Human papilloma virus (HPV) is associated with a subset of oropharyngeal squamous cell carcinoma and mouth or throat warts. However, there is currently limited information about oral HPV infections in Nigeria.
This study aimed to provide information on the occurrence and circulating genotypes of HPV among patients attending three (one government and two private) dental clinics in Ibadan, Nigeria.
An oral swab was collected from 231 dental clinic attendees in Ibadan between January 2016 and March 2017 and tested for HPV DNA by polymerase chain reaction targeting the E6/7 genes of the virus.
Twenty-three of the 231 swab samples were HPV DNA positive comprising 16 mono-infections and seven co-infections in 13 males and ten females. Genotype 16 was present in ten patients, genotype 6/11 in five, Genotype 18 and genotype 33 in four each, genotype 31 in three and genotype 39 in one. Twenty-one cases were high-risk HPV genotypes, while two were low-risk. Samples had co-infection and five had low risk type 6/11 either as single or as co-infection. Persons who had engaged in oral sex as well as those aged 21-30 years has significantly higher prevalence.
This study showed that although HPV genotype 16 is the most common type among dental clinic attendees in Ibadan, other genotypes are also circulating and that oral sex is a risk factor for the infection. Therefore, introducing a multivalent HPV vaccine will reduce the risk of HPV-associated oropharyngeal carcinoma and other cancers in Nigeria.
Human papilloma viruses (HPV) are associated with anogenital cancer, including vulval, penile, vaginal, anal, and cervical cancer. These viruses have also been associated with a subset of head and neck cancers causing oropharyngeal squamous cell carcinoma.
The situation of HIV infection has caused an increase in the prevalence of oral HPV infection, especially among HIV-infected individuals in developed countries.
Human papilloma viruses are small, non-enveloped, double-stranded DNA viruses belonging to the Papovaviridae family. The family has five major genera: alpha, beta, gamma, mu, and nu, with those in the alpha genus having the most medical importance. Human papilloma viruses are further categorised into high risk or low risk based on their epidemiological associated malignancies.
Presently, the prevalence of anogenital HPV infection in Africa ranges from 9.8% in Central Africa to 19.5% in Western Africa
There is limited information on the prevalence and circulating genotypes of oral HPV in Africa and especially Nigeria. This information is essential to know if available vaccines will be effective in preventing oral HPV infection and associated oral cancers in Nigeria. This study was carried out to provide information on oral HPV and the circulating genotypes among patients presenting in dental clinics in Ibadan, Nigeria.
This study was approved by the Ethics Review Committee of the University of Ibadan and University College Hospital Ibadan with approval number 160057, and participants provided written informed consent. Confidentiality of the study participants was maintained; participants’ identities were codified, and only the authorised party could link the identity of the participants to the given code when the result is given to the dentist to notify the participants of their result.
This study was a descriptive cross-sectional study carried out among 231 patients attending three dental clinics comprising one government (University College Hospital Ibadan Dental Clinic) and two private dental clinics in Ibadan, Oyo State, Nigeria, from August 2016 to February 2017. Government dental clinics in Ibadan provide dental services for the population of Ibadan and its environment. The minimum sample size calculated was 196 but a total of 231 individuals was recruited for the study. The sample size was determined using the formula:
a prevalence of 15%
A total of 231 individuals who consented and were aged 18 years or older were recruited for the study. The participants were first-time attendees of dental clinics (one public and two private) in Ibadan, Oyo State, Nigeria, presenting with various dental complaints. A convenient sampling method was used.
Socio-demographic and risk behaviour information (presence of oral warts, smoking, number of sexual partners, previous oral infection, and engaging in oral sex) was collected from each participant using a semi-structured questionnaire. A dental technician trained in collecting oral swabs for HPV testing collected oral swab samples from all participants. Briefly, the tip of the swab stick was rubbed several times in the mouth and base of the tongue. The swab sticks were then placed in vials containing 500 µL of transport medium containing minimum essential medium and 2% bovine serum albumin with antibiotics (Gentamicin) in a secured capped vial. Samples were stored and transported at 4 °C to the laboratory. Initial processing in the laboratory was done within 24 h of sample collection, and included vortexing each vial containing transport medium, removing the swab; the medium was aliquoted and stored at –80 °C until analysed for HPV DNA by polymerase chain reaction (PCR).
Total DNA was extracted from each sample using a commercially available DNA purification kit (Jena Bioscience, Jena, Germany) per the manufacturer’s instruction. The extracted DNA was tested for the presence of the E6/E7 HPV viral gene by PCR using previously described primers (GP E6-3F, GP-E6-5B and GP-E6-6B) and protocol.
List of primres used and the amplicon size.
No. | Target | Primer name | Sequence | Amplicon size (base pairs) |
---|---|---|---|---|
1 | E6/& consensus | GP-E6-3F | GGG WGK KAC TGA AAT CGG T | 602–666 |
GP-E6-5b | CTG AGC TGT CAR NTA ATT GCT CA | |||
GP-E7-6B | TCC TCT GAG TYG YCT AAT TGC TC | |||
2 | HPV genotype 16 | HPV16-F | CAC AGT TAT GCA CAG AGC TGC | 457 |
HPV16-R | CAT ATA TTC ATG CAA TGT AGG TGT A | |||
3 | Genotype 18 | HP18-F | CAC TTC ACT GCA AGA CAT AGA | 322 |
HPV-18-R | GTT GTG AAA TCG TCG TTT TTC A | |||
4 | HPV genotype 31 | HPV31-F | GAA ATT GCA TGA ACT AAG CTC G | 263 |
HPV31-R | CAC ATA TAC CTT TGT TTG TCA A | |||
5 | HPV genotype 33 | HPV33-F | ACT ATA CAC AAC ATT GAA CTA | 398 |
HPV33-R | GTT TTT ACA CGT CAC AGT GCA | |||
6 | HPV genotype 35 | HPV35-F | CAA CGA GGT AGA AGA AAG CAT C | 358 |
HPV35-R | CCG ACC TGT CCA CCG TCC ACC G | |||
7 | HPV genotype 39 | HPV39-F | GAC GAC CAC TAC AGC AAA CC | 280 |
HPV39-R | TTA TGA AAT CTT CGT TTG CT | |||
8 | HPV genotype 42 | HPV42-F | CCC AAA GTA GTG GTC CCA GTT A | 277 |
HPV42-R | GAT CTT TCG TAG TGT CGC AGT G | |||
9 | HPV genotype 43 | HPV43-F | GCA TAA TGT CTG CAC GTA GCT G | 219 |
HPV43-R | CAT GAA ACT GTA GAC AGG CCA AG | |||
10 | HPV genotype 44 | HPV44-F | TAA ACA GTT ATA TGT AGT GTA CCG | 163 |
HPV44-R | TAT CAG CAC GTC CAG AAT TGA C | |||
11 | HPV genotype 45 | HPV45-F | GTG GAA AAG TGC ATT ACA GG | 151 |
HPV45-R | ACC TCT GTG CGT TCC AAT GT | |||
12 | HPV genotype 51 | HPV51-F | GAG TAT AGA CGT TAT AGC AGG | 233 |
HPV51-R | TTT CGT TAC GTT GTC GTG TAC G | |||
13 | HPV genotype 52 | HPV52-F | TAA GGC TGC AGT GTG TGC AG | 229 |
HPV52-R | CTA ATA GTT ATT TCA CTT AAT GGT | |||
14 | HPV genotype 56 | HPV56-F | GTG TGC AGA GTA TGT TTA TTG | 181 |
HPV56-R | TTT CTG TCA CAA TGC AAT TGC | |||
15 | HPV genotype 58 | HPV58-F | GTA AAG TGT GCT TAC GAT TGC | 274 |
HPV58-R | GTT GTT ACA GGT TAC ACT TGT | |||
16 | HPV genotype 59 | HPV59-F | CAA AGG GGA ACT GCA AGA AAG | 215 |
HPV59-R | TAT AAC AGC GTA TCA GCA GC | |||
17 | HPV genotype 66 | HPV66-F | TTC AGT GTA TGG GGC AAC AT | 172 |
HPV66-R | AAA CAT GAC CCG GTC CAT GC | |||
18 | HPV genotype 68 | HPV68-F | GCA GAA GGC AAC TAC AAC GG | 333 |
HPV68-R | GTT TAC TGG TCC AGC AGT GG |
Note: The three primers were used together and the amplicon size ranges from 602-666.
HPV, human papilloma virus.
The data anlalysis was done using Statistical Package for Social Sciences (SPSS) version 18.2 (IBM Corp. Armonk, New York, United States). All the data generated were analysed using descriptive statistics such as mean and standard deviation and results were analysed using chi-square at α = 0.05.
A total of 231 study participants were recruited for this study, out of which 129 (55.9%) were female. Their mean age was 42.59 (range: 18–62 years) (
Age distribution of dental clinic attendees in Ibadan, Nigeria, January 2016 to March 2017.
Category | Total |
---|---|
< 20 | 18 |
21–30 | 55 |
31–40 | 42 |
41–50 | 37 |
51–60 | 32 |
> 60 | 47 |
Male | 104 |
Female | 127 |
Married | 128 |
Single | 90 |
Widowed | 2 |
Divorced | 11 |
Note: Mean age = 42.59, standard deviation = 15.85, variance = 251.32.
Of the 231 oral swabs tested, 23 (9.9%) had HPV DNA, of which 21 were high-risk and two were low-risk HPV. Of the 23 HPV DNA-positive participants, 13 (56.5%) were male and 10 (43.5%) were female. Also, 18 (78.3%) of the 23 participants with HPV had previously engaged in oral sex. Participants in the age group 21–40 years had the highest rate of positivity (14.5%), whereas those aged 60 years or older had the lowest rate (2.1%) (
Distribution of human papilloma virus infection among dental clinic attendees in Ibadan, Nigeria, January 2016 to March 2017.
Variable | Category | Number tested | HPV DNA positive | % | Chi-square | |
---|---|---|---|---|---|---|
Presence of oral warts | 1.0629 | 0.303 | ||||
Yes | 36 | 2 | 5.6 | - | - | |
No | 168 | 19 | 11.3 | - | - | |
Smoking | 1.860 | 0.173 | ||||
Yes | 24 | 4 | 16.7 | - | - | |
No | 207 | 17 | 8.2 | - | - | |
Number of sexual partners | 4.208 | 0.121 | ||||
1 | 146 | 11 | 7.5 | - | - | |
> 1 | 54 | 9 | 16.7 | - | - | |
None | 31 | 1 | 3.2 | - | - | |
Previous oral infection | 0.696 | 0.404 | ||||
Yes | 108 | 8 | 7.4 | - | - | |
No | 123 | 13 | 10.6 | - | - | |
Gender | 1.371 | 0.242 | ||||
Male | 104 | 12 | 11.5 | - | - | |
Female | 127 | 9 | 7.1 | - | - | |
Marital status | 1.896 | 0.425 | ||||
Married | 128 | 9 | 7.0 | - | - | |
Single | 90 | 11 | 12.2 | - | - | |
Widowed | 2 | 0 | 0.0 | - | - | |
Divorced | 11 | 1 | 9.1 | - | - | |
Ever engaged in oral sex | 62.715 | 0.0001 | ||||
Yes | 46 | 18 | 39.1 | - | - | |
No | 185 | 3 | 1.6 | - | - | |
Age | 134.860 | 0.0001 | ||||
18–20 | 18 | 1 | 5.6 | - | - | |
21–40 | 97 | 14 | 14.4 | - | - | |
41–50 | 69 | 5 | 7.2 | - | - | |
≥ 60 | 47 | 1 | 2.1 | - | - |
Of the 23 participants with HPV infection, 16 had mono infections while seven had co-infections with either other high-risk or low-risk genotypes. The most common genotypes detected in this study were genotypes 16 and 18. HPV genotype 16 was detected in ten participants, eight of which were single infection and one co-infection with genotype 33, the other one were the low risk group (6/11). Genotype 18 was also detected in four participants, of which three were single infection and one co-infection with genotype 31. Genotypes 31 and 33 were each detected in three participants, two as single infection and one each as co-infection with other genotype. Genotype 39 was the least prevalent genotype detected in only one participants. A total of 21 participants had high-risk HPV DNA.
The prevalence of oral HPV infection and the circulating genotypes among dental clinic attendees in Ibadan, Nigeria, was determined in this study. An oral HPV prevalence of 9.9% was observed. Previous reports on the rates of oral HPV infection among asymptomatic individuals varied between different geographic areas. It ranged from 12.0% in South Africa to 5.0% in the United States. The prevalence obtained among dental clinic attendees in this study is lower than the 12% reported from South Africa among individuals attending HIV testing centres
The primers used for the detection of HPV DNA in this study target the E6/7 region of the virus. This region is part of the viral oncogenes and is usually integrated into the host genome. Thus, primers targeting this region are more sensitive than the L1 region most commonly used. Other studies have shown that PCR protocols targeting the L1 region of the HPV genome are likely to miss some infections if the viral DNA has been integrated, as the L1 region of the viral genome is usually deleted during viral integration.
In this study, people aged 60 years and older had the lowest prevalence (2.1%) of HPV infection, while those in the age group of 21–40 years had the highest rate (14.5%) (
Gender differences have been observed in the distribution of oral HPV.
This study detected low risk HPV genotype 6/11 and high-risk genotypes 16, 18, 31, 33 and 39 among dental clinic attendees in Oyo state, Nigeria. Among the previously reported circulating genital HPV types among women in Ibadan, Nigeria, according to Nejo et al. and Thomas et al.
Finally, the inclusion of the HPV vaccine into the national vaccination programme to cover both boys and girls in Nigeria will reduce the prevalence of oral HPV infection and, ultimately, HPV-associated oropharyngeal cancer in Nigeria. Presently, the most common vaccine in Nigeria (Cervarix marketed by GlaxoSmithKline) targets HPV 16 and 18 and Gardasil (marketed by Merk & Co.) targets only HPV 16, 18, 6 and 11 whereas this study and previous Nigerian studies have identified other genotypes. Thus, introducing HPV vaccine that will cover other circulating strains in addition to the ones they cover presently will make the vaccination programme more effective. The study by Herrero et al.
Patients with symptoms of oropharyngeal cancer were not included in this study to identify the virus genotypes associated with oropharyngeal cancer in this region. Information about the number of kissing partners of the participants was also not accessed in the study. The size of the study population is also a limitation of the study.
This study describes a high prevalence of oral HPV infection among dental clinic attendees in Ibadan, Oyo State, Nigeria, by detecting HPV DNA from the oral swabs of the study participants using PCR. A prevalence of 9.9% of HPV infection was identified and also showed that HPV type 16 and 18 are the most common types detected among the study participants. Oral sex was also significantly associated with HPV infection. The HPV vaccine for use in Nigeria should cover at least the commonest circulating genotypes to reduce the risk of HPV-associated oropharyngeal and other cancers in Nigeria.
We are grateful to the members of staff of Oyo State Dental Clinic, Ibadan, for their assistance in collecting samples analysed, as well as the participants in this study.
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them, as well as the participants in writing this article.
A.O.F. was responsible for conceptualisation of the idea, sample collection, sample analysis, writing of the draft manuscript and manuscript review. O.S.B. contributed to conceptualisation of the idea, sample collection, sample analysis and manuscript review. G.N.O. and O.D.O. were involved in conceptualisation of the idea, sample analysis and manuscript review, while J.A. contributed to conceptualisation of the idea, sample collection and manuscript review.
This study was partly funded by the College of Medicine Seed Award 2014.
The manuscript writing of the project described was supported by the Medical Education Partnership Initiative in Nigeria project funded by Fogarty International, the Office of AIDS Research, and the National Human Genome Research Institute of the United States National Institutes of Health, the Health Resources and Services Administration and the Office of the United States Global AIDS Coordinator under Award Number R24TW008878.
The authors confirm that the data supporting the findings of this study are available within the article.
The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding organisations.