Al-Azhar Assiut Medical Journal

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 15  |  Issue : 2  |  Page : 71--77

The prevalence of human papillomavirus genotypes (HPV-6, HPV-11, HPV-16, and HPV-18) in the unilateral and bilateral nasal polyps


Amani M Abd Elwahab1, Randa A Abdullah2, Gehan S Shalaby2, Soad Yehia2, Hanaa Aboeliazed3,  
1 Microbiology Department, Faculty of Medicine Al Azhar University for Girls, Egypt
2 ENT Department, Faculty of Medicine Al Azhar University for Girls, Egypt
3 Community Medicine Department, Faculty of Medicine Al Azhar University for Girls, Egypt

Correspondence Address:
Randa A Abdullah
Lecturar of Otolaryngology and Head and Neck Surgery, Alazhar University Hospitals, Nasr City, Cairo
Egypt

Abstract

Aim The aim of this study was to investigate the prevalence of different genotypes (HP-6, HP-11, HP-16, and HP-18) of human papillomavirus (HPV) in nasal polyps (NPs). Materials and methods This is a prospective, comparative, controlled study. A total of 24 patients with NPs along with 10 patients with deviated nasal septum without NPs (as controls) were enrolled. Biopsy specimens from the patients’ NPs and from nasal mucosa of controls were collected for the detection of the four HP genotypes using PCR-based DNA amplification using the genotype-specific primers (6/E5, 11/L1, 16/L1, and 18/L1). Results An overall 91% (22/24) of patients with NPs were found to be positive for HPV infection, whereas all participants of the control group were negative (P<0.05). Among the HP-positive cases, 19/22 (86.3%) were infected with a single genotype, whereas 3/22 (13.6%) demonstrated a coinfection of HP-11 and HP-18 genotypes. The HP-18 was found to be the most prevalent genotype [8/22 (36.36%)]. However, the occurrence of the HP-6 genotype was higher [6/22 (27.27%)] than that of the HP-11 [5/22 (22.7%)]; HP-16 was not detected in any of the unilateral or bilateral NP specimens. Conclusion Our findings suggest that the HPV infection is associated with the formation of NPs and further investigation is imperative to define a correlation between the two.



How to cite this article:
Abd Elwahab AM, Abdullah RA, Shalaby GS, Yehia S, Aboeliazed H. The prevalence of human papillomavirus genotypes (HPV-6, HPV-11, HPV-16, and HPV-18) in the unilateral and bilateral nasal polyps.Al-Azhar Assiut Med J 2017;15:71-77


How to cite this URL:
Abd Elwahab AM, Abdullah RA, Shalaby GS, Yehia S, Aboeliazed H. The prevalence of human papillomavirus genotypes (HPV-6, HPV-11, HPV-16, and HPV-18) in the unilateral and bilateral nasal polyps. Al-Azhar Assiut Med J [serial online] 2017 [cited 2018 Dec 17 ];15:71-77
Available from: http://www.azmj.eg.net/text.asp?2017/15/2/71/218849


Full Text



 Introduction



Nasal polyposis is a common chronic inflammatory disease of the nasal and paranasal sinus mucosa and is associated with non-neoplastic mucosal lesions. The trigger for nasal polyp (NP) formation is not yet clearly understood; however, the role of genetic predisposition has been suggested by some researchers [1]. Nasal polyposis reportedly affects 1–4% of the general population [2],[3]. NPs are outgrowths of the nasal cavity and paranasal sinus mucosa due to allergic or chronic inflammation [4]. NPs are characterized as multifocal polypoid mucosal swellings, typical bilateral, and arising mainly from the middle meatus and ethmoid sinus regions [4],[5]. Furthermore, these are regularly associated with chronic sinus infection [6],[7]. The average age of patients with NP is 50 years [8]. The respiratory epithelium is usually intact and frequently exhibits hyperplasia of goblet cells. On the contrary, the characteristic antrochoanal polyp typically constitutes a large solitary unilateral mass. These originate from the maxillary sinus, pass through the nasal cavity, extend into the choanae, and further into the nasopharynx. Antrochoanal polyps occur more commonly in children and young adults with an average age of 27 years [1], when there is a lack of other risk factors or associated diseases. It is noteworthy that allergic diseases seem to play no significant role in the pathogenesis of antrochoanal polyps [9]. Accordingly, they do not show morphological features typical for allergy-associated inflammatory NPs [8]. As compared with NPs, antrochoanal polyps do not present hyperplasia of goblet cells, although alterations of the ciliated epithelium are common. Even squamous cell metaplasia may be observed in antrochoanal polyps [10]. Although sinonasal polyposis is a well-investigated entity, the etiology still remains unclear. To date, only a few studies have been conducted to screen NPs in order to identify the causative factors, including the human papillomavirus (HPV) infection [11]. The HPV infections are related to the genesis of various benign and malignant human neoplastic diseases − for instance, the cervical cancer [12]. A subgroup of oropharyngeal cancer is increasingly recognized to be caused by persistent infection with high-risk HPV [13],[14]. As the NP formation is such a common condition, it becomes imperative for an otolaryngologist to precisely identify its cause, so that through prevention and control of the causative factor, the occurrence of NP may be minimized in the population. In the wake of this, the present study was conducted to recognize the association of HPV infection with NP formation, and also to study the prevalence of its various genotypes, including the high-risk and the low-risk (LR) ones.

 Materials and methods



Patients and controls

The study was conducted on individuals who were admitted in the Department of Otorhinolaryngology, Al-Zahraa Hospital, Al-Azhar University, Faculty of Medicine (Girls). A total of 34 participants were enrolled through the years (2014–2016) to determine the prevalence of infections by different HPV genotypes (HPV-6, HPV-11, HPV-16, and HPV-18) in the NPs. The investigation included one study group and a control group as mentioned below.

Study group

Twenty-four patients (age: 7−60 years; 11 male and 13 female) with the diagnosis of chronic rhinosinusitis, eight patients with unilateral and 16 patients with bilateral NP, were included. The patients had undergone endoscopic sinus surgery for the treatment of the NPs. The diagnosis of polyps involved patient history, clinical examination, nasal endoscopy, and computed tomography.

NP biopsies were extracted during the functional endoscopic sinus surgery. After surgery, a definitive diagnosis of the polyp was made by means of histopathological examination of the biopsy, which revealed no malignant or premalignant criteria in the specimens. The biopsy specimens were preserved in formalin and transported to the microbiology laboratory for further microbiological examination, followed by storage at −70°C under sterile condition until further use.

Control group

Ten patients (age: 16−66 years; four male and six female) who had undergone septoplasty (surgery for nasal septum deviation) without NPs were included in this study. The diagnosis of nasal septum deviation was made by recording patient history, endoscopic examination, and computed tomography.

Samples

NP biopsies were collected from the patients during the functional endoscopic sinus surgery and after surgery; the biopsy was preserved in saline and sent to the microbiology laboratory for microbiological examination. The specimens were stored at −70°C under sterile condition until the study tests were performed.

DNA extraction from tissues of nasal polyp and nasal mucosa

The tissues were subjected to DNA extraction using a commercially available kit by Qiagen (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany) as per the manufacturer’s protocol. Briefly, the tissues were digested with 180 µl of the ATL buffer (provided with the kit) and 20 µl of proteinase K (20 mg/ml) by overnight incubation at 56°C. After digestion, 200 µl of the AL buffer and 100% ethanol were added. The solution was transferred to a QIAamp spin column and centrifuged for 1 min, followed by washings with 500 µl of the AW1 buffer by spinning for 1 min, and with AW2 buffer (spinning for 3 min). The DNA was eluted by applying 200 µl of the AE buffer at room temperature by means of centrifugation for 1 min.

Polymerase chain reaction

The extracted DNA was subjected to PCR as previously described by Zaravinos et al. [15]. The primers used for detection of each genotype are shown in [Table 1].{Table 1}

Electrophoresis

The amplified PCR products were resolved on 2% agarose gel with ethidium bromide. A standard DNA ladder was also loaded alongside the test samples to confirm the amplification of DNA of correct molecular size.

Statistical method

The data were analyzed using SPSS program (version 18; SPSS Inc., Chicago, Illinois, USA). The frequency of genotypes detected in the samples was calculated to describe the qualitative data, and the comparison of the study groups was made using the χ2-test. Mean and SD were estimated to describe the central tendency and the dispersion of parametric data, and Student’s t-test was used to compare between the two mean. The level of significance was taken as P value 0.05 or less and the results are presented in tables.

 Results



Demographic line data

The baseline data did not show any statistically significant difference among the two study groups, in terms of distribution of age and sex ([Table 2]).{Table 2}

Distribution of unilateral and bilateral polyps among the cases according to the sex of the patient

As shown in [Figure 1],[Figure 2],[Figure 3],[Figure 4], there were two (18.2%) patients with unilateral polyp and nine (81.8%) patients with bilateral NP among the total 11 male patients in our study group, and there were six (46.2%) patients with unilateral NP and seven (53.8%) patients with bilateral NP among the 13 female patients with a nonsignificant P value greater than 0.05.{Figure 1}{Figure 2}{Figure 3}{Figure 4}

Prevalence of human papillomavirus infection among the study and control groups

We analyzed a total of 24 patient samples in the study group, of which 22 (91.7%) were found to be positive for HPV infection, whereas two (8.3%) tested negative. Of the total 10 samples from the control group, none tested positive for HPV infection (P<0.05) ([Figure 2]).

Prevalence of human papillomavirus infection among the unilateral and bilateral polyps

HPV infection was detected in six (75%) samples of the unilateral NP group, whereas two (25%) samples were observed to be HPV-negative cases. On the contrary, the entire bilateral NP samples [16 (100%)] tested positive for HPV infection (P<0.05) ([Table 3]).{Table 3}

Prevalence of single and multiple human papillomavirus genotype infections among the positive cases

The samples analyzed in our study comprised polyp cases that exhibited single or coinfections of HPV genotypes. The single HPV genotype infection was observed in 19/22 (86.4%) study cases, whereas 3/22 (13.6%) cases had a dual infection of HPV-18 and HPV-11 genotypes (P<0.05) ([Table 3]).

Prevalence of different human papillomavirus genotypes through the positive cases

Among the 22 HPV-positive samples, eight (36.37%) cases showed positivity for the HPV-18 genotype, six (27.27%) were positive for HPV-6 genotype, and five (22.72%) were positive for HPV-11 genotype. However, the HPV-16 genotype was not detected in any of the HPV-positive cases (P>0.05) ([Table 3]).

Prevalence of different human papillomavirus genotypes with the laterality of the polyp

As shown in [Table 3], six (37.5%) HPV-18-positive cases, four (25%) HPV-6-positive cases, and four (25%) HPV-11-positive cases were identified among the bilateral polyp specimens. In contrast, two (33.3%) HPV-6/E5-positive cases, two (33.3%) HPV-18/L1-positive cases, and one (16.6%) HPV-11/L1-positive case were observed among the unilateral polyp samples (P>0.05).

 Discussion



The present study aimed to analyze the prevalence of HPV infection in NP cases. It was observed that 91% of the specimens that were collected from the patients with NPs demonstrated HPV infection, whereas none of the samples were positive for the virus in the control group. Our findings are in accordance with Zhou et al. [11], who reported 92.3% of NP patients demonstrating HPV infection. The correlation between the HPV infection and the development and prognosis of NPs is not well understood. It was reported [17] that 27.14% of patients with NP were positive for HPV infection. Pei et al. [18] conducted a genotypic analysis of 204 NP samples using flow-through hybridization and gene chip method and detected HPV in 82 (40.2%) patients, whereas all healthy controls were negative for the virus. Some authors published that about 13% HPV infections result in NPs, 4% in the adjacent turbinate [19] and 50% in other pathologies [20]. However, such findings are in contrast to the report by Hoffmann et al. [21], in which HPV was detected in none of the NP specimens analyzed. This contrary result may be attributed to the limited number of samples investigated or to the different method used for HPV detection, as the flow-through hybridization and gene chip techniques are known to be highly sensitive and specific for HPV genotyping [15].

In our work, all of the bilateral NP cases (16 cases) were found to be positive for HPV DNA (100%), whereas only six (75%) of the total eight unilateral NP cases were HPV positive (P<0.05). These findings are in contrast to the previous report by Knör et al. [22], who published that the HPV infection has a significantly higher association in antrochoanal polyp cases (53.5%) than in the bilateral NPs (15.1%).

Our results showed that HPV-18 was the most prevalent genotype [8/22 (36.36%)] among the NP patients, followed by HPV-6, which was detected in 6/22 (27.27%) cases, and lastly HPV-11, which was observed in 5/22 (22.7%) patients. Syrjänen [23] reported 11 (78.5%) HPV-positive patients from the total 14 papilloma patients, one of them was positive for HPV-6, five for HPV-11, two for HPV-16, and three for HPV-11 and HPV-16 coinfection. Other researchers [18] have reported that the most prevalent HPV genotypes among both single and multiple HPV infections were the LR HPV genotypes, HPV-11 (45.28%), followed by HPV-58 (16.04%) and HPV-52 (10.38%). However, a few studies [11],[17] suggested that LR HPV-5 and LR HPV-6 are the major prevalent HPV genotypes in a Chinese population with NPs. Thus, the previous reports along with the data of our present research work confirm the hypothesis that the NPs are benign lesions. Certain discrepancies may be due to the difference in the studied population or the methods for HPV detection in the specimens. In our study, we performed PCR for the detection of HPV in the samples and found that none of the NP specimens were positive for the HPV-16 genotype. This indicates that there were no malignant or premalignant cases in our study group, which was further confirmed by histopathological examination of all specimens. There are reports [24],[25],[26] that describe the HPV genotype 16 as one of the high-risk HPV genotypes, which is more frequently associated with invasive carcinoma of the oral cavity, oropharynx, larynx, and the nasal cavity. Yamashita in 2015 [27] detected HPV-16 in squamous cell carcinoma and inverted papilloma (IP) and compared with the IP and chronic rhinosinusitis groups and found that persistent infection and integration of high-risk HPV-16 play an important role in the malignant transformation of IP. In contrast, Cnor et al. (2015) [22], unexpectedly detected the oncogenic HPV-16 genotype as the dominant one in their study, exhibiting 76% positivity in the studied NP samples and 61.9% detection in antrochoanal polyp patients. In our study, 19/22 (86.4%) of the HPV-infected cases showed single infection, whereas 3/22 (13.6%) expressed coinfection of the HPV-18 and HPV-11 genotypes; this is in agreement with a previous report [18], in which single infection showed a prevalence of 74.39% positivity compared with a prevalence of 25.61% for multiple infection cases. The authors reported that the incidence of dual infections of HPV (90.48%) was much higher as compared with the triple (4.76%) and quadruple HPV genotype infections (4.76%), suggesting that the various HPV genotypes have no competitive inhibition in their hosts.

The present study showed that six (37.5%) of the bilateral NP cases had an infection of HPV-18, four (25%) had HPV-6 and four (25%) had HPV-11 genotypes, two (12.5) showed dual infection of HPV-11 and HPV-18, and there were no positive cases of HPV-16 genotype. In the unilateral NP group, there were about two (33.3%) HPV-6-positive patients, two (33.3%) HPV-18-positive cases and one (16.6%) HPV-11-positive case, only one (16.6%) case with HPV-11 and HPV-18 coinfection, and no HPV-16-positive case (P>0.05); this result is in agreement with a previously published report [28]. Contrary to our findings, a research group [22] reported the high-risk HPV-16 (76%) to be the prevalent genotype among the 166 NP specimens analyzed using PCR for the HPV DNA detection. In addition, 8% of high-risk type HPV-56, 8% of LR type HPV-11, and 8% HPV-11/16 coinfections were also detected. Within the HPV-positive antrochoanal polyp cases, the predominant genotype was the HPV-16 (61.9%), followed by the HPV-11/16 coinfections in 23.8% and HPV-11 single infection in 14.3% of the samples. The identified HPV genotypes in nasal turbinate specimens were deadly coinfections of HPV-16, HPV-11, and HPV-11/16.

Upon comparison of the various studies conducted by different research groups on the prevalence of HPV infection and its genotypes among the unilateral and bilateral NPs, the diverse findings may be attributed to distinct factors, such as race, environmental condition, geographical region, laboratory technique used, tissue fixation method, the number of samples, and the inclusion criteria for the control group.

There was no significant difference in the demographic data of the patients having NPs or with the incidence of the HPV infection, as the patient age and sex were not associated with the variable HPV prevalence. The same observations were also made by some authors [18],[28], who reported no correlations between HPV infection or HPV subtypes and the clinicopathological characteristics of the patients, such as age, sex, the number of surgery, and disease course. However, some researchers [29] studied HPV prevalence and reported that the male : female ratio for the bilateral NPs was nearly 2 : 1 and for the antrochoanal polyp cases it was 1 : 1, with no significant difference. This is in contrast with another study [30], in which a U-shaped age-specific pattern with a higher prevalence in younger and older women compared with the middle-aged women was observed for the HPV infection. Thus, the establishment of a correlation between HPV infection and the patient age requires more understanding.

However, Chen et al. [31] recorded a higher prevalence of antrochoanal polyp in young adults and children as they found that about 28% of the antrochoanal polyp cases were children in their study. Conversely, other authors [32] approved that bilateral NPs present much later in life, being rare in children, as observed in their study of 4986 adult patients with NPs.

The present analysis revealed a nonsignificant difference in the laterality of polyps among the female patients, whereas a higher occurrence of bilateral NPs was observed in male population; this is in agreement with Settipane and Chafee [32],[33], who proved that bilateral NPs were dominant in male population, whereas Orvidas [29] reported no sex predominance among antrochoanal polyp patients. A study [10] demonstrated a higher incidence of antrochoanal polyps (87.5%) in male population; however, the inclusion criteria and less number of participants could be a reason behind their results.

 Conclusion



Our findings indicate an association of HPV infection with unilateral or bilateral NPs. The single genotype infections were more prevalent compared with multiple coinfection. Further investigations are required to explore the correlation of HPV infection with the prognosis of the NPs.

Acknowledgements

The authors thank the ENT and the microbiology team at Al Zaharaa Hospital, Faculty of Medicine of Girls, Al-Azhar University, for their skillful assistance and help.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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