This study provides an estimate of the prevalence of HPV in women participating in routine cytology screening in BC. This is the largest typing study of its kind in Canada to date and one of the largest single-center studies worldwide. While the HPV prevalence of screened women is not necessarily equivalent to that of all women [11
] in BC, the high participation rate of the CCSP (70% [12
]) argues that it provides a good estimate for the province. The use of direct sequencing theoretically allows the detection of all known HPV types and provides a level of detail not attainable using existing hybridization probe sets or the Digene Hybrid Capture 2 system.
Several international studies have examined the prevalence of HPV in women. The diversity of population samples, sample media and HPV typing methods make it difficult to identify studies that are exactly comparable to each other. It is not surprising that our HPV type distribution differs from that of a large US study based on self-sampled vaginal swabs [13
]. Low-risk HPV types that are more prevalent in the vagina and vulva [14
] will not be well represented in our samples, as these are almost exclusively cervical smears. It is also reported that self-collected vaginal sampling methods are generally less sensitive than cervical smears for the detection of HPV [15
]. Overall HPV infection rates in population-based studies where all women were included found HPV prevalence rates from 2% in Hanoi, Vietnam [17
], to 40% in Mozambique [18
]. Our overall HPV infection rate was 16.8%, close to that of an Ontario study (13.3%) [19
]. A recent large study in The Netherlands typing high-risk HPV in the population found a rate of 5.6% [20
] and shows a similar trend for age as observed in our study (Fig. a, b). Our prevalence is also at a similar level to that seen in a recent, large meta-analysis for Asian women at 14.4% for cytologically normal samples [21
]. Prevalence of HR HPV ranged from 4.4% [22
] to almost 20% [23
] in these studies, in keeping with our rate of 13.9%. Similarly to many other studies, HPV16 was the most common high-risk type in BC. We found a higher prevalence of HPV16 (10.7%) than other studies, which showed less than 1% to just over 5%. Several studies [17
] used GP5+/6+ primers, but detected HPV types by hybridization followed by enzyme-based immunoassay. Our method subjects the PCR products to an additional, albeit linear, amplification in the sequencing reaction, likely enhancing the sensitivity of detection. Comparison of cycle sequencing, line blotting and hybrid capture showed that sequencing is the most sensitive [29
HPV positivity increases, as expected, from normal (12.3%), to benign (19.6%), to LGIL (69.3%), to HGIL (81.0%). The trend for HPV 16 also makes sense, going from normal (8.7%), benign (7.6%), LGIL (35.2%) and HGIL (52.4%). The relative proportion of HPV16 (HPV16/totalHPV), however, is unexpectedly high in normal samples (71%), when compared to benign (39%), LGIL (51%) and HGIL (65%). It is unlikely that contamination could account for this difference, because all samples were processed on multi-well plates and were not separated according to cytology. We propose that there is a real biological explanation for this observation that likely relates to the sensitivity of PCR and sequencing to detect HPV. We may be detecting transient, sub-clinical HPV exposures in addition to overt HPV16 infections that would be detected with less sensitive techniques.
We found that 33% of HPV positive samples contained multiple HPV types, within the range of 12–62% seen in other studies [28
]. Direct sequencing may underestimate the MI rate; however, our conservative over selection of potential MIs (all sequence traces with any sign of mixed types were subcloned) should compensate for this. Our higher observed prevalence of HPV16 is not likely to be a result of our intensive characterization of MI samples.
The percentage of HPV positive samples that had multiple infections was higher in the cytologically normal HPV positive samples, possibly reflecting clonal outgrowth of cells infected with a single HPV type in the pre-cancerous lesions. This may imply that multiple types of HPV simultaneously infect the same woman but not necessarily the same individual cells, or it may reflect the preferential persistence of one HPV type. Multiple infections may be more recent infections that have had less time for one or some of the types involved to be cleared.
We did not exclude women who were tested as a follow-up to a previous abnormal smear. The smear-takers were high-volume sites; this could bias toward young sexually active women who are seeking birth control. Compared to the CCSP in 2004, our sample has a higher proportion of young women, who would be more likely to have current HPV infections. Sellors and colleagues showed a lower rate (9.6% for high-risk HPV) in older women than in younger women [31
]; we also observe this trend. Thus, our study could slightly over estimate the prevalence of HPV infection relative to the general female population of BC.
Differences between recruitment methods can complicate direct comparison of our findings to those of other Canadian studies [11
]. An Ontario study [19
] using Digene Hybrid Capture 2 and PCR showed a prevalence range of 25–6% depending on age; Montreal University students had an HPV rate of 29% [33
], similar to the prevalence we observed in this age group. A recent international analysis by IARC [28
] illustrates the differences in type distribution in different countries. We detect HPV90, but this type was not included in the probe set used by IARC [28
]. Types seen more commonly in Asian countries (such as 51, 52 and 58) were not significantly increased in BC, despite its large Asian population. Comparison to worldwide data [28
] demonstrates our ability to detect most or all known HPV types, despite the tendency of GP5+/6+ primer set to underestimate HPV 52 [34
Prophylactic vaccines are now available against HR HPV types 16 and 18. Efficacy evaluations to date for these vaccines show 100% protection against development of LGIL and HGIL associated with the HPV types targeted. A minimum estimate of the impact of a vaccine protecting against HPV16 and HPV18 would be the proportion of a lesion for which representative samples are positive for 16, 18, or 16 and 18, but not other HR types of HPV. In our data, the proportion of LGIL and HGIL samples that meet this criterion is 29.9% for LGIL and 55.6% for HGIL (data not shown). Conservatively, we predict that vaccinating against HPV16 and 18 would, in an effectively vaccinated group, prevent the development of one-third of LGIL and more than half of HGIL, and an even larger proportion of cervical cancer. If these estimates are expressed as a percentage of those LGIL and HGIL that had detectable HR HPV, a likely more realistic estimate (57.2% of LGIL and 70.0% of HGIL) of the percentage of these lesions that are attributable to vaccine-related HR types is obtained. Including additional HPV types in future vaccines (such as 56 and 90 in BC) would further increase the percentage of cervical lesions prevented. These data provide a baseline from which to monitor changes in HPV prevalence that result from future use of HPV vaccines in BC and may inform the use of HPV testing as a first-line screening alternative to cytology.