Human papillomavirus genotype profiles and cytological grades interlinkages in coinfection with HIV

Introduction The study aimed to examine and characterize human papilloma virus (HPV) cytological grade trends and genotypes among HPV/HIV co-infected/cases and HPV monoinfected/control women attending Kisii Teaching and Referral Hospital, Kenya. Methods HIV positive co-infected with HPV (HPV/HIV) and HIV negative women monoinfected with HPV profiled as co-infected/cases and monoinfected/control arms respectively were enrolled. HPV subtypes were assayed by Xpert® HPV system (GXHPV-CE-10) alongside pathological cytology analysis of cervical tissue samples. Results Low grade intraepithelial lesion (LSIL) was the most predominant cytological grade across cases and controls with a prevalence of 32 (38.1%) while high grade squamous intraepithelial lesion (HSIL) was highest among HPV/HIV co-infected with a prevalence of 23 (27.38%). Among the monoinfected (controls) the predominant lesion was low grade intraepithelial lesions (LSIL) with a prevalence of 23 (27.38%). HPV type 16 had the highest prevalence 26 (30.8%) among the VIA positive women in the overall study participants followed by combinations of HPV types (16, 18/45) at 19 (22.6%). Conclusion High risk HPV types 16 and 18/45 were the most predominant in the established cytological grades and among the co-infected women. Routine screening using both cytological and HPV testing should be embraced and/or reinforced as early screening and preventive strategies in the covered geographical region population. Provision of the currently available vaccines to these women at an early age would provide effective protection since the HPV type profiles in this population are covered by such vaccines.


Introduction
Human Papillomavirus (HPV) is classified as high risk or low risk depending on its association with infection progression to cancer [1].
Human Immunodeficiency Virus (HIV) infection among women already infected with HPV infection has been correlated to having an association with a greater prevalence with broader ranges of the high risk human papilloma virus (HR-HPV) genotypes [1][2][3]. The presence of HIV is thought to increase the risk and susceptibility to infections with oncogenic human papilloma virus (HPV) which subsequently accelerates the natural history of Invasive Cervical Carcinoma (ICC) [2,3]. Cytological findings point to the fact that there is greater rate of persistence in high risk HPV type infections in HIV infected women leading to high-grade squamous intraepithelial lesions (HSIL) which culminate to invasive cervical carcinoma [4]. Infections with HIV is thought to be responsible for alteration of the spectrum of HPV types in normal cervical epithelium as well as in invasive cervical carcinomas resulting to a complicated morbidity in the interplay between the two viruses [5].
According to data at Kenya Medical Research Institute (KEMRI) which serves as regional cancer registry, approximately 80% of reported cases of cervical carcinoma are diagnosed at advanced stages, when little can be achieved in terms of curative treatment [6,7]. Even with HIV infection and cervical cancer co-infections being a major public health concern, molecular characterization of HPV types and trends specific data in HIV/HPV co-infections are scarce. In Kenya, to the best of our knowledge there is no such data in Kisii region as part of the broader Nyanza geographical terrain. Such gaps in the coinfections and genotype dynamism, translates to many setbacks in screening and prophylactic vaccination strategies among the vulnerable groups [4,8,9]. Studies have recommended further research in deciding the most appropriate screening approaches for different populations [2,8,9]. In order to better predict the impact of current vaccines on cervical cancer and for the improvement of screening programs, regional data on distribution of human papillomavirus (HPV) types in women with or without cervical cancer is crucial [10,11]. Thus, in view of the evidence gap, the study aimed to examine and characterize HPV cytological grade trends and genotypes among VIA positive HPV/HIV co-infected and HPV monoinfected women attending Kisii Teaching and Referral Hospital, Kenya in a quest to generate and enhance regionally adapted insights for better public health cues and planning information.  [14]. Going by such prevalence and underpinnings, we arrived at an independent case control study with HIV positive women coinfections with HPV as cases and HIV negative women monoinfected with HPV as controls [15,16] while adopting the double proportion formula in arriving at adequate sample size [15]. The minimum required sample size was therefore 32.675; 33 study participants for each group/arm making a total minimum of 66 participants for the two groups/arms.

Methods
The study participants were recruited by invitation. Three female nurses and one female clinical officer with prior knowledge and experience on visual inspection of the cervix with acetic acid (VIA) were trained on recruitment process of the participants and how to obtain satisfactory cervical smears. They also worked in conjunction with a residence gynecologist who oversaw routinely the process of sample collection as is recommended [17]. A research assistant was also trained to consent the participants per the bioethically approved study protocol. After consenting, the women underwent visual inspection of the cervix using 5% acetic acid so as to pick the VIA positive clients [18,19]. The VIA positive women had their cervical smear samples collected for both cytology and HPV genotyping.
Additional laboratory and clinical data such as other infections and pregnancy test results was obtained from enrolled participants medical records at the facility. All qualified participants' data were coded and anonymized.

Cervical specimens for cytology and High-Risk (HR) HPV
genotyping: all VIA positive women had cervical cell samples exfoliated and preserved for confirmatory testing with Pap and HPV genotyping [20][21][22][23][24]. Briefly samples of cervical cells (cervical swabs) were exfoliated from the zone of transformation on the cervix were carefully exfoliated [22,23] To guarantee sampling of adequate cells for both cytology and genotyping a cervical broom-like device (Rovers Medical Devices, Netherlands) designed to make contact with ectocervix and endocervix simultaneously was carefully used [20,21].
Immediately after exfoliation of the cervical cells, a thin smear was made on the microscope slide using the broom and fixed for cytology [18,19,25]. The cervical cells left on the broom after the smear preparation on slide were immediately collected in PreservCyt ® Solution (Hologic Corp.) per manufacturer protocols [26][27][28][29]. There was no need of multiple sample collection or multiple visits since Food and Drug Administration (FDA) approves one Thin Prep Pap Test as the only liquid based technique for both Pap and HPV testing directly [24]. The Pap samples were prepared in duplicate to allow two pathologists to read them independently via microscopy as earlier detailed [22,23,30,31]. The cytology cells slides were classified, categorized and reported according to the revised standardized 2001 Bethesda classification [31,32]. The women with VIA negative screening results were advised to repeat VIA screening once in every three years as is recommended [18,19].
For HR-HPV genotyping, all samples were analyzed by Xpert ® HPV assay system (GXHPV-CE-10). Based on the manufacturer's instructions, samples were shaken for several seconds, and 1 ml total volume of PreservCyt was poured into an Xpert ® HPV cartridge and subsequently loaded into Cepheid Xpert Diagnosis instrument that uses a second generation real-time PCR [27,28]. Interpretation of assay results followed the manufacturer's guidelines as previously described and per the recommended WHO standards [26][27][28][29]. The applied Xpert ® HPV assay specifically identifies types HPV 16  followed by HPV type category (16, 18/45) with a prevalence of 6 (7.14%). In cases, HSIL cytological grade had second highest total HPV prevalence of 30 (35.7%) in type categories 16 with a prevalence of 9 (10.7%) followed by type categories (16, 18/45) with a frequency of 6 (7.1%). In these cytological grades the most prevalent HPV types were (16) and (16, 18/45). This study shows an increase in prevalence of HPV type 16 from normal cytology to LSIL and HSIL having 4  Table 4.

Discussion
The study examined and characterize HPV cytological grade trends and genotypes among VIA positive HPV/HIV co-infected and HPV monoinfected women attending Kisii Teaching and Referral Hospital, Kenya in a quest to generate and enhance regionally adapted insights for better public health cues and planning information for instance thus, bringing to the fore understandings that would prove valuable in deciding on the most appropriate screening strategies and HPV vaccination programs approach. Overall, there was 27.38% prevalence of high grade squamous intraepithelial lesions (HSIL) among the HIV positive HPV co-infected women which was the predominant lesion. The overall prevalence of HR-HPV in the VIA positive women in this population was 20.48% while the highest prevalence of high risk HPV genotypes was type 16 while genotypes (16), (18/45) and (16, 18/45) which accounted for 66.67% of the cases. In the cytological grading of participants who were HR-HPV positive regardless of HIV status, the prevalence increased in the order; f normal cytology, ASCUS, HSIL to LSIL albeit, there was marginal difference between LSIL and HSIL cytological grade prevalence. There was diverse variance in prevalence trends of the predominantly prevalent HPV types in the cytological grades ASCUS, LSIL, HSIL across numerous findings reported this far but outstandingly, normal cytology was the least observed cytological grade in regard to HR-HPV infection [34][35][36]. It has been reported, that the age of the participant, history of other infections and number of pregnancies increases the severity of dysplasia and HPV prevalence [36] thus the most probable factors that cause the difference in dysplasia in these studies.
Related findings have also noted that HPV types did not influence severity of dysplasia rather the duration of HPV infection and immunological responses to infections by the host determine cervical tissue transformation to ICC after HPV infections [36]. There could also be differences in the natural history of the infections in women above reproductive age since the immunological responses to infections, inflammation and anti-cancer responses are reduced with increase in age [37]. The aspect of geographical disparities and ethnicity may also contribute to these discrepancies in cytological grades as shown by differences in genotype specific HPV in some populations from different regions and with differences in ethnicity inclination [38] whereas differences in prevalence of HR-HPV types has highly been associated with geographical regions and the extent of neoplasia in cervical cells [39,40]. Abnormal cytology in the current study was double in cases compared to that of controls indicating how critical and essential intensification of cervical cancer screening in the HIV positive women in this region would be. Moreover, the prevalence of co-infected women with HSIL was more than threefold compared to the monoinfected. If the co-infected women were to progress to cervical cancer, then there would be a greater chance and therefore they entail a population segment that would require more screening at an earlier stage.
The overall prevalence of HR-HPV in the VIA positive women was lower than that observed by Ghedira et al [41] with women in Nigeria urban setting, a varying geographical and ethnic setting in the African context having a prevalence that was higher [42]. In current study the HR-HPV prevalence was high among women with abnormal cytology compared to normal cytology. Although the prevalence of HR-HPV in both normal and abnormal cytology in the current study are far much below those reported earlier [41] where the prevalence of HR-HPV in women with squamous intraepithelial lesions (SIL) was higher in normal cytology, the two studies are in agreement that the prevalence of HR-HPV is higher in abnormal cytology [41]. HR-HPV genotype present has been associated with the cytological abnormality with HPV 16 and 18 being associated with abnormal cytology [43]. Studies have shown that increased immune expression of p53 genes reduces HPV 16 infection rates a clear indication that differences in genetic expressions in an individual will alter the immunological responses and that will affect infectivity rate [44]. In LSIL, HPV type category (16, 18/45) had the highest prevalence followed by HPV 16 and comparative findings on HPV type 16 has been pointed also been reported in the African context [41].
In the present study, HPV type 52 has a low prevalence while type 53 was not detected. This trend is amenable with Bangkok Metropolitan women findings which indicated that the rate of HPV detection was related to severity of cytological grades with the rate following an ascending order from 13.0%, 30.8%, 39.5%, 56.3% and 100.0% for ASC-US, ASC-H, LSIL, HSIL and SCC respectively [45]. In our findings, HPV 16 prevalence increased with increase in severity of lesions ASCUS 2 LSIL 6 and HSIL 14 which was comparative to the Metropolitan findings [45]. The idea of severity of HPV cytological lesions in infection has also been reflected by the fact that abnormal cytology had a higher prevalence rate of HPV compared to normal cytology [46] which correlates to HPV genotype where HPV genotype 16 as a single infection or combined with others increase with increase of severity of lesions [47,48].

Conclusion
High risk HPV types 16 and 18/45 were the most predominant in the established cytological grades and among the co-infected women.
There was a higher prevalence of HR-HPV genotypes in high grade lesions in co-infections which was twice that in mono-infections.
Routine screening using both cytological and HPV testing should be embraced and/or reinforced as early screening and preventive strategies in the covered geographical region population. Such routine screening services would substantially mitigate progression of the preinvasive lesions to cervical carcinoma since most women were within the low grade squamous intraepithelial ranges especially for the HIV positive. Provision of the currently available vaccines to these women at an early age would provide effective protection considering the presented findings ascertain the HPV type profiles in this population are covered by such vaccines.

What is known about this topic
 HPV genotypes has been reported to be highly prevalent and widely varied among HPV monoinfected and HIV/HPV co-infected women in the country and beyond;  Regional variations in the prevalence of HPV types exist in the country.  Tables and figure   Table 1: recruitment summary of the cases and controls from 410 via positive women Table 2: cervical cytological grades in HPV infected women