K. Choi, and T. S. Park. 2003. Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray. Gynecol. Oncol. 90:51-56.[CrossRef][Medline]
13
13. Kahng, J., and H. J. Lee. 2008. Clinical efficacy of HPV DNA chip test in the era of HPV vaccination: 1,211 cases, a single institution study. Korean J. Lab. Med. 28:70-78.[CrossRef][Medline]
14
14. Kim, J. Y., J. K. Yoon, M. J. Citardi, P. S. Batra, and H. J. Roh. 2007. The prevalence of human papilloma virus infection in sinonasal inverted papilloma specimens classified by histological grade. Am. J. Rhinol. 21:664-669.[CrossRef][Medline]
15
15. Kim, K. H., M. S. Yoon, Y. J. Na, C. S. Park, M. R. Oh, and W. C. Moon. 2006. Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip. Gynecol. Oncol. 100:38-43.[CrossRef][Medline]
16
16. Kjaer, S. K., A. J. van den Brule, G. Paull, E. I. Svare, M. E. Sherman, B. L. Thomsen, M. Suntum, J. E. Bock, P. A. Poll, and C. J. Meijer. 2002. Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study. BMJ 325:572.[Abstract/Free Full Text]
17
17. Kjaer, S. K., G. Breugelmans, C. Munk, J. Junge, M. Watson, and T. Iftner. 2008. Population-based prevalence, type- and age-specific distribution of HPV in women before introduction of an HPV-vaccination program in Denmark. Int. J. Cancer 123:1864-1870.[CrossRef][Medline]
18
18. Kleter, B., L. van Doorn, L. Schrauwen, A. Molijn, S. Sastrowijoto, J. ter Schegget, J. Lindeman, B. ter Harmsel, M. Burger, and W. Quint. 1999. Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus. J. Clin. Microbiol. 37:2508-2517.[Abstract/Free Full Text]
19
19. Lee, G. Y., S. M. Kim, S. Y. Rim, H. S. Choi, C. S. Park, and J. H. Nam. 2005. Human papillomavirus (HPV) genotyping by HPV DNA chip in cervical cancer and precancerous lesions. Int. J. Gynecol. Cancer 15:81-87.[Medline]
20
20. Lee, H. S., K. M. Kim, S. M. Kim, Y. D. Choi, J. H. Nam, C. S. Park, and H. S. Choi. 2007. Human papillomavirus genotyping using HPV DNA chip analysis in Korean women. Int. J. Gynecol. Cancer 17:497-501.[CrossRef][Medline]
21
21. Lehr, H.-P., M. Reimann, A. Brandenburg, G. Sulz, and H. Klapproth. 2003. Real-time detection of nucleic acid interactions by total internal reflection fluorescence. Anal. Chem. 75:2414-2420.[Medline]
22
22. Li, R. Z., J. F. Shi, Q. Z. Zhou, R. F. Wu, N. Li, L. N. Wu, Y. Q. Zhou, Q. Wang, Z. H. Liu, B. Liu, and Y. L. Qiao. 2006. Evaluation of gene chip technology for high risk type human papillomavirus in cervical cancer screening. Zhonghua Yi Xue Za Zhi 86:307-311.[Medline]
23
23. Liu, C. H., W. L. Ma, R. Shi, Y. Q. Ou, B. Zhang, and W. L. Zheng. 2003. Possibility of using DNA chip technology for diagnosis of human papillomavirus. J. Biochem. Mol. Biol. 36:349-353.[Medline]
24
24. Luo, C. W., C. H. Roan, and C. J. Liu. 2007. Human papillomaviruses in oral squamous cell carcinoma and pre-cancerous lesions detected by PCR-based gene-chip array. Int. J. Oral Maxillofac. Surg. 36:153-158.[CrossRef][Medline]
25
25. Menzo, S., K. Marinelli, P. Bagnarelli, S. Rolla, and M. Clementi. 2007. Human papillomavirus infections: new perspectives for prevention and treatment. New Microbiol. 30:189-212.[Medline]
26
26. Muñoz, N., F. X. Bosch, X. Castellsagué, M. Díaz, S. de Sanjose, D. Hammouda, K. V. Shah, and C. J. Meijer. 2004. Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int. J. Cancer 111:278-285.[CrossRef][Medline]
27
27. Nuovo, G. J., D. Bartholomew, W. W. Jung, I. K. Han, T. Um, D. F. Grabarz, D. J. Lee, and R. T. McCabe. 2008. Correlation of Pap smear, cervical biopsy, and clinical follow-up with an HPV typing microarray system. Diagn. Mol. Pathol. 17:107-111.[CrossRef][Medline]
28
28. Park, T. C., C. J. Kim, Y. M. Koh, K. H. Lee, J. H. Yoon, J. H. Kim, S. E. Namkoong, and J. S. Park. 2004. Human papillomavirus genotyping by the DNA chip in the cervical neoplasia. DNA Cell Biol. 23:119-125.[CrossRef][Medline]
29
29. Perrons, C., R. Jelley, B. Kleter, W. Quint, and N. Brink. 2005. Detection of persistent high risk human papillomavirus infections with hybrid capture II and SPF10/LiPA. J. Clin. Virol. 32:278-285.[CrossRef][Medline]
30
30. Qu, W., G. Jiang, Y. Cruz, C. J. Chang, G. Y. Ho, R. S. Klein, and R. D. Burk. 1997. PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems. J. Clin. Microbiol. 35:1304-1310.[Abstract]
31
31. Rajan, S., T. H. Shen, J. Santhanam, N. H. Othman, N. Othman, and T. T. Hock. 2007. Comparison of DR. HPVtrade mark chip kit with hybrid capture II assay for the detection of human papillomavirus in clinical samples: a preliminary study. Trop. Biomed. 24:17-22.[Medline]
32
32. Schiffmann, M., and D. Solomon. 2003. Findings to date from the ASCUS-LSIL triage study (ALTS). Arch. Pathol. Lab. Med. 127:946-949.[Medline]
33
33. Schiffmann, M., P. E. Castle, J. Jeronimo, A. C. Rodriguez, and S. Wacholder. 2007. Human papillomavirus and cervical cancer. Lancet 370:890-907.[CrossRef][Medline]
34
34. Smith, J. S., L. Lindsay, B. Hoots, J. Keys, S. Franceschi, R. Winer, and G. M. Clifford. 2007. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. Int. J. Cancer 121:621-632.[CrossRef][Medline]
35
35. Villa, L. L., R. L. Costa, C. A. Petta, R. P. Andrade, J. Paavonen, O. E. Iversen, S. E. Olsson, J. Hye, M. Steinwall, G. Riis-Johannessen, A. Andersson-Ellstrom, K. Elfgren, G. Krogh, M. Lehtinen, C. Malm, G. M. Tamms, K. Giacoletti, L. Lupinacci, R. Railkar, F. J. Taddeo, J. Bryan, M. T. Esser, H. L. Sings, A. J. Saah, and E. Barr. 2006. High sustained efficacy of a prophylactic quadrivalent human papillomavirus types 6/11/16/18 L1 virus-like particle vaccine through 5 years of follow-up. Br. J. Cancer 95:1459-1466.[CrossRef][Medline]
36
36. Wang, D., L. Coscoy, M. Zylberberg, P. C. Avila, H. A. Boushey, D. Ganem, and J. L. DeRisi. 2002. Microarray-based detection and genotyping of viral pathogens. Proc. Natl. Acad. Sci. USA 99:15687-15692.[Abstract/Free Full Text]
37
37. Wu, T. C. 2007. Therapeutic human papillomavirus DNA vaccination strategies to control cervical cancer. Eur. J. Immunol. 37:310-314.[CrossRef][Medline]
38
38. zur Hausen, H. 2002. Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer 2:342-350.[CrossRef][Medline]
39
39. zur Hausen, H. 2000. Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis. J. Natl. Cancer Inst. 92:690-698.[Abstract/Free Full Text]

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Journal of Clinical Microbiology, May 2009, p. 1428-1435, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02080-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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