Epidermodysplasia verruciformis, and they are associated with the development of nonmelanoma skin cancer at sun-exposed skin sites in these individuals. Organ transplant recipients also have greater susceptibility to β-HPV infection of the skin and an increased risk of developing nonmelanoma skin cancer. In both immunosuppressed and immunocompromised individuals, cutaneous HPVs are ubiquitously disseminated throughout healthy skin and may be an intrinsic part of the commensal flora. Functional analysis of E6 and E7 proteins of specific cutaneous HPVs has provided a mechanistic comprehension of how these viruses may induce carcinogenesis. Nevertheless, additional research is crucial to better understand the pathological implications of the broad distribution of these HPVs.Cutaneous human papillomaviruses (HPVs) include β- and γ-HPVs, in addition to a small fraction of α-HPVs. β-HPVs were first isolated from patients with the rare genetic disorder  L1 major capsid gene sequence, the majority of the over 200 viral types characterized to date cluster within the alpha (α)-, beta (β)-, or gamma (γ)-HPV genus http://www.nordicehealth.se/hpvcenter/reference_clones/), and it is expected that these numbers will further increase once partial sequences of putative novel types are described Human papillomavirus (HPV) represents a diverse group of viruses infecting mainly epithelial and mucosal tissues Epidermodysplasia verruciformis (EV) that is characterized by extensive warts throughout the body  were the most extensively investigated. OTRs slightly resemble EV patients in that they are often covered with wart-like skin lesions and actinic keratosis (AK). Furthermore, OTRs have up to a 100-fold increased risk of developing NMSC compared to the general population Among OTRs, cutaneous warts were detected in 43% of individuals at 3 months to 9 years following transplant The incidence of NMSC is also related to long sun exposure. Boyle et al. Although cumulative sun exposure is the major risk factor for NMSC, recent studies have revealed a role for HPV as a cofactor in association with UV radiation in cSCC in OTRs. OTRs have a higher cutaneous HPV prevalence rate in cSCC (up to 90%) than in normal skin 11-32%) % 32. MorAmong immunosuppressed OTRs, the occurrence of multiple cutaneous HPV infections is common Nevertheless, given the high incidence of cSCCs in OTRs, identifying a clear link between β-HPV infection and cSCC would have important implications for therapy and prevention Cutaneous β- and γ-HPV DNA can be detected beginning in early infancy and may be detected in 70% of children by 4 years of age β-HPVs are widespread in immunocompetent (IC) individuals within the general population: when plucked hairs from different body sites are tested, the prevalence is approximately 90% p<0.01) and Bangladesh (p<0.05) Advanced age has been shown to be the most important factor influencing the presence of β-HPV DNA in IC individuals β-HPV persistence was more commonly observed in adults (92%) than in children (66%), and although multiple β-HPVs can infect persistently, no specific type seems to predominate in such infections The IARC recognizes the need for further research on cutaneous HPVs to better understand the widespread distribution of these viruses. β-HPVs may also play a role in the pathogeny of NMSC in healthy individuals vs 54% among IC), SCC (84% vs 27%) and basal cell carcinoma  Regarding the HPV status in NMSCs from immunosuppressed and IC individuals, viral prevalence was higher in the former group for all lesion types analyzed: premalignant lesions  cohort study We further observed that male external genital lesions (EGLs) are not associated with β-HPV infections HPVs are small, nonenveloped viruses with circular double-stranded DNA of approximately 8000 bp. The viral genome is divided into three regions: the long control region (LCR) contains cis-responsive elements for viral and cellular proteins that regulate viral gene expression and replication; the early region (E), which encodes proteins crucial to viral transcription and replication; and the late region (L), which encodes the viral capsid structural proteins The HPV life cycle is tightly associated with the differentiation of the stratified squamous epithelium. HPV infection begins with entrance of the virus into the basal layer of the epithelia due to microtrauma As previously discussed in this review, β-HPVs most likely play a role in the initiation of cSCC rather than in the maintenance of the transformed phenotype in vitro to E6-AP (E6-associated protein), p53 degradation was observed in the presence of only HPV 49 Studies have shown that β-HPVs 38 and 49 are able to immortalize primary human keratinocytes, whereas HPVs 10, 14, 22, 23, 24 and 36 do not have this ability in vitro to pRb but are unable to induce pRb degradation when expressed in human keratinocytes The E7 proteins from the cutaneous HPVs 154, 22, 23, 24, 36, 38 and 49 bind Although most research on oncogenic potential and disease association has focused on α-HPVs, there is interest in identifying a role of non-α HPV types in the pathogenesis of benign and malignant lesions . ChallenNunes EM, Talpe-Nunes V and Sichero L critically discussed and wrote the manuscript.


About 23% of patients develop CIN2+ after LEEP treatment due to residual or recurrent lesions. The majority of patients with HPV infection were HPV negative before treatment, but 16,4% were still HPV 16 positive after treatment, indicating that conization do not necessarily clear HPV infection rapidly. The aim of this retrospective study was to evaluate the possible correlation existing between the appearance of recurring high-grade lesions and the viral genotype 16, and other risk factors such as residual disease.One hundred eighty-two HPV positive patients underwent LEEP for CIN2+. The follow-up post treatment was carried out every 6 months. Abnormal results during follow-up were confirmed histologically and considered recurrent high-grade intraepithelial cervical lesions (CIN2/CIN3 or CIS).P < 0.5) with the Easy Fischer Test. We calculated the Odds Ratio (OR) of women with peristent HPV 16 infection and positive margin, to have a recurrence.Statistical analysis was performed by using the SPSS software package for Windows . Descriptive statistics are expressed as frequency, arithmetic mean, standard deviation (S.D.) and percentages. We calculated significance  with 94% (17/18) of the recurring disease occurring within 18 months of follow up. From this study it was found that the persistence of genotype 16 is associated with a greater rate of relapse post-conization of CIN 2+ lesions, with respect to other genotypes. Our study further supports those studies that demonstrate that the risk for residual disease or relapse is not to be overlooked, also when the margins are negative, but persistent HPV infection is present. In our case study, 40% of relapses were in women with negative margin, but with persistent HPV 16 infection. Even more so, the margins involved in HPV16 positive subjects is another prediction factor for relapse.Our results show the importance of genotyping and that persistent HPV 16 infection should be considered a risk factor for the development of residual/recurrent CIN 2/3. The treatment of Cervical Intraepithelial Neoplasia grade 2+ (CIN2+) (CIN3-CIS) consists in a conservative surgical approach using loop electrosurgical excision procedure (LEEP) that guarantees surgical radicality and preserves functional integrity of the uterine cervix, considering that young women are the most affected by these pathologies. The treatment for CIN 2/3 or carcinoma in situ (CIS) with LEEP is efficacious and most patients require no further treatment. However, about 23% of patients develop CIN2+ after conservative treatment due to residual or recurrent lesions .It has been seen that the women who do not eliminate the virus have greater recurrence rates. Furthermore, in the ASC-US LSIL triage study   and in tBased on these data, in this study we wanted to investigate if the HPV 16 genotype could be a risk factor with respect to high-grade lesion recurrence after excisional treatment with LEEP and the management of the residual disease in cases of cone with positive margin.The aim of this retrospective study was to evaluate the possible correlation existing between the appearance of recurring high-grade lesions and the viral genotype 16, and other risk factors such as residual disease.The study protocol was approved by the Institutional Review Board of the Department and was conducted. in accordance with the 1975 Declaration of Helsinki.Patients with histological diagnosis of CIN2+;Patients who had an HPV test before and after treatment;Patients who had no anti-HPV vaccination;Patients without pathologies of the immune system;Patients who had completed at least two-years of follow-up.We studied the clinical files of 230 patients who, from April 2015 to April 2017, underwent LEEP for CIN2+ at the Colposcopy Outpatient Service of the Gynaecological/Obstetrics Unit at the Policlinico Universitario, Catania  and who satisfied the following inclusion criteria:Moreover, patients were excluded if they were HPV negative or had any suspicion of infiltrating neoplastic pathologie.Only 192 patients satisfied the inclusion criteria; their clinical data was collected, of which: patient’s age, type of pathology, HPV strain, treatment method used, resection margins, follow-up cervical histology, date of follow-up, pretreatment viral genotype, post-treatment HPV genotype, and recurrent cases.All the patients underwent conservative surgical treatment of cone excision (conization) electrosurgery with conization (LEEP) of 20 mm width and 12, 15 or 20 mm depth .The histological examination of the cervical cone established a definitive histological diagnosis and evaluated the extension of the cone margins, defined as positive if the distance between the CIN lesion and the margin of the resection was less than 1 mm.The follow-up was carried out every 6 months during the first year after treatment and then once to year  and the successive follow-ups at 6 (T1), 12 (T2), 18 (T3) and 24 (T4) and 30 (T5) months). Abnormal results during follow-up were confirmed histologically and considered recurrent high-grade intraepithelial cervical lesions (CIN2/CIN3 or CIS).At each follow-up examination the patients were examined by conventional Pap test, HPV test and colposcopy, moreover, cytological eso-endocervical samples were taken and placed in ThinPrep Solution. The samples were sent to the laboratory for extraction of total DNA for the genotyping of viral DNA by means of genetic amplification followed by hybridization with genotype-specific probes able to identify most HPV genotypes of the genital region .The commercial method used was NucliSenseasy MAG system bio Merieux SA, Marct l’Etoile, France.The HPV genotype was classified as follows: 1) negative, 2) HPV 16, 3) hr HPV .Colposcopy was carried out using a Zeiss OPM1F colposcope  and applying acetic acid and a Lugol iodine solution. Any colposcopic abnormality was classified based on the nomenclature proposed by the International Federation for Colposcopy and Cervical Pathologies (IFCPC) in 3 grades of increasing anomalies based on severity: Zone of Abnormal Transformation (AnTZ) grade 1 (AnTZ1) and grade 2 (AnTZ2), or cancer. We evaluated the visibility of the squamo-columnar junction and specific biopsies were taken from the portio and/or endocervical curettage to guide the diagnosis in cases of an abnormal Pap test or suspicion of residual/recurrent disease at colposcopy.Statistical analysis was performed by using the SPSS software package for Windows . Descriptive statistics are expressed as frequency, arithmetic mean, standard deviation (S.D.) and percentages.P < 0.5) with the Easy Fischer Test.We calculated significance  of women with peristent HPV 16 infection and positive margin, to have a recurrence.The mean age of the 192 patients was 39.3 ± 8.7 years (range 22 to 73 years). During the follow-up, 18 (9.8%) of the 182 patients relapsed. The average time between LEEP and the diagnosis of recurrent disease was 18 months (range 6 to 30 months).The 192 patients had CIN3-CIS, as determined at biopsy or histological examination of the cone. In total, 182 (94.7%) of the 192 patients were positive for the HPV test before surgery, 10 patients that were negative at the HPV test excluded by the study.Of the 182 patients with a positive HPV test, 104 (57.1%) belonged to genotype 16, 78 (42.8%) to other genotypes. (Table p < 0.001), 29 cases (15.9%) HPV 16 showed virus persistence, only 9 case of virus persistence was found in the hr HPV group (Table Of the 182 patients positive at the HPV test at T0 (before LEEP), 144 (79.1%) were negative at 6 months (T1) (A negative margin was found in 162/182 (89%) patients, while 20/182 (10.9%) patients had a positive margin. Nine patients with genotype 16 had positive margin, while in the hr HPV group, only 6 patients had positive margin.From the Table The women with CIN2+ with residual or recurring lesions are at 5 times greater risk of cancer with respect to the general population . VariousOn the other hand, the viral clearance at follow-up after conization is significatively associated with efficacy of the surgical treatment, as confirmed by Cricca et al. . Authorsp < .05) of relapse post-conization of CIN 2+ lesions, respect to other genotypes with OR = 11.33 (CL95% = 1.25–102.93).From this study it was found that the persistence of genotype 16 is associated with a greater rate (17/18) (94.4%) .Viral clearance at follow-up can also occur in cases of positive margins , only thMoreover, in our study, no cases of relapse were found in women who were negative at the HPV test at 6 months, independently from the type of involvement of the margin, indicating a negative predictive value of 100% for relapse during follow-up .Our results show the importance of genotyping  and thatFurthermore, some researchers believe that when the DNA of post-treatment HPV is absent for 3 to 6 months after conization, especially in patients with a negative margin of the cone, the patients can go back to general-population screening .Furthermore, the possibility of re-infection after treatment should be considered. Infact, Jung Mi Byun et al.  during fThe prevalence of the diverse genotypes of HPV seen in our cohort is similar to other Italian and European populations . HPV 16 In the absence of viral infection, the risk of relapse is minimal. On the other hand, the cone with negative margins in the presence of persistent HPV 16 infection has a high incidence of relapse and thus persistent infection with HPV 16 should be considered a risk factor for the development of CIN2+ relapse.HPV vaccination for HPV 16 type may be useful in preventing recurrence of CIN 2/3 and CIS.There are some limitations in our study that include the small sample size, the retrospective design and the limited long-term follow-up. However, follow-up is still underway.


Oropharyngeal cancers associated with high-risk type human papillomavirus (HR-HPV) infection have better prognosis than virus negative cancers. Similarly, the HPV status in laryngeal cancer (LC) may be associated with better outcome.Samples from 88 patients with LC were investigated using the polymerase chain reaction (PCR) and p16 immunohistochemistry for HR-HPV analysis. The cut-off point for p16 overexpression was diffuse (≥75%) tumor expression with at least moderate (+ 2/3) staining intensity.n = 5, 5.7%; CS = 100%), HR-HPV DNA-positive/p16 overexpression-negative , HR-HPV DNA-negative/p16 overexpression-positive (n = 0), and HR-HPV DNA-negative/p16 overexpression-negative . HR-HPV DNA-positive/p16-positive cases tended to have integrated HPV infection and high viral load, compared with HR-HPV DNA-positive/p16 overexpression-negative cases.The 5-year cumulative survival (CS) rate was 80.7% in all patients with LC. According to a combination of HR-HPV DNA status and p16 overexpression, subjects with LC were divided into four groups: HR-HPV DNA-positive/p16 overexpression-positive (LC patients with HPV infection and high levels of p16 expression might have an improved survival outcome; however, it is necessary to recruit additional LC cases with HPV infection to determine the definitive characteristics of HPV-mediated LC and estimate survival outcome. These results may contribute to the development of a useful method for selecting patients with a potentially fair response to treatment and ensure laryngeal preservation. High-risk human papillomavirus (HR-HPV) infection has become an increasingly common cause of oropharyngeal squamous cell carcinoma (OPSCC) , 2. HPV-We previously demonstrated that both HPV DNA-positive status and p16 overexpression are useful for detecting transcriptionally active HPV infection in head and neck cancer . HPV infThe aim of this study was to clarify the rates and clinical characteristics of LC with transcriptionally active HPV infection diagnosed by HPV-DNA testing and p16 immunohistochemistry.18F-fluorodeoxyglucose-positron emission tomography CT imaging.This study involved 88 treatment-naïve LC patients without distant metastasis. All patients were diagnosed with laryngeal squamous cell carcinoma by pathological examination of samples and were treated at the Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, between January 2008 and December 2017. The final prognosis of patients was judged in July 2018. Classification of the tumor, node, metastasis (TNM) stage was carried out according to the AJCC Staging Manual (7th edition). To determine the clinical stage and to detect concomitant multiple primary cancers, the patients underwent physical and endoscopic examinations of the upper gastrointestinal tract, ultrasonic inspection of the neck, computed tomography (CT), and This study was approved by the Institutional Review Board of the University of the Ryukyus and was carried out in accordance with the 1975 Declaration of Helsinki, as revised in 2008. Informed consent was obtained from all LC patients before enrollment.The principal treatment for the primary lesion with curative intent was conventional radiotherapy (RT) alone or laser resection in T1, concurrent chemoradiotherapy (CCRT) in T2, CCRT or surgery in T3, and surgery in T4, regardless of the presence of HPV. Nodal lesions were managed with CCRT or neck dissection combined with resection of the primary lesion. All patients who received RT  had CT-assisted three-dimensional radiation treatment planning in the treatment position with mask immobilization. The protocol for CCRT was as reported previously . When thAll tissue samples from primary lesions without any radiation or chemotherapy were analyzed with the polymerase chain reaction (PCR) for HPV DNA detection using fresh frozen samples, which were obtained at biopsy or surgical resection, and p16 immunohistochemistry using FFPE samples. Cases with negative HPV detection results on PCR and p16 overexpression (≥75% positive cells and at least moderate staining intensity) were further evaluated for HPV status using in situ hybridization (ISH) with HPV DNA probes (DNA ISH) , 21.In brief, DNA was isolated from the tumor samples using a Gentra Puregene Tissue Kit . The presence and integrity of DNA were verified in all samples by PCR β-globin gene amplification using the primers PC04 and GH20 . The genImmunohistochemistry for p16 was performed using a CINTec® p16 Histology Kit  according to the manufacturer’s protocol . ImmunolThe scoring criteria for p16 immunoreactivity used in the present study were as follows: 0, no staining; 1, 1 – < 25% of the tumor cells positive for p16; 2, 25 – < 50% positive; 3, 50 – < 75% positive; 4, ≥75% positive and weak staining intensity; and 5, ≥75% positive and at least moderate staining intensity. The term “p16 overexpression” (p16-positive) was defined as a score of 5 in the present study.Biotinyl tyramide-based ISH was performed using the GenPoint™ HPV biotinylated DNA probe and the GenPoint tyramide signal amplification system for biotinylated probes according to the manufacturer’s protocol . The GenPoint HPV biotinylated DNA probe reacts with HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 in 4-μm-thick sections of FFPE samples. Detection of the hybridized probe was performed using the GenPoint detection system according to the manufacturer’s protocol, with the included primary streptavidin-horseradish peroxidase (HRP), biotinyl tyramide, secondary streptavidin-HRP, and 3,3′-diaminobenzidine l . The slides were counterstained with hematoxylin.E2 and E6 open reading frames were used  of the pB-actin early plasmid, which was a gift from Karl Munger, carrying the complete HPV-16 early gene region . Viral DNA load was assessed by calculating E6 copy number. An external standard curve was created using known serial dilutions  of human genomic placental DNA  for cellular DNA quantification, and β-globin was amplified as described previously. The amount of DNA was calculated by plotting the Cq values against the logarithm of the standard curve. The physical status of HPV-16 was assessed based on a previously published method [E2/E6 ratio ≥ 1 indicates the predominance of the episomal form, whereas a ratio of E2 copy number/total E6 < 1 indicates the presence of both integrated and episomal forms (mixed form).To evaluate the viral load and physical status of HPV-16, qPCR was performed as described previously , 20. Brid method , 21. An E6 mRNA expression was detected in samples from patients identified as HPV-16-positive with p16 overexpression. Total RNA was extracted from frozen tissues using RNAiso Plus  according to the manufacturer’s instructions. Total RNA (500 ng) was used to synthesize first strand cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser  according to the manufacturer’s instructions. To measure HPV-16 E6 mRNA expression, qPCR was performed using the CFX96 Touch™ Real-Time PCR Detection System  and TaqMan PCR Master Mix II . Primers and TaqMan probes  of the pB-actin early plasmid and the pCAG-mGFP-actin plasmid, which was a gift from Ryohei Yasuda, carrying the complete coding region of β-actin . The expression of the E6 gene in each sample was normalized by the amount of the internal control β-actin.ere used , 20. TheE6/E7 mRNA was performed using an RNAscope® 2.5 HD Reagent Kit  according to the manufacturer’s instructions. In brief, serial 4-μm thick sections of FFPE samples were deparaffinized in xylene and rehydrated using a graded alcohol series. Endogenous peroxidase was blocked with the RNAscope® Hydrogen Peroxide Reagent at room temperature for 10 min. Target HPV RNA retrieval was performed in RNAscope® Target Retrieval Reagent at 100 °C for 15 min. The slides were digested with RNAscope® Protease Plus at 40 °C for 30 min. The HPV-16/− 18 E6/E7 RNA probe (RNAscope® Probe-HPV16/18) was added to the sections and a coverslip was applied. The slides were transferred to a humidified chamber for hybridization at 40 °C for 2 h. Then, the coverslips were removed and the slides were washed with RNAscope® Wash Buffer Reagent at 40 °C. Signal amplification was performed according to the manufacturer’s instructions. Detection of the hybridized probe was performed using 3,3′-diaminobenzidine (RNAscope® 2.5 HD Reagent Kit). The slides were counterstained with hematoxylin. Positive control slides (HeLa cells expressing HPV-18 E6/E7 mRNA) were included in RNA ISH. Positive staining was identified as brown punctate dots seen in the nucleus and/or cytoplasm.RNA ISH for HPV-16/− 18 P < 0.05 was considered significant.Pearson’s chi-square test was used to compare the characteristics of LC patients according to HPV DNA and p16 overexpression status. Cumulative survival (CS) was calculated using the Kaplan-Meier method and was compared between two groups using the log-rank test. All analyses were performed with SPSS Statistical Package . n = 5, 5.7%), HR-HPV-positive/p16-negative , HR-HPV-negative/p16-positive , and HR-HPV-negative/p16-negative . The clinical characteristics of the patients are summarized in Table Of the 88 patients with LC, there were 16 18.2%) HR-HPV-positive cases, 2 (2.3%) low-risk type HPV (HPV-6)-positive cases, and 5 (5.7%) p16-positive cases. All p16-positive cases harbored HR-HPV DNA. The patients were divided into four groups according to the presence of HR-HPV DNA and p16 overexpression as follows: HR-HPV-positive/p16-positive  was 58.4–7.7 × 104 copies/ng genomic DNA, while HPV-16-positive/p16-negative cases (4 cases) had 0.3–2.1 copies/ng genomic DNA , 1 with a score of 3 (50 ≤ p16-positive < 75), and 1 with a score of 1 (1 – < 25% of the tumor cells positive for p16). Thus, all 5 patients with a score of 5 harbored HPV DNA, while 2 cases with < 75% p16 expression had no HR-HPV infection.A 41-year-old man, with a smoking history of 40 pack years and alcohol drinking of 20 g/day, presented to a referral hospital with a 1-year history of hoarseness. Physical examination revealed a transglottic tumor located mainly in the supraglottic region; CT showed a transglottic mass with invasion of the thyroid cartilage and multiple swollen lymph nodes in the neck bilaterally  and 1 (level II) in the right side of the neck. CCRT after surgery was carried out with 50.4 Gy irradiation to the entire neck, and 2 cycles of nedaplatin plus 5-fluorouracil as adjuvant therapy . As of 7The HPV viral load and physical status of this patient are shown as Case 1 in Table E2/E6 ratio was 0.11; thus, the HPV-16 integration status was judged to be mixed type. E6 mRNA expression was also detected with RT-PCR as well as RNA ISH (Fig. PCR for HPV DNA revealed the presence of HPV-16 infection Fig. f. qPCR sStrong p16 expression was observed in metastatic lymph nodes Fig. h as wellThere have been contradictory reports regarding the presence of HPV and p16 expression in LC , 14, 23.In this study, HPV DNA was detected only by ISH DNA, and not PCR, in 2 cases. PCR-based analysis is usually more effective for HPV detection and is cheaper than DNA ISH. There are several reasons for this outcome. One explanation is that breakpoints or deletions may have occurred in L1 at the time of HPV integration and HPV DNA only existed in the integrated form in the tumor. Another explanation is that it is difficult to obtain a sufficient amount of specimen for analysis before treatment in LC, because laryngeal cancer lesions are sometimes very small. Indeed, the clinical T stages in 2 cases were T1a and T1b, respectively Table . In diagA systematic review of 1668 LC patients  showed aBecause the rate of HPV-mediated LC is much smaller than that of OPSCC, and LC cases with T1 and T2 usually have a fair treatment response and survival outcome, analysis of HPV infection in LC may be clinically relevant in patients with advanced disease who desire laryngeal preservation. In our series, CS in HPV-mediated LC was better than in other LC cases, but there was no significant difference in CS between HPV-mediated LC and non-HPV LC, likely because the number of HPV-mediated LC cases was limited. Although several studies refute the utility of HPV testing in LC, further study is needed to clarify survival outcome with the combination of HPV DNA detection and p16 overexpression. In previous studies in OPSCC, 3–4% of cases showed p16 overexpression without HPV infection and had a worse treatment response than cases with HPV-mediated OPSCC , 28. TheAlthough the number of HPV-mediated LC cases is much smaller than that of OPSCC, several cases had carcinogenesis based on HPV infection. Survival outcome might be better for cases with HPV-mediated OPSCC; thus, the recruitment of large numbers of HPV-mediated LC cases is needed to outline definitive criteria for HPV-mediated LC. These results may contribute to developing a useful method for selecting patients with the potential for a fair response to treatment and to facilitate laryngeal preservation.


We conducted in 2018 a descriptive, quantitative, population-based, cross-sectional survey estimating the prevalence of cervical high-risk human papillomavirus (HR-HPV) infection and associated risk factors among adult women living in N’Djamena, Chad.La Renaissance Plus”, N’Djamena. Medical, socio-demographical and behavioral informations were collected. HPV DNA was detected and genotyped in endocervical swab using Anyplex II HPV28 genotyping test .Five of the 10 districts of N’Djamena were randomly selected for inclusion. Peer educators contacted adult women in community-churches or women association networks to participate in the survey and come to the clinic for women’s sexual health “253 women  including 3.5% of HIV-positive women were prospectively enrolled. The prevalence of HPV infection was 22.9%, including 68.9% of HR-HPV infection and 27.6% being infected with multiple genotypes, providing a total HR-HPV prevalence of 15.8% (95% CI%: 11.3–20.3). The most prevalent HR-HPV genotypes were HPV-58, HPV-35, HPV-56, HPV-31, HPV-16, HPV-45, HPV-52 and HPV-18. HPV types targeted by the prophylactic Gardasil-9 vaccine were detected in nearly 70% (67.5%) and HPV-58 was the most frequently detected. HIV infection was a risk factor strongly associated with cervical infection with any HPV [adjusted Odds ratio (aOR): 17.4], multiple types of HPV (aOR: 8.9), HR-HPV (aOR: 13.2) and cervical infection with multiple HR-HPV (aOR: 8.4).These observations highlight the unsuspected high burden of cervical HR-HPV infection in Chadian women, and point the potential risk of further development of HPV-associated cervical precancerous and neoplastic lesions in a large proportion of women in Chad. The high rate of preventable Gardasil-9 vaccine genotypes constitutes the rationale for introducing primary vaccine prevention against cervical cancer in young female adolescents living in Chad. Human papillomavirus (HPV) infection is the most common viral sexually transmitted infection (STI) worldwide and high-risk (HR)-HPV genotypes are responsible for 5.2% of all cancers worldwide, 2.2% of cancers in developed countries and 7.7% of all cancers in developing countries –3. In suAlthough all HR-HPV genotypes have oncogenic potency, some of them are more frequently involved in cancers than others. These include HPV-16 and HPV-18, accounting for about 70% of all cervical cancers worldwide, and HPV-31, HPV-33, HPV-45, HPV-52, HPV-58 and HPV-68 which are among the ten most frequently isolated HR-HPV genotypes from cervical tumor biopsies , 10. TheBefore implementing vaccination with Gardasil-9 vaccine in African countries, it is essential to establish the epidemiological distribution of the main circulating genotypes in the general population. Indeed, these indications are critical because they allow evaluating the preventive efficiency of vaccination against HPV with the current HPV vaccine in specific populations. A wide heterogeneity in the distribution of circulating HR-HPV genotypes exists from one country to another and also between two regions within the same country throughout the African continent . GeneralIn Chad, a country of around 15 million people, including more than 3 million women aged more than 25 year-old , 23, no The study was a descriptive, quantitative, population-based, cross-sectional survey, using a face-to-face questionnaire to collect data in 2018 among adult women living in N’Djamena, Chad.dx.doi.org/10.17504/protocols.io.wgefbte.The laboratory protocol of this study was been deposited in protocols.io website and is available at: La Renaissance Plus”, N’Djamena, which is one of the main settings for women’ sexual health in Chad, and to participate in the study. Adult women living in N’Djamena regularly attend the clinic “La Renaissance Plus” for gynecological examinations and for obstetrical services.The capital city N’Djamena comprises 10 districts, which include a variable number of neighborhoods. Twenty-three sites in neighborhoods of 5 out of the 10 districts randomly selected were further chosen for study inclusion, as depicted in the The inclusion criteria were agreeing to participate in the study, having given signed informed consent, being aged between 25–65 years, being sexually active, having no genital troubles at physical examination and having completed the questionnaire. Exclusion criteria included age less than 25 year-old and more than 65 year-old and not willing to participate to the study or to answer the face-to-face questionnaire to collect data.After signed the informed consent form, the selected women benefited from free HIV and hepatitis B (HBV) and C (HCV) testing, by multiplex HIV/HCV/HBsAg immunochromatographic rapid test  , clinicaLa Renaissance Plus”, by experienced counsellors using a face-to-face questionnaire to collect socio-demographic characteristics and behavioral data, including age, marital status, social occupation, education level, residence location in N’Djamena, history of STI, HIV status and also sexual behavioral characteristics such as the number of lifetime sexual partners and the age at first sexual intercourse.At inclusion, a standardized interview was conducted at the clinic “hôpital Européen Georges Pompidou, Paris, France, for molecular analyses.After completing the socio-demographic data collection questionnaire, a nurse performed cervicovaginal sampling using a flocked swab . Briefly, specimens for molecular testing were obtained by inserting the swab into the vaginal canal until the cervix mucosa, gently rotating 5 times and then removed and immediately placed into its container and frozen at -80°C before DNA extraction procedure. Finally, cervicovaginal swab were transported in frozen ice packs, to the virology laboratory of the DNA was extracted from the cervical swab specimen using the DNeasyBlood and Tissue kit  as recommended by the manufacturer. After extraction, DNA was concentrated and eluted in 100 to 200μL of the kit elution buffer before genotyping.Comité Français d’Accréditation (COFRAC) according to the ISO 15189 Norma for the biological markers "HPV detection" and "HPV genotyping".The detection and the genotyping of HPV were carried out using the real-time PCR assay Anyplex II HPV28 , . AccordiP) were calculated using Pearson’s χ2 test or Fisher’s exact test for categorical variables and the non-parametric Mann-Whitney U-test for quantitative variables. Logistic regression models using univariate and multivariate analyses were performed to determine the association of each independent variable  with the HPV type-specific cervical infections . All variables statistically significant (P < 0.05) in univariate analysis were computed into a multivariate logistic regression analysis. Crude Odds ratio (cOR) and adjusted Odds ratio (aOR) were calculated, as appropriate along with their 95% CI. Finally, a risk factor was defined as an independent variable giving in univariate analysis a cOR  strictly higher than “1” with a P-value lower than 0.05. An aOR  strictly higher than “1” with a P-value lower than 0.05 defines a risk factor in multivariate analysis. A protective factor was defined as an independent variable for which the cOR  is strictly lower than “1” with a P-value lower than 0.05 in univariate analysis. An aOR  strictly lower than “1” with a P-value lower than 0.05 characterizes a protective factor in multivariate analysis .From June to August 2018, 260 women from the 23 inclusion sites participated to the study [1La Renaissance Plus” were consecutively and prospectively included in the study and their socio-demographic, sexual behavior, clinical and biological characteristics are summarized in the After physical examination, 7 women were excluded because of genital troubles . Finally, a total of 253 women  referred to the clinic “Using multiplex HIV/HCV/HBsAg rapid test, 9 study women  were diagnosed infected by HIV-1, 19  by HBV (positivity for HBsAg) and 8  were seropositive for HCV.Several women  were young, aged from 25 to 29 years, engaged in life couple with a male partner , with a relatively high education level ; but most of them were unemployed . The majority of study women  reported having only one regular sexual partner in their life, while about 10% reported to have up to 5 different sexual partners. Generally, women included in this study became sexually active at 16 to 20 years of age , whereas some of them  started their sexual life earlier, before 16 years. Finally, none of the women included in this study had ever been screened for cervical cancer and nor vaccinated against HPV infection.Overall, 58 adult women were positive for genital HPV DNA giving a total HPV prevalence of 22.9% (95% CI: 17.7–28.1). Of those who were HPV DNA positive, 68.9%  harbored cervical HR-HPV infection, providing an overall HR-HPV prevalence of 15.8% (95% CI: 11.3–20.3), as shown in the The Gardasil-9 vaccine HR-HPV type 58 was the predominant genotype , followed by the HR-HPV types 31, 35 and 56 and the LR-HPV types 42 and 44 with a prevalence of 10.3% (6/58). The 9-valent vaccine HR-HPV types 16, 45 and 52 and also the LR-HPV types 53 and 70 were present with a prevalence of 8.6% (5/58). These HPV genotypes were followed by the HR-HPV types 18, 51, 59 and 68, the LR-HPV types 6 and 54 and the probably oncogenic HPV types 73 and 82 with a prevalence of 6.9% (4/58). The HR-HPV-39 was present only in 3 women (5.2%) and a minority of study women were infected with LR-HPV types 40 and 43  and HR-HPV-33 . Finally, none of the HPV positive samples was simultaneously positive for HPV-16 and HPV-18 .Concerning the distribution of cervical HPV type-specific prevalence according to age groups, there was no significant difference between each of the 4 age groups. Even so, women aged from 25 to 29 years were most infected with any HPV types and LR-HPV types than both the 3 others age groups . LikewisP = 0.00006). High-risk types was similarly distributed with higher rates (36.2%) in women who started sexual intercourse between 16 to 20 years of age compared to women who had their sexual debut below 16 years (20.7%) and those who began after 20 years of age (12.1%), (P = 0.007).The distribution of cervical HPV type-specific prevalence according to the age of sexual intercourse onset is depicted in the versus 20.5%; 95% CI: 15.4–25.6; P = 0.0005), HR-HPV types , multiple infections with any type of HPV  and multiple HR-HPV types  (The impact of HIV in HPV type-specific infection in the study was assessed. Seven out of the 9 HIV-infected women (77.8%) were positive for cervical HPV DNA and 85.7% (6/7) of these infections were caused by HR-HPV genotypes, with the predominant genotype being HPV-56 . Moreover, when compared to HIV-negative, HIV-positive women were significantly more infected with any type of HPV  .The associations between each one of the type-specific HPV infections with their potential predictive risk factors were assessed by logistic regression analysis, as shown in the P = 0.001), multiple types of HPV , HR-HPV  and cervical infection with multiple HR-HPV . Likewise, the variable “being student” was statistically associated with the cervical carriage of any type of HPV  and HR-HPV . Another statistical association seems to emerge between the variables "being 50 years of age or older" and getting infected with any HPV . “Being 50 years of age or older” seems to have a slightly protective effect against cervical infection with any HPV, but the statistical power of this protective effect was canceled.In univariate analysis, the variable “HIV infection” was significantly associated with cervical infection with any type of HPV , multiple types of HPV , HR-HPV  and cervical infection with multiple different HR-HPV . Finally, the other explicative variables taken into account in this analysis, such as “age at inclusion”, “marital status”, “education level”, “number of lifetime sexual partners” and “age at first intercourse” were found to be not significantly associated with any of the four outcomes variables characterizing cervical HPV infections in the study women.The variable “HIV infection” and the three subclasses of the variable “Occupation”  were computed into the final multivariate analysis. Only the variables “HIV infection” succeeded to maintain its statistical link with the HPV outcomes variables. Indeed, HIV infection was strongly associated with cervical infection with any HPV  of the HPV-positive cervical samples contained one of the 4 genotypes  covered by the Gardasil-4 vaccine and less over (1.7%) contained simultaneously several of these 4-valent vaccine-genotypes. Regarding the Gardasil-9 vaccine, 50% (29/58) of HPV-positive cervical samples harbored at least 1 HPV type prevented by the 9-valent HPV vaccine, with 17.8% (5/28) of them which contained multiple HPV genotypes . Moreovea priori good predictive efficacy of the prophylactic Gardasil-9 vaccine could be envisioned for the primary prevention of cervical HPV infection in Chad. Thus, since HPV DNA molecular screening in Chad remains currently opportunistic or unavailable for many women, prophylactic HPV vaccination using a multivalent vaccine may help in achieving cervical cancer elimination.The present study depicts for the first time the molecular epidemiology of cervical HPV infection in adult women living in N’Djamena, the capital city of Chad. One of five (22.9%) of study women had cervical HPV, which was mostly constituted of cervical HR-HPV (68.9%), 27.6% of HPV positive women harboring multiple genotypes. In this series, HIV seropositivity (3.5%) constituted the main risk factor significantly associated with an increasing risk of being infected by cervical HPV. The majority (≈70%) of women infected by cervical HR-HPV showed high-risk genotypes covered by the 9-valent Gardasil-9 vaccine, with the HR-HPV-58 being the predominant genotype, followed by HPV-31, HPV-16, HPV-45, HPV-52 and HPV-18. Remarkably, most of these high-risk genotypes  were not covered by the Gardasil-4 vaccine. Taken together, these observations highlight the unsuspected high burden of cervical HR-HPV infection in women more than 25 years living in Chad, with a high rate of preventable Gardasil-9 vaccine genotypes. These molecular findings demonstrate that cervical HR-HPV infection and associated risk for cervical cancer involve a large proportion of adult women living in Chad. According to our findings, with the hypothesis of a rate of genital shedding of HR-HPV at 15.8%, it may be estimated that at least 460,000 Chadian women aged more than 25 years thorough the country may be at risk for cervical cancer during their life. Finally, an “La Renaissance Plus” of N’Djamena, we observed a high frequency (68.9%) of cervical HR-HPV genotypes in HPV DNA positive women. Such high prevalence of HR-HPV appears notably higher as compared to those commonly reported in unscreened African women aged from 25 years of age and older, with cervical HR-HPV infection rates never exceeding half of the HPV-positive women. Indeed, cervical HR-HPV prevalences in adult women in sub-Saharan Africa vary frequently across regions of the same country and also from one country to another, and vary between 5.4% in Djibouti [In this large series of never screened adult women attending the obstetrics and gynecology clinic Djibouti , 10.0% aDjibouti , 37, 12.Djibouti , 18.5% aDjibouti , 40, 19.Djibouti , 20.3% iDjibouti , 22.2% iDjibouti , 25.0% iDjibouti , 45, 39.Djibouti  and finaDjibouti . HoweverDjibouti , to 67.9Djibouti . Only stDjibouti –51 than Djibouti .Concerning HPV genotype distribution, women carrying HR-HPV infection showed remarkable high rates (≈70%) of high-risk types targeted by the Gardasil-9 vaccine, with HPV-58 being the most represented genotype. Moreover, five other high-risk types included in the 9-valent vaccine  belonged to the four most detected genotypes; HPV-16 and HPV-18 being respectively the third and the fourth most represented. In the other hand, two high-risk types were not covered by the Gardasil-9 vaccine, HPV-35 and HPV-56.a priori, prevented using the Gardasil-9 vaccine. In the context of the important population of females living in Chad, estimated in 2018 at around 7,600,000 [Most previous studies conducted in sub-Saharan African countries depict a wide heterogeneity in the distribution of the main HR-HPV in women , 38–48. ,600,000 , our epiIn the present series, two main groups characterized by their risk factors for HPV infection were identified. Firstly, HIV-infected women were found to be 17-times more at risk for cervical infection with any type of HPV and more than 8-times for multiple types of HPV than HIV-negative women. Regarding cervical HR-HPV infection, HIV-infected women were 13-times more at risk than HIV-negative women and more than 8-times for infection with multiple high-risk types. These findings are consistent with previous reports demonstrating the aggravating effect of HIV in the burden of HR-HPV infection in women and particularly in the African settings –55. SecoOur observations highlight that women living in Chad constitute a neglected high risk group for cervical HR-HPV infection and consequently for cervical cancer. These findings constitute the first report ever provided on the epidemiological burden of HPV infection in Chad. The very high prevalence of cervical HR-HPV in adult women clearly demonstrates that cervical HR-HPV infection in Chad constitutes a major public health problem which remains largely unsuspected. Therefore, there is an urgent need for implementing a cervical cancer prevention program in Chad, as recommended by the WHO . AccordiLa Renaissance Plus” of N’Djamena, although they were not part of the patients from the clinic, avoiding the obvious bias of recruitment by health care facilities. Furthermore, 9 (3.5%), 19 (7.5%) and 8 (3.2%) study participants were HIV-, HBsAg- and HCV-specific antibody- positive, respectively, in accordance with the high endemicity of these three major chronic viral infections in Chad [La Renaissance Plus " of N’Djamena, which is considered by the public as the benchmark of good practice in the area of obstetrics and gynecology. Finally, in regard of our small sample size some risk factors may have been underestimated in the statistical analyses. Despite these possible limitations, our study constitutes the first report providing objective information on molecular epidemiology of cervical HPV in symptom-free adult women living in N’Djamena, and thus enables us to show all the interest of the prophylactic vaccination by the Gardasil-9 vaccine in eligible young adolescent women, that could be very possibly generalized to the whole country.We tried to limit the selection bias of the study population in order to make this survey as much representative as possible of the female population in Chad. Thus, the study inclusions were initially carried out by random sampling of districts in N’Djamena, with further selection of neighborhoods inclusions site. Study women were referred to the clinic “ in Chad , 61. Ind in Chad , as obse in Chad . In addiIn conclusion, an unsuspected high prevalence of cervical HR-HPV infection, worsened by HIV, was observed in women more than 25 years living in N’Djamena, Chad. The study women recruitment according to a population-based approach allows to extrapolate our observations to the general female population, and to make the hypothesis that the burden of HR-HPV shedding may be particularly high in Chadian women, and consequently, the risk of further development of HPV-associated cervical precancerous and neoplastic lesions. In addition, the HPV genotypes distribution in genital secretions of study women indicates the probable existence of a particular molecular epidemiology of HPV in Chad, with rare genotypes being predominant such as HPV-58. Finally, our results demonstrate that the majority of cervical HR-HPV corresponds to preventable Gardasil-9 vaccine genotypes, making the rationale of introducing primary prevention against cervical cancer in young female adolescents living in Chad by prophylactic vaccination. Taken together, our findings point for the first time the unsuspected high exposure of Chadian women to oncogenic HR-HPV, making thus cervical cancer, its diagnosis and prevention, some of the most important public health challenges that Chad will faced in a near future.S1 FileMicrosoft excel baseline database containing all information from the study women analyzed in the study and related to their sociodemographic and clinical characteristics and also information associated to the results of the HPV testing and genotyping.(XLSX)Click here for additional data file.S2 File(DOC)Click here for additional data file.


Microbiota composition was characterized by 16S rRNA gene sequencing. HPV DNA was detected in 33% of placenta, 23% cervical and 33% oral samples. HPV16 was the most frequent type in all regions. HPV infection was associated with higher microbiota richness (p = 0.032) in the mouth but did not influence microbial diversity or richness in other samples. HPV infection was associated with higher abundance of Lactobacillaceae (p = 0.0036) and Ureaplasma  in the placenta, Haemophilus (p = 0.00058) and Peptostreptococcus (p = 0.0069) genus in the cervix and Selenomonas spp. (p = 0.0032) in the mouth compared to HPV negative samples. These data suggest altered bacterial microbiota composition in HPV positive placenta, cervix and mouth. Whether the changes in bacterial microbiota predispose or result from HPV remains to be determined in future studies.We investigated the association between HPV infection and bacterial microbiota composition in the placenta, uterine cervix and mouth in thirty-nine women. HPV DNA genotyping of 24 types was conducted using Multimetrix HPV has long been regarded mainly as a sexually transmitted disease (STD) but recent studies have shown that HPV infection can be acquired by vertical transmission as well as via the placenta from mother to child7. Traditionally, HPV infection in the placenta has been considered to increase the risk of pregnancy complications11 but HPV DNA has also been discovered from placentas from healthy pregnancies14 and transabdominally obtained placental samples16.Human papilloma virus (HPV) is a known oncovirus and it has potential to cause carcinoma in the genital, anal and oropharyngeal regionsProteobacteria18. Parnell et al.19 have reported that the microbiota composition varies depending on the location within the placenta and that the placental microbiota may change according to maternal factors such as gestational diabetes mellitus20. However, not all published data support the notion of intrauterine colonization22.In addition to viruses, humans have diverse bacterial microbiota which has been recognized to play an important role in human health. Recently published data suggest that even the placenta may harbor a unique microbiota which is mainly composed of 24. Individuals with HPV infection more frequently have higher diversity of cervico-vaginal microbiota and HPV clearance in turn has been associated with increased number of antigen-representing Langerhans cells25. A study by Gao et al.26 showed more L. gasseri and Gardnerella vaginalis in HPV infected women’s cervico-vaginal microbiota. HPV persistence is considered a critical event in malignant transformation. We have previously shown that bacterial vaginosis, even when asymptomatic, predicted HPV persistence23. Di Paola et al.27 propose that Atopobium spp. and G. vaginalis may serve as microbial markers for HPV persistence in Italian women while Adebamowo et al.28 showed that Mycoplasma hominis was associated with HPV persistence in Nigerian women. Furthermore, changes in the vaginal microbial composition, connected to lower levels of Lactobacillus spp., have been associated with more severe cervical intraepithelial neoplasia and higher diversity in microbiota might lead to more severe disease29.Healthy vaginal microbiota is known to be predominantly dominated by lactobacilli. Shifts in vaginal microbiota balance may result in altered composition referred to as polybacterial dysbiosis and to disease such as bacterial vaginosis, both of which have been connected to vaginal HPV infection31. Furthermore, there are differences in the microbiota between normal oral mucosa and patients with oral squamous cell carcinoma32. It has even been suggested that the oral microbiota may have an effect on developing pancreatic cancer, possibly mediated by P. gingivalis. P. gingivalis has also been linked to chronic inflammation and atherosclerosis34. Accumulating evidence thus suggests that both HPV and bacterial dysbiosis might play a significant role in malignant transformation. Nonetheless, our knowledge about the interactions between HPV infection and the bacterial microbiota and its impact on human health is still rudimentary.Recent research has revealed that malignancies may arise in various human organs in reaction to bacterial inflammation or by bacteria’s own direct oncogenic mutations to human cells. Bacteria have been linked with conditions including liver cancer, esophageal cancer, pancreatic cancer, gall bladder cancer and colorectal cancerWe aimed to investigate whether an existing HPV infection has influence on the bacterial microbiota composition in the placenta, the uterine cervix or the mouth. This could lead us to understand part of the mechanisms modifying HPV clearance and persistence leading to malignancy.The HPV genotypes of all the samples including sex of the infant, delivery mode and gestational age are presented in Table Firmicutes (58.3% vs. 66.6%) followed by Proteobacteria (21.1% vs. 15.7%), Actinobacteria (13.8% vs 10.0%) and Bacteroidetes (6.5% vs. 6.1%) in HPV negative and positive respectively , Enterococacceae (15.6% vs 13.8%), Veillonellaceae (8.8% vs 7.4%), Corynebacteriaceae (6.3% vs 0.8%) and Moraxellaceae (6.1% vs 0.1%) were the predominant groups in both HPV negative and positive samples, respectively. At genus level, Staphylococcus (22.9% vs 29.5%); unclassified Enterococcaceae genus (15.6% vs 13.7%), Corynebacterium (6.3% vs 0.1%), and Acinetobacter (6.0% vs 0.1%) were the most abundant in HPV negative and positive placenta samples.To study the impact of potential contaminants, pooled placental negative controls yielded 726 reads and the identified OTUs n = 11) representing 6 different genera listed in Supplementary Table 1 represeely Fig. . At famiLactobacillaceae (p = 0.0036) and Lactobacillus genus (p = 0.0023), were observed in HPV positive group compared to HPV negative placenta samples . We also performed detailed analyses of the Lactobacillus species associated with HPV positivity in the placenta samples. In the HPV positive group, we detected L. zeae, L. reuteri and Unclassified Lactobacillus. To further investigate the composition, we selected LEfSe analyses at OTU level to reveal the Lactobacillus group in HPV positive placenta samples to be combined by L. iners, L. crispatus, L. jensenii, L. gasseri and L. reuteri. In addition, in HPV negative group L.iners was evident. LEfSe analysis showed that Ureaplasma and Lactobacillus genus were significantly enriched in HPV positive placenta samples compared to HPV negative ,  were found between HPV positive (n = 6) and negative (n = 13) placenta groups Fig. . Howeverles Fig. . Interes5), Fig. .Staphylococcus (p = 0.012) and Lachnospira genus (p = 0.050) in LR-HPV genotype compared to those observed in HR-HPV genotype. Although not with significant statistical differences, a higher diversity and lower richness were observed in HR-HPV genotype compared to LR-HPV genotype. LEfSe analysis showed that Ureaplasma and Lactobacillus genus were significantly enriched in HR-HPV genotype placenta samples while Lachnospira genus was enriched in LR-HPV genotype.Despite the lower number of HPV positive samples, comparing HPV positive samples between low-risk HPV types  and high-risk HPV types  genotypes we observed higher abundance of Firmicutes (91.0% vs. 90.7%) and Actinobacteria (7.3% vs. 5.1%), followed by Fusobacteria (1.2% vs. 0.1%), Bacteroidetes (0.2% vs. 3.3%) and Tenericutes and Proteobacteria in HPV negative and positive samples, respectively. No statistically significant differences in species richness or diversity were detected between HPV positive and negative cervical samples . The HPV positive cervical samples mainly harboured L. crispatus and L. jensenii whereas in HPV negative cervix group L. iners and L. reuteri were more abundant. The relative abundance of Coriobacteriaceae (p = 0.083) was slightly increased in HPV positive cervical samples. On the other hand, Peptostreptococcaceae (p = 0.0065) and Enterococcaceae (p = 0.022) families were significantly increased in HPV negative compared to HPV positive cervical samples  genus as compared to HPV negative cervical samples whereas Haemophilus (p = 0.00058) and Peptostreptococcus (p = 0.0069) were significantly increased in HPV negative group at genus level. At genus level, Lactobacillus was predominant in both HPV groups; negative and positive (89.7% in HPV negative and 86.7% in HPV positive). At species level, L. iners, mainly the OTU 133075, was the predominant Lactobacillus group in both HPV negative and positive samples .On the family level, the most abundant groups belonged to les Fig. . The HPVCoriobacteriaceae genus was significantly enriched in HPV positive group compared to HPV negative , while Haemophilus (LDA score > 4.18) were enriched in HPV negative compared to HPV positive cervix , Proteobacteria (24.6% vs. 18.6%), Actinobacteria (12.5% vs. 11.8%), Bacteroides (8.7% vs. 10.6%) followed by Fusobacteria (3.8% vs. 4.5%) and TM7 (0.6% vs. 1.0%) in HPV negative and positive samples, respectively. The predominant microbial families belonged to Streptococcaceae, Pasteurellaceae, Veillonellaceae, Micrococcaceae, Prevotellaceae, Neisseriaceae, Gemellaceae and Fusobacteriaceae. At genus level, Streptococcus, Haemophilus, Veillonella, Prevotella, unclassified Gemellaceae, Fusobacteriumand Actinomyces were the predominant bacterial genera in both groups.The most abundant phyla in the mouth were TM7 was increased (p = 0.026) in HPV positive oral samples compared to the HPV negative group by ANOVA test. Increased relative abundance of TM73 (p = 0.011) at family level as well as Selenomonas spp. (p = 0.0032), Megasphaera spp. (p = 0.026) and TM73 (p = 0.018) at species level was detected in HPV positive oral samples. Haemophilus spp. was higher (p = 0.019) in HPV negative compared to HPV positive.At phylum level, the relative abundance of Bifidobacteriaceae and Finegoldia genera were significantly enriched in HPV positive samples as compared to HPV negative , while Haemophilus genus was enriched in HPV negative compared to HPV positive  of bacterial microbiota as compared to HPV negative samples (p = 0.0319) Fig. . No diffive Fig. .Figure 3Lactobacillus genus (p = 0.0023) and Ureaplasma , Coriobacteriaceae (p = 0.083) in cervix and Selenomonas spp. (p = 0.0032), Megasphaera spp. (p = 0.026) and TM73 (p = 0.018) in the mouth. In cervical samples Peptostreptococcaceae (p = 0.0065) and Enterococcaceae (p = 0.0069) were significantly enriched in HPV negative samples.HPV infection is associated with altered bacterial microbiota in the placenta, uterine cervix and mouth. Distinct differences were detected in the relative abundance of specific microbial groups on family and genus levels while the overall microbial richness and diversity appeared to be associated with HPV infection with the exception of higher richness in HPV positive oral samples compared to HPV negative samples. HPV positive placenta samples showed increased abundance of 18. Still there has been controversy about whether the findings are genuine or a result from contamination35. Some studies have not found specific placental microbiota22. Several studies have also reported HPV DNA in the placenta, even in samples collected transabdominally and sometimes associated with adverse effects in pregnancies37.It has recently been suggested that the fetus does not develop in a sterile environment based on discovery of a distinct microbiota in the placentaCyanobacteria, Chloroplasts and Rhizobiales and sequences that could not be classified to domain level at OTU picking were removed from the results to control any possible bias. The presence and localization of HPV infection of our placental samples (Finnish Family HPV Study samples) have been examined before by Sarkola et al.12 and Koskimaa et al.7 and co-workers. Sarkola and coworkers confirmed the PCR positive HPV results by localizing the HPV16 and HPV6 DNA in syncytiotrophoblasts with tyramide amplified in-situ hybridization.In this study we attempted to meticulously rule out the possibility of false positive results of either with bacterial microbiota or HPV DNA. We took special care and performed several steps to ensure that our findings are relevant and not result from contamination. The reagents used for PCR amplification were sequenced as well as negative controls to rule out possible reagent contamination. With placenta samples, negative placental controls reached 726 reads and identified 11 OTUs. We considered >1000 reads as a limit for inclusion. A total of 19/39 placenta samples (49%) reached that and therefore were included in this study. At OTU picking we further excluded singletons and OTUs with a relative frequency below 0.01. Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, which is in line with recent previous studies39. With HPV positive samples, we saw a significant increase in the amount of Lactobacillus genus which was mainly composed by L. iners, L. crispatus, L. jensenii, L. gasseri and L. reuteri species. Lactobacillus is the most abundant phyla in the vaginal microbiota40 but has also been detected in placenta in previous studies without analyzing the presence of HPV38. The most abundant Lactobacillus species in cervix and placenta were L. iners , L. crispatus , L. gasseri  and L. jensenii  without taking into account the HPV status of the samples.We recognized placental microbiota composed of Ureaplasma spp. (Ureaplasma parvum 99% identity) was enriched in HPV positive placenta sample group. Ureaplasma spp. has previously in several studies been associated with chrorioamnionitis, which jeopardizes normal pregnancy and might lead to preterm delivery45. However, our results did not support the view that HPV presence would explain pregnancy complications and risk pregnancies as all had normal pregnancy without any complication. Nevertheless, the growing evidence of bacteria and virus in the placenta warrants further investigation from the point of view of a potential causal role in pregnancy complications including chorioamnionitis and preterm delivery46.In the present study, 47 and may be regarded as a predictor of imbalanced vaginal flora, especially decreased amount of Lactobacillus48. In the present study, we did not detect differences in the amount of Lactobacillus but rather an increase of Peptostreptococcaceae, Enterococcaceae and Haemophilus in HPV negative cervical samples. Peptostreptococcus has previously been associated with high-grade invasive cervical carcinoma when encountered together with HPV infection29. Interestingly, Enterococcus has also previously been linked to HPV-16 positive cervical cancer biopsies49 in contrast to our present study. Still, these previous findings have been made in single studies only and Enterococcus has even been observed from asymptomatic and healthy female vaginal flora50. It is currently not clear whether the vaginal microbiota play an important role in acquiring and persistence of HPV infection51. We have also previously presented data that some of the women from Finnish Family HPV Study, who presented HPV16 positive, developed incident cervical intraepithelial neoplasia within 14 years of follow-up52. Whether Lactobacillus plays a protective role in HPV clearance remains also to be determined54. Moreover, LEfSe test showed higher abundance of unclassified Coriobacteriaceae genus in HPV positive group cervical samples which was mostly attributable to Atopobium vaginae. A.vaginae has previously been reported to be associated with HPV persistence in cervico-vaginal region51 and bacterial vaginosis56. Still, A.vaginae has also been suggested in some cases to be a part of healthy vaginal microbiota60. This would suggest that present HPV infection and the observed changes in cervical bacterial microbiota may independently or synergistically create an environment favoring HPV persistence, dysbiosis and neoplastic transformation.HPV infection has previously been linked to higher richness in vaginal microbiota64. HPV infection has primarily been considered as a sexually transmitted disease but recent research has begun to challenge this notion and so far we also know that vertical transmission during delivery or later in life, horizontal transmission and even autoinoculation have been established as possible ways of acquiring HPV infection65. Based on the results of the current study, we are able to conclude that bacterial microbiota can be one of the factors influencing the composition of overall microbiota in different anatomical sites.In this study, we observed a number of women with multiple HPV infection simultaneously at different body sites. Other studies have also observed this phenomenon but the cause behind this is currently not knownActionbacteria, Bacteroidetes, Firmicutes, Fusobacterium, Streptococcus, Veillonella and Proteobacteria66. In our study the presence of HPV infection increased the amount of Selenomonas spp., Megasphaera spp. and TM73 in the oral microbiota. Selenomonas is a part of oral normal microbiota and oral biofilm but is also associated in disease, especially Selenomonas noxia in (aggressive) periodontitis (gum disease) and increased caries activity69. Megasphaera spp. is a part of dental plaque, so called early colonizer and has been discovered in endodontic abscess specimens70. The microbiota changes we found in oral HPV positive samples have not been earlier connected with HPV infection but may affect oral health. The association between oral HPV infection and oral health has not been extensively investigated but our data suggests that the oral microbiota composition in the presence of present HPV infection may favor potential pathogens.The mouth harbors one of the most diverse microbiota in the human body and the most abundant bacterial phyla in the oral cavity include Our study has several limitations. In our material of 329 mothers and deliveries and 315 placentas, only 13 placenta samples were HPV positive. This small number of HPV positive samples most likely explains why differences in the overall richness in the microbiota were not found. Secondly, the amount of usable DNA residues from bacteria in placenta samples is low which renders drawing definitive conclusions of the overall composition of microbiota difficult.19.To our knowledge, this is the first study to show that HPV infection is associated with altered bacterial microbiota composition in the placenta and the mouth. We used the same protocol to indicate microbiota from oral, cervical and placenta samples. The diversity and composition of the bacterial microbiota was characterized by sequencing of the 16S rRNA gene and because a single bacterium can harbor multiple sequences of the 16S rRNA genes we have better possibilities to detect them as a sign that there has truly been a bacterial colonization. Still we were only able to investigate a small proportion of the placenta samples and needed to exclude some samples because the low number of reads to strengthen the reliability of our data. This definitely has an impact on our results but it has been an issue also in recent published articles investigating the placenta microbiotaIn summary, we report a novel finding of association between microbiota and HPV infection. Our data may be interpreted to corroborate the hypothesis of a distinct microbiota of placenta. These initial findings still need to be replicated in larger studies to either confirm our findings or to reveal new perspectives into this neglected field of viral bacterial interactions. Whether the changes in bacterial microbiota predispose or result from HPV and if it influences HPV persistence, remains also to be determined in future studies.71 were utilized in this study. The original study was designed to evaluate the interactions of HPV infection in families. A total of 329 pregnant women in their third trimester were enrolled in the study together with their male spouses (n = 131) and offsprings to come (n = 313) with 6-year follow-up.Samples collected in the Finnish Family HPV Study62. The subjects were selected according to the HPV status of the placenta and mode of delivery. Altogether 13 placentas with HPV DNA were available. Thirteen HPV DNA negative placentas from vaginal deliveries with 13 HPV DNA negative placentas from caesarean section deliveries were selected as controls. The placenta samples obtained after delivery included all the tissue layers from the maternal side of placenta and the HPV DNA was localized in syncytiotrophoblasts12. Furthermore, the oral and cervical samples of these women had been collected as mucosal scrapings with a brush  and immediately frozen −70 °C until used.For the present substudy, DNA samples from the placenta, the uterine cervix and the mother’s mouth taken during the last trimester were included. The presence of HPV DNA has been examined earlierThe clinical study and its amendments were found acceptable by the Ethical Committee of the Intermunicipal Hospital District of Southwest Finland . The methods were carried out in accordance with the relevant guidelines and regulations. Informed consent was obtained from all the subjects participating in the study.72.HPV DNA was extracted by the high salt-methodet al.7 and Syrjänen et al.62.HPV testing was performed with nested PCR using MY09/MY11 as external primers and GP05+/GP06+ as internal primers followed by genotyping with Multimetrix® kit , which detects 24 HPV types  as described in Koskimaa Isolated DNA concentrations were measured using a Qubit® 2.0 Fluorometer  and normalized to10 ng/μL. The V3-V4 region of 16S rDNA gene was amplified by PCR using Illumina adapter overhang nucleotide sequences following Illumina protocols. After 16S rDNA gene amplification, the multiplexing step was performed using Nextera XT Index Kit  and PCR product was checked in a Bioanalyzer DNA 1000 chip . Libraries were sequenced using a 2 × 300 pb paired-end run (MiSeq Reagent kit v3) on a MiSeq-Illumina platform . To rule out and control for possible contaminations, PCR amplification and libraries controls were also sequenced as negative controls.73 and filtered with samtools version 1.3.1–50. Clean FASTQ files were converted to FASTA files and UCHIME program version 4.2 was used to remove chimeras.Quality control of the FASTQ files was performed using Fastx tool kit version 0.0134 to remove reads with quality less than Q20, once the sequences were clean based on quality scores, we trimmed traces of the 16S rRNA primers and sequencing adapters using cutadapt version 1.2.5. After primer removal, sequences with <300 nucleotides read length were trimmed using perl scripting. Sequences were mapping against the human genome BWA version 0.7.174. Singletons and OTUs with a relative frequency below 0.01 were removed. Sequences that could not be classified to domain level, or were classified as Cyanobacteria, Chloroplasts  and Rhizobiales  were removed from the dataset.An open reference OTU picking method using 97% identity to the Greengenes 13_8 database was performed using QIIME pipeline (version 1.9.0)http://cgenome.net/calypso/) was used with total sum normalization (TSS) for the statistical analysis, and also, Cumulative Sum Scaling normalization (CSS) for multivariate tests . Linear discriminant analysis effect size (LEfSe) was used to detect unique biomarkers (LDA score > 3.0) in relative abundance of bacterial taxonomy. P-values ≤ 0.05 were regarded as statistically significant.Alpha diversity indices (Chao1: richness and Shannon: diversity) and beta diversity using UNIFRAC (phylogenetic) and Bray Curtis distance (non-phylogenetic) among samples were studied and PERMANOVA was used to test significance. Calypso software version 8.10 (The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Tables 1 and 2


Cervical cancer is the second cause leading of malignancy-related death among Mexican women. The present study determined the population-based prevalence of high risk Human Papillomavirus (HR-HPV) infection and associated cofactors in female beneficiaries of the Institute of Security and Social Services for State Workers (ISSSTE) attending the Program for HPV Screening and Early Detection of Cervical Cancer and registered in the Women’s Cancer Detection System (SIDECAM).In a cross-sectional study, cervical samples from 115,651 female users of the program for HPV screening and early detection of cervical cancer recruited in 23 ISSSTE care centers were analyzed for HR-HPV. Logistic regression analyses, adjusting for potential confounders, were performed to determine the association of HR-HPV infection with sexual health and behavior variables and with positivity to cervical premalignant lesions by cytology.The overall prevalence of HR-HPV infection among female ISSSTE beneficiaries in the 2013–2015 period was 13%. A bivariate analysis of relevant variables for HR-HPV infection showed a statistically significant association for age, number of sexual partners, use of hormonal contraceptives and smoking. A statistical association was found between infection by HR-HPV with the use of hormonal contraceptives, number of sexual partners and smoking and association of HPV 16 and other non-16/18 HR-HPV infection with number of lifetime sexual partners and tobacco use adjusted for age, history of hormonal contraception, number of sexual partners and tobacco use with the exception of exposition variable itself. Similarly, an association was found between HR-HPV infection, regardless of the virus genotype, with positivity to cervical premalignant lesions adjusted for age, number of lifetime sexual partners, history of hormonal contraception and tobacco use.HR-HPV prevalence in female ISSSTE Women’s Cancer Program users is similar to the population-based prevalence previously reported in Mexican women without cervical alterations. The ISSSTE robust screening and early detection program, based on cytology studies and HPV co-testing, allows us to know the prevalence of HR-HPV infection among female users of the service. According to the latest systematic analysis from the Global Burden of Disease Study 2015, 530,000 new cervical cancer (CC) cases were attributable to Human Papillomavirus (HPV) infection [Worldwide prevalence of HPV infection in women with no cervix abnormalities is 11–12%, with the highest rates being found in sub-Saharan Africa 24%), Eastern Europe (21%), and Latin America (16%) . Age-spe%, EasterIn Mexico, the official standard NOM-014-SSA2–1994 supported by national government covers CC prevention, detection, diagnosis, treatment, control, and epidemiological surveillance. The program for early CC detection consists of a Papanicolaou smear test, along with biomolecular tests for HPV detection as a support for cervical cytology and direct visualization with acetic acid (only when Papanicolaou smear is not available). This detection test should be performed on any asymptomatic women between 25- to 64-year-old or women under 25 and over 64 years of age who show reduced morbidity and the presence of some risk factor associated with CC [About 10%  of the Mexican population is affiliated to the Institute of Security and Social Services for State Workers (ISSSTE); of these, 26.9%  are women in ages from 25 to 64 years, the target group of the Program for HPV Screening and Early Detection of Cervical Cancer and registered in the Women’s Cancer Detection System (SIDECAM); in turn, 1 602,754 ISSSTE affiliates were users of the program in 2013. This study is aimed to assess the prevalence of high-risk- (HR)-HPV infection and associated cofactors among users of the Program for HPV Screening and Early Detection of Cervical Cancer and registered in the SIDECAM.A cross-sectional study was conducted, with data analysis from a biological data bank of cervical tissue samples built between 2013 and 2015 from 115, 651 female users of the Program for HPV Screening and Early Detection of Cervical Cancer and registered in the SIDECAM attending primary care, reproductive and family planning services in 23 ISSSTE care centers.The Program for HPV Screening and Early Detection of Cervical Cancer is the population screening program of the ISSSTE aimed at asymptomatic women between 25- to 64-year-old or women under 25 and over 64 years of age who show reduced morbidity and the presence of some risk factor associated with CC. The algorithm that is followed for the HPV screening and early detection of cervical cancer is presented in Fig. Sociodemographic, lifestyle, and reproductive data were retrieved from the SIDECAM database for each subject: age, number of lifetime sexual partners, history of consumption of hormonal contraceptives, smoking habit, symptoms referred by the patient, signs observed by colposcopists, PCR-processing laboratory and PCR result for HPV-16, HPV-18, or non-16/18 HR-HPV genotypes.Information related with the reason for follow-up, result of the last Papanicolaou, diagnosis of premalignant lesion according to Papanicolaou had missing data in the SIDECAM database. The analysis of differential distribution of the reason for follow-up, result of the last Papanicolaou and diagnosis of premalignant lesion between HR-HPV-positive and negative groups was realized in 4176; 11,430 and 11,191 women screened for HR-HPV infection within the Program for HPV Screening and Early Detection of Cervical Cancer and registered in the SIDECAM, respectively.Healthcare staff assigned to the family planning module of each ISSSTE unit sample endocervical exudates with the PreservCyt vial  for HR-HPV detection and either liquid cytology or Papanicolaou smear. The samples are then sent for analysis to the appropriate ISSSTE pathology unit.Cervical epithelial scrapings taken from each patient are stored at 4 °C until automated HR-HPV genotyping analysis is done with the Cobas 4800 HPV Test  in the PCR reference laboratory of each ISSSTE regional office. Cobas 4800 is a fully automated real-time PCR system that separately detects HR-HPV-16 and -18 genotypes, as well as ten other HR genotypes  and two “probable high risk” strains (66 and 68). HPV testing is performed according to the manufacturer’s directions. For purposes of describing the result of the HR-HPV test in our study, we will describe it as HPV 16 positive, HPV 18 positive and non-16/18 HR-HPV positive.A bivariate analysis was performed to determine proportion differences of sociodemographic, sexual behavior and obstetric-gynecological history variables, frequency of patient symptoms, and signs reported in cytological studies between groups with and without HR-HPV infection, using the chi-square for categorical variables. Age and the number of sexual partners were recorded as continuous, numeric variables and then analyzed as categorical variables. History of hormonal contraception, smoking habit, patient symptoms and signs according to the cytological study, reason for follow-up, results of last Papanicolaou smear, and diagnosis of premalignant lesion in the population under study were both collected and analyzed as categorical variables. A test for trend across ordered groups (Chi2 test np trend) was used for verifying whether the number of negative and positive for HR-HPV cases followed a linear trend frequency in each group of the analyzed variables.In the analysis, the subjects were classified as HR-HPV positive when tested positive for any of the 14 HR-HPV types, including HPV-16 and HPV-18. Among these positives, the subjects were subclassified as positive for HPV-16 or HPV-18, regardless of whether they were also positive for another HR-HPV type. The “non-16/18 HR-HPV” group included individuals who were positive for other HR-HPV types , but neither HPV-16 nor HPV-18.To determine the association between HR-HPV infection, either generic or segregated by genotype, with some risk factor found to be associated in the preliminary bivariate analysis, a logistic regression analysis with odds ratio (OR) calculation and 95% confidence intervals (CI) was performed. Then, a logistic regression analysis was performed adjusting for potential confounders to determine the association of HR-HPV infection, either generic or segregated by genotype, with positivity to cervix premalignant lesion. All statistical analyses were performed with the software STATA v.14.0 .The overall prevalence of HR-HPV infection understood as the percentage of women with at least one high-risk HPV genotype detected, in the ISSSTE-affiliated population in the 2013–2015 period was 13%, corresponding to 15,040 beneficiaries. The prevalence of coinfection by HPV 16, HPV 18 and non-16/18 HR-HPV; HPV 16 and HPV 18 only; HPV 16 and non-16/18 HR-HPV and HPV 18 and non-16/18 HR-HPV was 0.06, 0.03, 0.8 and 0.3%, respectively.The prevalence of single HPV 16 infection was 1.3 and 2.2% with coinfection by other HR-HPV. Single HPV 18 infection prevalence was 0.54 and 0.9% with coinfection with other HR-HPV. The prevalence of single non-16/18 HR-HPV infection and with coinfection with HPV 16 or HPV 18 was 10 and 11%, respectively.The prevalence of HR-HPV, HPV-16/18 and non-16/18 HR-HPV by age group shows a bimodal distribution with an increased prevalence for the youngest women in the population aged 18–39 with a progressive drop in each age interval and a second bump of positivity for the oldest women aged 65 and above. The prevalence of general HR-HPV, HPV 16 and non-16/18 HR-HPV was higher in the age group of 18 to 24 years  for age, number of sexual partners, history of hormonal contraceptives, and smoking habit ; ectropion (p = 0.01), abnormal bleeding (p = 0.01), and Trichomonas sp. infection (p = 0.006), was found between HR-HPV-positive and negative groups. The most frequent reason for follow-up in positive HR-HPV cases was a previous HPV diagnosis (68%) and the prevalence of HR-HPV infection was 15% in cases with negative result for SICL in the last Papanicolaou. The prevalence of HR-HPV infection was 30% (142/466) in cases with positive diagnosis of SICL  for age, number of lifetime sexual partners, smoking, and molecular HR-HPV diagnosis. No significant differences were observed when comparing diagnosis of cervical premalignant lesions between both groups with respect to the history of hormonal contraception nor with ectropion or leucorrhoea in colposcopic examination (data not shown).A bivariate analysis of relevant variables with biological relevance between groups with and without premalignant cervix lesion according to Papanicolaou smear showed significant differences (p < 0.05). Number of lifetime sexual partners greater than 6 and tobacco use were found associated with HPV 16 infection and other non-16/18 HR-HPV infection in all evaluated models adjusting for age, and in the multivariate model adjusting for age, number of lifetime sexual partners, history of hormonal contraception, and smoking, except for the exposition variable itself. No association was found with HPV-18 infection  , 13, simInterestingly, HPV-16/18 prevalence was similar to that reported in a previous study in Mexico , 17, 18.Previous studies found a similar association of a higher number of sexual partners –21, histAlthough the association between HR-HPV infection and hormonal contraception found in the multivariate analysis was weak, the proposed association is biologically plausible because the estrogens and progestagens have been reported to interact with hormone receptors, mainly progesterone receptors, in the cervical tissue; additionally, sex steroid hormones are thought to enhance the expression of HPV-16 E6 and E7 oncogenes, promoting the degradation of p53 tumor suppressor genes and enhancing the ability of viral DNA to transform cells .Smoking is another risk factor associated with HR-HPV infection; this could be explained considering that tobacco smoke contains well-known carcinogens that could have a direct transformation effect on cervix tissues and/or cause immunosuppression, allowing HPV infection to persist and progress to cancer .The association herein found between HR-HPV infection and premalignant lesion in the cervix confirms the already known etiological role of HPV in this disease –32.While no cancer records exist to date in Mexico at the national, regional, nor state levels, great efforts have been made within the program for screening and co-testing for CC in the ISSSTE to integrate such screening with an comprehensive care model with a process-oriented approach, going from the traditional cytology-only scheme to a combination of cytology and HR-HPV genotyping, aiming to implement liquid-based cytology in the future, along with HR-HPV genotyping and immunohistochemical tests . To this end, the ISSSTE has reinforced its infrastructure in terms of cytology laboratory services, molecular biology laboratory, and colposcopy rooms; it also increased its personnel  and installed capacity, as reflected in the number of cytology and HR-HPV tests performed per year, to avoid follow-up interruption in women screened as positive.This study provides information about the prevalence of HR-HPV infection among the female ISSSTE beneficiaries attending in the Program for HPV Screening and Early Detection of Cervical Cancer and registered in the Women’s Cancer Detection System as part of primary care services. It can help policymakers to make better-informed decisions on resource allocation when trying to determine the best screening and triage practices in Mexico.The main strength of this study is its large-population-based approach. This is the second largest study in Mexico to assess the prevalence of HR-HPV (both HPV-16/18 and non-16/18 HR-HPV). As such, it can also be used in the future to evaluate the impact of HPV vaccination within the ISSSTE female user population. On the other hand, the main limitation of our study is the difficulty to generalize its findings to other populations.HR-HPV prevalence in female users of the Program for HPV Screening and Early Detection of Cervical Cancer of the ISSSTE is similar to the population-based prevalence previously reported in Mexican women without cervical alterations. Age, number of sexual partners, history of hormonal contraception, and smoking habit were found to be positively associated with HR-HPV infection; and number of sexual partners and smoking with HPV-16 infection and infection by non-16/18 HR-HPV. The ISSSTE has a robust early detection program based in cytology studies and HPV testing, that allows us to know the prevalence of HR-HPV infection in the female user population.


The ASCO Expert Panel Steering Committee feels this letterhelps amplify the guideline.We appreciate the insight and comments of Swanson, Gimei, andHuchko1 state that “ASCOrecommends VIA [visual inspection with acetic acid] scale-up in settings where HPV[human papillomavirus] testing is considered not feasible, as a necessary step to createinfrastructure for future HPV testing. We disagree with this recommendation,” inreference to the ASCO recommendation (only in the basic setting) that “if HPV DNAtesting for cervical cancer screening is not available, then VIA should be offered withthe goal of developing health systems and moving to population-based screening with HPVtesting at the earliest opportunity.”2 Swanson et al state that “given the increasing availabilityof feasible, acceptable HPV DNA tests which can be self-collected by women outside aclinic, we suggest that resources may be better spent developing community-based HPVtesting for primary screening, rather than scaling up widespread VIA.” The ASCOrecommendation was based on a review of evidence on HPV testing and VIA. It recognizesthat in some basic settings, there is little infrastructure  for public health systems with sufficient development to haveoutreach workers, and it strives to make recommendations both for current situations andfor what planners could put in place in future. The guideline states that research onVIA has shown mixed results and that “the goal [is] moving to population-basedscreening with HPV testing at the earliest opportunity.”2 In addition, the guideline reviewsemerging literature on self-collection and largely agrees with the potential use of itin some settings; ASCO will update the guideline pending additional evidence onself-collection.Swanson et al1 suggests that implementing the ASCO recommendation that“women who are postpartum should be screened with VIA 6 weeks after delivery inbasic settings”2 is“missing a key opportunity to interact with an at-risk population.” TheASCO recommendation was the result of extensive expert panel discussion, and because ofsimilar concerns , this was aformal consensus-based (rather than evidence-based) recommendation . The guidelineauthors believe this provides flexibility in settings where women are less likely tohave postpartum visits, as Swanson et al describe for Uganda; the target age for therecommendation for basic (as well as limited and enhanced) settings is women age 30years or older, and pregnancy is not as common after age 30 years in basic settings asit is for women living in maximal resource settings, who are more likely to accessservices postpartum. We do agree that “we need to create strategies linking thepost-partum cervical cancer screening to other health visits”  and with the statement by Swanson et al “optimizing useof existing infrastructure will be essential to effective national screening programs,especially in basic settings with competing health priorities.”In addition, the letter by Swanson et al