The goal of accurate detection of infectious agents is to provide consistent and meaningful results in the research and clinical setting to help target and focus resources in disease prevention and control. Over the past years, WHO has worked with the scientific community, national regulatory authorities, other standards-setting bodies, and users through a series of consultations to review the scientific basis of biological reference materials. WHO reference reagents, which may serve as interim standards, and the published catalogue of WHO biological reference materials includes over 300 materials (a list of reference materials may be found at www.who.int/biologicals
). It is recognized that some international standards may be used for qualitative rather than quantitative purposes. It may also be necessary to establish materials that might act as a reference panel to aid in the evaluation of diagnostic tests. Indeed, a reference panel of 10 individual genotypes of HIV-1 has been previously established to help assess the specificity of nucleic amplification technology-based assays for HIV-1 (44
In addition to evaluating the performance of various HPV DNA detection methods, the present international collaborative study evaluated the feasibility of generating HPV DNA international standard reagents and the suitability of recombinant plasmids containing full-length HPV-cloned genomes for this purpose. Historically, international standards in the form of nucleic acids have been isolated from pools of virus-infected biological material, such as the hepatitis C RNA standards established in 1997, hepatitis B DNA standards established in 1999, and HIV-1 RNA standards established in 1999 (45
). International standards must fulfill several criteria, including the following: demonstrate consistent performance, demonstrate long-term stability under selected storage conditions, contain sequences found often in the real target nucleic acid (i.e., full viral genomes if possible), perform in detection assays like the naturally occurring target, and be readily available and renewable. Ideally, standards should mimic properties of actual biological samples under measurement and allow evaluation of the full laboratory sample processing procedures. HPV clinical samples are not plasma derived, and the creation of international standards represented by pools of cervicovaginal specimens is not feasible and not as reliable and reproducible as recombinant HPV nucleic acid standards. At a minimum, HPV DNA standards should contain full HPV genomes to allow identification of any genomic region that may be targeted in detection assays and should be presented in a background matrix of human genomic DNA. Indeed, the proposed materials fulfilled these requirements. The studied materials also contained a matrix of epithelial DNA derived from a cervical carcinoma cell line (C33A), which is of human origin and is HPV DNA negative and offers a source of renewable and reproducible matrix material. Studies need to determine if HPV DNA candidate standards can be further developed to assess the full spectrum of sample processing schemes that would mimic diagnostic sample preparation including DNA extraction, precipitation, or centrifugation procedures. This would require the further development of culture models that would harbor authentic episomal HPV genomes. Future panel assessments will need to also include pooled biological specimens to relate standard materials to clinically relevant levels of HPV DNAs.
The overall detection limits observed among participating laboratories, across all HPV detection systems employed, were significantly different between HPV types 16 (dilutions between 10−5 and 10−7) and 18 (dilutions between 10−6 and 10−10) in this study. These dilutions corresponded to a detection limit ranging for HPV 16 from approximately 104 to 102 genome equivalents per assay and for HPV 18 from approximately 103 to 10−1 genome equivalents per assay. Although initial characterizations by both reference laboratories suggested similar levels of HPV types 16 and 18 DNA within the panel dilutions, a subsequent analysis by qPCR revealed a difference between HPV types 16 and 18. In general HPV 18 plasmid material appeared at least 1 order of magnitude more concentrated than the HPV 16 plasmid material. This is reflected in the qPCR results shown in Fig. and in the apparent lower detection limits observed for HPV 18 in the majority of participating laboratories using a variety of HPV DNA assays. In part for this reason, the data obtained in this panel evaluation were displayed as a function of the dilution that the original material was subjected to rather than as an estimate of genome equivalents. Data from this study demonstrate that future evaluations of candidate HPV recombinant DNA standards will require rigorous examination of longitudinal stability. In addition, it may be best to designate HPV international standard reagents using arbitrary international units rather than genome equivalents.
In this panel, the determination of sensitivity for the five HR HPV types (HPV types 31, 33, 35, 45, and 52) and one low-risk HPV type (HPV 6) was not addressed using a dilution series. HPV 6 was included in the panel because it is a component of one vaccine preparation aimed at preventing genital warts and a proportion of low-grade cervical dysplasia. The HR HPV types 31, 33, 35, 45, and 52 were included in this panel to assess potential cross-priming, or hybridization, or competitive amplification within a particular HPV DNA detection system. The results showed that in the model proposed here, HPV 16 and 18 DNA detection was not compromised by the codetection of these additional HR DNAs using two selected concentrations of other HR HPV types. This result is somewhat surprising since HPV types 16, 31, 33, 35, and 52 phylogenetically belong to the same species 9, while HPV 45 is the nearest relative of HPV type 18 (species 7) (3
). Results also suggest that HPV types 33 and 45 were equally detected by all HPV DNA tests evaluated in this study. HPV 31 was the least accurately detected by participating laboratories (Table ). Approximately half of participating laboratories failed to detect high concentrations of HPV 31 and, to a lesser extent, to detect HPV types 35, 52, and 6. The failure to detect HPV types 31, 35, 52, and 6 could reflect inherent assay differences in sensitivity and specificity that have been previously reported (22
) (Table ).
The results of this study support the concept that recombinant HPV DNA constructs can be used to develop international standard reagents. The international collaborative study group recommended that the focus of international standard reagents be first on HR types HPV 16 and 18 and not on low-risk HPV types not related to cancer, and then expand to the most prevalent HR HPV types as follows: HPV types 31, 33, 35, 45, 52, and 58. It should be noted that an initial assessment of HPV type 58 in this panel was not conducted due to the fact that it is cloned within the L1 gene segment and would have required reengineering. This effort will be undertaken for future generation of an HPV 58 international standard.
The use of HPV DNA standards will vary depending on the setting in which they are applied. For example, in clinical vaccine trials, where women are under evaluation for prophylaxis of HPV infections and related disease, highly sensitive HPV DNA assays are desirable (8
). In contrast, the management of genital HPV-related clinical disease has demonstrated that less sensitive HPV detection limits may be appropriate (25
). For genital HPV infections, the high prevalence of HPV DNA versus clinical disease has demonstrated that overly sensitive HPV detection would result in excessive triage of women for diagnosis and treatment. Establishment of appropriate sensitivity for any HPV assay used in clinical settings requires evaluation in very large, preferably randomized, trials and issues of cost-effectiveness as related to use in public health settings must be considered. With the introduction of highly sensitive technologies to detect HPV, quantitative assays may be useful for establishing clinically relevant sensitivity. An intrinsic part of using such technology should be the use of well-characterized standards or proficiency panels.
For HPV DNA international standards, it is desirable to develop monovalent or individual HPV type standards. This will allow unequivocal calibration of individual HPV DNA material and will allow assessment of potential detection interference when multiple HPV types are present. The HPV DNA international standard unit remains to be established and could be defined using genome equivalents, micrograms, copy numbers, or other units. International standard units for hepatitis B DNA reagents, for example, were arbitrarily assigned a potency of 106
international units (IU)/ml, as agreed based on the sensitivity of assays used at the time that the international standard was established (24
Because cervical cancer prevention is a high priority for public health interventions in many countries, WHO supported the preparation of this panel of candidate HPV reference reagents aimed at facilitating interlaboratory comparisons and detection worldwide. HPV types 16 and 18 were the focus of this panel because they are responsible for the majority of cervical cancer cases worldwide and are the primary targets of current prophylactic HPV vaccines. The results of this pilot study show that the majority of participating laboratories accurately detected HPV types at the highest concentrations represented in the panel. Both the individual laboratory proficiency with a given test and the HPV DNA detection system itself are contributors to the interlaboratory variations observed here. For instance, a single HPV detection method used by seven laboratories demonstrated several orders of magnitude of variation in sensitivity for HPV 16 detection. Similar observations have been reported in proficiency studies of hepatitis B (36
), hepatitis C (20
), HIV (19
), herpes simplex virus (28
), and Chlamydia trachomatis
) using nucleic acid detection. These data underscore the need to critically consider information on HPV type-specific prevalence in epidemiology studies and point to the utility of developing HPV DNA standards. In addition, the outcome of this study underlines the need for standard operating procedures, quality control panels, and reference reagents. To address these needs the following should be considered: (i) large batches of analytical reference HPV DNA reagents, similar to those used in the present studies, must be prepared according to international guidelines for worldwide use; (ii) written standard operating procedures that describe all steps in the handling, processing, and storage of the reference reagents must be provided with the reagents; (iii) active quality assurance programs must be promoted that use external quality control panels of known HPV type and concentration; and (iv) designation of regional WHO HPV reference laboratories could serve laboratories and act to facilitate high levels of performance in HPV DNA detection.
The potential benefits of available reference reagents are many. The sensitivity and specificity of HPV DNA assays can be determined, validated, and monitored. In addition, performance of HPV DNA detection methods as related to international standards will facilitate comparisons of data from multiple studies. Thus, the availability of international HPV DNA standards will contribute to the field of HPV prevention, diagnosis, and treatment. In particular, such standards, if available worldwide, will allow for reference calibration of HPV DNA tests, thereby enabling manufacturers to further validate and develop HPV detection reagents and kits, and will allow reliable disease monitoring and improve health care worldwide.