Human papillomavirus (HPV) testing is replacing cytology in primary screening. Its limited specificity demands using a second (triage) test to better identify women at high-risk of cervical disease. Cytology represents the immediate triage but its low sensitivity might hamper HPV testing sensitivity, particularly in low-income and middle-income countries (LMICs), where cytology performance has been suboptimal. The ESTAMPA  study will: (1) evaluate the performance of different triage techniques to detect cervical precancer and (2) inform on how to implement HPV-based screening programmes in LMIC.Women aged 30–64 years are screened with HPV testing and Pap across 12 study centres in Latin America. Screened positives have colposcopy with biopsy and treatment of lesions. Women with no evident disease are recalled 18 months later for another HPV test; those HPV-positive undergo colposcopy with biopsy and treatment as needed. Biological specimens are collected in different visits for triage testing, which is not used for clinical management. The study outcome is histological high-grade squamous intraepithelial or worse lesions (HSIL+) under the lower anogenital squamous terminology. About 50 000 women will be screened and 500 HSIL+ cases detected . Performance measures  of triage techniques to detect HSIL+ will be estimated and compared with adjustment by age and study centre.The study protocol has been approved by the Ethics Committee of the International Agency for Research on Cancer (IARC), of the Pan American Health Organisation (PAHO) and by those in each participating centre. A Data and Safety Monitoring Board (DSMB) has been established to monitor progress of the study, assure participant safety, advice on scientific conduct and analysis and suggest protocol improvements. Study findings will be published in peer-reviewed journals and presented at scientific meetings.NCT01881659 The study design in which additional samples from all women screened with HPV and cytology are collected at screening simulating a reflex-testing scheme whenever possible, will allow the evaluation of triage techniques without influencing the outcome of the study, as tests are performed or evaluated after disease confirmation.This is the largest cervical screening study in Latin America with more than 500 histologically confirmed high-grade squamous intraepithelial lesions (HSILs) expected that will permit evaluation of several triage tests, alone or in combination, for HPV-positive women.A large number of women are being screened with HPV testing in Latin America, where the majority of women would not otherwise benefit from high-quality cervical screening.The multicentric nature of the study will allow capturing experiences from areas which are geographically, culturally and socio-economically distinct from each other and with different health systems/areas that may face common challenges but that require different approaches in accordance with their context.Colposcopy and collection of biopsies was not performed in HPV-negative women , potentially introducing verification bias when assessing absolute performance measures of screening tests to be used in primary screening; however, the study design will allow unbiased evaluation of triage tests.More than 500 000 new cases and nearly 300 000 deaths of cervical cancer occur every year, more than 90% in low-income and middle-income countries (LMICs).It is now clear that a group of about 12 human papillomavirus (HPV) types are the causal agents of cervical cancer and that HPV16 and HPV18 are responsible for about 70% of tumours. HPV is a very common infection usually acquired shortly after initiation of sexual activity, but most infections are cleared by the immune system within 2 years of acquisition and only a few persist and progress to cancer.3Highly effective and safe vaccines against HPV16 and HPV18 have been developedCurrently, there are several tests for HPV detection with high sensitivity and reproducibility that can detect more cervical disease at an earlier stage and offer long-term reassurance of low risk of cervical precancer and cancer. These tests are now being used or considered to replace cervical cytology in primary screening as they allow extension of the screening interval, with consequent savings that can compensate the possibly higher cost of the test compared with cytology. In addition, HPV testing can be done on self-collected samples, increasing screening uptake.Screening with HPV has the problem that transient HPV infections are very common, particularly among young women, where the majority of infectious will regress spontaneously. Even among women over 30 years of age, HPV infection tends to regress and only in a fraction of women with persistent infection, it can lead to true cancer precursors and cervical cancer. Thus, one main issue to resolve is which tests or strategies can better select HPV-positive women, who are most likely to have or develop the significance disease (triage) in the near future, for further evaluation and treatment.Here we detail the design of a multicentric study that aims to evaluate visual methods, cytology-based and novel molecular-based techniques that can be used to triage women who test positive for HPV and that can lead to the establishment of HPV-based efficient, affordable and sustainable screening programmes.The aims of the study are: (1) to investigate the performance of emerging cervical cancer screening and triage techniques among women 30 years and older and (2) to evaluate the feasibility of different approaches for implementation of organised HPV-based screening programmes.The primary objective is to estimate performance characteristics  of multiple techniques alone or in combination for detection of histologically confirmed cervical high-grade squamous intraepithelial lesion (HSIL) or worse lesions (HSIL+) under the Lower Anogenital Squamous Terminology LAST  among HPSecondary objectives are: (1) similar performance analyses among all recruited women and restricted to those with negative cytology; (2) estimations of colposcopy referral, overdiagnosis and overtreatment rates by a single technique or a combination of techniques; (3) establishment of a biological specimens’ bank with an associated database to evaluate future cervical cancer screening and triage techniques; and (4) assessment of the feasibility of implementing organised HPV-based screening programmes within local health systems. The methodology to assess this objective will be reported separately.The key hypotheses of the study are:Cervical cancer screening by HPV testing followed by triage with one or more additional tests identifies the majority of women at high risk of having precancerous cervical lesions who need treatment to prevent cervical cancer.The number of screened women lost to follow-up in the screening process  could be reduced if: (1) women are well informed of the process and trust the healthcare system, (2) healthcare professionals are well trained and ready to offer care and (3) the follow-up is centrally organised with capacity to contact screenees.HPV-based cervical cancer screening could be implemented in Latin America if a screening platform is developed, and affordability and sustainability of the screening programme can be guaranteed ahead of implementation.The study is being conducted in 12 study centres in Argentina, Colombia, Paraguay, Bolivia, Costa Rica, Honduras, Mexico, Peru and Uruguay.Recruitment started in May 2013 and will be completed in December 2020. However, as the study includes a follow-up visit after 18 months of initial screening, final completion of the study is envisaged for July 2022.Different tests and approaches will be evaluated for triage of HPV-positive women. Some of them may also be evaluated as stand-alone without triage test: one with enough sensitivity to be used for primary screening but good specificity so that no triage is required.The alternatives for triaging HPV-positive women include visual methods, cytology, high-risk and type-specific HPV persistence, HPV genotyping, HPV oncoproteins and other novel molecular biomarkers.Visual inspection of the cervix after the application of 5% acetic acid (VIA) is inexpensive, simple and can be carried out by primary care personnel . The sensitivity and specificity of VIA are limited and highly dependent on training and experience of examiners, who require continued training and supervision.12VIA may be used as triage of HPV-positive women in remote areas where high-tech methods for diagnosis may not be available, identifying who can be treated with ablative treatment, who should be referred to a higher healthcare level and who can be followed up without treatment.14In this study, VIA will be standardised to allow evaluation of whether a lesion is present or not and if the observer considers that the subject would be a candidate for immediate ablative treatment, but VIA results will not be used for clinical management except when cancer is suspected and the woman should be immediately referred to colposcopy. Visual inspection after the application of iodine lugol, the other naked-eye visual method, will be evaluated in some study centres under a different protocol.Conventional cytology or Pap has been used since the 1950s in primary screening and has succeeded in decreasing cervical cancer in several HICs, particularly where women have had cytology frequently and high adherence to diagnosis and treatment has been achieved. This has not happened in LMIC, where usually coverage has been low and access to diagnosis and treatment has been limited.Liquid-based cytology (LBC) uses the same principles and criteria of Pap and is replacing Pap in HICs because it offers the opportunity for reflex testing and significantly reduces the number of inadequate samples,18HPV testing followed by triage with cytology (Pap or LBC) is the immediate screening modality to be used in HPV-based screening settings with high cytology capacity. However, the limited sensitivity of cytology might detriment HPV testing sensitivity, especially in places where strict quality assurance cannot be ensured.It has been postulated that prior knowledge of HPV status may improve the performance of cytology since the interpreter will be more meticulous when reading a smear of an HPV-positive woman. However, results from studies have not been fully consistent; some have shown increases in the proportion of abnormal resultsIt has been proposed to use 1 year HPV persistence  to follow HPV-positive/cytology-negative women in places where cotesting is recommended like in USA or after using cytology for triage of HPV-positive women, like in Argentina.Stratifying women on the presence of HPV16 and HPV18, which are responsible for about 70% of cervical cancer and their precursors, will identify women at the highest risk of CIN3 or worse lesions (CIN3+). In a large cohort study in the USA, among women older than 30 years, the 18 year cumulative incidence of CIN3+ among one-time HPV16-positive women was 8.5% compared with 3.1% for other oncogenic HPV-positive women negative for HPV16,26Some of the HPV screening tests already provide separate results on HPV16 and/or HPV18, and we will evaluate the performance of HPV16 and HPV18 among HPV-positive women who will be screened with the COBAS HPV test (Roche) in some study centres.Additionally, the potential to improve the accuracy of HPV testing and triage with different combinations of HPV types is under evaluation,E6 and E7 oncoproteins are the main effectors of the HPV oncogenic activity. The molecular structure, functions and expression levels are different between oncogenic and non-oncogenic HPV types.An HPV E6 strip test, the OncoE6 Cervical Test targeting E6 HPV16 and HPV18  has been used in studies around the world with promising results.p16ink4a, a cyclin-dependent kinase inhibitor markedly overexpressed in cancerous and precancerous cervical tissue. It corresponds to a cellular correlate of increased expression of the viral E7 oncoprotein that disrupts the retinoblastoma protein (pRb) pathway, leading to compensatory overexpression of p16ink4a.p16ink4a can be measured using ELISA and by immunostaining of histology and cytology slides as done by the CINtec test .Another important biomarker under intensive study is p16ink4a clearly increased with severity of cytological  and histological  abnormalities. The authors noted limited reproducibility due to insufficient standardised interpretation of the immunostaining. One study comparing Pap, HPV testing and HPV triaged with p16ink4a found that the latter scheme maintains the sensitivity gained by the HPV test but with a referral rate to colposcopy similar to that of a Pap-based programme.35A meta-analysis of the performance of this test,p16 but also commits the cell into proliferation which leads to overexpression of Ki-67. Since overexpression of p16 (tumour suppression protein) and Ki-67 (proliferation marker) are mutually exclusive under normal conditions, the detection of overexpression of both simultaneously by dual-immunostaining could identify cells with deregulated cell cycle. This dual-staining test can substantially simplify and standardise the evaluation of stained slides.37Another biomarker of the cell cycle is Ki-67 which is a proliferation marker. As described, E7 leads to accumulation of DNA methylation is a mitotically transmitted epigenetic motif that reflects molecular events in host cells contributing under certain conditions to cervical carcinogenesis. It can be quantitated with good accuracy in exfoliated cells and could be applied directly to the residual sample after HPV test.Women are invited to screening using different approaches, such as: door-to-door census of the recruitment area, invitation by community leaders or media campaigns.Women eligible for inclusion are those aged 30–64 years and residents of a specified catchment area, mentally competent to understand the consent form, able to communicate with study personnel and physically able to have a pelvic exam. Women who have not initiated sexual life, who have a history of cervical cancer or treatment for cervical precancer within the previous 6 months and those with hysterectomy or serious pre-existing medical conditions or plans to move out of the study area in the next 12 months are excluded. The participation of pregnant women and those who have given birth less than 3 months before screening are deferred until they are 3 months or more postpartum or up to the end of the enrolment period. Similarly, women with heavy vaginal bleeding or severe cervical infection are deferred until the condition is resolved.This is a screening study in which HPV testing and Pap will be carried out in up to 50 000 women aged 30–64 years in multiple centres across Latin America. HPV-positive women and those with abnormal Pap will be referred to colposcopy with biopsy if needed. Identified high-grade cervical lesions will be treated with large loop excision of the transformation zone (LLETZ). Women with no evident disease will be recalled 18 months since initial screening for another HPV test and those positives will undergo colposcopy with biopsy, followed by treatment as needed.The study is organised in four main clinical visits: Visit 1: Initial Screen, Visit 2: Initial Colposcopy, Visit 3: Follow-up Screen 18 months after the initial screen and Visit 4: Final Colposcopy on HPV positives in the follow-up screen .After obtaining informed consent and providing contact information and basic socio-demographic data to nurses or doctors trained on data confidentiality, women agreeing to participate are assigned a unique study ID number, and a clinician performs a pelvic exam and refers women to adequate care if clinically required. Next, after inserting a speculum without lubricant, the clinician collects cervical cells in the following order: (1) a Dacron swab that will be placed in a cryovial without preservation medium and frozen within 24 hours of collection; and (2) two consecutive collection brushes; the first brush is used to prepare a Pap smear and then washed in a vial with PreservCyt (Hologic) medium (PC); the second one is directly washed in a second PC vial. After being vigorously shaken inside the vials, the brushes are discarded and the vials are kept at room temperature or at 4°C until testing or aliquoting.The frozen dry swab will be used for oncoprotein tests, the first vial for HPV testing and for LBC preparation, the remains and the second vial will be aliquoted for HPV testing quality control (QC) and other molecular tests under evaluation.HPV testing is performed at local laboratories selected for the study. All HPV-positive women, those with abnormal cytology and those testing positive in any QC test become the study cohort, and are referred to colposcopy for diagnosis followed by treatment of cervical lesions if needed.Women who are not referred to colposcopy are given their results, explanations on the significance of a negative HPV result and recommendations for future local regular care.At this visit, a risk factor interview, including sexual behaviour information, is administered.A pelvic exam with collection of cervical cells using a swab that is washed in a PC vial is done before applying acetic acid to the cervix. This sample is used for a new HPV test and to perform other tests which could not be performed on the enrolment sample due to insufficient sample volume.In some centres, after cervical sampling and immediately before colposcopy, a trained nurse, midwife or a doctor performs VIA. After application of 5% acetic acid, the clinician records the presence of acetowhite areas and documents his/her assessment on the need for treatment. The clinician does not discuss his/her findings with other team members to avoid inducing bias; except when cancer is suspected and the woman should be immediately referred to colposcopy.After the VIA, the colposcopist inspects the cervix under the colposcope after application of acetic acid and collects 2–3 biopsies of all acetowhite areas observed. The colposcopist then ranks the biopsies according to the probability that the acetowhite area, from which each biopsy was collected, contained the worst lesion on colposcopic impression. No blind biopsies or biopsies from women without observable acetowhite areas are collected.Collection of a 10 mL blood specimen from the arm is done at this visit, following usual procedures for blood collection. The blood sample will be used for HPV and cervical disease assays that could be available in the future, including markers of genetic susceptibility.The clinical management of women attending colposcopy is defined by the enrolment cytology and the colposcopy results as shown in Treatment with LLETZ should be offered without prior histology confirmation to women with a ‘positive major’ colposcopy, whenever possible. Biopsies should be collected if the colposcopy is considered ‘positive minor’ or where LLETZ without confirmed CIN2 is not allowed. If a transformation zone type 3 (TZ3) is observed, an endocervical sample should be collected and an excision type 3  diagnostic or therapeutic should be performed.The clinical management of women with discordant results: lesions less than CIN2 (<CIN2) or those with negative colposcopy but with HSIL+ cytology should be revised at multidisciplinary team (MDTs) meetings, that are attended by the cytologist, pathologist, colposcopist and the local principal investigator at least. During the MDT, cytology, histology and colposcopy results, considering the age and parity of the woman, should be discussed to finally recommend treatment or recall for a second HPV test 18 months later. In the benefit of women, who may be lost to follow-up, recommendation for LLETZ will be prioritised above 18 months recall.Biopsies should be collected from all women with positive colposcopy. An endocervical sample should be collected from women with TZ3 and if the result of this sample is HSIL+, treatment (excision type 2 or 3) should be offered. Women with histology <CIN2, negative colposcopy and those with a TZ3 and <HSIL on the endocervical sample should be recalled at 18 months. MDTs should be carried out when the colposcopy is positive major but the histology is <CIN2.The colposcopic impression using a standard colposcopy nomenclature and specific colposcopy features  are recorded in colposcopy study forms.At the treatment visit, first a colposcopy is done followed by LLETZ. The reason and type of LLETZ as well as the colposcopic impression are recorded in treatment study forms. A pregnancy test is administered to participants before treatment with LLETZ and local consent forms for surgical procedures are sought. Self-reported adverse events are documented.careBrush (QIAGEN) to be inserted in a specimen transport medium to be tested with hybridisation techniques or a swab to be inserted into a tube with no preservation medium to be washed in PC and tested with PCR techniques. Alternatively, a clinician will perform a pelvic exam and collect the sample using a cytobrush to be washed in a PC vial. The clinician-collected sample will be used for HPV testing and future triage tests. HPV-negative women exit the study and return to local regular screening. HPV-positive women will be referred for a final colposcopy round for diagnosis and treatment.All women who were not treated after an initial positive screen are invited to a final follow-up screening with HPV testing 18 months after enrolment. Women will be allowed to attend this visit up to 30 months after enrolment or until the study ends. To increase attendance to this visit, women may self-collect a vaginal sample either at the health centre or at home (depending on the follow-up strategy). If this was the case, women will be explained what self-sampling is and how to self-collect a sample using a graphical brochure. Women will be given either a The clinical management of women attending final colposcopy will be defined only by the colposcopic impression. Biopsies will be collected if the colposcopy is positive and an endocervical sample will be collected if a TZ3 is observed. Women with local CIN2+ histology will be treated with LLETZ and those with HSIL+ endocervical cytology will be treated with excision type 3; afterwards these women exit the study and return to routine healthcare. Women with histology <CIN2 or endocervical cytology <HSIL or negative colposcopy also exit the study and return to routine healthcare.All women exiting the study are given a report with their screening and diagnosis results and clear indications on how to continue with routine follow-up care. Study colposcopists who usually work on a hospital that covers the area selected for ESTAMPA , have committed to follow-up treated women. It has been agreed that follow-up may be done by HPV testing or Pap with colposcopy of those HPV-positive or with abnormal smears, and that the first follow-up visit should be done at 6 or 12 months in accordance with local regular care.Cervical tissues collected by biopsy or LLETZ are fixed in buffered formalin at the colposcopy clinic and are transported at room temperature to a local pathology laboratory. Tissues are processed, cut and stained under standardised study procedures. Three cuts per tissue block are mounted into: (1) a regular H&E stained slide to be used for immediate histological diagnosis by a local pathologist and corresponding clinical management; (2) an electro-charged slide for p16 immunohistochemistry (IHC) stained at request and (3) a regular slide to be later H&E stained if needed.The local study pathologist interprets the first H&E stained slide giving a diagnostic report under the CIN classification as follows: negative, atypical metaplasia, CIN1, CIN2, CIN3, AIS and invasive cancer . All slides are stored at room temperature and will be sent to IARC for final study diagnosis by a review panel when required. Blocks are stored at each site but will be available for the study, in case if additional tests to confirm or clarify the study outcome are necessary.p16 IHC to clearly define histological HSIL. Under LAST, cervical HSIL includes p16-positive CIN2, CIN3 and AIS.Until recently, histologically confirmed cervical disease was classified as CIN grades 1, 2 and 3, with CIN3 being the most reproducible cancer precursor and CIN2 being an intermediate representing a mixture of HPV infections (CIN1) and cancer precursors (CIN3). A new nomenclature describing disease of the lower anogenital tract was proposed after a consensus exercise of a large number of international experts collaborating on the LAST project. The LAST classification incorporates the current knowledge of HPV biology and the use of biomarkers to improve diagnosis and recommends the use of The primary endpoint of the study is histologically confirmed HSIL or worse lesions (HSIL+). Two secondary endpoints will be considered: (1) locally diagnosed CIN2+ as CIN2 is the current treatment threshold and (2) locally diagnosed CIN3+ as CIN3 is more representative of cervical precancer under the CIN classification routinely used. Lesions detected after initial and follow-up screening will be counted as outcomes.p16 IHC slides in addition to the regular H&E, and will give results as follows: negative, LSIL, HSIL and cancer. As local histology is reported under the CIN classification: negative, CIN1, CIN2, CIN3 and cancer; concordance will be reached for the paired results: <CIN2 and<HSIL or CIN2+ and HSIL+ .All study histology is reviewed by international experts on cervical pathology without knowledge of screening results. Reviewers may request to interpret The adjudication process involves two experts at a time. The first expert reviews the original diagnosis and only discordant diagnoses are reviewed by the second expert. Agreement diagnosis between the local and the first expert pathologist or between the two experts is considered adjudicated. When both experts do not agree, final adjudication is done in a face-to-face meeting, at which if agreement is not reached the second expert diagnosis prevails. For the purpose of the study, the final histology diagnosis could be <HSIL or HSIL+ .Performance estimates  to detect histological HSIL+ for each triage candidate alone or in combination, and their potential to be used in primary screening whenever applicable, will be estimated see . McNemarWhen evaluating tests in primary screening, relative measures of performance  will be used.Sensitivity analyses will be done to account for the impact of women not attending colposcopy or follow-up visits and those with inadequate screening results.Challenges faced when implementing HPV-based screening and different approaches used to increase study participation and adherence to screening will be reported separately.The sample size is based on the ability to detect differences in sensitivities of a paired triage tests for the detection of the primary study endpoint (histology-based HSIL+) with a type I error of 0.05 according to Connor.10.1136/bmjopen-2019-035796.supp1Supplementary dataClinical and laboratory data are collected at study centres using standardised paper forms that are entered into a centralised web-based system specifically developed for the study in Spanish, with appropriate security, privacy and automated backup system. The capture is standardised but allows site-specific customisation. The information system also monitors timely implementation, quality of inputs, progress and highlight activities where special attention is needed to guarantee the study outcome. All data are treated as confidential and kept for as long as required by law.There was no patient or public involvement in the design of this trial.The study protocol was approved by the Ethics Committee of the International Agency for Research on Cancer (IEC Project 12–27-A7), the Pan American Health Organization (PAHO) Ethical Committee and Ethical Committees in each of the study participating centres  has been established to monitor progress of the study, assure participant safety, advice on scientific conduct and analysis of the study and suggest improvements or modifications to the protocol. The DSMB is formed by international experts on HPV infection, cervical cancer and screening: a gynaecology oncologist, a medical public health specialist, an epidemiologist and a statistician, as well as, two Latin American women: a medical scientist and women’s rights advocate.Scientific reports on each triage candidate and of triage combinations, using all study data, will be published in peer-reviewed scientific journals. The DSMB will advise on the evaluation of novel emerging technologies as the study goes on. In addition, support will be given to local investigators to propose and lead analyses that could use all study data, data from one centre or from several centres.The ESTAMPA study represents a large collaboration organised among Latin American cervical cancer researchers to jointly contribute to cervical cancer prevention. About 50 000 women in the region will be screened, many of those for the first time, with a highly sensitive HPV test with efforts concentrated on treating all women with detected HSIL+.The study will contribute to establish the clinical management of HPV-positive women under different scenarios, those where high-tech molecular biomarkers can be used as triage and those where low-tech VIA may be the only suitable triage test.The introduction of HPV testing in primary screening is imminent in the region. In fact, Argentina and Mexico have been offering HPV screening for several years within the public health system. In the other countries, HPV testing is available in the private sector and is slowly becoming available or there are plans to introduce it at reduced cost in public systems. Clinical guidelines on how to follow-up HPV-positive women are under development, and the use of cytology as triage as done in HIC need to be proven acceptable in Latin America, where cytology has demonstrated to have very low sensitivity and attempts to organise cytology-based screening programmes have largely failed. More sensitive and affordable triage tests to be used in Latin America are awaited, the study will contribute with evaluating the most promising ones.A few limitations of the study design should be mentioned. First, capacity (personnel and facilities) differences among study centres could influence results. To prevent this, special attention is given to training health professionals involved in the study: clinicians collecting samples, colposcopists, pathologists, molecular biologists among others. The training includes good clinical practices, research governance and up-to-date specialty training to standardise procedures all over the study. Second, we are not performing colposcopy nor collecting biopsies in HPV-negative women, potentially introducing verification bias for evaluation of techniques in primary screening but not in triage. However, as cotesting with Pap is being done, and although the rate of cytological abnormalities among HPV-negative women will be usually low, we will have a small group of them having colposcopy helping us to estimate disease among HPV negatives. We also do not collect random biopsies at colposcopy when acetowhite lesions are not evident, thus, it is possible that some lesions are being missed. To minimise missing lesions, the study protocol includes: (1) 2–3 biopsies of any acetowhite areas observed are collected at colposcopy with ranking of severity of biopsies collected to further evaluate the use of multiple and severity of biopsy collection; and (2) a second round screening at 18 months for women with no evident disease at initial screening, providing an additional opportunity of detecting any missed disease at initial screening.The main strengths of our study are: (1) the design will allow the evaluation of a series of triage techniques without influencing the outcome of the study, as tests are performed/evaluated usually after cervical disease status of HPV-positive have been determined; (2) the sample size will allow the evaluation of multiple combinations of techniques and to adjust for centres heterogeneity when needed; (3) the study is centrally coordinated by IARC with participation of 12 study centres, each of them with strong expertise in different cervical screening aspects  and oppoIt is also important to highlight that the study is mostly being run within public health services already in place, with the exception of HPV testing that has been implemented in some university or hospital laboratories for the study. Thus, the study will substantially contribute to further scale-up of HPV testing as recommended by Pan American Health Organization by developing a ‘screening platform’ for implementation of HPV-based cervical screening programmes in the future.


Sneathia, Prevotella, and Megasphaera. In addition, the correlation analysis demonstrated that twice as many women with non-Lactobacillus-dominant vaginal microbiota were infected with oncogenic HPV types, compared with L. crispatus-dominated vaginal microbiota. The data suggest that HPV infection, especially oncogenic HPV types, is strongly associated with a non-Lactobacillus-dominant vaginal microbiota, regardless of age and vaccination status.Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases. To define the HPV-associated microbial community among a high vaccination coverage population, we carried out a cross-sectional study with 345 young Swedish women. The microbial composition and its association with HPV infection, including 27 HPV types, were analyzed. Microbial alpha-diversity was found significantly higher in the HPV-infected group , compared with the HPV negative group. The vaginal microbiota among HPV-infected women was characterized by a larger number of bacterial vaginosis-associated bacteria (BVAB),  HPV infection is the main cause of cervical cancer and is related to many other cancers, including head and neck cancer3. Depending on their oncogenic potential, mucosal HPV types can be divided into oncogenic HPVs, such as those observed in cancer cases, and non-oncogenic HPVs, mainly found in condyloma4. The two most common HPV types in cervical cancer are HPV16 and 18, which are responsible for ~70% of cervical cancer cases worldwide6. At a youth clinic in Stockholm, Sweden, we have previously shown an overall cervical HPV prevalence of over 70% among young girls in Sweden9. The HPV vaccination program was gradually introduced to Sweden from 2007. Since 2012, all girls between the ages of 10 and 12 years are offered free vaccination with the quadrivalent Gardasil vaccine against HPV6, 11, 16, and 18, in a school-based vaccination program and catch-up vaccination. The vaccination ratio has increased dramatically from 10.7% (2008–2010) to 82.1% (2017–2018)7. The prevalence of HPV types covered in the vaccine has dropped significantly in vaccinated women compared with non-vaccinated women, underlining the importance and success of the vaccination program8. However, the total HPV prevalence caused by HPV types that are not covered by the vaccine is still high, indicating that more interventions to reduce these HPV infections are still needed8. Further, the influence of the HPV vaccine on vaginal microbiota has not been thoroughly investigated, especially in a high vaccination coverage country.Infection with human papillomavirus (HPV) is among the most common sexually transmitted diseases in the world, with the highest prevalence among women below 2513. The vaginal microbiota is primarily dominated by one of the four most common Lactobacillus species: Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii16. In addition, some women may have vaginal microbiota dominated by bacterial species other than Lactobacilli, such as Prevotella, Gardnerella, and Sneathia18. The general clinical diagnostic approaches worldwide for bacterial vaginosis (BV), which is also characterized by a lack of Lactobacilli but a higher quantity of aerobic and anaerobic bacteria, are the Amsel criteria and the Nugent score, based on wet smear diagnosis and Gram staining. However, the sensitivity and specificity for both methods are moderate19. Molecular diagnosis, such as 16S rRNA gene sequencing, enables the microbiota determination at the species level. Bacterial vaginosis associated bacteria (BVAB), including BVAB 1, 2, and 3, have been identified from the vaginal fluid of women with bacterial vaginosis and could serve as potential vaginosis biomarkers22. Unfortunately, BVAB have not been included in the 16S amplicon sequencing-based vaginal microbiota studies related to HPV, probably due to taxonomic information missing from the popular 16S rRNA databases, with most studies on BVAB being based on qPCR sequencing.An increasing number of studies suggest that vaginal microbiota play an essential role in women’s health, specifically in sexually transmitted diseases, pelvic inflammatory disease, and adverse obstetric outcomesL. crispatus is observed more frequently in women without HPV infection and cancer lesions, whereas L. iners and non-Lactobacillus species are more common in HPV-infected women and patients with cancer lesions26. However, there are no data available yet in the Nordic countries on the vaginal microbiota composition and its relationship with HPV infection, using a sequencing method. Thus, we initiated a cross-sectional study to assess the association between the vaginal microbiota and 27 HPV types in Sweden. In addition, because HPV infection has the highest prevalence among young women, we designed our study to focus on women below 30 years old.Cross-sectional studies and very few longitudinal studies from other countries showed that n = 18) or incomplete clinical information (n = 15). Samples with low DNA concentration and low reads in sequencing (n = 55) were excluded from downstream microbiota analysis. Eventually, 169 samples from women visiting the youth clinic and 88 samples from women attending the cervical screening were included for analyses , while Chao 1 analysis showed a significant difference between the two sources  based on the Bray-Curtis distance demonstrated that all the samples were mainly separated into ies Fig. . Detail L. crispatus and L. iners were found to be the most dominant species among both HPV-uninfected and HPV-infected participants  , but no significant difference among other groups was observed. The PCoA analysis showed no clear separation of samples with HPV-uninfected, infected with both oncogenic and non-oncogenic HPVs, and only oncogenic HPVs , women infected with HPV39, 42, and 56 had significantly higher diversity compared with HPV-uninfected group in the Shannon analysis, which indicates that infection by these three HPV types tends to be related to higher diversity in vaginal microbiota , compared with women below 18, and women from the youth clinic , compared with the cervical screening samples . After adjustment for the other variables listed in the table, the risk of oncogenic HPV infection was OR = 2.0 (95% CI 1.0–3.9) in non-Lactobacillus-dominated samples, compared with L. crispatus-dominated samples. The difference became more pronounced when only women with multiple HPV and multiple oncogenic HPV types were included in the analysis . Although our study lacks clinical data such as vaginal pH or data on vaginal infections other than HPV, compared to other vaginal microbiota studies, our study design limits the effect from confounders such as the variability of hormone and immunity levels due to a large age span. Further, this young group gives us a unique group with high HPV infection prevalence7. The samples from cervical screening, aged 23 years and above, have an HPV prevalence of 34.1% 7. Their samples were collected during the clinical visit either by clinical staff or self-collected with a vaginal swab . Vaginal swab was Inserted ~2–3 cm into the vagina and swirled for ~30 s. In addition, 139 study participants between 23 and 29 years were enrolled from the population-based cervical screening at maternal health clinics (n = 133), and from follow-up screening at the gynecological clinic, Uppsala university hospital (n = 6), Uppsala, Sweden. These samples were collected by clinical staff and referred to as the cervical screening samples. Collection methods were compared prior to the study and showed no difference in microbiota composition33. For all the samples, the collection swabs were immediately put inside FluidX tubes  containing 0.8 ml DNA/RNA-shield . Information as to age, HPV vaccination status and antibiotic usage within the past 3 months was ascertained from study participants. All participation was voluntary and anonymized, and written informed consent was obtained. This study was performed according to ethical permission approved by the Stockholm Regional Ethics Committee and Uppsala Regional Ethics Committee in Sweden.In total, 172 previously described young women and 34 new participants, between the ages of 14 and 22, who visited a youth clinic in Stockholm were included  as the negative and positive extraction controls, respectively. The purified DNA was stored at −20 °C until further HPV genotyping and microbiota sequencing.The collection fluid containing the vaginal microbiota was bead-beaten with Matrix E beads , followed by digestion with Proteinase K  and purification with magnetic beads according to the manufacturer’s guidelines for the ZR-96 Genomic DNA MagPrep kit , as previously describedDNA was polymerase chain reaction (PCR) amplified with broad-spectrum GP5+/6+ primers targeting the HPV L1 region, as well as HPV16 and HPV33 primers targeting the E6 region. The HPV16 E6 primers consist of HPV16E6-Forward (TCAAAAGCCACTGTGTCCTGA), HPV16E6-3-Reverse35. The PCR conditions contained initial denaturation at 94 °C for 15 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 38 °C for 90 s and extension at 71 °C for 80 s, and a final extension step at 71 °C for 4 min. The PCR product was further evaluated using a multiplex bead-based assay on a MAGPIX instrument , as reported previously7. Probes for detecting HPV16 E6p (GTCTTGTTGCAGATCATCAAG) and HPV33E6p (GAAACTGCACTGTGACGTGTA) were added35. Twenty-seven types of HPV were analyzed, including fifteen oncogenic HPVs , six probably oncogenic HPVs , and six non-oncogenic HPVs 7.(GCTGGGTTTCTCTACGTGTTC). HPV33 E6 primers including HPV33E6-Forward (TCGTTGGGCAGGGCGCTGTG), and HPV33E6-Reverse (CTCGTGTCCTCTCATGGCGTT) as previously described33. For each batch of sample sequencing library preparation, we included DNA-free water and Zymo Microbial Community DNA Standard  as the negative and positive PCR controls, respectively. Thereafter, the 16S amplicons indexed from the samples and controls (including extraction and PCR controls) of the same batch were pooled together in equal molar amounts and subjected to an Illumina MiSeq sequencing platform  with MiSeq Reagent Kit v3.The V3-V4 regions of the 16S rRNA genes were amplified from extracted DNA with Illumina sequencing index-binding primer pairs 341F/805R, as previously describedq value of 15, maximum N base of zero, and minimum qualified read length of 12036. Thereafter, the processed reads were sent to DADA2 pipeline (v. 1.13.1) for further quality trimming, de-noising, merging, and chimera removal. In this step, the paired-end reads that have more than two expected errors ) or derived from PhiX (rm.phix = TRUE) were discarded37. The low-quality tail from each read was trimmed off to keep the mean q value >20 ). After DADA2 de-noising, the paired-end reads were merged with at least a 30-bp overlap. Chimera checking was conducted on the merged reads and the recovered ASVs were summarized and used to generate the sequence table for the sequencing run. The sequencing content from the sequencing library controls was used to assess the performance of each sequencing run. Finally, all sequence tables of the qualified sequencing runs were parsed into a total sample sequence table, by collapsing rows of the identical ASVs from the raw sequence tables.After base calling and de-multiplexing, the fastq files generated from each run were subjected to quality trimming, de-noising, merging, and chimera removal, before being processed into sequencing tables. The degenerated primers were firstly trimmed off from the paired-end reads and a preliminary quality trimming was then conducted with cutadapt (v. 2.0) with the setting of minimum 38. ASVs that show positive correlation in their reads fraction and DNA concentration in negative controls but not in samples, and ASVs that have high prevalence in controls but not in samples, were discarded. After the decontamination step, the controls were excluded from the sequence table for downstream data analysis.In order to remove the ASVs from sequencing backgrounds , a decontamination procedure was conducted on the merged sequence table, containing both samples and controls. As the substantial fraction of DNA from vaginal swab samples is human DNA, we employed a combined method considering both frequency and prevalence, to identify contaminant ASVs by using Decontam (v. 1.1.2) on R39. As Lactobacillus spp. are dominant in most of the vaginal microbiota, we improved the classification of the ASVs annotated as Lactobacillus by manually BLAST searching them in the National Center for Biotechnology Information (NCBI) database41. Moreover, bacterial vaginosis-associated bacteria (BVAB) 1, 2, and 3 were searched from ASVs containing 16S primer-binding regions in the whole genomic sequences and 16S sequences downloaded from other published works48. The classification of ASVs was further validated by searching BVAB qPCR primer sequences from the BVAB ASVs49. For the classification of Lactobacillus species and BVAB, a maximum of one mismatch was allowed from the alignment.A preliminary classification was conducted in R by using DADA2 functions “assignTaxonomy” and “addSpecies” to search ASVs against the Silva database (v. 128)To facilitate the calculation of Faith’s diversity and UniFrac distance matrix for the downstream analyses, a phylogenetic tree of the ASVs generated in this study was constructed. A multi-alignment of all ASVs was firstly conducted using MAFFT (v.7.407) with the default parameters. The phylogeny was determined using FastTree2 (v.2.18). The generated tree in newick format was then loaded to FigTree for visualization and double-checking whether the phylogenies of the ASVs correspond to their taxonomic annotations.The sequence table was rarefied to 4902 reads per sample, before conducting a diversity calculation in R by using the package “vegan” (v. 2.5-6). Alpha diversities  were calculated using the functions from the same package, which were visualized in violin plots using the package “ggplot2” (v. 3.2.1). PCoA was conducted based on Bray-Curtis distance matrix and weighted UniFrac distance matrix, using the package “phyloseq” (v. 1.26.1).To test the differences among microbial alpha diversities, the Wilcoxon rank-sum test and Kruskal–Wallis test were employed when comparing diversities between two groups and more than two groups, respectively. Benjamini–Hochberg correction was conducted to decrease the false discovery rate for multiple tests. The same test method was also conducted to identify taxa that are significantly different in proportional abundance between the cohorts. The association between microbiota community structure and HPV, oncogenic HPV, or multiple HPV types was analyzed by multivariable logistic regression, adjusted for age, HPV vaccination status, sample source, and presented as OR and 95% CI, with 95% CI not including OR = 1, indicating statistically significant differences. The relationship among HPV infection, vaginal microbiota, age, sample source, and vaccination status were compared by Fisher’s exact test.Further information on research design is available in the Supplementary InformationReporting Summary


Chikungunya virus (CHIKV), a serious health problem in several tropical countries, is the causative agent of chikungunya fever. Approved antiviral therapies or vaccines for the treatment or prevention of CHIKV infections are not available. As diverse natural phenolic compounds have been shown to possess antiviral activities, we explored the antiviral activity of α-Mangostin, a xanthanoid, against CHIKV infection.The in vitro prophylactic and therapeutic effects of α-Mangostin on CHIKV replication in Vero E6 cells were investigated by administering it under pre, post and cotreatment conditions. The antiviral activity was determined by foci forming unit assay, quantitative RT-PCR and cell-based immune-fluorescence assay. The molecular mechanism of inhibitory action was further proposed using in silico molecular docking studies.In vitro studies revealed that 8 µM α-Mangostin completely inhibited CHIKV infectivity under the cotreatment condition. CHIKV replication was also inhibited in virus-infected mice. This is the first in vivo study which clearly showed that α-Mangostin is effective in vivo by significantly reducing virus replication in serum and muscles. Molecular docking indicated that α-Mangostin can efficiently interact with the E2–E1 heterodimeric glycoprotein and the ADP-ribose binding cavity of the nsP3 macrodomain.The findings suggest that α-Mangostin can inhibit CHIKV infection and replication through possible interaction with multiple CHIKV target proteins and might act as a prophylactic/therapeutic agent against CHIKV.The online version contains supplementary material available at 10.1186/s12985-021-01517-z. Chikungunya fever (CHIKF), caused by chikungunya virus (CHIKV) is frequently associated with a high prevalence of chronic arthralgia. After the acute phase, polyarthritis can be recurrent and may persist for several years after infection . This isSimilar to other members of the alphavirus genus, the CHIKV starts its life cycle by entering the target host cells via receptor-mediated endocytosis . The virGarcinia mangostana pericarp (GMP) extracts, including α-Mangostin, β-Mangostin and γ-Mangostin have been better elucidated [Garcinia mangostana (Mangosteen) Linn belongs to the Clusiaceae family and is cultivated mainly in Malaysia, Thailand, Indonesia, Philippines, Sri Lanka and India [Natural polyphenolic compounds like flavonoids and xanthonoids possess wide ranging bioactivities such as anti-inflammatory, anti-oxidative, anti-bacterial, anti-fungal as well as antiviral activities –16. Amonucidated . Garcinind India . α-Mangond India , 22. Thend India . There aVero E6 cells and the CHIKV strain of African genotype  were used. Modified Eagle’s medium was used to maintain the Vero E6 cell line supplemented with 5% Fetal bovine serum  and anti mycotic antibiotic solution . α-Mangostin, a natural compound used for this study was obtained from the National MolBank compound repository of CSIR-IICT, Hyderabad. The purity of the compound was determined to be 83% by HPLC.For induction of chikungunya disease, female C57BL/6 mice (n = 9) of 4–5 weeks age were obtained from the institutional animal breeding facility. Animals were randomly selected, marked to permit individual identification, and kept in their cages for at least one week prior to the start of the dosing to allow for acclimatisation to laboratory conditions. All animal experiments were done in a biosafety level-2 animal facility at the ICMR-NIV and the study was approved by the Institutional Animal Ethics Committee (IAEC) and Institutional Bio-safety committee . Animal To evaluate the cytotoxic effect of α-Mangostin on Vero E6 cells, MTT assay was used as reported previously .For studying the effect of pretreatment of cells with α-Mangostin, the cells were pretreated with different concentrations of the α-Mangostin for 4 h followed by infection, while in case of cotreatment, the virus was mixed with different concentrations of α-Mangostin and immediately used for infecting the cells for 1 h. From the virus stock with the known titre of FFU/ml, a multiplicity of infection (M.O.I) of 0.01 was used in all the antiviral assays. After infection, the culture supernatant was replaced and incubated for 24 h. To study the effect of α-Mangostin post infection, the cells were first infected and after 1 h incubation, the virus was removed and 2 washes of sterile PBS was given to cells to remove unbound virus particles. After washing, various concentrations of α-Mangostin were added to wells according to standard protocol in triplicates and incubated for 24 h. After 24 h, the plate was freezed for further assays. The vehicle control (VC) contained DMSO diluted with the working media in the same ratio as in the highest concentration treatment group (8 µM). In all conditions, after the incubation, the culture supernatant was assessed for viral load in terms of viral genomic RNA levels by real time RT-PCR and infectious virus particles by focus forming unit (FFU) assay. The magnitute of the change in viral load was measured on a logarithmic scale and log reduction to percentage reduction was calculated as reported previously . For assFFU assay was performed as reported earlier with modifications . The pri5) were seeded in a 24 well plate  with a coverslip placed in each well. The cells were allowed to form a confluent monolayer and were infected with CHIKV and incubated for 12 h. After incubation, the immunofluorescence assay (IFA) was performed as reported in a previous study [Approximately equal number of Vero E6 cells . One step real time RT-PCR was performed as reported previously . The pri. Mice were infected with 1 × 106 pfu of CHIKV by intramuscular (i.m.) route and viral RNA copies were assessed in serum and muscles on the 3rd, 5th, and 7th day post infection (dpi). α-Mangostin was diluted in normal saline with 0.5% sodium salt of carboxy methyl cellulose (CMC) as the vehicle, and two doses, 20 mg/kg and 40 mg/kg, were administered at 1st, 2nd and 3rd dpi via the intraperitoneal (ip) route. As per the recommended volume (10 ml/kg), of a vehicle for the IP route, the mice (body weight ~ 25 gm) were administered 250 µl (1.25 mg) of CMC solution. Mice from the VC group received the same amount of the CMC solution without mangostin to observe any possible effect of the vehicle. Sodium carboxy methyl cellulose was used since it is a good dispersing agent, viscosity modifier and emulsifier [Female C57BL/6 mice of 4 week age were used to investigate the anti-CHIKV activities of α-Mangostinulsifier . At the Histopathological evaluation was performed on the muscle tissues of the hind limb from the Normal , CHIKV infected , and CHIKV infected mice treated with different doses of α-Mangostin (20 mg/kg and 40 mg/kg). Hind limb tissues, excluding the femur bone, were fixed in 10% formaldehyde as per the standard procedure and processed for histopathology. Paraffin sections of 5 µm thickness were prepared, stained with haematoxylin and eosin.https://zhanglab.ccmb.med.umich.edu/LOMETS/). The modeled protein structures were further validated using PROCHECK (http://servicesn.mbi.ucla.edu/PROCHECK) and ProQ (https://proq.bioinfo.se/cgi-bin/ProQ/ProQ.cgi).The 2-dimensional structure of α-Mangostin was generated using ChemDraw® Professional 16.0.1.4 software. All available crystal structures of the CHIKV target proteins in the Protein Data Bank (PDB) including the CHIKV envelope glycoprotein complex in its mature form (3N42.PDB), CHIKV nsP3 macrodomain bound with ADP-ribose (3GPO.PDB) and the CHIKV peptidase C9 domain of nsP2 (3TRK.PDB) were retrieved. For modeling the structures of the nsp1 v-methyltranseferase domain, nsp2 helicase domain and nsp4 RdRp domain, the protein sequences of the CHIKV targets were obtained from the NCBI GenBank (accession nos. ARB19731). As no homologous protein structure templates were available in PDB for these CHIKV proteins, all structure modelling studies were carried out using the on-line Local Meta-Threading-Server LOMETS, for protein structure prediction   (p < 0.001). When the virus was mixed with α-Mangostin and used for infection (cotreatment) 100% reduction of the virus titer was noted at 8 µM concentration  (p < 0.001).Vero E6 cells were infected with CHIKV and treated with different concentrations  of the α-Mangostin for before, after and simultaneous infection and the FFU assay and Quantitative RT-PCR was performed at 24 h. In FFU assay, a dose dependent reduction in viral titers with respect to CHIKV was noted for pretreatment, co-treatment and post-treatment conditions  decrease in copy number of CHIKV RNA for pre and cotreatment conditions using 8 μM α-Mangostin respectively compared to the untreated cells while a < 1 log reduction in the CHIKV RNA copy number was observed under post-treatment conditions (p < 0.05 for 4 µM) and (p < 0.001 for 8 μM)  of viral RNA compared to untreated animals (VC) (p < 0.05). At 5th dpi, reduction in viral RNA copy number with the low dose of α-Mangostin was 1.8 log10 (p < 0.01) and with the high dose of α-Mangostin group was 2.23 log10 (99.4% reduction) (p < 0.001). At 7th dpi, with the high dose treatment, the reduction in viral RNA was retained at the same level (2.22 log10) compared to VC (p < 0.001)  CHIKV RNA copies respectively in the tissue supernatant. At 5th dpi, 2.44 log10 and 3.1 log10 (99.91% reduction) reduction in CHIKV RNA copies was observed in mice treated with low dose and high dose of α-Mangostin. On 7th dpi. no significant reduction in viral RNA was observed in the low dose α- Mangostin treated mice while in high dose α- Mangostin treated group, reduction of 3.4 log10 (99.96% reduction) CHIKV RNA copies was observed compared to VC group.The reduction in the CHIKV copies in muscle tissue from α-Mangostin treated mice was observed from day 3  compared to muscle tissues of normal mice. Compared to the control , presence of mononuclear inflammatory cellular infiltration in between the muscle bundles and coagulative and degenerative changes in the muscle fiber (with loss of continuity in length) are clearly observable in magnified close-ups while these signs are improved in the dose of 40 mg/kg treated α-mangostin group. Treatment with low dose α-Mangostin showed improvement in inflammatory signs in muscle tissue at 7th dpi compared to VC group. High dose α-Mangostin treated mice muscle tissues showed the regeneration after treatment (from 5 dpi onwards) (Magnification X100)  and stabilize the complex with strong non-covalent molecular interactions. Hydrogen bond interactions were noted with E1: Tyr233, Tyr51 and E2: Tyr237. While pi-alkyl interactions were observed between E2: Arg36 and the cyclic ring of the ligand. Alkyl interactions were formed between E2: Pro240, E1: Ile55,Lys 241 and the prenyl group of α-Mangostin  as it completely lacked interaction with the catalytic dyad residues Cys 1013 and His1083 and only one hydrogen bonding interaction was observed with Trp1084 which was in the immediate vicinity to the catalytic residue  which is known to play an important role in enzyme activity  which is. Hence, In summary, this study for the first time reports anti-CHIKV potential of α-Mangostin in an in vivo system. The in vitro findings indicate that α-Mangostin can inhibit CHIKV, under pre and cotreatment conditions. In silico studies suggested that α-Mangostin can efficiently interact with multiple CHIKV proteins during different stages of the virus life cycle. Our findings clearly demonstrated that α-Mangostin treatment in CHIKV infected mice reduces viral burden and might help to alleviate disease symptoms in CHIKV-infected mice. Proof of the mechanism of action through likely interactions with the CHIKV mature envelope protein complex and nsP3 macrodomains need to be corroborated by measuring virus internalisation, vesicle acidification or through nsp3-specific functional assays.Overall the findings from the present study may form the basis to support clinical trials to investigate the possibility of using α-Mangostin to treat chikungunya fever in patients.Additional file 1: Figure S1. Cytotoxicity of α-Mangostin against Vero E6 cells. MTT assay was used to evaluate the cytotoxicity of the compounds. Vero E6 cells were cultured with different concentrations of α-Mangostin for 24 hours. After incubation, MTT solution was added and incubated in the dark at 37°C for 3 h with 5% CO2.. After incubation, the medium was discarded and 100 μL of acidified isopropanol was added to each well and incubated at 37°C for 1 h. The readings were taken in a microplate reader at a wavelength of 570 nm with reference filter at 690 nm. Percentage cytotoxicity or viability was calculated in comparison with cells untreated with α-Mangostin. All experiments were conducted in triplicates.Additional file 2: Figure S2. Molecular interaction of α-Mangostin with nsP2 peptidase C-9 domain (3TRK.pdb) (a) Solvent surface view (probe radius 1.8Å) of docked pose of α-Mangostin (in blue stick model) with nsP2 peptidase C-9 domain (b) 2D interaction diagram of α-Mangostin with nsP2 peptidase C-9 domain showing different intermolecular interactions. All the interactions are visualised and analysed using Biovia Discovery studio client 2017.Additional file 3: Figure S3. Molecular interaction of α-Mangostin with modeled CHIKV targets. The solvent surface rendered view (probe radius 1.6Å) and 2-dimensional interaction diagram showing α-Mangostin (in blue stick model) interacting with (a) nsP2 helicase domain (b) nsP4 RdRP domain and (c) nsP1 methyltransferase domain. All the interactions are visualised and analysed using Biovia Discovery studio client 2017.


People living with HIV (PLHIV) are at a high risk of developing HPV-related cancers. HPV-related malignancies occur frequently and/or are high among PLHIV, with cervical cancer as a designated AIDS-defining condition. We aimed to explore the evidence on the interplay of HIV and HPV-related cancers in sub-Saharan Africa (SSA).The scoping review was guided by Arksey and O’Malley’s framework. We searched for literature from the following databases: PubMed; World Health Organization (WHO) Library; Science Direct; Google Scholar and EBSCOhost . Studies reporting on evidence HIV and HPV-related cancers interplay in SSA were eligible for inclusion in this review. The Mixed Methods Appraisal Tool (MMAT) tool was used to assess the risk of bias of the included studies. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) was used for reporting the search results. Thematic analysis used to reveal the emerging themes from the included studies.A total of 74 potentially eligible articles were screened. Of these, nine  were eligible for data extraction. The studies reported about a total of 16,351 participants in different settings. The nine included studies showed evidence of cervical cancer among HIV-infected women and distribution of HPV infection and cervical abnormalities among HIV-positive individuals. In the four studies generalizing about HIV and anal cancer, only one reported about HPV. Two studies generally reported about HIV and head and neck cancers and one reported about interaction of HIV with vaginal cancer, vulvar cancer, and penile cancer, respectively.HIV positivity is associated with increased prevalence of HPV infection on different anatomic sites, which will result in increased burden of HPV-related cancers among PLHIV. Furthermore, primary studies with robust study designs aimed at investigating the risk developing HPV-related cancers among PLHIV are recommended.Systematic review registration: PROSPERO CRD42017062403 The International Agency for Research on Cancer (IARC) classified both human papillomavirus (HPV) and human immunodeficiency virus (HIV) type 1 as carcinogens , 3. IndiAs evidenced by the HIV and AIDS Cancer Match Study, cervical cancer incidence among HIV-infected women in the USA has been unchanged since the introduction of ART at 64.2 cases per 100,000 person-years from 1990 to 1995 compared to 86.5 cases per 100,000 person-years from 1996 to 2002 . From coGardasil) or the bivalent (Cervarix) HPV vaccine [The introduction of HPV vaccine in the immunization programs in high-income countries had played a key role in the reduction of the incidence of cervical cancer using either the quadrivalent  Library, Science Direct, and Google Scholar, and within EBSCOhost platform, the following databases were searched: Academic Search Complete, Health Source: Nursing/Academic Edition, and CINAHL with full text for published studies. The search took place from April 2017 to July 2017. We used the following keywords on the search databases: HPV-related cancers, HIV, prevalence, incidence, and sub-Saharan Africa (to search titles of the eligible studies). Boolean terms AND/OR were used to separate the keywords during the search. There was no language and date restriction. Medical Subject Headings (MeSH) terms were included in the search. The literature search strategy in the databases was developed by KMBL in consultation with a librarian. Reference sections of included full articles were reviewed to identify additional eligible articles. Detailed description of the database search strategy is attached in Additional file Firstly, KMBL searched the eligible titles guided by the eligibility criteria from the databases and imported them to a new Endnotes X7 library created for this scoping review. Duplicates were removed before the start of the abstract screening phase. Both abstract and full article screening tool were developed in Google forms using the inclusion criteria. Secondly, two skilled reviewers (KMBL and DK) screened all retrieved abstracts against the eligibility criteria. To be selected for the next stage of the review, a study had to be conducted among humans and report of data on HIV and assessment of the interplay with HPV-related cancers are needed. If the reviewer was uncertain of the eligibility of the study population while the intervention, outcome, and study setting were eligible, the article was not excluded but rather carried onto the next stage of the selection process. Disagreement at this stage were resolved via discussions until a consensus was reached. Again, two independent reviewers (KMBL and DK) did a full article screening and a third reviewer (TPM-T) was involved to resolve the disagreements. To measure the inter-rater agreement between reviewers, we used Cohen’s kappa coefficient (κ) statistic using Stata 13.0SE .Studies involving individuals of all agesStudies focused on HIV and HPV-related cancersStudies reporting evidence from SSA countries , Congo .Quantitative study designsThis study included the following:Studies that do not focus on HPV-related cancersStudies that did not focus on HIV and HPV-related cancersStudies that reported about HIV onlyStudies that reported evidence from countries outside SSAThis study excluded the following:We abstracted the data from the included studies using a piloted form designed in Google forms. The following information was extracted from included studies: author and date, study setting, intervention, age, percentage of males/females, gender, study aim, and study designs.Cervical cancerAnal cancerVaginal and vulvar cancersHead and neck cancersPenile cancerThematic analysis was performed to identify the interplay of HIV and HPV-related cancers in SSA from the included studies. The included manuscripts were manually coded into categories, which were grouped into the following five themes:The quality assessment of included studies was performed using Mixed Methods Appraisal Tool (MMAT)–Version 2011, with the purpose of evaluating the risk of bias . Two revp value < 0.05). In addition, McNemar’s chi-square statistic suggests that there is not a statistically significant difference in the proportions of yes/no answers by the reviewer with a p value > 0.05  –96 and (The studies were done and reviewed from different study settings. About 11.1% (1/9) study generalized about SSA with no specific country mentioned , 44% , except for one which generalized on children and adolescents . The ageA total of 120 studies were excluded from this study as they did not meet the inclusion criteria for this study. Of those 74 studies that underwent full manuscript review, 65 of them were found to have no valuable data for analysis in this study. A total of 53 articles did not report anything about study setting of interest which is SSA –79, 7 diFrom the included ten studies, two primary studies underwent methodological quality assessment using the appropriate section of the Mixed Methods Appraisal Tool (MMAT)–Version 201 1. Both stThe scoping review revealed findings about HPV-related cancer and their interplay with HIV. These cancers are cervical cancer, anal cancer, vulvar and vaginal cancers, head and neck cancers, and penile cancer.The nine included studies reported about cervical cancer, HPV, and HIV –98. The n = 43) compared to the HIV-negative group. Additionally, Hr-HPV infection was significantly high (27/86 = 31%) in HIV-positive women compared to HIV-negative women (20/86 = 23%). The study further highlighted that among the HIV-negative group, one woman with HPV and no lamin A/C developed cervical lesions and two women with no HPV but with total absence of lamin A/C developed cervical lesions [Recent review performed by Palesky has shown that HIV positivity is associated with increased prevalence of cervical HPV infection and cervical intraepithelial neoplasia (CIN) . Further lesions . A revie lesions .A review by Firnhaber et al. showed that HIV-infected women have a higher prevalence of HPV infection and cervical abnormalities compared to HIV-seronegative women throughout the world , and thiFive of the included studies generally reported about anal cancer, HPV, and HIV , 96, 98.One of the nine included studies reported about vulvar and vaginal cancers and HIV . A reporTwo from the included studies generalized about oropharyngeal cancer and HIV , 98. It One of the nine included studies generalized about penile cancer and HIV . AccordiThe study was aimed at exploring the evidence on the interplay of HIV and human papillomavirus-related cancers in SSA. This scoping review provided a general overview and evidence on the interplay of HIV with cancer of the cervix, anus, vulvar, vaginal, penile, and head and neck in SSA. This scoping review presented evidence from six countries within SSA; only two studies were conducted in urban areas and not specified in most of the studies. Again, generally there is a lack of studies conducted specifically on males as opposed to those conducted on females. The findings demonstrated a gap in literature on HPV-related cancers among HIV-infected individuals in many countries within SSA and lack of research in rural areas and poor participation among men. Our results show that there is limited evidence to confirm the presence of HPV genotypes on different anatomic cancer sites among PLHIV. It also shows scarcity of studies aimed at evaluating the risk of developing HPV-related cancers among HIV-infected people. Although evidence on interplay of HIV between vulvar cancer, vaginal cancers, head and neck cancers, and penile cancer has been identified, it was not sufficient to make a generalization and conclusion due to the limited evidence.This study shows that the prevalence of cervical HPV infection and CIN is increased with HIV positivity . In agreThis study has been shown that knowledge on the prevalence of anal cancer in HIV-infected men and women in resource-limited countries is very limited, yet on the other hand, HPV-related anal cancer is increasing in both HIV-infected women and men despite ARV treatment . In lineDespite the limited literature in SSA, in the USA, the estimated cancer risk among HIV-infected individuals as compared to the general population has been estimated to be as high as 29 times increased for anal cancer, 4 times for penile cancer, 6 times for vulvar and vaginal cancer as well as cervical cancer, respectively . The finAn important strength of this study is that there has been a comprehensive search of relevant studies to be included in this scoping review with no limitation on publication dates and language. This scoping review methodology has allowed for the inclusion of different study designs, and a systematic approach was used to identify relevant studies, charting, and analysis of outcomes , 23. StuDespite the strengths reported for this study, there is a limitation as well. The study was limited with the exclusion of qualitative studies as the research question is more epidemiological, hence the use of PEO framework. Qualitative research was not qualified to be included in this scoping review; therefore, it might have resulted in omission of data.This study shows limited evidence on the interaction of HIV and HPV-related cancers especially on anal cancer across all genders, vaginal cancer, vulvar cancer, head and neck cancers, and penile cancer. We hope this study results will prompt further studies to provide better insights on the natural history of HPV-related cancers. More studies to evaluate the risk of developing HPV-related cancers among PLHIV are recommended. Additionally, more future studies to be conducted at different countries within SSA and in rural areas are needed. It also calls for recruitment of more males to participate in research.This study revealed the need for policy makers in public health to take note on the development of HPV-attributable cancers among PLHIV when planning and making decisions, bearing in mind that HIV-infected individuals who live longer on ART may be at increased risk of persistent HPV infection . Our stuThis study enabled the authors to provide evidence on the interplay of HIV and HPV-related cancers in SSA. Although this study demonstrated shortage of evidence from published studies on the interaction between HIV and HPV-attributable cancers on all identified anatomic sites, high number of studies reported the association of HIV positivity with increased prevalence of HPV infection on different anatomic sites, which will result in increased burden of HPV-related cancers among PLHIV. Therefore, a meta-analysis study is recommended to confirm this association. Furthermore, primary studies with robust study designs aimed at investigating the risk of developing HPV-related cancers among PLHIV in SSA are recommended. Furthermore, primary studies with robust study designs aimed at investigating the risk of developing HPV-related cancers among PLHIV in SSA are recommended.Additional file 1: Table S1. Electronic database search results for title screening.Additional file 2: Table S1. Calculation of degree of agreement.Additional file 3: Table S1. MMAT quality assessment tool.Additional file 4: Table S1. PRISMA checklist.


The 9th meeting of Advances Against Aspergillosis in beautiful Lugano, Switzerland clearly had the most drama of any of the previous meetings, exceeding even the 1st one, in San Francisco, when we, the Co-Organizers, weren’t sure that although we had a great educational idea, and had put together a great list of speakers and topics, we might have few attendees, and go bankrupt! ; those 267 papers comprised 2026 pages of full papers. The full papers are intended to be largely reviews. The abstracts of all accepted submissions are published in the program distributed at the time of the meeting (there have been 1594 such abstracts to date). Registration records from past meetings have indicated the attendees are infectious disease specialists, hematologists, oncologists, transplantation specialists, allergists, chest physicians, intensivists, other internists and pediatricians, clinical microbiologists, pharmacists, toxicologists, financial and pharmaceutical industry and hospital executives, governmental employees in healthcare and regulatory fields, veterinarians, environmental specialists, public health specialists, mycologists, immunologists, geneticists, epidemiologists, plant pathologists, other basic scientists, nurses, Fellows, Residents, and MD and PhD students. They come from dozens of countries.Aspergillus and Mucorales are ubiquitous in nature, found on decaying matter, in soil and water, and have industrial and agricultural impacts, as well as medical . While pfections . The funAspergillus products that trigger asthma, and on the role of fungi in unresponsive asthma. Other papers deal with Aspergillus regulatory metabolic networks, and on important non-fumigatus Aspergillus species. The molecular basis of Pseudomonas-Aspergillus competition is reviewed- so important in some human lungs, as is how volatile species are part of the intermicrobial competition. Regarding the Mucorales, there are reviews relevant to Rhizopus biochemical physiology, and to host defenses against these pathogens. The critical utility of animal models in understanding pathophysiology of fungal infections, and their treatment, is covered. In the realm of diagnostics, how siderophores can be useful in clinical detection of fungal pathogens is discussed, as is the latest in in vitro drug susceptibility methodology. Finally, how the International Society for Human and Animal Mycology is exerting collaborative efforts to advance the field is made evident. The open access policy of the Journal of Fungi makes all these important works so accessible to the entire scientific community, including those who did not have the benefit of the individual personal interactions, so important in scientific meetings, in Lugano.All such diverse attendees will be interested in the subject matter of this Journal of Fungi Supplement. It is full of important compendia. There are papers detailing immunity to pulmonary mycoses, on I promised, above, a narrative about the drama: As chance would have it, the planes of two of the Co-Organizers, Bill Steinbach and I , coming from the US, and of Derry Green, in charge of the meeting planner operation  coming from the UK, arrived in Zurich airport, just before the start of the meeting, within minutes of each other . The Zurich arrival group had arranged to rendezvous in the airport after their arrivals, and then connect by rail to Lugano, during which time we could chat leisurely about last-minute preparations, and the inevitable small last-minute issues that always come up. en masse because they were fearful of arriving in the middle of an exploding epicenter, and whether they would be allowed entry into Switzerland or transit airports en route, or fearful they would be put into quarantine once they returned to their home countries, or because their institutions had forbidden travel outside of their immediate home areas. What ensued was a series of panicky nonstop discussions, and nonstop phone calls between the Zurich arrival group and Dr. Denning, as we all tried to figure out what to do.When we had our rendezvous, we were by that time gathering information that the coronavirus was beginning to close in on us. The epicenter outside of China at the time appeared now to be northern Italy, which, in a way, logistically, surrounds Lugano. The alternate route to Zurich, in order to get to Lugano, is Milan, which was the plan for many speakers and attendees. There was talk in the media that all of northern Italy was headed for a lockdown, and that the Milan airport and train station would be soon closed. There was also news that the first 1 or 2 cases were hospitalized in Lugano. Hence we weren’t sure that there would be a partial lockdown in Lugano, even taxis in Lugano after our rail arrival to get us to our hotels and the congress center, or whether the congress center might be closed by the Lugano authorities. Most immediate and ominous was news that scheduled speakers, and attendees, were cancelling What we eventually worked out was to have some absent speakers give their talks “live” from their home locales via the internet, and others  to record their talks and email them, to be projected at the meeting. This meant, we had to find, with essentially no notice, a local audiovisual and computer crew who could cope with needs unanticipated in the meeting planning. In addition, there were innumerable incoming phone, email and fax messages with questions from attendees and speakers about whether the meeting would proceed and what would happen, including absent poster presenters who were trying to arrange colleagues who had already arrived to present the posters of those who now couldn’t or wouldn’t come, and scholarship awardees, from developing countries, who had never travelled internationally before, not to mention into a presumed epidemic area. We also had to cope with suddenly decreased numbers for the ancillary congress events, and food and drinks for meeting breaks that had been booked.As it turned out, roughly half the scheduled speakers and poster presenters, and half the audience, never made it to Lugano. However, very few scheduled talks were not able to be given, using the remote tools described above, although the original schedule had to be re-arranged for many. Kudos to all the speakers and poster presenters who did make it to the meeting, or cooperated with the alternate plans. Attendees who made it were lavish in their positive evaluations of what the meeting did provide for them. I would nominate, on behalf of my 2 Co-Organizers and myself, that the true heroine of the egress from the chaos would be Derry, and her team, and their improvisation and efforts to go ahead, as closely as possibly could be done, with the original plan.I thank Cornelia Lass-Florl, Chair of the AAAM9 Scientific Committee (and Chief Editor of the Supplement), and all the Committee. I thank the crew of the Journal of Fungi, whose careful work made this Supplement possible; the Guest Editors , drawn from the meeting faculty; and the reviewers chosen for these submissions, for all their efforts. I thank the many corporate and foundation sponsors; without their support, this conference would not have been possible. I have special thanks to my 2 fellow Co-Organizers, whose unstinting willingness to take on the many necessary tasks, in the amazing absence of any quarrels among us, since 2003, make these meetings possible. Our founding vision of what we wanted the meetings to achieve has been, in our opinion, realized. We look forward with uncertainty to the 10th Meeting, which had originally been scheduled to take place in Lyon in 2022, as we do not know how the world will look after COVID-19 (follow our website to see what will be planned for the meeting). The format may be modified, if necessary, but the diseases caused by these filamentous fungi have not lessened in importance as a result of the coronavirus, and in fact, these opportunistic pathogens are riding in on the wave of coronavirus infections, in intensive care units and hospitals ,5,6,7.


The roles HPV plays in the host were gradually identified through the interaction between it (including its early genes) and host RNA. In recent years, increasing numbers of studies in HPV-related cancers have been published showing the relationship between HPV and host RNA. Here, we present a database named HRRD, which contains the regulatory relationship between HPV and RNA . The information was extracted from 10,761 papers in PubMed . In addition, the sequence map of HPV (198 genotypes) is also contained. HRRD was designed as a user-friendly web-based interface for data retrieval. It integrated the information of interaction between HPV and RNA, which reflects the relationship between HPV and host. We hope HRRD will further provide a comprehensive understanding of HPV in carcinogenesis and prognosis. HRRD is freely accessible at  After HPVs infect the basal cells of epithelia, they employ cellular machinery to replicate and survive in host cells2. With the persistent infection, HPV and the released viral genome will lead to squamous intraepithelial lesions, even cancers3, such as cervical cancer4 and head and neck cancer5. HPV not only plays an important role in carcinogenesis, but also participate in host tumor immunity, prognosis, drug resistance and so on. All these functions are achieved through the interaction between HPV and host RNA.Human papilloma viruses (HPVs) are a group of double-stranded circular DNA viruses with about 8000 nucleotides in size. More than 200 HPV types have been characterized to date, including some high-risk types . HPV genome is composed of six early gene  open reading frames (ORFs), two late gene (L1 and L2) ORFs and a non-coding long control region (LCR). Distinct genes perform different functions6. HPV activates miR-34 to participate in the cell apoptosis7. In addition, the expression of HOTAIR was significantly down-regulated by E7 to cause cervical cancer8. In cancer cells, HPV participates in cancer metastasis, prognosis, immune response and so on. For instance, HPV activates TGF-β and IL-10 expression to activate the host immunity9. HPV promotes tumor metastasis by affecting various miRNAs in HPV-related cancers10. Besides, there are many other RNA changes caused by HPV and its oncogenes in HPV-related cancers. By affecting the expression of host RNA, HPV can lead to the occurrence and development of cancers, and has a huge impact on the treatment, immunity and prognosis of cancers. Moreover, it has also been reported recently that host RNA can affect the expression of HPV in converse.Host RNA can be divided into coding RNA and non-coding RNA. mRNA is the main type of coding RNA, while miRNA and lncRNA are two kinds of non-coding RNA which are popular in the field of research. HPVs influence host cells  by regulating the expression of these RNAs Fig. . In norm11 is a database about papilloma virus (including HPV). HPVdb12 is a Human Papillomavirus T cell Antigen Database. HPVbase13 shows the integration information of HPV. However, these databases only focus on the biological characteristics of HPV itself, such as its type and sequence information, but do not collect any information about the HPV-infected host. Despite of numerous experimental studies in the field, there are no computational resources with a unique focus on the relationship between HPV and host RNA. Hence, it is important and necessary to establish a proper multi-comparative platform to show this mutual relationship. Here, we present HRRD, a database of the relationship between HPV and host RNA, which contains the existing studies on HPV and host RNA. It will contribute to providing a comprehensive web-based resource for HPV and host, and data support for HPV-related research.In the recent past, some databases about HPV have been published. PaVEhttps://www.eclipse.org), one of the open-source integrated development environment (IDE). The database manage system was MySQL (https://www.mysql.com) which stores the relationship data model. Java Server Pages (JSP) in the web tier was used to present dynamic responses to clients. The client tier was made up of static and dynamic html pages including forms, tables, charts, and embedded JavaScripts. The model-view-controller (MVC) model web application was managed by Struts (https://struts.apache.org). All plots and web crawler were generated with python (version 3.8.1). HRRD is implemented and hosted on a Windows operating system, with Tomcat and MySQL (version 5.6.19), for serving static and dynamic content and providing the features for data management, respectively , mRNA, lncRNA (long non-coding RNA), gene. Articles with the combination of keywords were searched on PubMed . Web crawler got the research literatures with the keywords above. After deleting the epidemiological survey, review and the duplications, there were 10,761 articles remaining . The whole genome information of HPV (198 genotypes with specific sequence information) was downloaded from NCBI in genbank format.The information of the relationship between HPV and host RNA was extracted from articles, containing the PMID number, cancer type, HPV genotype, HPV oncogene, RNA type and name, method, material and the regulatory relationship between HPV and host RNA (action direction & action type). The information is obtained by reading the literature manually. The collected data were checked and unified, and finally summarized into Excel form. There are totally 784 pieces of information extracted from the articles, including 13 pieces of lncRNA, 195 pieces of miRNA, and others are mRNA.The sequence information of different HPV was extracted from the genbank files, including the full-length genome of HPV and the location of its oncogenes. The information of HPV sequence was stored in the database in TXT format.The principal function of HRRD is query. The information about the relationship between HPV and host RNA is stored in the database. In the Relationship page, there are three input boxes with examples for users to search. Users can search by one to three conditions like HPV genotype, HPV gene or RNA name to get a list containing the relevant content of users searched. The list would contain following information: HPV genotype, HPV gene, RNA type and name, action direction and type, cancer type, PMID, method and material. Users can jump to the article summary in PubMed by clicking the PMID number. “HPV to host” means HPV affects host RNA expression, while “host to HPV” means host RNA influences HPV expression. “Activation” refers to up-regulated expression, while “Suppression” means down-regulated expression. The method shows whether this message was concluded from experimental verification or data analysis Fig. C. The reThe genome information of HPVs is stored in the database. In the Sequence page, the table shows all species of HPV stored in HRRD. Users can input different genotype of HPV in the search box and click the search button to search HPV in the table below. Users can go to the result page by the plot button or clicking the HPV ID in the table Fig. B. The reOther pages contain some other ancillary functions. A simple Home page was designed Fig. A with a To our knowledge, HRRD is the first database designed for the relationship between HPV and RNA specifically. To ensure the accuracy of data, the information was manually extracted from relevant literature. HRRD describes the sequence map of HPV and incorporates some links to HPV-related databases so that users can search and get relevant information quickly and easily. We chose RNA as our research object, because RNA is unstable and susceptible to changes and mutates by viruses, and it is the first step in protein synthesis. For example, if researchers detect HPV infection in cancer, HRRD can help to find out whether HPV affects the key RNA and causes the carcinogenesis. In addition, if researchers want to find some key RNA in HPV-related cancer, HRRD can help to do preliminary screening and query. With the plenty resources, it will provide candidate biomarkers for HPV-related carcinogenesis and prognosis and identify potential targets for molecular therapy. We anticipate it will facilitate an insight into HPV and host.Despite the database has contained data up to December 1st, 2019, a rapidly growing number of relevant researches are emerging from then. HRRD will be updated regularly by scanning newly published literatures. To ensure the timeliness of data, we plan to use data mining and machine learning to obtain information from the literatures. At the same time, we will update the software with modified function and expanded the scope of application. These updates will be implemented in the near future.