Prevalence of human papillomavirus genotypes among women with cervical cancer in Ghana

The West African region was estimated to bear the second highest burden of cervical cancer, with a mortality to incidence ratio of 81.2 % and cervical cancer was estimated to be the most frequent cancer among women in Ghana as at the time of this study [1]. The age standardised ratio (ASR) of 39.5 per 100,000 women [1]. An earlier study at the Korle-Bu Teaching Hospital also reported that 58.3 % of gynaecological cancers seen at the hospital in the year 2000 were cases of cervical cancer [2]. At the same hospital, a 10 year (1991–2000) study of cancer mortality pattern revealed that cervical cancer was one of the four leading causes of cancer deaths (Age Standardized Cancer Ratio of 8.74 %) in females [3].

A number of studies have shown that persistent infections with certain human Papillomavirus (HPV) genotypes known as high risk HPV type (HPV-16, -18, -31, -35, -39, -45, -51, -52, -56, -58, -59 and -68) are necessary in the aetiology of cervical cancer. These high risk HPV genotypes have been shown to be associated with greater than 99.0 % of all cervical cancer cases [4–7]. Of these high risk HPV genotypes HPV16 and HPV18 are the two prevalent HPV types in cervical carcinoma globally and are associated with approximately 60.0 % and 10.0 % of cervical cancer cases respectively [8–10]. However, epidemiological studies have shown a global geographical variation in the type specific and relative HPV prevalence among different populations; these range between 2 and 44 % [11]. Fortunately, there are indications of high efficacy of available vaccines against HPV infections and a greater possibility of preventing cervical cancer. However, it not clear how the population specific HPV genotype distribution, extent of multiple-infections and the HPV types involved in these multiple-infections will change following a successful implementation of these efficacious vaccines. Evidence is building on the occurrence of cross protection against non-vaccine HPVs and the possibility of the occurrence of HPV genotype replacements among vaccinated populations. In order to determine the occurrence of such changes, knowledge of the pre- and post-vaccination HPV genotype distribution is very important.

However, empirical data on the overall and HPV genotype specific prevalence in cases of cervical cancers in Ghana were very few at the time of this study. Additionally, and in order to evaluate the potential benefits of an introduction of HPV vaccination in Ghana, this study was designed to detect, genotype and determine the extent of multiple HPV infection using archival formalin-fixed paraffin-embedded cervical tumour specimen collected from the Pathology Department of Korle-Bu Teaching Hospital during the years 2004–2006. For the detection of HPV, a nested-multiplex PCR method that had been shown to be very sensitive and specific, with detection rates of between 91.8 % for cervical intraepithelial neoplasia (CIN I) and 99.3 % for CIN III was used [12].

As shown in Fig. 1, the observed distribution of cervical cancer cases was in line with global observations reported for the study period [16] and the age group with the highest burden of cervical cancer in this study was consistent with global data on cervical cancer (Table 4). Similarly, the prevalence of HPV DNA (89.8 %, 95 % CI 85.7–93.4 %) among these cases of cervical cancer, which was based on the amplification of the viral E6 and E7 oncogenes, was comparable to that (89.4 %) reported by a study of cervical tumour samples in the neighbouring Cote d’Ivoire [17]. Additionally, a comparable HPV prevalence of 93.9 % was reported among cases diagnosed as CIN II or higher in the study from which the HPV detection and genotyping methods were adapted [14]. However, in an earlier study of 50 similarly processed samples collected between January and December 2003 at the same hospital and using the same HPV detection methods, an HPV positivity of 98.0 % was reported [15]. Additionally, data reported for Ghana in a multi-centre study, which used biopsy samples collected between October 2007 and March 2010 and a different HPV detection and genotyping methods, indicated an overall HPV prevalence of 93.9 % [18]. In respect of the genotype specific prevalence (Table 2), HPV18 was the commonest genotype detected in this study as was the case the earlier study in Ghana [15], however, HPV16 was the commonest for the Ghanaian data reported in the multi-centre study [18].

Although these overall HPV prevalence were within the expected range of 90–100 % [19] and that there are reports of within country variation in HPV genotype specific prevalence [11], the differences between these studies may have been influenced by the following facts. The first is the difference in the type of specimen used in these studies. Specifically, this and the study by Attoh et al., [15] used archived formalin-fixed paraffin-embedded tissue blocks while the study by Denny et al., [18] used freshly collected biopsy samples. Also, the processing of the archived tissue blocks used in this studyand that by Attoh et al., [15] were not standardized and therefore different levels of inhibitors may have been present in the samples and in extracted DNA. The second was that the cases in these studies were received from different locations across the country and therefore the differences in prevalence may be a reflection of the inter-country variations. Thirdly, the variations in the times of samples collection, without overlaps, (2003, 2004–2006 and 2007–2010) and the relatively small number (n = 50) of samples used in the study by Attoh et al., [15] may have contributed to the differences in the data of these studies.

Inrespect of the genotype specific prevalence, the high prevalence of HPV59 and its frequency in multiple infections in this study remains unclear. However, the differences in these three studies most probably are a reflection of the variability in the HPV prevalence in the Ghanaian population and therefore there is the need for a well-controlled randomized population based HPV prevalence study in Ghana. On the other hand, a comparison of these HPV distributions with those of Ghana’s neighbouring populations strongly supports the existence of geographical difference in the prevalence of HPV genotypes and the possibility that HPV16 may not be the most prevalent genotype in these African countries. For instance, a study in Benin reported HPV59 (24.6 %), HPV35 (22.5 %), HPV16 (17.6 %), and HPV18 (14.8 %) as the common HPV genotypes detected [15]. Also, a study in Cote d’Ivoire reported HPV16 (45.0 %), HPV18 (21.0 %), HPV45 (9.0 %), HPV35 (8.0 %), and HPV31 (3.0 %) as the common genotypes [17] while in a study in Burkina Faso, HPV52 (14.7 %), HPV35 (9.4 %), HPV58 (9.4 %), and HPV51 (8.6 %) were the common genotypes [20]. Furthermore, study form other regions in and outside Africa confirm the assertion that although HPV16 and HPV18 are the commonest HPVs in cervical cancers globally, they are not always the two commonest HPVs in every country. For instance, in Tanzania, HPV16 and HPV58 were the first two prevalent genotypes while HPV18 was the fifth [21]; In Mozambique, HPV35 was the most prevalent HPV genotype while no HPV18 genotype was detected among women diagnosed with HSIL or carcinoma [22]. Liaw et al., [23] reported HPV52 and HPV58 as the most prevalent type in parts of China.

Interestingly, HPV18, HPV59 and HPV45, which are of the same phylogenetic family [7], as expected were the common genotypes in adenocarcinoma (Table 5). On the other hand, HPV16 and its phylogenetic related family members, HPV31 and HPV35 were respectively the fourth, fifth and eighth prevalent HPV in this study. Furthermore, most of the multiple-infections observed in this study involved HPV18 and HPV59. These suggest that a phylogenetic dependency in the colonization of cervical epithelium might contribute to HPV prevalence, as was observed by Conesa-Zamora et al., [24] for HPV18 and HPV45. Therefore, these may suggest a phylogenetic related HPV prevalence in Ghana, although the bases for such specificities are still not clear.

Although, the frequency of multiple-infections varies with the type of HPV detection method used [25], the 52.2 % multiple-infections observed in this study as compared to that of the earlier Ghanaian study, 19.6 % [18], are discussed in light of the fact that most (about 96 %) of the cases in this study (Fig. 1) were invasive cancers (IAC and ISSC) which are known to be associated with high multiple-infections [25]. Also, data from neighbouring countries have shown similar high frequencies of multiple-infections. A 52.9 % rate of multiple-infection was observed in a study in Burkina Faso [20], while a 40.2 % rate was reported by a in Benin [19]. These may suggest a high rate of multiple-infection in the West African region. However, these may be a population specific observation as was shown by two studies in Spain, a 25.6 % rate of multiple-infection among HSIL cases in Southern Spain [24], while a 34.0 % was observed in a cohort of women in Madrid [24, 26].

Since both cross-protection of the available HPV16/18 vaccines and its clinical relevance determined with the data available for vaccine efficacy have shown additional protection against HPVs -31, -33, -45, -51, -52, -56 and -58 [27, 28], the expected impact of HPV vaccination on cervical cancers in Ghana may be further increased. Specifically, if the infection by HPV59 depends on a prior infection by HPV18 [24] since they most occurred together in this study, then a lower prevalence of both HPV18, HPV 59 and lower frequency of multiple infection may result after the introduction of the HPV16/18 vaccines.

Another finding worth commenting on was the observation that low risk HPV types (HPV-6/11, -42 and -44) were solely detected as single infections in 8 cervical cancer cases (1 CIS, 7 ISCC). These were least expected and may be misleading in suggesting a higher oncogenic potential for these low risk HPV types since low risk type HPVs are rarely observed as single infections in invasive cancers [7]. However, due to the limitation associated with DNA extract and PCR using formalin-embedded paraffin-embedded specimen (presence of inhibitors from sample fixatives), it was possible that the other multiple-infecting high risk HPV genotypes present were not detected since DNA extracts from paraffin-embedded formalin-fixed tissue samples have been reported to intermittently fail to amplify by PCR [29]. Specifically, because PCR inhibitors may have been be present at varying concentrations, the concentration of the target DNA and its quality may have been greatly reduced after tissue processing and/or the target viruses may have been heterogeneously distributed in the cancerous tissues [29–31]. The limitation of this study includes the fact that it was not powered to determine the associations between HPV genotypes and the diagnosis categories of cervical cancer. Also, the HIV statuses of the patients, which may influence HPV prevalence, were not determined.

Overall, the findings of this study indicate a peculiar HPV profile for Ghana, which has important implication for the introduction of HPV vaccination and forms part of a growing body of evidence of the pre-vaccination HPV prevalence for Ghana. However, rigorous epidemiologic data and well-controlled randomized trials are needed in order to estimate the extent of protection or prevention of cervical cancer that may be achieved with the introduction of HPV vaccination in Ghana; particularly in light of the high multiple-infection prevalence observed in this study. These also underscore the need for community based screening of women for both cervical cancer and HPV infection, which should include HPV genotype analysis as part of the screening schemes for women with high-grade neoplasia and cervical cancer in Ghana. These will help throw more light on the high rate of multiple-infections, particularly those involving HPV59.