External anogenital warts are the most common, macroscopically visible, clinical manifestation of HPV infection of the lower genital tract. The vast majority of the cases were monoinfection of the low-risk HPV genotypes 6 and 11. It has been proposed that low-risk HPV genotypes do not integrate their DNA into the chromosomes of the infected cells, hence such low-grade lesions have a low-risk of progression to malignancy. However, around 19–33% of the EGW cases are coinfected with oncogenic, high-risk HPV genotypes [6, 22]. While most of the studies and screening programs focus on the HPV related malignant disease, the epidemiology of external anogenital warts are not well characterized; data about the prevalence of different HPV genotypes related to EGW, especially in Mid-European countries, are underrepresented and incomplete . The present study aimed to determine the prevalence of different HPV genotypes in surgical external anogenital wart samples’ of a cohort, diagnosed with hard-to-treat EGW. Formalin-fixed paraffin-embedded samples of 94 patients (66 females and 28 males) with EGW were recruited in the study. The explanation of the significantly smaller sample size of the men’s group could be due to the different attitudes of visiting doctors. Unlike females who routinely take part in screening programs and undergo follow-up with gynecologists, males usually seek consultation with specialists if they present symptoms. Asymptomatic individuals often not identified, representing an invisible proportion for healthcare systems. The observed overall 100% prevalence of low-risk HPV infection indicated that EGW lesions never appear in the absence of low-risk HPV infection. According to our results, among the examined Hungarian patients the most common HPV genotypes were HPV 6 and 11. The vast majority of the samples, 72.34%, were diagnosed with monoinfection of HPV 6 and 11 genotypes, while these two genotypes either alone or in combination with other HPV genotypes, occurred in 98.93% of the cases. Apart from the applied method, data regarding the prevalence of HPV 6 and 11 genotypes are comparable to the data reported from other European countries. By applying a highly sensitive method we were able to identify a wide spectrum of HPV coinfections. In our study, 27.65% of the observed cases were multi infections and out of these cases, 26.90% were co-infected with more than two HPV genotypes. There were no significant differences in the presence of multi infections among females and males. Comparing multi infections and mono infections by the mean ages, we could conclude, the proportion of multi infections were more frequent at lower mean age, as 34.46 compared to 38.94 years.
Focusing on high-risk genotypes we could conclude that oncogenic, high-risk HPV genotypes never occurred as monoifection. Our study revealed, that out of the 27.65% coinfected samples 19.4% of the cases occurred with ≥ 1 high-risk HPV genotype. Although data were not significant, by analysing the prevalence of high-risk genotypes according to different age distributions, in line with other studies, we found that the proportion of ≥ 1 high-risk genotype is more frequent at ages under thirty . This could be explained by lifestyle attributes. Furthermore, our results also indicated more HPV genotypes among female patients. Interestingly, we observed slight differences according to the prevalence of most frequently appearing high-risk genotypes compared to the data reported by other European countries; instead of HPV 16 being the most common high-risk type, HPV 56, 59, 66, and 73 occurred to be the most frequent genotypes presenting with the same prevalence of 3.19% each, of all EGW cases [21, 22]. In the case of the other tested genotypes, the prevalences are comparable to the data reported in European countries; the observed slight differences could be mostly attributed to the variation in the methods used for HPV detection.
Nowadays, wide spectrum of diagnostic HPV tests are available on the market, used in routine diagnostics, such as INNO-LiPA, Linear Array HPV Genotyping test, PapilloCheck HPV test, Digene Hybrid Capture 2, CLART HPV2, etc. Nevertheless, these methods show wide differences regarding genotype-specific sensitivity and sample types that are validated in the applied method [29–32].
In our study, a sensitive, genotype-specific method, HPV Direct Flow Chip Test was used. The technology is based on the amplification of a short DNA fragment of HPV L1 ORF, enabling the use of FFPE samples. The test is CE IVD marked in compliance with European Union diagnostic medical device manufacturing standards. Though the most acceptable sampling method from the external lesions is using cytobrush, it is reported that these lesions often don’t provide proper cell retrieval, resulting in insufficient material for genotyping . Considering these, it is an important advantage, that the applied method is validated for FFPE sample material as well, providing suitable material for the analysis. The sensitivity and specificity of the applied method had been proved in independent studies [29–31]. Regarding these studies, although the HPV Direct Flow Chip showed a very high genotype-specific concordance with the other methods (Linear Array, Hybrid Capture 2, INNO-LiPa systems) in the pair-matched results, the HPV Direct Flow Chip yielded significantly higher rates of HPV infection than other methods used in the study, especially in case of FFPE samples, moreover, the HPV Direct Flow Chip test proved more effective than other HPV detection systems in samples with low viral load .
Based on the analysis of Mayenaux and Chaturwedi the incidence of HPV infection could be kept in check; though the continuously increasing number of risk factors, as the growing number of lifetime sexual partners, earlier sexual debut (< 16 ages), homosexual relationships, history of other STDs, smoking and human immunodeficiency virus (HIV), could only be counteracted by screening programs and vaccination .
Currently, 3 licensed HPV vaccines are available using L1 capsid antigens of 2, 4, or 9 HPV genotypes. All the 3 vaccines include HPV 16 and 18, which cause the majority of HPV related cancers. While the quadrivalent (Gardasil 4) and nonavalent (Gardasil 9) also target low-risk types HPV 6 and 11, which are primarily associated with external anogenital warts. In addition, the nonavalent vaccine includes 5 other high-risk HPV types above, such as HPV 31, 33, 45, 52, and 58. A very high efficacy of the quadrivalent and nonavalent HPV vaccines against HPV 6 and 11 associated disease was reported in multiple randomized, controlled trials [34–36]. There is accumulating evidence that population-based vaccination targeting HPV 6 and 11 as well can result in dramatic declines in genital warts incidence [33, 35]. Regarding our results, we found that the prevalence of HPV 6 and/or HPV 11 was 98.93% among all EGW cases indicating a potentially great benefit from the vaccine. Though it must be highlighted that out of the 11 detected high-risk genotypes the nonavalent vaccine covered only 3 genotypes, while out of the 10 detected low-risk genotypes, it covered 2 genotypes, suggesting the importance of follow up of patients diagnosed with EGW. In Hungary, the bivalent vaccine has been available since 2007, while the quadrivalent vaccine, that was available from 2006 was replaced with the nonavalent vaccine in 2015. As part of the national immunization program, in 2014 the bivalent HPV vaccine was introduced to 13-year-old girls by a school-based vaccination program. In 2018 the bivalent vaccine was replaced and since then the nonavalent vaccine has been applied, benefiting protection against EGW as well. The acceptance of the vaccine is increasing among girls. It was around 80% in 2019, based on the result of the State Public Health and Medical Officer. It is a great advance, that males at 13 years of age have also been recruited in the national HPV vaccination program from 2020. Though the attitude seems positive, educating the population could further improve the coverage of the vaccine .