Introduction

The human papillomaviruses (HPV) of the genus alpha commonly infect the anogenital tract without necessarily causing pathologic lesions. In women, 90 % of infections clear spontaneously. However, following persistent infection with one or more high-risk (HR) HPV types, precursor cervical intraepithelial neoplasias (CIN) and cervical cancer might develop. Additional risk factors, such as a high number of sexual partners (e.g. in prostitution and with promiscuity) or immunosuppression (e.g. in HIV-positive persons or transplant recipients), are well-known cofactors for higher HPV susceptibility and persistence [1]. During pregnancy, a physiological Th1–Th2 shift occurs leading to a systemic dominance of humoral immunity and suppression of cell-mediated immunity, which may favour the increased susceptibility to intracellular pathogens like HPV [2, 3].

Cervical cancer is the fourth most common cancer in women worldwide [4], and is estimated to be the most frequent cause of malignant disease in women of all ages in Ghana, a lower-middle-income country. The estimated age-standardized incidence rate amounts to 35.4 per 100,000 women, which is the fourth highest rate compared to all 15 Western African countries [5].

Cervical cancer is also estimated to be the first cause of deaths from malignant disease in Ghanaian women. Regarding all 15 Western African countries, Ghana takes the seventh highest position with an age-standardized mortality rate of 18.9 per 100,000 women [5].

Epidemiologic data on HPV prevalence among African women with normal and abnormal cervical cytology [6–8] as well as those obtained with histological specimens from the cervix [9–11] are still scarce compared to data available from other parts of the world [12–15]. According to the International Agency for Research on Cancer (IARC) in 2012, HR HPV types with sufficient evidence causing cancer of the cervix are HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59 (group 1 = carcinogenic to humans, HR). In addition, HPV-68, classified as probably carcinogenic (group 2A), and HPV-26, -30, -34, -53, -66, -67, -69, -70, -73, -82, -85, and -97, classified as possibly carcinogenic to humans (group 2B), are grouped as pHR HPV [1].

The current vaccines contain HPV-16 and -18, which have the highest oncogenic potential [16], and are responsible for up to 70 % of all cervical cancers in patients worldwide [17]. HPV-31, -33, -35, -45, -52, and -58 contribute to another 20 % of cervical cancers [17]. Nevertheless, these 20 % of cervical cancers show regional differences in HR HPV type prevalence [10, 18, 19]. Furthermore, HPV-16 and -18 are found in up to 38 % of low-grade and in up to 64 % of high-grade cervical lesions, respectively [20, 21].

With availability of the first generation HPV vaccines, quadrivalent Gardasil® (Merck & Co., Inc.) and bivalent Cervarix® (GlaxoSmithKline Biologicals), for almost 10 years now, there is an excellent tool for primary prevention of HPV infections and HPV-related diseases [22, 23]. The use of the second generation, nonavalent HPV vaccine GARDASIL® 9 (Merck & Co., Inc.), which was approved by the US Food and Drug Administration in 2014 and by the European Medicines Agency in 2015, in clinical practice has started. This new vaccine contains virus-like particles of low-risk (LR) HPV-6 and HPV-11 as well as the HR HPV types HPV-16, -18, -31, -33, -45, -52, and -58.

As further developments of HPV vaccines are going on, it is necessary to regard regional differences in HPV type prevalence to ensure the optimal protection by the vaccine. Our study was designed to contribute to improve the knowledge of the HPV type distribution in Ghana.

Materials and methods

Period and place of study

Between October 2011 and January 2012, a cross-sectional study investigating the HPV prevalence in pregnant women before delivery was conducted at St. Martin de Porres Hospital in Eikwe, Ghana. This hospital is a partner institution of the University Medical Center Göttingen (UMG). Since the year 2000 the Institute for Medical Microbiology of the UMG supports the running of the bacteriology laboratory [24].

The St. Martin de Porres Hospital in Eikwe is a general hospital located in a rural coastal village in the Western region of Ghana with 175 beds serving a population of about 380,000 people. Hospital service includes obstetrics and gynaecology (approximately 150 deliveries/month), general surgery, internal medicine, paediatrics, preventive care services, and a regional HIV treatment centre. At the time of the study, medical staff consisted of two specialists for obstetrics and gynaecology and four physicians in training.

HPV screening of vaginal swabs

On the initiative of the chief medical officer of the St. Martin de Porres Hospital, a gynaecologist, the Institute for Medical Microbiology of the UMG was requested for support to screen pregnant women for HPV infection to get information about the local HPV prevalence. Many women especially from rural areas do not attend antenatal clinic regularly but most of them come to the hospital for delivery. This was considered as appropriate possibility to have an almost unselected group of women at almost the same stage of pregnancy.

Vaginal swabs of Ghanaian pregnant women, routinely collected before delivery to rule out bacterial infections, which can cause early onset sepsis, were screened for 12 HR, 13 pHR and 18 LR HPV types.

The study was approved by the ethical committee of the University Medical Center Göttingen, Germany (number 14/10/11), and the authority of the St. Martin de Porres Hospital in Eikwe, Ghana.

Patient selection and inclusion criteria

Pregnant women to be included in this study were admitted from Monday till Friday between 8 am and 4 pm. During this time, a translator (trained nurse) for the two most common local languages, Nzema and Akan, was available to obtain informed consent and to fill out a questionnaire together with the pregnant women asking for origin, journey time, demographic data, and obstetric history. Inclusion criteria were gestational age ≥39 weeks or delivery within ≤1 week. The study population consisted of 177 pregnant women attending the antenatal clinic of the hospital.

Vaginal swab collection

Part of antenatal care is a microbiological screening for pathogenic bacteria like B streptococci by vaginal swab taken by a trained nurse. The vaginal specimen was obtained approximately 2 cm cranial of the introitus vaginae by using the ESwab system (Copan Liquid Amies Elution Swab; Copan Italia S.P.A.). It consists of an applicator swab with flocked nylon fibre tip and a polypropylene screw-cap tube containing 1 mL of modified liquid amies preservation medium. Following collection, the swab was put in the tube, immediately sent to the laboratory, and there inoculated and streaked at a chocolate and a blood agar plate for bacteriological culture. Thereafter, the swab was put again in the tube and kept at 4 °C until further processed for HPV testing. No additional swab was collected for HPV testing.

Human papillomavirus typing

A portion (200 μL) of the swab specimen was subjected to nucleic acid purification by using the MagNA Pure LC 2.0 instrument in combination with the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics GmbH) according to the instructions of the manufacturer.

To obtain the highest sensitivity, HPV DNA detection and typing were done using two different broad spectrum PCRs (A6/A8- and BSGP5 + 6 + -PCR), which are based on different primers and amplification protocols (nested vs. single round PCR) with slight variations in their sensitivity for the detection of different HPV types.

Hybridization of PCR products was performed to 43 (12 HR, 13 pHR, and 18 LR) type-specific probes by using bead-based multiplex genotyping on a Luminex Lx100 as described before [25–28] (M. Schmitt, personal communication). The probe sequences are summarized in Supplementary Table 1. The tested HR HPV types were: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59. The tested pHR HPV types were -26, -30, -34, -53, 66, -67, -68, -69, -70, -73, -82, -85, and -97. The tested LR HPV types were: HPV-6, -11, -27, -40, -42, -43, -44, -54, -55, -57, -61, -71, -72, -81, -83, -84, -89, and -177. Any A6/A8 PCR sample, that tested positive in agarose gel electrophoresis, but did not hybridize in the Luminex assay, was sequenced as described before [29].

Quantification of the β-globin gene by real-time PCR was performed to ensure that sufficient cellular material for investigation had been collected and additionally serving as an internal control to rule out the presence of inhibitory factors [28]. A vaginal smear with four (=2 cells) or less β-globin gene copies was considered as not evaluable.

Statistical analysis

Statistical analysis was performed with STATISTICA, version 10, for Windows (StatSoft GmbH, Hamburg, Germany). Pairwise comparisons between groups were conducted, using Student’s t test for continuous variables, Chi-Quadrat-test for categorical variables and Fishers exact test for dichotomous variables.

Kendall’s tau (τ) coefficient was used to test for dependency of non-parametric variables.

P values were regarded as significant at P < 0.05 and Bonferroni Holms procedure was used to adjust the P value to obtain an overall significance of P < 0.05.

Results

Human papillomavirus prevalence

Of the 177 vaginal swabs from pregnant women, 165 (93.2 %) were of good quality (ß-globin-positive) and were included in the study. HPV was found in 55 of the 165 vaginal swabs representing a prevalence of 33.3 % (95 % CI 26.3–41.1 %). The only HIV-positive woman in the study was HPV-negative.

Human papillomavirus type prevalence

The vaginal swabs were investigated for 43 different HPV types, which included 12 HR, 13 pHR, and 18 LR HPV types (Supplementary Table 1).

Ninety-three HPV infections, caused by nine different HR HPVs, nine different pHR HPVs, and 12 different LR HPVs, respectively, were detected in 55 females (Supplementary Table 2).

Infection with a single HPV type occurred in 35 women (21.2; 95 % CI 15.4–28.4 %), and infections with multiple HPV types were identified in 20 women (12.1; 95 % CI 7.7–18.3 %; Table 1).

Table 1 Detected single and multiple HPV infections in 55 HPV-positive pregnant women according to age group
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In 23 women (13.9, 95 % CI 9.2–20.4 %), 30 HR HPVs were detected [single infection in eight women (4.9, 95 % CI 2.3–9.7 %); multiple infections in 15 women (9.1, 95 % CI 5.4–14.8 %)]. In 22 women (13.3, 95 % CI 8.7–19.7 %), 23 pHR HPVs were detected [single infection in eight women (4.9, 95 % CI 2.3–9.7 %); multiple infections in 14 women (8.5, 95 % CI 4.9–14.1 %)]. In 31 (18.8, 95 % CI 13.3–25.8 %) women, 40 LR HPVs were detected [single infections in 19 women (11.5, 95 % CI 7.3–17.6 %); multiple infections in 12 women (7.3, 95 % CI 4.0–12.6 %)] (Table 1).

The most prevalent HPV types were LR HPV-6 (eight patients, 4.8, 95 % CI 2.3–9.7 %), HR HPV-52, pHR HPV-67, and LR HPV-61 (seven patients each, 4.2, 95 % CI 1.9–8.9 %), pHR HPV-53 and LR HPV-54 (six patients each, 3.6, 95 % CI 1.5–8.1 %), HR HPV-45 (five patients, 3.0, 95 % CI 1.1–7.3 %), and HR HPV-18 and LR HPV-42 (four patients each, 2.4, 95 % CI 0.8–6.5 %). HR HPV-16 was found in two patients only (1.2, 95 % CI 0.2–4.8 %) (Fig. 1). HR HPV-33, -39, -56, pHR HPV-26, -70, -85 and 97, and LR HPV-11 were not detected.

Fig. 1
figure 1

Prevalence (%) of HR, pHR, and LR HPV types in 165 pregnant women. aClassification of HPV types according to the International Agency for Research on Cancer (IARC) in 2012; group 1, carcinogenic to humans, HR; group 2A, probably carcinogenic to humans, pHR; group 2B, possibly carcinogenic to humans, pHR; group 3, not classifiable as to their carcinogenicity to humans, LR [1]. The bars show the prevalence of each HPV type listed in the respective line. HPV types included in the nonavalent vaccine Gardasil 9 are marked in bold. HPV human papillomavirus, HR high-risk, pHR probably/possibly high-risk, LR low-risk, IARC International Agency for Research on Cancer

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Human papillomavirus status and age

The age of 146 (88.5 %) of the 165 women was available. Of these 146 women, 49 were HPV positive and 97 were HPV negative. The median age was 26 years (range 14–48 years; mean age 26.3 ± 6.6). The median age of the 49 HPV-positive and the 97 HPV-negative mothers was 23 years (range 14–39 years; mean age 23.9 ± 5.5) and 27 years (range 15–48 years; mean age 27.5 ± 6.8), respectively. HPV-positive women were significantly younger than HPV-negative women (P = 0.0018; Fig. 2). The HPV frequency in women <25 years (n = 60) was 45.0 % (95 % CI 32.3–58.3 %) compared to 25.6 % (95 % CI 17.1–36.3 %) in women ≥25 years (n = 86). There was a negative association between the number of identified HR, pHR, and LR HPV types in a woman and her age (τ = −0.16, τ = −0.15, and τ = −0.23, respectively), and the infections were significantly more frequent in younger women (P = 0.0032, P = 0.0084 and P < 0.0001, respectively). Triple infections were only detected in women <25 years.

Fig. 2
figure 2

Age distribution of HPV-negative and HPV-positive women. The lower and the upper end of the boxes represent the 25th and 75th percentile, respectively. The minimum age was 15 years in HPV-negative and 14 years in the HPV-positive group. ***P = 0.0018, according to Student’s t test. HPV human papillomavirus

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The frequency of LR HPV-6 and pHR HPV-67 was particularly high in women <25 years compared to women ≥25 years (7:1 and 6:1, respectively). Both HPV types were detected mainly in co-infections, which were found only in women <25 years. The HPV type distribution in co-infections is detailed in Table 1.

Human papillomavirus status, parity, and age

Among 148 (89.7 %) of the 165 pregnant women, information about their parity was available. Primipara (n = 47) were significantly more frequent HPV positive with 53.2 % (95 % CI 38.2–67.6 %) compared to multipara (n = 101) with 21.8 % (95 % CI 14.4–31.3 %; P = 0.0001). Among 131 (79.4 %) of the 165 mothers, both age and parity were recorded. HPV-positive multipara (n = 21; mean age: 21.2 ± 4.5) were significantly younger than HPV-negative multipara (n = 69; mean age: 29.5 ± 6.2; P = 0.0086). There were no significant age differences in HPV-negative (n = 20; mean age: 21.2 ± 4.5) and HPV-positive primipara (n = 21; mean age: 21.0 ± 3.9; P = 0.9702).

Discussion

This study investigated the HPV status in Ghanaian pregnant women from a rural area before delivery. Routine vaginal swabs were screened for 12 HR, 13pHR, and additionally for 18 LR HPV types [1].

The prevalence of HR HPV types in vaginal and cervical specimens has been shown to be similar as carcinogenic HPV types infect the entire lower genital tract equally [30]. A vaginal swab can be considered as alternative to a cervical swab [30, 31] and may be preferred by the woman, as it is less invasive since no speculum is needed.

In our study, 93.2 % of the swabs contained sufficient cellular material as demonstrated by ß-globin DNA detection. LR HPV types are known to be more prevalent in the vaginal squamous cell epithelium than in the metaplastic or columnar tissue of the cervix [30]. This may explain the frequent detection of LR HPV types in the present study. HPV-6 was the most prevalent HPV type with 4.8 %. HPV-61 with 4.2 % and HPV-54 with 3.6 % belong also to the most frequent detected HPV types (Fig. 1; Supplementary Table 2).

A meta-analysis by Liu et al. [14] of the risk of HPV infection in pregnant women from Asia, Europe and North America revealed a significantly increased risk (summary odds ratio 1.42) resulting in a prevalence of 16, 13, and 30 %, respectively. Furthermore, a higher HPV prevalence in the younger age group of pregnant women was shown (24 % in women <25 years of age and 14 % in women ≥25 years of age) [14], which is in line with the widely accepted higher prevalence in younger women. Our results confirmed the findings of the meta-analysis. After sexual debut, HPV prevalence rises sharply up to a region specific maximum and then declines rapidly after the age of 20–25 years as a result of developing immunity and elimination of HPV as well as less sexual activity [32, 33].

Brandful et al. [7] found a HPV prevalence of 64.5 % in healthy, HIV-negative women between 18 to 41 years of age at various stages of pregnancy attending the antenatal clinic from the university hospital in Accra, Ghana. This prevalence is much higher than we detected in pregnant women with rural residence. Brandful et al. did not discuss the results in relation to other prevalence studies in pregnant women but only in reference to Attoh et al. [9] a study performed on cervix carcinomas in Ghanaian women. The higher prevalence could be explained to some extent by a younger mean age compared to our study population. However, the mean age is not provided [7]. According to the Ghana Demographic and Health Survey 2014, the percentage of women who reported having in the past 12 months a sexually transmitted infection, genital discharge, sore or ulcer was 27.2 % in women with urban residence and 21.8 % in women with rural residence, respectively [34]. This might have been also contributed partly to the higher HPV prevalence in the study of Brandful et al. [7]. Ultimately, the reasons for the higher HPV prevalence remain largely unclear.

Taking into account the about 1.4-fold increased risk of HPV infection in pregnant women calculated in the meta-analysis by Liu et al. [14], the prevalence of 33.3 % observed in our study correlates with a prevalence of 25 and 27 % described for non-pregnant women with normal cytological findings from Nigeria [35], and Benin [36], respectively. Recent data by Ogembo et al. 2015 estimate the HPV prevalence in Western Africa (Benin, Côte d’Ivoire, Gambia, Ghana, Guinea, Mali, Nigeria, Senegal) with 28.7 % (95 % CI 27.1–30.8 %) [21].

Yar et al. [8] investigated cervicovaginal swabs of non-pregnant HIV-positive and HIV-negative women from metropolitan Kumasi, and found a HPV prevalence of 86.9 and 56.0 %, respectively. HIV is a long known risk factor for a higher HPV prevalence [1]. The higher HPV prevalence in HIV-negative women (mean age 40.9 ± 11.3 years), compared to the prevalence we found in pregnant women (mean age 26.3 ± 6.6 years), might be explained in part through a higher prevalence of sexually transmitted infection, genital discharge, sore or ulcer in women with urban compared to rural residence (27.2 vs. 21.8 %) [34]. Yar et al. [8] did not specify any underlying disease in the study group of HIV-negative women. It is only mentioned that these women attended the outpatient department of the hospital. It is conceivable that these women might have suffered from non-communicable diseases, which may lead to immunosuppression to some degree, contributing to a higher HPV prevalence. However, it remains speculation.

The prevalence of identified HR HPV types in Ghanaian pregnant women differs considerably from the worldwide ranking [13]. The most frequently detected HR and pHR HPV types in our study were HPV-52 and HPV-67 (4.2 % each) and HPV-53 (3.6 %). In contrast, HPV-16 and HPV-18 were relatively rare (Fig. 1 and Supplementary Table 2), which is in line with the findings of Brandful et al. from Ghana placing HPV-16 and HPV-18 on rank eight [7].

Yar et al. [8] found in cervicovaginal swabs of HIV-negative, non-pregnant women the following most prevalent HR and pHR HPV types in descending order: HPV-58 (10.9 %), -35, -70, -82 (10.0 % each), and -31 (7.0 %). Except pHR HPV-70, which was not detected in our study, and pHR HPV-67, which was not investigated by Yar et al., all the other HPV types were also detected in our study but less prevalent. The prevalence of HR HPV-52 and pHR-53 (5.0 % each) and the low prevalence of HPV-16 (2.0 %) and HPV-18 (1.0 %) [8], are similar to our results from the rural coastal Eikwe. Differences in HPV type distribution may be partially due to the different geographical locations.

Three studies performed on cervical cancer specimens, all from Accra, Ghana, from 2003 by Attoh et al. [9], 2004–2006 by Awua et al. [11] and 2007–2010 by Denny et al. [10], respectively, found a HPV prevalence of 98.0, 89.8, and 93.9 %, respectively. HPV type-specific prevalence was quite different. Attoh et al. [9] detected most frequently HPV-18 (84 %), HPV-16 (24 %), HPV-45 (6 %), and HPV-39 (4 %). Awua et al. found HPV-18 (47.4 %), HPV-59 (42.2 %), HPV-45 (37.4 %), and HPV-16 (10.0 %) to be the most prevalent HPV types [11]. In contrast, Denny et al. detected most common HPV-16 (55.4 %), HPV-18 (19.0 %), HPV-35, and -45 (6.6 % each) [10]. The different prevalence of HPV-16 and HPV-18 is striking, but cannot be explained by different squamous cell carcinoma/adenocarcinoma ratios either by the geographical location. However, differences in HPV genotyping might have been contributed.

Irrespective of the HPV-16/-18 difference, the high ranking of HPV-16, -18, and -59 in cervical cancers in comparison to HPV-52, -53, and -67 in the pregnant women of our study is not unexpected because of the enrichment of particularly high risk types in cancers [37, 38]. This may also explain the fact that HPV-53 belonged to the most frequently detected HPV types in our study, but was found as single infection in only one cervical cancer from Nigeria in the study by Denny et al. [10].

HPV-67 was not included in the studies of cervical cancer in Ghana [9–11]. The carcinogenic potential of HPV-67 is still poorly defined. Its classification as pHR type is based on a limited number of HPV-67-positive CIN and cervical cancers [39–42]. One reason for the limited data on HPV-67 is most probably the lack of HPV-67-specific probes in commercially available HPV genotyping systems used in major studies on HPV prevalence [10]. In a recent meta-analysis by Bzhavala et al., HPV-67 was detected in 0.2 % of normal cervical specimens versus 0.3 % in invasive cervical cancers [38], which may hint to a certain carcinogenic potential. HPV-67 is the only member of species alpha9 including HPV-16, which so far is not categorized as carcinogenic [1]. HPV-67 is not included in the just recently licensed nonavalent HPV vaccine (Gardasil 9) [43], but may be a candidate to be added in future HPV vaccines, if more information about its prevalence and carcinogenic potential is available.

Although the number of women included in the study was relatively low, this is to the best of our knowledge the first systematic study on prevalence of different HPV types in rural Ghana.

In conclusion, investigations of vaginal swabs from pregnant women taken during routine health programs, like antenatal clinic, may be a good option to screen for HPV and get more information about the local HPV type epidemiology in resource constrained countries. In attempts to make future HPV vaccines more effective for regions with a high prevalence of cervical cancer like Sub-Saharan Africa countries, more prevalence studies should be performed ideally on a national basis. The genotyping in HPV prevalence studies should include HPV-67 to get more information about the real prevalence.