Papillomaviridae comprise a diverse family of non-enveloped, small circular double-stranded DNA viruses, capable of infecting mammals and birds [¹]. In humans these viruses cause pathologies that range from benign warts to malignant cancer. It is now accepted that HPV is the causative agent of more than 90% of all cervical cancers [²]. HPV has also been found to be associated with anal cancer [³] and anogenital warts [⁴]. There is increasing evidence that HPV is associated with head and neck squamous cell carcinoma unrelated to smoking and/or alcohol consumption [^5-7].

HPV types are divided into low- and high-risk groups with regards to their association with malignancy [⁸]. Low-risk HPV types 6 and 11 are most commonly detected in genital and anal warts, representing 90% of these cases [⁴]. Oncogenic HPV types 16 and 18 account for 70% of HPV-related cervical cancers. An increase in HPV viral loads has been correlated with disease progression in cervical cancer [⁹]. Similarly, elevated HPV viral loads were detected in HPV16-associated oropharyngeal squamous cell carcinomas [¹⁰]. These aforementioned associations highlight the importance of detecting, distinguishing, and quantitating both low risk and oncogenic HPV infections for monitoring and treating disease development and progression [⁹,¹⁰].

PCR represents a sensitive method for the detection of HPV DNA. Currently, standard nested HPV PCR can be performed using degenerative primers followed by direct sequencing of the PCR product. Alternatively, PCR products can be hybridized to DNA of known HPV types, to determine the type of HPV present in the PCR-amplified sample (e.g. Roche HPV Amplicor system). Both assays are time consuming and do not allow for the quantitation of viral DNA to address the role of viral load in disease progression.

Real time quantitative PCR (qPCR) allows for quantitation of DNA over 8 orders of magnitude [¹¹]. While qPCR assays have been developed for HPV16 and/or 18 [^12-16] relatively little has been done to develop assays that are capable of detecting other HPV types. Recently, two qPCR assays have been described that have increased the types of HPV that can be quantitatively detected [¹⁷,¹⁸]. In one assay, molecular beacon probes were used to distinguish low risk and oncogenic HPV types in a single multiplex reaction [¹⁸] although it did not allow for the specific determination of the HPV type. In another assay, TaqMan probes were designed to detect HPV16, HPV31, HPV 18/45 or HPV33/53/58/67 [¹⁷] in two separate multiplex reactions, did not allow the distinction between HPV33, 52, 58 or 67 and was unable to differentiate HPV18 from 45. More recently, a multiplex qPCR assay has been described that can quantitatively detect 7 oncogenic HPV types [¹⁹]. The assay is comprised of 2 reactions that detect only oncogenic HPV and no low risk HPVs. Thus, a single tube, multiplex reaction that can recognize common low and oncogenic HPV types involved in disease would be advantageous to the monitoring and detection of HPV infection. This would minimize reagents as well as decrease the chance for error that is inherent in performing multiple single or multiplex reactions.

This report describes two real time qPCR assays that can be used in tandem for detection of both common and uncommon HPV infection. Many distinct HPV types have been associated with disease hence, the first qPCR assay is a degenerate assay that targets the HPV E1 region of known HPVs and should allow for detection of any of these types. The second qPCR assay targets the coding region of the hypervariable loop V of the L1 gene of HPV6, 11, 16 and 18. These HPV types are associated with the majority of HPV related disease and are represented in the currently available quadrivalent vaccine. The primers and probes used in the development of the assay were designed to target and differentiate between these HPV types. In initial studies the vaccine has proven effective for prophylaxis against the initial infection by these types of HPV [²⁰,²¹]. Vaccine efficacy has been assessed by either cytology and/or qualitative PCR. The qPCR assays described in this study will provide a more efficient quantitative means of monitoring of HPV types in populations vulnerable to HPV-associated disease.

The causal relationship between oncogenic HPV and malignancy indicates the importance of being able to quickly detect and quantify specific HPV types when it is suspected that a lesion may harbor an HPV infection. This would aid in the assessment of precancerous lesions and impact on the decision to treat the lesion before it progressed to a more severe form of disease or allow the lesion to resolve on its own. In addition, low risk types of HPV also impact negatively on quality of life indicating the importance of tracking and identifying HPV types involved in the development of warts. Assays that are capable of distinguishing between common types of HPV responsible for cancers as well as those involved in the development of warts would facilitate the understanding of HPV epidemiology and pathology. In order to do so, two qPCR assays were developed for use in tandem. An E1 degenerate broad spectrum Sybr green-based assay was developed that would detect any HPV. In addition, the real-time qPCR that utilizes specific TaqMan probes labeled with different fluorophores represented an attractive method for quickly identifying and quantifying the presence of multiple viruses in a single sample, simultaneously, [²⁵] and could be adapted to type-specific detection of HPV. Multiplexing such a reaction reduced the possibility of introducing errors inherent in performing multiple single type specific reactions for an individual sample, it also reduced the amount of time and reagents required to perform the assay. This would reduce the cost of performing the reaction thus making the assays more cost effective and increasing the accessibility of the assay to the application of clinical samples in general. While comparable L1 MY09/MY11 degenerate priming, these 1-step single tube qPCR assays have the additional advantage of being high throughput allowing for the detection of HPV in a a large number of samples in a relatively short amount of time when compared to the standard assay involving nested PCR followed by hybridization.

Assays were developed that detect HPV types 6, 11, 16 and 18; types targeted by the prophylactic quadrivalent vaccine. Each type-specific TaqMan probe contained a fluorophore that differed significantly in its excitation/emission spectra compared to other type-specific probes in the assay, and worked with equal efficiency allowing for the detection of multiple probes in a single reaction tube and reducing the time and reagents necessary to perform multiple individual reactions. The presence of large amounts of cellular genomic DNA did not affect the specific amplification and detection of amplicons. This assay had a linear range of detection over 6 orders of magnitude, allowing identification/typing of viral genomes and detection of viral load [¹¹]. A recent study performed to determine HPV 6, 11, 16 and 18 seroprevalence in the US determined that many sexually active females become exposed to these HPV types, suggesting that continued evaluation is warranted to determine the impact of vaccination[²⁶]. The qPCR assays described here may be instrumental in determining the efficacy of the quadrivalent HPV vaccine, particularly in vulnerable patients and will be instrumental in investigating HPV epidemiology in longitudinally collected samples. Increasing oncogenic HPV viral loads have been associated with development of malignancy [⁹]. While to our knowledge these associations have not yet been determined for other HPV associated non malignant conditions, it is reasonable to expect that viral loads precede disease development in that realm as well. These assays may be of significant use in deciphering the importance of HPV replication prior to disease development. The use of these qPCR assays have significant utility in distinguishing the presence of specific types of HPV. The one tube multiplex assay containing type/fluorophore specific TaqMan probes directed against HPV 6,11,16 and 18 provides a quick reliable method of distinguishing between HPV types and quantitating viral load while minimizing reagent costs and reducing errors.

In cases where other HPV types are involved, the "broad spectrum" HPV qPCR assay is instrumental in detection of HPV types that may be present but are not detected by the multiplex assay. While HPV16 and 18 have been shown to be responsible for the majority of HPV-related cancers, there are additional oncogenic types of HPV involved in malignancy. We used E1 degenerate primers in a sybr green-based qPCR assay that could amplify all HPV types. The E1 consensus region is approximately 150-200 bp, and is much more amenable to real time qPCR than the MY11-MY9 region which is 600 bp. To date, we are unaware of other primers sets used for HPV degenerate qPCR, however, the L1 based spf1/2 primers could serve as an alternative [²⁷]. These primers generate a 65 bp fragment and have been used to detect 43 types of HPV in a hybridization line assay. The E1 assay is truly broad spectrum and was capable of detecting and quantitating 35 cutaneous and mucosal HPV types including HPV6, 11, 16 and 18 from previously characterized clinical specimens and provides an additional assay to augment the multiplex qPCR assay. Table 3 and 5 highlights the fidelity of these complimentary methods and clearly illustrates that in HPV 16 positive oral cancer samples, that were by multiplex were also positive by both singleplex and E1 broad spectrum assay. Further, similar levels of HPV 16 were detected by each of these methods. We have shown that we consistently detect the same range of HPV types as the standard MY09/11 and gp5+/6+. Theoretically we can detect more types because we are not limited to HPV types available on pre-blotted membranes used for hybridization.

The ability to detect and quantify specific HPV types in clinical samples allows for assessment of HPV-related disease. While used on cloned DNA/plasmids for types 6,11,16 and 18, the multiplex assay also performed optimally on well-validated anogenital and skin specimens. Our test shows both high sensitivity and specificity 89% and 90%, but it should be noted that the specificity estimate is based on only 9 true negatives, and thus, has a large confidence interval. Our sensitivity estimate's narrow confidence interval indicates that the true sensitivity of our test is likely higher than 77%. Takac et al [¹⁸] in their development of a realtime assay that detected 15 oncogenic and 5 low risk risk HPV types also had similar levels of sensitivity and specificity (95.45% and 91.57%, respectively). Among the clinical isolates there were HPV types 31, 33, 52,58, 67 and 35, not cross reactivity was detected with HPV 16. Likewise clinical isolates with HPV types 45, 59 did not cross react with HPV 18 nor did HPV 55 cross react with HPV 6/11 nor did HPV 6 cross react with 11 (Table 2, related types are indicated by symbols, Figure 4B and 4C). This highlights the specificity of the assay. There was nearly 100 percent agreement with regard to detection of HPV 6, 11, 16 and 18 and no cross reactivity with other types although there was disagreement in one of the specimens. Multiple HPV types including HPV 6 and 16 had previously been detected in sample 950496 using a nested PCR/line blot technique. While HPV 6 was detected by our multiplex assay, HPV16 was not detected. This discrepancy may be due to levels of HPV16 below the level required for detection by our qPCR assay. Compared to traditional nested PCR, the real time qPCR assays described here were able to distinguish between specific mucosal types of HPV and also had the added benefit of quantitating viral loads in cervical lavages and in head and neck tissue biopsied from a small cohort of oral cancer patients with high risk factors (smoking/drinking) as well as from non-smoker/non-drinker cancer patients. In the HIV-positive women approximately 31% of the cervical fluids were HPV-positive with multiple concurrent HPV infections. Interestingly, in the oral cancer group that had a history of smoking and/or drinking, only 15% of subjects were positive for HPV. In contrast oncogenic HPV could be detected in almost all of the cancer subjects in the non-smoking/non-drinking group suggesting that HPV plays a significant role in the development of oral cancers in the absence of additional risk factors. This is consistent with findings from this lab as well as others, showing low rates of oncogenic HPV infection in oral cancers of smoker/drinkers and high rates of infection in oral cancers obtained from non-smoker/non-drinkers [²⁴,²⁸].

With real time PCR technology becoming an increasingly common technology for clinical labs, this assay will allow for the rapid identification and quantification of pathogenic types of HPV. As viral replication often precedes the development of virus-associated pathology, the ability to quantitate HPV in persons 'at risk' for HPV associated disease may be an informative prognostic indicator. The novel qPCR assays described here may provide insight into the epidemiology and natural history of HPV infections within individuals and groups, providing prognostic capability and leading to the development of intervention strategies to prevent disease progression.

We developed a single tube real time PCR assay capable of differentially detecting and quantifying 4 types of HPV represented in an FDA approved vaccine, types 6,11,16, and 18.

The ability of the assay to distinguish between these 4 types of HPV in a complex mixture of DNA and in the presence of human genomic DNA was validated. A "broad spectrum" sybr green-based qPCR assay that utilizes E1 degenerate primers that is capable of detecting multiple HPV types was also developed and validated. We showed that our assays performed as well as traditional nested PCR followed by sequencing/hybridization in distinguishing between different types of HPV in anogenital and oral fluids and tissues from high risk/diseased subjects.

HPV: Human papillomaviruses; HAPB: human apolipoproteinB; qPCR: quantitative PCR; ATCC: American Type Culture Collection

The authors declare that they have no competing interests.

WTS and JW-C prepared the manuscript and developed and performed the PCR assays. EA and MC identified and obtained oral cancer tissue. EMK and SC-U and identified and obtained cervical lavage samples from HIV infected individuals. JP identified and obtained well characterized HPV-positive anogenital samples used in validating the assays. AD performed the statistical analysis. All authors read and approved the final manuscript.

This work was supported by NIH/NIDCR grant BRF-ACURE-Q-06-00160.T00