Possible Synergistic Interactions Among Multiple HPV Genotypes in Women Suffering from Genital Neoplasia

Objective: Persistence of HPV infection is the true cause of cervical disorders. It is reported that competition may exist among HPV genotypes for colonization. This survey was designed to establish the multiple HPV genotype status in our community and the probability of multiple HPV infections involvement.
Methods: All multiple HPV infections were selected for investigation in women suffering from genital infections referred to private laboratories in Tehran, Iran. A total of 160 multi HPV positive specimens from cervical scraping were identified by the HPV genotyping methods, "INNO-LiPA and Geno Array". Result: In present study, HPV 6 (LR), 16 (HR), 53 (pHR), 31 (HR) and 11 (LR) were included in 48.8% of detected infections as the most five dominant genotypes. HPV 16 was detected at the highest rate with genotypes 53, 31 and 52, while HPV 53 appeared linked with HPV 16, 51 and 56 in concurrent infections. It appears that HPV 16 and 53 may have significant tendencies to associate with each other rather than with other genotypes. Analysis of the data revealed there may be some synergistic interactions with a few particular genotypes such as "HPV 53".
Conclusion: Multiple HPV genotypes appear more likely to be linked with development of cervical abnormalities especially in patients with genital infections. Since, there are various patterns of dominant HPV genotypes in different regions of world, more investigations of this type should be performed for careHPV programs in individual countries. Creative Commons Attribution License

Introduction

Infection with high-risk Human Papillomavirus (HR-HPV) genotypes are responsible for cervical cancer. Frequency and the distribution of HPV in each community have its own dominant types with a specific pattern that may be comparable in some genotypes with other areas. Although, HPV 16 is usually reported as one of the most prevalent types in most countries. Hence, it is necessary to have prevalence evaluated of HR-HPVs and LR-HPVs in cervical cancer prevention and Care HPV programs (Quint et al., 2012; Shayanfar et al., 2013; Sohrabi et al., 2014; Sohrabi et al., 2014). It is reported various parameters such as age of the infected patients, sexual behavior, and number of the partners, environmental conditions, smoke and some other risk factors are the main causes of HPV infection. Some of these influential parameters can cause enhancement of multiple HPV genotypes such as having different sexual partners (Chaturvedi et al., 2011; Mzarico et al., 2015 ;Tran et al., 2015). General tendency has been reported in previous studies for all HPV types being in similar cluster. It is frequently reported cervical concurrent infections with more than one HPV genotype are common, especially among young women (Chaturvedi et al., 2005; Mendez et al., 2005; Vaccarella et al., 2010). It is continuously are discussed these infections occur randomly or the result of interactions between HPV types (Chaturvedi et al., 2011; Chaturvedi et al., 2005 ; Plummer et al., 2012). Some studies have formally evaluated synergistic interactions across multiple HPV genotypes on cervical malignancies and risk of genital infections. In these reports higher rate have mentioned in those patients with abnormal cervical cytology especially those which included in α-9 family (Alpha-PV 9), particularly HPV16, 31 and 35 (Trottier et al., 2006; Wentzensen et al., 2013; Bernard et al., 2010; Dickson et al., 2013; Tommasino et al., 2014). It is emphasized the association between HPV16 and cancer is significantly stronger than that between either HPV31 or HPV35 (Pirog et al., 2014). This survey aims to find out the status of multiple HPV genotypes in our community among identified women suffering from multiple HPVs infections, also to understand whether certain HPV types have the tendency to be involved in multiple HPV infections or not.

Materials and Methods

Patients

Based on our previous study all patients who had multiple HPV genotypes were entered in this study (Sohrabi et al., 2016). These patients were all women referred to private clinical laboratories for diagnosis of genital infections and regular checkups in Tehran, Iran from January 2012 till December 2013. They were totally160 multi HPV positive specimens from cervical scrapping (Liquid Based Cytology) and genital lesions. All clinical data were collected from patients’ medical records.

HPV Genotyping

HPV DNA was extracted by the High Pure PCR Template Preparation Kit (Roche Diagnostics®, Mannheim, Germany). Extracted DNA was stored at −20°C until experiment. HPV genotyping was performed by approved HPV commercial diagnostic technologies as PCR Hybridization methods by either GenoArray Test kit (Hybribio) and INNO-LiPA HPV Genotyping Extra (INNOGENETICS®, Belgium) according to the manufacturer’s instructions. The GenoArray allowed for the detection of the following 21 HPV genotypes: 14 high risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68), five low risk genotypes (HPV6, 11, 42, 43, 44and CP8304 [81]), and probably HR types (HPV 53). However, the INNO-LiPA Extra I and Extra II were able to detect high risk types (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73, 82), low risk types (HPV6, 11, 40, 42, 43, 44, 54, 61, 62, 67, 81, 83 and 89), and probably HR types (HPV 53).

Definition of Multiple Infections

All specimens with two and more detected HPV genotypes considered as multiple HPV infections.

Statistical Analysis

In order to determine any significant relationship between the HPV genotypes, an independent tests (Chi-Square) were used. All tests were performed at the significant level of 0.05 using SPSS software version 23.

Results

Overall Prevalence of HPV Genotypes

Of the 426 HPV genotypes were identified from 160 multi HPV specimens which 32 HPV genotypes (high risk-HPVs:16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73, 82; low risk-HPV: 6, 11, 40, 42, 43, 44, 54, 61, 62, 67, 69/71, 81 and 89 and probably HR-HPVs: 53) were diagnosed by genotyping assays Figure 1. The distribution and frequency rate of Multiple HPV infections were determined for all low and high risk HPV genotypes. Comprehensive HPV genotypes interactions are shown in Figure 1. In present study, HPV 6 (LR), 16 (HR), 53 (pHR), 31 (HR) and 11 (LR) were involved in 48.83% from all detected infections as the most five dominant genotypes with 59, 57, 33, 31, and 29 cases, respectively. None of these dominant genotypes were involved with HPV40, 43, and 62 (Figures 1 and 2).

Figure 1

Associations Between LR-LR (A), HR-LR (B) and HR-HR HPV Genotypes Involved in Patients with Multiple Infections

Figure 2

Frequency of Detected HPV Genotypes in Multiple HPV Infections

Frequencies of Multiple HPV Genotypes

All detected genotypes were analyzed regarding their tendencies to the other genotypes in another aspect. In order to find out the rate of multiple HPV genotypes distribution with each other, each detected HPV genotypes were compared with others. The significant directly relationship between HPV genotypes in multiple HPV infections especially in dominant genotypes are shown in Table 1. Frequency rate for the three most dominant HR types 16, 53 and 18 have been compared with each others. HPV 16 was detected at the highest rate with genotypes 53, 31 and 52, while HPV 53 was with HPV 16, 51 and 56 in multiple HPV genotypes (Figure 3). HPV 16, 53 obviously showed higher tendencies to each other rather than other types Figure 3. These detected HPV multiple genotypes were belong to 92, 42, 17, 7, 1 and 1 cases as two, three, four, five, six and seven HPV multiple infections, respectively. Rate of multiple HPV infections were reduced in those specimens contain more than two types. As a result, there were no statistically significant differences between these multiple HPV genotypes (p≥ 0.05).

Table 1

Comparison Frequency of HPV Genotypes Rates which Involved in Multiple HPV Infections

HPV Genotypes	No. of Detected HPV Genotypes in Subjects	No. of Involved HPV Genotypes in Multiple Infections
16 (HR)	95	23
18 (HR)	36	13
26 (HR)	15	10
31 (HR)	57	22
33 (HR)	3	3
35 (HR)	15	11
39 (HR)	36	14
45 (HR)	22	14
51 (HR)	46	18
52 (HR)	39	15
56 (HR)	45	17
58 (HR)	26	14
59 (HR)	21	11
66 (HR)	38	16
68 (HR)	13	9
70(HR)	1	1
73 (HR)	5	4
82 (HR)	3	3
53 (pHR)	63	17
6 (LR)	82	20
11 (LR)	40	18
40 (LR)	1	1
42 (LR)	16	11
43 (LR)	1	2
44 (LR)	13	9
54 (LR)	5	5
61 (LR)	2	1
81	20	11
62	2	2
67	1	1
69/71	10	8
89	2	2

Figure 3

Comparison Frequency of Dominant HR- HPV Genotypes 16, 53,18

Discussion

It is reported multiple HPV genotypes are the major cause of the cell transformations in infected cases for progressing toward the cervical intraepithelial neoplasia (CIN I-IV) and association with abnormal cytology (Beca et al., 2014; Salazar et al., 2015). In this regard, the influence of newly diagnostic HPV technologies have been obviously had an important tools in the increasing of HPV genotypes. Therefore, apply high quality genotyping methods have now been necessarily more than ever (Sohrabi et al., 2015 ; Sorabi et al., 2016; Sorabi et al., 2016). Characterization of HPV genotypes in detected multiple infections had various frequencies in present study. Our results revealed all HPV genotypes are participating in genital infections in our community. HPV genotypes in multiple infections could be random or may be synergistic interactions across concurrent infections for each genotype in cervical disorders. Those HPV scatters in Figure 1 illustrated multi-type infections in some particular types, which supposed of the synergistic interactions between them. On the other hand, some types had interaction with many HPV genotypes but with low frequency rate. It could be meaning and suggesting for a random pattern rather than some specific tendency for these types. However, a specific tendency can be observed in those types having higher frequency rate. Analysis of scatters (Figure 1) demonstrated that LR-HPVs have low association with themselves, while HR-HPVs have strong and high association with themselves and even with LR-HPV types. The pattern of the dominant HPV genotypes are evaluating in most regions of the world in last decade. Furthermore, newly pHR-HPV genotypes have recently been raised in genital infections and also in cervical malignancies (Dickson et al., 2013; Shafaghi et al., 2013; Puryasin et al., 2015). No strong evidence has not been reported for HPV53 in cervical cancer in spite of frequent reports (Beca et al., 2014; Salazar et al., 2015). We observed stronger association of other HPV genotypes (HPV 52, 56, and 16) with HPV 53 in this study. The role of HPV53 and some other pHR types have been studied in cervical cancer and compared their effect with HR carcinogenic types. This type repeatedly reported as dominant type in many countries in the world (Shafaghi et al., 2013; Puryasin et al., 2014; Meloni et al., 2014; Sun et al., 2014). Phylogenic analysis proved HPV 53 has been located in 2B carcinogens with HPV26, 66, 67, 68, 70 and 73 which is belonging to the family of the α-Papillomaviridae the same group as HR-HPV types. It was reported these pHR-HPV genotypes were biologically active when presented as a single infection in cervical cancer affecting the same cellular pathways as any of the fully recognized carcinogenic HR-HPV types (Miller et al., 2012; Gill et al., 2014). Its potential effect has been interested in some reports (Padalko et al., 2015). However, our results demonstrated the rate of interactions were strongest between HR-HPV genotypes. Although, these results illustrated random patterns (multi HPV genotypes) were more likely in genital disorders. It still needs to be worked on: “is the pattern of multiple HPV genotypes, random or synergistic interaction?”. However there may be some synergistic interactions in a few particular types specifically “HPV 53” in those patients with multi HPV infections. It is suggested that more surveys should be emphasized in different populations to draw better conclusion for probability of synergistic interactions and its influence on the rising the rate of multi-type infections.

Disclosure

The authors who have taken part in this study declare that they do not have anything to disclose regarding the conflict of interest with respect to this manuscript.