INTRODUCTION

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Human papillomaviruses (HPVs) are important human carcinogens (30). There is now overwhelming evidence from both epidemiological and functional studies that specific high-risk HPV types are one of the major etiological agents responsible for anogenital cancer. A role for HPV in nonmelanoma skin cancer (NMSC) has also been proposed (16, 17) but remains controversial with the possible exception of a role in the rare disorder epidermodysplasia verruciformis (EV). This condition is characterized by a genetic predisposition to widespread cutaneous infection with HPV types not usually pathogenic in the healthy population and the subsequent progression of these warts to squamous cell carcinoma (SCC) on sun-exposed sites (13).

There is also accumulating evidence that HPV may participate in the pathogenesis of NMSC in immunosuppressed renal transplant recipients (RTRs). Over 90% of patients develop viral warts and up to 40% of patients develop NMSC within 15 years of transplantation, a 50- to 100-fold increased risk compared with that for the general population (3). An association between viral warts and skin cancer in RTRs was first noted in Australia (29), and there is now both clinical and histological evidence which indirectly supports the progression of viral warts through increasingly dysplastic squamous lesions to invasive SCC (1, 4, 5, 11). In the early posttransplantation years, warts are usually of the common or palmoplantar type (11). With increasing time after transplantation, increasing age, and higher levels of sun exposure, flat warts on sun-exposed sites develop, and these may become confluent, particularly over the dorsum of the hands and forearms. Histologically, such warts often show cytological atypia, and it is from such areas that SCCs often arise (4).

The study of HPV in NMSC has been limited by the methods used for detection and typing of HPV from the skin. Most available methods rely on the amplification of DNA by PCR. However, the existence of at least 80 distinct HPV types and their genomic diversity dictate that only closely related HPV types will be detected if type-specific primers are used. This has led to the application of degenerate PCR (21). By this technique the combined studies from several groups suggest that diverse cutaneous, mucosal, and EV HPV types may be found in benign and malignant skin lesions, particularly from immunosuppressed individuals, and that more than one HPV type may be detected within an individual lesion (2, 8, 20, 22). Nonetheless, discrepancies still exist in the published data, which may in part reflect the different sensitivities and specificities of particular degenerate primers for the detection of HPV types (16, 17).

We have recently examined these discrepancies by evaluating the sensitivities and specificities of three degenerate primer sets which have previously been used individually to detect HPV DNA in benign and malignant skin lesions from RTRs (27). By comparing the primer sets HVP2-B5 and F15-B15 (20, 21) and MY09-MY11 (15) and the nested set CP62-CP69 and CP65-CP68 (2) in PCRs with serial dilutions of cloned HPV and with a series of mucosal and cutaneous warts, we observed that the combined panel of primers allowed detection of a broader range of HPV types than was possible with the individual primer sets. However, it was apparent that such an approach, although advantageous, would require further modification. In particular, while the sensitivity for detection of EV types was high, that for detection of mucosal types was lower and that for detection of cutaneous types was lower still. Furthermore, sequence data for HPV isolates from some lesions indicated the presence of multiple HPV types which could not be individually identified with the primer sets used.

In the present study, we describe a modified degenerate PCR technique which seeks to address the problems inherent in existing methodologies. This approach has the capacity to detect a broad spectrum of cutaneous, mucosal, and EV HPV types to a high degree of sensitivity. We have used this technique to analyze a series of viral warts from RTRs.

	MATERIALS AND METHODS

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HPV plasmids. Twenty-eight plasmid clones containing HPV genomes were used as representatives of cutaneous (HPV type 1 [HPV-1], -3, -4, -10, and -41), mucocutaneous (HPV-2 and -57), mucosal (HPV-6, -11, -16, -18, -26, -31, -32, -33, -34, -66 and -72), and EV (HPV-5, -8, -14, -19, -20, -22, -23, and -36) HPV types (see Table 2).

Patients. Patients comprised 23 immunosuppressed RTRs (18 men and 4 women). Ten had a history of NMSC, 3 had a history of premalignancies only, and 10 had had no premalignant or malignant skin lesions, despite the presence of a transplanted kidney for at least 10 years.

Tissue samples. Specimens comprised 51 cutaneous warts. All lesions were histologically confirmed to be warts and were derived from biopsy specimens collected at the time of surgery, snap frozen, and stored at 70°C. Three large warts were subdivided into between two and four sections, and each section was analyzed independently. On the basis of clinical and/or histological criteria, the warts fell into two categories. The first category comprised verrucae vulgares or common warts (n = 23), which were considered to be those warts with typical morphological features and no evidence of dysplasia histologically. The second category of warts (n = 28) were those warts that occur on sun-exposed sites with atypical clinical features and/or evidence of epithelial dysplasia histologically (4). In addition, 20 benign warts obtained from 15 immunocompetent patients with warts were also analyzed (see below).

DNA extraction. Samples were finely minced with sterile scalpels on a petri dish, washed twice in phosphate-buffered saline, and resuspended in lysis buffer containing proteinase K to a concentration of 0.1 mg/ml. Following lysis overnight at 37°C, samples were pelleted and the supernatant was removed and placed in a fresh tube. DNA was then extracted by a standard phenol-chloroform-isoamyl alcohol technique followed by ethanol precipitation (14).

PCR primers. On the basis of our preliminary data, established degenerate primer pairs were modified in order to improve the detection of cutaneous, mucosal, and EV HPV types (Table 1). These primers were located within the highly conserved L1 (major capsid protein) open reading frame (ORF) of the HPV genome. By using the multiple alignment program Clustal V and the Genetics Computer Group (GCG) package of software programs, nested primers were designed within the existing degenerate primer pairs in the case of all HPV phylogenetic groups (6) with the exception of cutaneous group B2 (see below). The resulting primers were analyzed with the Oligo 4 Primer Analysis Software program (National Biosciences Incorporated).

(i) Cutaneous HPV types. The degenerate primer pair HVP2-B5 was described by Shamanin et al. (20, 21) for detection of HPV from all groups with the exception of the phylogenetic clade comprising HPV-4, -48, -50, -60, and -65, for which the primer pair F14-B15 was used. Of the primer pairs evaluated in our preliminary studies, these preferentially detected cutaneous HPV types, but only at a viral copy number of 5 to 5,000 copies/cell (27). We therefore designed the following primers nested within HVP2 and B5 to increase the sensitivity and specificity for detection of indicated cutaneous types: CN3F-CN3R, group A2 (HPV-3, -10, -28, -29, and -77); CN2F-CN2R, group A4 (HPV-2, -27, and -57); CN1F-CN1R, group E (HPV-1, -41, and -63). The single-round primer pair C4F-C4R was designed to detect the cutaneous group B2 (HPV-4, -48, -50, -60, and -65) in order to improve the sensitivity obtained with F14-B15.

(ii) Mucosal HPV types. The primer pair MY09-MY11 was originally designed by Manos et al. (15) for the detection of HPV in genital lesions. In preliminary studies it detected mucosal HPV types at approximately 10 fg to 1 pg (0.05 to 5 copies/cell). To increase the level of sensitivity, a second established primer, GP6, was seminested within MY11 (24).

(iii) EV HPV types. Berkhout et al. (2) have described several pairs of nested primers which preferentially detect EV HPV types. Of these, one particular set comprising CP62-CP69 as an outer primer pair and CP65-CP68 as an internal nested pair was chosen for the purposes of this study since they were highly sensitive, detecting EV HPV types at levels of 10⁴ copies per cell. In order to facilitate the detection of mixed EV-associated HPV infections potentially present in skin, as predicted by our preliminary data and those of others (2, 27), additional nested primer pairs within CP62-CP69 were designed for the major EV HPV clusters, as follows: EN1F-EN1R, cluster a1 (HPV-5, -8, -12, -36, -47, and -ICPX1); EN2F-EN2R, cluster a2 (HPV-14, -19, -20, -21, and -25); and EN3F-EN3R, clusters b1 and b2 (HPV-9, -15, -22, -23, -37, -38, -RTRX1, -VS42, -VS73, -RTRX3, -RTRX6, -VS92, and -VS102).

PCR amplifications. PCR amplifications were performed exactly as described for previously published primer pairs. For the newly designed primers, amplification reactions were performed in 50 µl of a reaction mixture containing 1 U of AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, Conn.), GeneAmp 10× PCR Buffer II and 2 mM MgCl₂ (supplied by the manufacturer), a 0.2 mM concentration of each deoxynucleotide triphosphate (Advanced Biotechnologies Ltd.), and 50 pmol of each primer. These conditions were established with magnesium and annealing temperature titrations. Thirty cycles of PCR were performed (95°C for 1 min, 50°C for 1.5 min, and 72°C for 2 min), followed by extension at 72°C for 5 min. All PCRs were performed on a Perkin-Elmer 480 thermal cycler.

Sequence analysis. Amplified PCR products that appeared as visible bands after ethidium bromide staining were purified after separation in a 2% agarose low-melting-point gel (QIAquick Gel Extraction Kit; QIAGEN) and were directly sequenced by fluorescent dideoxynucleotide chain termination cycle sequencing on a Perkin-Elmer 2400 thermal cycler (ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit; Perkin-Elmer) with both forward and reverse primers. The products were analyzed on a Perkin-Elmer 377 ABI Prism automated sequencer. The nucleotide sequences obtained were analyzed with the Sequence Navigator computer software (Macintosh). Sequences of 140 bases or more with fewer than 5% unidentified bases were processed. The forward and reverse complement sequences were aligned, and the homology of the consensus sequence was compared with those of known HPV types available through the GenBank database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md.) by using the GCG Blast program. In accordance with established guidelines, a nucleotide sequence was regarded as an HPV type if it shared over 90% homology with a known type and a related type if the sequence homology was less than 90% (6).

Use of differential PCR to detect mixtures of viruses. On the basis of our previous study, it was predicted that a proportion of lesions were likely to harbor more than one HPV type (27). In order to determine whether the panel of primer sets would detect their target when a mixture of viruses was present, HPV plasmids from three different phylogenetic groups were mixed together. The primers designed to detect these types were used sequentially to amplify the mixture. Since mixtures with EV HPV types in particular had previously emerged in clinical samples from immunosuppressed patients (27), EV HPV types 5, 14, and 23 from clusters a1, a2 and b1-b2, respectively, within group B1 (6) and the corresponding primers EN1, EN2, and EN3 were chosen for the purposes of this experiment. Each plasmid was used at a final concentration of 50 ng/ml in the plasmid mixtures.

Comparison of differential PCR versus PCR and cloning for detection of mixtures of viruses. An alternative strategy for the detection of mixtures of HPV types within individual lesions is to clone the PCR products generated by the general degenerate primers for cutaneous, EV, and mucosal types and then to sequence a proportion of the resulting clones. We compared the ability of the multiple nested primer and direct sequencing approach adopted in this study with PCR and cloning to differentially detect mixtures of HPV types. By using as a model the mixtures of EV HPV plasmids described above, the gel-purified PCR amplification products generated by the nested PCR with CP62-CP69 and CP65-CP68 were cloned with the PCR-Script Cloning Kit (Stratagene). At least five resulting colonies were picked and amplified with primers CP65 and CP68 to identify positive clones. The PCR products were then gel purified and sequenced as described above.

Confirmation of results obtained for clinical specimens with L1 degenerate primers with E6 type-specific primers. Type-specific primers for the E6 ORFs of HPV-10, -23, -24, and -27 (Table 1) were designed by using E6 nucleotide sequence data (Los Alamos National Laboratory HPV Sequence Database, 1997). The HPV types were randomly chosen from among those that had occurred at least once in the series of clinical specimens. By using the E6 ORF primers, PCR amplification of several representative clinical samples was used as additional confirmation of the results obtained with the L1 degenerate nested primers. Amplification reactions were performed in 50 µl of reaction mixture as described above. Forty cycles of PCR were performed (95°C for 1 min, 55°C for 1 min, and 72°C for 1 min), followed by extension at 72°C for 5 min. In plasmid titration experiments, each E6 primer set detected its relevant target plasmid to a sensitivity of at least 0.01 fg.

Comparison of results obtained by an alternative degenerate PCR-based methodology. De Villiers et al. (9) have recently refined their PCR-based HPV DNA detection and genotyping method to include a set of degenerate EV HPV-specific nested primers first described by Berkhout et al. (2), in addition to their 16 established pairs of degenerate PCR primers (22). DNA from some of the clinical samples analyzed as described above had previously been analyzed by this methodology. Although DNA was extracted from the residual tissue for the purposes of this study, such that identical aliquots of DNA were not analyzed by the two laboratories, it was of interest to us to compare the results obtained by both methods for the same lesions as further validation of our nested primer approach.

Comparison of results with those for warts from immunocompetent individuals. In order to exclude the possibility that our HPV detection methodology was overly sensitive or that the results were attributable to PCR artifacts, a biological control in the form of a series of warts from immunocompetent patients was also analyzed. Lesions comprised 20 benign warts from 15 patients, and the clinical spectrum was extended to include not only cutaneous warts but also examples of lesions from anogenital, oral, and conjunctival mucosae. HPV DNA detection and genotyping were performed exactly as described above for warts from RTRs.

	RESULTS

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Sensitivity and specificity of degenerate primer sets. Analysis of serial dilutions of individual cloned HPV plasmids confirmed the increased specificity and sensitivity of the modified degenerate PCR approach described here (Table 2). Representative HPV types from the main cutaneous, mucosal, and EV phylogenetic groups were each detected to a sensitivity of at least 10 fg (0.05 copies per cell), although this varied (0.001 to 10 fg, equivalent to 5 × 10⁶ to 0.05 copies per cell).

Detection of mixtures of EV-associated HPV plasmids by differential PCR or PCR and cloning. By analyzing mixtures of EV HPV types 5, 14, and 23, we observed that the respective primer sets EN1, EN2, and EN3 successfully identified the EV HPV type for which they were specifically designed. Following PCR cloning of the same plasmid mixtures, all three HPV types included were also successfully detected. It proved necessary to sequence at least four clones from each respective mixture in order to ensure detection of both HPV types present. The capacity of differential PCR to detect mixed EV HPV infections in clinical lesions is illustrated in Fig. 1. The electropherogram presented in Fig. 1A is the result of PCR amplification of lesion iv from patient 11 with the general EV HPV primers CP62-CP69 and CP65-CP68, followed by sequencing with CP68. The poor quality of the sequence is evident, in particular, the large number of ambiguous bases, and this almost certainly reflects the superimposed sequences of at least two HPV types. In contrast, Fig. 1B and C show the results for the same lesion following PCR with the nested primer pair EN2 and EN3, respectively. In both cases the sequences are clear with no ambiguous bases and represent HPV-25 and -38, respectively.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Mixed EV HPV types are detected with cluster-specific nested primers. The electropherograms in panels A to C were from lesion iv from patient 11. (A) The sequence was obtained with the general EV HPV primer CP68 and shows features suggestive of the presence of at least two distinct HPV types (see text). (B) The electropherogram was obtained with EN2R, the reverse primer for the pair designed to detect EV cluster a2. This sequence appears to be a single type and was subsequently proved to represent HPV-25. (C) The electropherogram was obtained with EN3R, the reverse primer for EV HPV clusters b1 and b2. This sequence also appears to be a single type and represents HPV-38.

Clinical specimens from RTRs. By using the combination of primers, HPV DNA was found in all of the warts analyzed. Examples of PCR amplifications from selected clinical samples are shown in Fig. 2. Cutaneous HPV types predominated, being found in 43 of 51 (84.3%) warts; EV HPV types were found in 41 (80.4%) lesions, and mucosal types were found in 14 (27.4%) warts. Mixed infections, in which two or more HPV types were detected, occurred in 48 of 51 (94.1%) warts. The majority of mixed infections contained two or three different HPV types, but six distinct types were identified in one wart (lesion i, patient 12). Mixed infections were predominantly with cutaneous and EV HPV types.

View larger version (113K): [in this window] [in a new window]   FIG. 2.   Gel image of representative PCR amplification products from clinical samples after amplification with selected primer pairs. Lanes 1 to 6, lesion iii from patient 12 with nested general EV HPV-specific primer pair (CP62-CP69 and CP65-CP68); lanes 1 and 2, PCR negative buffer controls (first and second rounds, respectively); lane 3, buffer extraction control; lane 4, PCR-negative DNA control (human placental DNA); lane 5, patient sample (HPV-RTRX5); lane 6, positive control (10 fg of HPV-5 plasmid); lanes 7 to 12, lesion iv from patient 6 with nested cutaneous primers for group E (CN1F and CN1R); lanes 6 and 7, PCR-negative buffer controls; lane 9, buffer extraction control; lane 10, PCR negative DNA control; lane 11, patient sample (HPV-41); lane 12, positive control (10 fg of HPV-1 plasmid); lanes 13 and 14, lesion i from patient 5 with nested cutaneous primers for group A4 (CN2F and CN2R); lane 13, patient sample (HPV-57); lane 14, positive control (10 fg of HPV-2 plasmid); lanes 15 and 16, lesion ii from patient 3 with nested cutaneous primers for group A2 (CN3F and CN3R); lane 15, patient sample (HPV-10); lane 16, positive control (10 fg of HPV-3 plasmid); lanes 17 to 22, lesion from immunocompetent patient 2 with cutaneous primers for group B1 (C4F and C4R); lane 17, PCR-negative buffer control; lanes 18 and 19, negative extraction controls (buffer and HPV-negative tissue); lane 20, PCR-negative DNA control; lane 21, patient sample (HPV-4); lane 22, positive control (10 fg of HPV-4 plasmid); lanes 23 to 28, lesion from immunocompetent patient 12 with mucosal seminested primers (MY11 and GP6); lanes 23 and 24, PCR-negative buffer controls; lane 25, buffer extraction control; lane 26, PCR-negative DNA control; lane 27, patient sample (HPV-11); lane 28, positive control (10 fg of HPV-16 plasmid); lanes M, molecular size markers (100-bp DNA ladder). Bands of 600 and 100 bp are indicated.

Clinical specimens from immunocompetent individuals. We were particularly interested to compare the spectrum of HPV types detected within transplant-associated warts with those in warts from immunocompetent individuals in order to exclude the possibility that the observation of multiple, diverse types within single lesions from RTRs is an artifact of highly sensitive PCR primers (Table 5). In only one lesion was more than one HPV type found; this was also the only lesion in which an EV HPV type was identified and was clinically a somewhat unusual recurrent nipple wart. For all other lesions the HPV types detected were those expected at cutaneous (HPV-2, -3, -4, -10, and -57) and mucosal (HPV-6, -11, -16, -32, and -57) sites.

Confirmation of results. DNA from three samples which were found to harbor HPV-27 with the degenerate L1 ORF primers and DNA from four samples which were HPV-27 negative were amplified with the HPV-27 E6 type specific primers, and in all cases the results confirmed the results obtained with the L1 primer. Similarly, the presence of HPV-23, -24, and -10 was confirmed in the warts tested. The HPV genotypes in eight warts were also independently tested by a second laboratory, the Deutsches Krebsforschungszentrum, Heidelberg, Germany. The technique used consisted of a degenerate PCR methodology with 16 degenerate primer pairs and a pair of nested EV HPV primers (9). In five of the warts examined, the methods were concordant for the presence of at least one HPV type (Table 2). For the remaining three lesions, the HPV types identified by the two laboratories differed. However, it is noteworthy that for one of the warts with apparently discrepant results (lesion i, patient 17), the HPV types detected by the two laboratories (HPV-VS92-1 and HPV-9, respectively) are very closely related.

	DISCUSSION

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Previous studies of benign warts in RTRs by degenerate PCR and other methodologies have failed to detect HPV DNA in as many as 40% of lesions, suggesting either a failure to detect known HPV types or the presence of as yet unidentified HPV types (17). By using the modified degenerate nested PCR technique described here, we have achieved an HPV DNA detection level of 100% for the 71 viral warts analyzed. These data suggest that such a technique is sufficiently sensitive and discriminatory for detection of the wide spectrum of HPV types that may be harbored by the lesions of immunosuppressed individuals. Particularly notable is the capacity of this approach to identify mixed infections, such that combinations of cutaneous, mucosal, and EV HPV types were successfully detected within individual warts. We have confirmed the validity of this technique by comparison with PCR and cloning, by using type-specific primers from a different ORF, by comparison with results obtained from a different laboratory, and finally, by comparison of the spectrum of HPV types detected with a control series of warts from immunocompetent individuals.

Comparison with previous data on warts from transplant recipients. Over 20 case reports or series published to date have included data on the HPV types found in warts from RTRs, and the spectrum of types identified has proved to be remarkably variable. These discrepancies most likely reflect the different experimental approaches used (17). The majority of studies have used DNA hybridization techniques (Southern blotting, dot blotting, and in situ hybridization), which, in general, tend to be less sensitive and specific than PCR-based methods. Many of these series report a spectrum of HPV types in warts from RTRs similar to those found in the immunocompetent population, particularly HPV types 2 and 4 and, to a lesser extent, types 1, 3, and 10. Fewer studies have found EV or mucosal HPV types. The problem of cross-hybridization inherent in these techniques is exemplified by the discrepancies in the rates of detection of HPV-2 and -3. Previous studies may have overreported the presence of HPV-2 as a probable consequence of cross-hybridization with probes for the closely related type HPV-27, which we found to occur more frequently than HPV-2. This has also been confirmed with a recent series of warts from immunocompetent individuals, in which HPV-27 and -57 were also the most commonly detected HPV types (19). Similarly, HPV-3 may have been overreported through cross-hybridization with HPV-77 and HPV-28, which are closely related and which we found to be more prevalent than HPV-3.

Interlaboratory variation in HPV typing results. It is well recognized that variations in HPV typing methodologies between different investigators may account for discrepancies in published data on warts from transplant recipients and patients with nonmelanoma skin cancers (16, 17, 27), and this is the first study to examine this possibility directly by analyzing the same lesions by two such methodologies. In the majority (five of eight lesions), there was concordance in the HPV types identified, while the results for three lesions were nonconcordant and in several lesions additional HPV types were found by our laboratory. Differences in the sensitivity and specificity profiles for the panel of primer pairs used in each laboratory are a likely explanation (9, 27), and these data highlight the desirability of developing a unified typing methodology for laboratories that study HPV in the skin. However, it is also important that for each of the eight samples, DNA was extracted independently from duplicate portions of the same lesion. Since we have observed that different portions of the same lesion processed under identical conditions may harbor different HPV types, this may also have been a factor that contributed to nonconcordance.

Detection of multiple HPV types. The frequent existence of mixed infections in warts from transplant recipients as shown in our study is striking and is in contrast to the findings for warts from immunocompetent individuals. It is unclear whether this represents coinfection of single cells with multiple HPV types or the presence of a mixture of cells infected with single virus types. Similarly, it is not known whether only one or all of the HPV types present are responsible for the phenotype or whether lesions are polyclonal with several HPV types simultaneously being transcriptionally active. Published data relating to these issues are so far limited and inconsistent. In one study in which several genital HPV types were detected within a single anal wart from an RTR, localization by in situ hybridization suggested that each HPV type maintained regional separation within the lesion (7). Unger et al. (28) also reported the presence of multiple HPV types within anogenital warts in immunosuppressed human immunodeficiency virus-positive patients clustered in geographically distinct areas. These data may lend support to the hypothesis of a polyclonal origin for viral warts; i.e., they arise from multiple founder cells that carry distinct HPV types. In contrast, by double fluorescence hybridization, HPV-1 and -63 were both detected within nuclei from a plantar wart (10), although only the cytopathogenic effect of HPV-63 was evident in the infected cells. Our data for three warts divided for the purposes of HPV genotyping might provide further evidence of regional separation of individual HPV types within lesions. Furthermore, differences in HPV detection between our methodology and that of the Deutsches Krebsforschungszentrum might also be partly explicable on this basis since DNA was extracted from different portions of the same lesion independently. Rigorous evaluation by in situ hybridization of cutaneous warts from transplant recipients will be necessary to further our understanding of the presence of multiple HPV genotypes within single lesions in terms of the spatial localization, relative viral load, and physical state of each type; we are undertaking such a study.

HPV typing and clinical features of warts from transplant recipients. Few previous studies have analyzed HPV in warts from transplant recipients with respect to their anatomical localization and morphological features, yet this may be particularly relevant in terms of establishing the relationship between HPV and NMSC. Malignancies are usually observed to colocalize with clinically and histologically atypical warts on sun-exposed sites rather than with common warts (verrucae vulgares) at other sites (11). In immunocompetent individuals, there seems to be a correlation between the morphology of common warts and the inducing HPV type (19). Overall, however, we found no significant differences in the prevalence of each of the major HPV groups found in common warts and the clinically or histologically atypical warts from sun-exposed sites, nor did a clear association emerge between one individual HPV type or any one group of HPVs and skin cancer risk.

Limitations of differential degenerate PCR methodology. Our aim in this study was to develop a robust method capable of detecting HPV types across a broad spectrum of HPV groups. The resulting differential PCR methodology, despite its proven usefulness in detecting multiple HPV types within individual lesions, has a number of inherent limitations. Perhaps the most obvious is that it may not detect mixtures of HPV types within a particular group. This may be the explanation for failure to identify the HPV type(s) present in the lesions from patients 10 (lesion v) and 11 (lesions ii and iii). However, to achieve this by a PCR and direct sequencing approach, type-specific primers would have to be used (i.e., at least 80 primer pairs), and these would be unlikely to allow detection of potentially novel HPV types. In circumstances in which the presence of more than one HPV type within a particular group is suspected, PCR cloning might therefore prove to be a useful adjunct. In addition, it is also possible that this methodology may still fail to detect certain known or novel HPV types with sufficient sensitivity, a likely explanation for failure to detect HPV in a portion of a wart from patient 9 (lesion iii). Nonetheless, the rate of detection of HPV DNA in 75 of 76 (98.7%) warts or portions of warts (and 100% of individual warts) is higher than that achieved in most previously published series.