Is human papillomavirus (HPV) infection during pregnancy associated with preterm delivery?In this multicenter cohort study of 899 pregnant women, persistent HPV-16/18 infection during pregnancy was significantly associated with preterm birth.If the identified association between HPV infection during pregnancy and preterm delivery is causal, HPV vaccination may reduce preterm birth and the associated burden. Preterm birth remains a leading cause of perinatal mortality and lifelong morbidity worldwide. The cause of most preterm births is unknown, although several infectious processes have been implicated.To assess whether human papillomavirus (HPV) infection, a frequent infection among women of childbearing age, is associated with preterm birth.The prospective HERITAGE cohort study was conducted at 3 academic hospitals in Montreal, Québec, Canada, among 899 pregnant women recruited between November 8, 2010, and October 16, 2016. Follow-up was completed on June 15, 2017. Statistical analysis was conducted from February 6, 2020, to January 21, 2021.Vaginal HPV DNA detection in the first and third trimesters of pregnancy and placental HPV infection.The main outcome was preterm birth (defined as a live birth or stillbirth between 20 weeks and 0 days and 36 weeks and 6 days of gestation). The association between HPV DNA detection and preterm birth was measured using logistic regression. Odds ratios (ORs) and 95% CIs were adjusted by inverse probability of treatment weights of the propensity score.The study included 899 women  with singleton pregnancies. A total of 378 women (42.0%) had HPV DNA detected in vaginal samples collected during the first trimester, and it was detected in 91 of 819 placentas (11.1%) at delivery. Fifty-five participants experienced preterm birth . Persistent vaginal HPV-16/18 detection was significantly associated with all preterm births  and spontaneous preterm births , as was placental HPV infection . Results were similar when restricting the analysis to participants without a history of cervical intraepithelial neoplasia treatment.The study’s results suggest that persistent HPV-16/18 infection is associated with an increased risk of preterm birth, independent of cervical treatment. Future studies should investigate the association of HPV vaccination and vaccination programs with the risk of preterm birth. This cohort study assesses whether human papillomavirus infection, a frequent infection among women of childbearing age, is associated with preterm birth. However, clinical studies have yielded mixed results regarding the association between HPV and preterm birth, possibly owing to HPV exposure misclassification, detection of HPV at inappropriate time points, and insufficient control for confounding.9 Given the high prevalence of HPV infection among women of childbearing age, the mechanistic plausibility of an association between preterm birth and HPV infection, the important burden of preterm birth, and the availability of effective HPV vaccines as a preventive method, confirmation of this association is of utmost interest. Therefore, we prospectively assessed whether vaginal HPV infections during pregnancy and placental HPV at birth were independently associated with preterm birth.Human papillomavirus (HPV) is the most common viral infection of the genital tract.10 In brief, pregnant women of at least 18 years of age were recruited during the first trimester of pregnancy. Women who were HIV positive or unable to provide written consent were not eligible for the HERITAGE cohort study. For this analysis, we excluded women with multiple pregnancies (twins or more), women who had spontaneous or induced abortions, and women with a history of cervico-isthmic incompetency who underwent a prophylactic cerclage in the first trimester. The study folllowed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. The institutional ethical and research review boards of the Centre Hospitalier Universitaire Sainte-Justine, the Centre Hospitalier de l’Université de Montréal, and the Saint-Mary’s Hospital Center approved this study. Each participant provided written informed consent.We conducted a prospective cohort study (the HERITAGE study) enrolling 1052 pregnant women from 3 university-affiliated health care centers in Montreal, Québec, Canada  between November 8, 2010, and October 16, 2016. The date of the last delivery was June 15, 2017. The study design and procedures have been described previously.10At recruitment, participants provided a self-collected vaginal sample for genotype-specific HPV testing. Participants who tested positive for HPV DNA at the first trimester visit provided additional swab samples for genotype-specific HPV DNA testing at 32 to 35 weeks’ gestation. Placental swab samples and biopsy specimens were collected immediately after birth from the fetal side of the placenta and the maternal side of the placenta after a standardized procedure designed to minimize the risk of contamination.Sociodemographic status, medical and sexual history, and tobacco and alcohol consumption were documented at baseline. Self-identified ethnic origin was collected and provided for the sole purpose of describing the study population. Pregnancy history and delivery information were extracted from electronic medical records.13Human papillomavirus detection and genotyping were performed with the Linear Array genotyping assay (Roche Molecular Systems) detecting 36 mucosal HPV genotypes, including genotypes 6, 11, 16, 18, 26, 31, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, and 89. In this study, HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82 were considered as high-risk HPV genotypes, and HPV genotypes 6, 11, 26, 34, 40, 42, 44, 53, 54, 61, 62, 67, 69, 70, 71, 72, 81, 83, 84, and 89 were considered as low-risk HPV genotypes.Preterm birth was defined as a live birth or stillbirth between 20 weeks and 0 days and 36 weeks and 6 days of gestation.14 First-trimester ultrasonography is part of routine prenatal care in the recruiting centers and was used to confirm gestational age. The outcome of our primary analysis was overall preterm birth, which included all births occurring between 20 weeks and 0 days and 36 weeks and 6 days of gestation. We also analyzed HPV infection as a risk factor for spontaneous preterm births specifically. This category included only births after spontaneous onset of labor or preterm premature rupture of membranes. Participants presenting with cervical dilation in the second trimester requiring emergency cerclage (n = 5) were included in the spontaneous preterm birth category irrespective of gestational age at delivery.Persistent HPV infection was defined as a variable with 5 mutually exclusive categories: negative for HPV, transient HPV infection with any type (positive at the first trimester only), persistent low-risk HPV only, persistent high-risk HPV but not genotypes 16/18, and persistent HPV-16/18 at both the first and third trimesters. For placental HPV infection, we used a binary categorization: negative or positive for any HPV genotype after we pooled all HPV DNA testing results obtained from swab samples and biopsy specimens.Exposure to HPV was conceptualized as (1) vaginal HPV infection detected in the first trimester; (2) persistent HPV infection, defined as vaginal HPV infection detected in both the first and third trimesters; and (3) placental HPV infection. We first categorized vaginal HPV infection detected in the first trimester as a binary variable: negative or positive for any HPV genotype. Subsequently, we constructed a 4-level variable with mutually exclusive categories: negative for HPV, positive for low-risk HPV only, positive for high-risk HPV but not genotypes 16/18, and positive for HPV-16 or HPV-18. 15 However, 113 participants (12.6%) had at least 1 variable with a missing value. Therefore, for the sake of precision, we imputed missing data by the mean if the missing value was a continuous variable or by the mode if the missing value was a categorical variable.Rates of missing values were lower than 1% for all variables except pregnancy-induced hypertensive disorders (10 of 899 [1.1%]), alcohol use (13 of 899 [1.4%]), gestational diabetes (21 of 899 [2.3%]), and history of treatment for cervical intraepithelial neoplasia (73 of 899 [8.1%]). Missingness was not associated with the observed values of the outcome or the exposure. Thus, we assumed that a complete case analysis would not have led to bias.16 We estimated a propensity score of any HPV infection during the first trimester and a propensity score of any placental HPV infection. The selection of factors associated with propensity scores was based on a priori knowledge of risk factors for preterm birth, regardless of whether or not they were associated with HPV infection.17 The following factors that have been consistently associated with preterm births across several studies were used to estimate propensity scores: maternal age (years), ethnic origin  and their 95% CIs were estimated using unconditional logistic regression. To control for confounding, adjusted ORs (aORs) and their 95% CIs were obtained using inverse propensity treatment weights (IPTW).P values were from 2-sided tests, and results were deemed statistically significant at P < .05. All analyses were performed using Stata/SE, version 14.2 (StataCorp LLC).All 18 we stratified analysis according to history of preterm birth. Moreover, considering that cervical treatment for intraepithelial neoplasia in and of itself increases the risk of preterm birth by altering the physical integrity of the cervix,19 we compared women with a history of cervical intraepithelial neoplasia treatment and women without. Finally, because smoking is hypothosized to augment the effect of HPV in precancerous and cancerous pathologic conditions,21 we explored whether the association between HPV infection and preterm birth differed between smokers and nonsmokers.To assess the robustness of our findings, we conducted 3 a priori–specified exploratory subgroup analyses. First, because a history of preterm birth can be considered a marker of unknown recurrent or chronic risk factors,After excluding HERITAGE cohort participants who lost or terminated their pregnancy, those with a multiple pregnancy, and women with a history of cervico-isthmic insufficiency who underwent a first-trimester prophylactic cerclage, the final sample included 899 women with singleton pregnancies and a valid first-trimester HPV DNA test result .Chlamydia trachomatis and Neisseria gonorrhoea in the first trimester as part of routine prenatal care, and none had positive test results. Compared with participants who had negative HPV test results (n = 521), those who had positive HPV test results (n = 378) were more likely to self-identify as White (289 [76.5%] vs 360 [69.2%]), smoke (57 [15.1%] vs 32 [6.2%]), have had a new sexual partner in the last year (50 [13.3%] vs 16 [3.1%]), have had a history of treatment for cervical intraepithelial neoplasia (37 [10.7%] vs 22 [4.6%]), and be nulliparous (199 [52.8%] vs 209 [40.3%]).Overall, 378 participants (42.0%) had positive HPV test results at the first-trimester recruitment visit, among whom 252 participants (28.0%) were infected with at least 1 high-risk HPV genotype and 167 (18.6%) were infected with more than 1 HPV genotype . Most paOverall, 55 participants experienced preterm birth, of which 38 were spontaneous and 17 were medically indicated. The median gestational age of preterm infants was 36.0 weeks . Of those infants, 41 were born after 34 weeks of gestational age, 6 were born between 32 and 34 weeks, and 8 were born before 32 weeks. As shown in Given the association between persistent HPV-16/18 and preterm birth, we explored how this association may vary depending on an a priori risk of preterm birth. We compared women with and without persistent HPV-16/18 according to history of preterm birth, history of cervical intraepithelial neoplasia treatment, and tobacco smoking . Althoug24 However, a similar high HPV DNA detection rate was observed on vaginal specimens in a cohort of women attending college in the same city.25We found that, even in a population considered to be at low risk based on sociodemographic and sexual history characteristics, HPV infection is frequent in pregnancy, and most HPV infections detected in the first trimester persist to the third trimester. The prevalence of HPV infection in our study was higher than previously described in several cohorts that also used broad-spectrum, sensitive polymerase chain reaction–based methods to detect HPV on cervicovaginal specimens.9 However, several studies included in the meta-analysis used abnormal Papanicolaou test results before or after pregnancy as a surrogate for HPV infection during pregnancy,31 some studies targeted only a limited number of HPV genotypes,33 and most studies did not take into account important confounders, such as age, previous treatment for cervical intraepithelial neoplasia, other genital infections, or other obstetrical risk factors.36 All of those factors may have been associated with a biased, lower estimate of the association between HPV infection and preterm birth.We did not find an association between vaginal HPV detection (any genotype) in the first trimester of pregnancy and preterm birth. However, the persistence of vaginal HPV-16/18 infection during pregnancy and the detection of any HPV on a placental swab sample were independently associated with the occurrence of both all and spontaneous preterm births. In a recent meta-analysis, HPV infections or HPV-related cytologic abnormalities were associated with preterm births but to a lesser degree than what was found in this study .19 It was not possible, in ecological or database studies, to differentiate between the role of HPV infection in and of itself and that of treatment for cervical dysplasia caused by high-risk HPV.37 In our population, the prevalence of cervical treatment was low, making it unlikely that it could explain the association between HPV and preterm birth. Moreover, excluding women who had a history of treatment did not change our conclusion. Another challenge of studying this topic is that HPV infections are more frequent among women who have other risk factors for preterm birth, such as young age, tobacco smoking, other genital infection, and low socioeconomic status,38 and thus may act merely as a marker for a pregnancy at higher risk of preterm birth. Through our detailed questionnaires and available clinical data, we were able to control for such factors using IPTW.Treatment for cervical dysplasia caused by high-risk HPV increases the risk of preterm birth.We can only speculate as to the pathophysiological mechanism underlying the association between HPV infection and preterm birth, independent of treatment for cervical dysplasia. Human papillomavirus infection of the keratinized epithelium (skin) and squamous or glandular epithelia  does not induce a measurable inflammatory reaction. However, HPV infection may be associated with overall changes in the vaginal microbiota, which in turn could trigger an immuno-inflammatory process leading to preterm birth. Further research should investigate this possibility in suitable animal models.4 We also detected several HPV genotypes, which enabled us to measure the association for distinct HPV genotypes. Furthermore, we used a standardized sampling protocol to avoid placental contamination. On both sides of each placenta, we collected 3 swab samples (including 1 under the amniotic membrane) and 4 biopsy specimens. Our prevalence of placental HPV (11.1%) was similar to that reported by a Danish study using in situ hybridization to localize HPV within trophoblastic cells.39 Using the IPTW technique, we generated a weighted sample in which the distribution of all measured confounders was similar between the groups of women studied. Last, having a detailed history of cervical dysplasia treatment enabled us to focus on HPV infection among women who had not been treated in the past.This study has several strengths. The main strength of this study is its prospective design that allowed for the detection of HPV throughout the exposure window period (pregnancy), during which HPV can exert a direct detrimental effect.This study also has some limitations. First, although the IPTW adjustment allowed for the full control of many confounders, some residual confounding is still possible. However, given the fact that the most important known risk factors for preterm birth were measured and taken into account either through restriction or adjustment by IPTW and that subgroups analysis showed consistent associations between HPV and preterm birth, it seems unlikely that residual confounding would explain our findings. Second, owing to a small number of events, we could not reach a conclusion on whether or not HPV infection conferred a higher risk of preterm birth in subgroups of women with an elevated baseline risk. Despite our use of a strict, standardized protocol for handling and testing the placental specimens, contamination of the placenta during birth can never be completely ruled out as an explanation for HPV-positive placental test results. However, the fact that HPV was detected on biopsy specimens and under the amniotic membrane indicates that true placental infection is more likely. Finally, we acknowledge that the study population of HERITAGE has several characteristics making it at low risk of preterm birth and that our conclusions may not translate to higher-risk populations.This is the first report, to our knowledge, to prospectively observe that persistent HPV-16/18 infection in pregnancy and placental HPV infection significantly increase the risk of preterm birth above infections by other HPV genotypes and separately from a history of cervical dysplasia treatment. If confirmed in larger and more diverse populations, these findings would support a role for HPV vaccination programs in the reduction of the burden of preterm births. This positive association would likely be observed during the next decade at the population level in settings with high-coverage HPV vaccination programs, when cohorts of girls vaccinated before sexual debut reach childbearing age.


Single-visit “screen-and-treat” strategies using visual inspection with acetic acid (VIA) and cryotherapy (liquid nitrous oxide ablation) in low-resource settings are commonly used to detect and treat precancerous lesions for cervical cancer prevention. This study compared VIA sensitivity and specificity in rural indigenous Guatemalan communities, to that of oncogenic human papillomavirus (HPV) testing for detection of precancerous changes, using cytology as the reference standard. Between 3–8 September 2017, trained nurses examined 222 women aged 23–58 years with VIA. Specimens for liquid-based cytology and HPV testing were obtained prior to VIA with a cytobrush and transported in PreservCyt to a US clinical laboratory. VIA and HPV test sensitivities were assessed as proportions of women with abnormal cytology that had abnormal VIA or HPV results, respectively, and specificities, as proportions with normal cytology with normal VIA or negative HPV tests. Of 222 women, 18 (8.1%) had abnormal cytology ). Excluding ASCUS, sensitivities of VIA and HPV were 20.0% and 100%, respectively. VIA-based screening may not be acceptable for detecting precancerous lesions, and field cryotherapy for preventing malignancy. The World Health Organization recommended in 2021 “…using HPV DNA detection as the primary screening test rather than VIA or cytology”. Cervical cancer incidence and mortality declined in high-income countries with the widespread use of cytological screening and treatment of precancerous lesions . Timely In low- and middle-income countries lacking access to standard-of-care cervical cytology nationwide, visual inspection with acetic acid (VIA), with onsite cryotherapy (liquid nitrous oxide ablation to freeze and destroy precancerous tissue at −50 degrees Celsius), (“test-and-treat”), have been recommended ,7. VIA rWomen living in underserved rural indigenous communities in Alta and Baja Verapaz, Guatemala with little access to clinical facilities are advised by community health workers 14–21 days prior to community-based screening activities that VIA will be offered at no cost by Partner for Surgery, a non-governmental organization, which provides multiple health services in rural villages, including cervical cancer screening and precancerous lesion cryotherapy treatment to women aged more than 21 years. VIA screening in these and other rural villages in Guatemala, with recruitment conducted in this way, is routinely offered by Partner for Surgery several times per year. VIA examinations take place in community centers in the villages, which allows privacy for the women to be interviewed  and examined by Guatemalan registered nurses trained and experienced in VIA and liquid preparation collection.A sterile, disposable clear plastic vaginal speculum is inserted in the vagina and freshly prepared 4% acetic acid is applied to the cervix for cytology. After one minute, the cervix is inspected using a light source (a hand-held flashlight). VIA examinations are considered positive for precancerous changes when a well-defined, dense whitened area with regular margins is visible at the squamocolumnar junction or in the transformation zone. VIA tests are considered negative if no acetowhitening is observed. Cryotherapy is offered to women with abnormal VIA exams. Those whose VIA examination suggests malignancy  are referred on the day of the examination to specialized care in a provincial secondary care or capital  tertiary facility offering subsidized but not free care.VIA examination procedures took place in the villages visited from 3–8 September 2017 , with invitations 14–21 days before, in an identical way as is routine (described above), except that specimen collection was performed immediately after speculum insertion, before application of acetic acid. Specimen collection for cytology and HPV testing was completed prior to VIA using spatulas and cyto-brushes. Specimens were placed immediately after being obtained into PreservCyt, a methanol-based transport solution. Containers were labeled with a number to link with the patient identifiers, VIA exam, and other information. Specimens were transported to the Palm Beach Pathology Laboratory , a US Clinical Laboratory Improvement Amendments (CLIA) approved laboratory, where all laboratory testing was performed. The cytological specimens were processed using Thin Prep Processor 2000  liquid-based thin-layer preparation, and results were reported using Bethesda System for Reporting Cervical Cytology. HPV testing  was performed by nucleic acid amplification testing using Aptima HPV 16 18/45 Genotype Assay . Results of cytological tests and HPV tests were provided to Partner for Surgery staff within 45 days of specimen collection.Partner for Surgery advised patients to seek specialized care in government facilities if they had abnormal results , positive HPV tests, etc.). The referral was to include VIA results and other results provided to Partner for Surgery staff to distribute to patients. After linkage to cytological and HPV test data, and the distribution to Partner for Surgery staff and to participants as described above, all identifiers were removed from the VIA, cytology, HPV test, and interview data, and the data were provided for analysis to authors.This was a cross-sectional analysis of the de-identified data collected during the brief interviews of women who presented for VIA , and results of VIA, HPV testing, and cytological examination. Abnormal cytology results were defined as those that reported HSIL, LSIL, carcinoma, or atypical squamous cells of unknown significance (ASCUS). Due to cytological and HPV testing being more likely to signify persistent or progressive HPV infection (vs. transient infection that would clear spontaneously) in immunocompetent older women than in younger women ,10, we cVIA and HPV test sensitivity and specificity were assessed for all participants and restricted to only women aged 30 years and older using cytological exam results as the reference standard. Analyses were performed excluding and including ASCUS results. VIA and HPV test sensitivities were calculated as the proportions of women with abnormal cytology specimens whose VIA test was abnormal, or whose HPV test was positive, respectively . SpecifiData used for estimation of sample size consisted of results of de-identified HPV tests collected during VIA activities in Guatemala indigenous communities, as part of the National Cancer Institute (NCI) international HPV infection prevalence surveys in 2013. In that dataset, 29.6% had HPV detected in their samples. For 80% power to detect a statistically significant difference between a minimum HPV test sensitivity of 95% and a maximum VIA sensitivity of 60%, with two-sided significance level of 95%, we would require 22 women with abnormal cytological examinations  ,12. AntiNurses examined 223 women aged 23–58  years. Parity ranged from 1–12 deliveries, and 96% had delivered at least 1 child; 11  of VIA examinations were considered abnormal, including 1 with a lesion consistent with cancer, in a woman who had had VIA and cryotherapy in 2015. Oncogenic HPV types were detected in 38  women. Of the 222 women with usable cytological specimens, 10  had abnormalities, including the carcinoma, 5 LGSIL, and 4 HGSIL. Another 8 had ASCUS. HPV prevalence was significantly higher among women aged less than 30 years than among older women . Youngerp = 0.007) (1 LGSIL and the 1 with cancer) (p = 0.005), respectively. VIA did not detect any participants with ASCUS, but HPV testing detected five of eight; sensitivities of HPV and VIA for detecting abnormal cytologies, including ASCUS, were 83.3% and 11.1%, respectively (p < 0.0001). When confined to participants aged ≥30 years, HPV test sensitivity (excluding ASCUS) remained 100%, and the 95% CI still did not overlap with VIA sensitivity, which declined to 16.7%; both specificities rose (to 90.5% for HPV and to 97.4% for VIA), still significantly different (95% CIs did not overlap)  participants with HGSIL, LGSIL, and the 1 with cancer, while VIA tests detected only 2 of 10  a. Negatioverlap) b. DiagnoCryotherapy was used to treat four women with abnormal VIA exams who did not have abnormal cytology or HPV detected. Another woman was referred based on abnormal VIA and had a negative HPV test as well as an unsatisfactory cytological specimen and was not included in this analysis.In this small study, sensitivities of VIA and HPV testing differed considerably relative to cytology, more than anticipated. Only 2 of 10 women with abnormal cytological examinations had abnormal VIA; 1 of these 2 had had an abnormal VIA and cryotherapy 2 years before and had progressed to invasive cancer despite the cryotherapy. Even though only 10 participants had cytology examinations meeting the criteria for carcinoma, HGSIL or LGSIL, the difference in sensitivity between VIA and HPV achieved statistical significance , due to These findings suggested that alternatives to VIA should be sought, and that HPV testing should be considered. The assessment that opens a 2008 review of new approaches to cervical screening LAC was grim: “Efforts to control cervical cancer in the LAC have been largely unsuccessful.” . The agecareHPV test , a lower-cost DNA test to be used in settings without clean water or electricity, was the most cost-effective strategy [In considering HPV-based screening as an alternative to VIA, concerns arise related to the possible loss of cost-effectiveness with HPV screening, and the loss of the “screen-and-treat” efficiency of VIA, in the context of resource-constrained settings due to the potential requirement to send specimens to a laboratory and requirement for a return visit for treatment. However, the ASPIRE trial, which assessed the feasibility and acceptance of HPV self-collection vs. VIA in a cohort of women from Kisenyi, Uganda, explored cost-effectiveness , reportistrategy .This study had several limitations. Abnormal cytology and VIA examination prevalence in the population were lower than expected; the low number of positive cytology and VIA examinations reduced the power to detect significant differences by age-group a and theSince sexual debut may be early in these communities, as the high parity suggests, and cervical cancer is the principal cause of cancer mortality in Guatemala in 15–44 year old women, Partner for Surgery routinely offers VIA screening to women aged over 21 years. Although the prevalence of high-risk HPV infection in younger participants suggested that some of these infections may have regressed spontaneously, some of the participants with high-grade dysplasia were less than 30 years old, supporting the Partner for Surgery approach. Moreover, even the analysis confined to women aged 30 years and older, although it excluded some participants with high-grade dysplasia, showed significantly higher HPV sensitivity than VIA. Finally, overall test accuracy, which tends to overstate lower-sensitivity test performances in low prevalence populations , did notDespite these limitations, our study suggested that VIA-based screening with cryotherapy may miss women with treatable precancerous lesions. The failure of cervical cancer mortality to decline  in Guatemala in the decades in which VIA has been widely used  suggesteThis analysis of anonymized data comparing VIA “test-and-treat” screening to HPV testing using cytological examination as a reference standard suggested that VIA performed by well-trained and highly skilled staff may be unreliable because of lower sensitivity. Furthermore, failure of cryotherapy to arrest the progress of pre-cancerous lesions in one of the indigenous women living in rural underserved communities in Guatemala suggested that cryotherapy in the field may need to be reconsidered as well. HPV testing, which can be performed in real time in the field on self-obtained samples , is bein


Squamous cell carcinoma of unknown primary (SCCUP) is a challenging diagnostic subgroup of oropharyngeal squamous cell carcinoma (OPSCC). The incidence of SCCUP is increasing in parallel with the well-documented increase in OPSCC and is likewise driven by the increase in human papillomavirus (HPV). The SCCUP patient often presents with a cystic lymph node metastasis and undergoes an aggressive diagnostic and treatment program. Detection of HPV in cytologic specimens indicates an oropharyngeal primary tumor origin and can guide the further diagnostic strategy. Advances in diagnostic modalities, e.g., transoral robotic surgery and transoral laser microsurgery, have increased the successful identification of the primary tumor site in HPV-induced SCCUP, and this harbors a potential for de-escalation treatment and increased survival. This review provides an overview of HPV-induced SCCUP, diagnostic modalities, and treatment options. Annually, more than 90,000 people are diagnosed with oropharyngeal squamous cell carcinoma (OPSCC) worldwide . A subgrIn parallel with the increasing numbers of SCCUPs induced by HPV, HPV testing of the cervical lymph node metastasis is increasingly applied as a part of the diagnostic workup . HPV detThe aim of this review is to provide an overview of HPV-induced SCCUP, including diagnostic workup strategies for the primary tumor and different treatment options in HPV-induced SCCUP patients.HPV is a widespread sexually transmitted disease, e.g., it is the most common sexually transmitted disease in the United States , and in A substantial number of primary tumors identified in SCCUP patients are found in the oropharynx and are HPV-induced, with HPV16 being the predominant high-risk subtype ,28,29. THPV is a double stranded DNA virus that integrates with the host genome in the cell nuclei where transcription of E6 and E7 carcinogenic proteins is activated ,38. The Within the oropharynx, a specialized reticulated lymphoepithelial mucosal tissue is found in the crypts of the palatine and lingual tonsils, which are particularly susceptible to HPV infection  and takeIn contrast to HPV, tobacco use causes direct DNA damage and formation of oncogenic DNA adducts that can be traced in human tissue, as also demonstrated in head and neck cancer . SmokingA rigorous and aggressive diagnostic workup program is normally accomplished in order to successfully identify the primary tumor and includes thorough physical examination, fine needle aspiration (FNA), radiologic examinations, and examination under general anesthesia ,8,11.As patients with HPV+ SCCUP often present with an enlarged cervical lymph node, it is imperative to discriminate between underlying malignancy and benign conditions, e.g., branchial cleft cysts. This evaluation can be performed using an FNA from the lymph node, since radiologic imaging is insufficient in distinguishing between a branchial cleft cyst and the cystic metastasis from HPV+ SCCUP . FNA hasn = 31) found that there was no significant association between p16 and HPV status and imaging results [The radiological examinations used are typically CT or MRI, both providing identical reported primary tumor identification rates of 9–23% ,60, disr results . PET-CT  results ,69,70. T results ,71, whic results ,72,73,74n = 47 being p16+, the study unfortunately did not reveal how many of the patients were classified as SCCUP before enrolment, but the average dimension of identified tumors was 2.3 cm [In OPSCC, where the tumor is successfully visualized on CT scans, it is hypothesized that the modality is able to distinguish between HPV− and HPV+. This is because HPV− tumors are significantly more often invading the adjacent pharyngeal musculature than HPV+ tumors , and HPVs 2.3 cm .After radiological imaging and clinical examination, the patient is normally scheduled for an examination under general anesthesia (pan-endoscopy). The identification rate is 22–31% after direct pharyngo-laryngoscopy ,78,79, 2An addition to the clinical examination of SCCUP patients in the pursuit of successful primary tumor site identification is narrow band imaging (NBI). The endoscopic tool can visualize neo-angiogenic formations that are characteristic of malignant tumors by restricted light wavelength. In a recent meta-analysis comprising SCCUP patients, the use of NBI showed a sensitivity and specificity of 83% and 88%, respectively, and the overall identification rate was 35%, disregarding HPV status which was not reported [In 2009, the FDA approved the use of TORS for early-stage OPSCC . The proThe utility of TORS and TLM-assisted lingual mucosectomy has also been proven as a new diagnostic modality for SCCUP patients, with successful identification rates of 70–78% of the primary tumor sites ,86,87,88Synchronous tumors have been demonstrated to occur in 6–18% of palatine and lingual tonsil cancers ,81,91,92Referring to a European multi-center study, HPV+ SCCUP generally possesses a better prognosis than its HPV− counterparts, with significantly better overall survival and progression-free survival . A SwediThe identification of the primary tumor site is mandated for an optimal treatment.n = 136) has highlighted the importance of identifying the primary tumor site as this leads to improved overall survival, cause-specific survival, and disease-free survival, also when stratified for HPV status [A retrospective study  from Ireland has demonstrated that ECE in association with p16+ does not significantly influence recurrence-free survival and disease-free survival when the majority of patients received adjuvant RT [Recent literature also suggests that ECE in the case of HPV+ does not signify the same risk of an inferior treatment outcome as its HPV− counterpart. A recent study  demonstrated that cN2 and cN3 HPV+ patients had similar survival regardless of treatment choice, be that surgery, surgery with adjuvant RT, or surgery with adjuvant CRT or definitive CRT or definitive RT on multivariate analysis, while de-escalation therapy in HPV− patients was associated with worse outcome [Furthermore, a retrospective database study from the Commission on Cancer National Cancer Database in the United States . However, the relatively few annual SCCUP patients and the diverse diagnostic and treatment modalities used across institutions are limiting the possibilities of a multi-center, multi-national, and randomized clinical trial.n = 22) and recruitment not limited by HPV status, yet similar designs should be implemented as a research purpose at multiple institutions. TORS has been proven as a safe procedure with limited effect on QoL [The addition of TORS and TLM as a new diagnostic and treatment modality has proven a useful and highly effective tool in the search for the primary tumor. In several trials, the identification rate has increased with the addition of TORS and TLM-assisted lingual mucosectomy compared to traditional diagnostic approaches ,9,34,112t on QoL ,114; howt on QoL . This ist on QoL . Future t on QoL , should In conclusion, HPV+ SCCUP is a challenging disease entity for patients, clinicians, and researchers with a lack of consensus regarding diagnostic procedures, treatment, and understanding of the pathophysiology in disease development. Fortunately, awareness is increasing as indicated by the recent 8th AJCC cancer staging manual classifying SCCUP based on HPV status rather than a uniform disease entity, as it was considered up until recently. Different diagnostic workup algorithms and treatment options should also be based on HPV status. The HPV-induced SCCUP disease could be seen as a subgroup of OPSCC, but with a tumor so small that it is missed during radiological imaging and histological examinations of resected specimens, supported by the observed increase in identification rates with the implementation of advanced modalities as TORS, TLM, and PET-CT. Several ongoing trials are aiming at optimizing diagnostics and treatment modalities, but given the rarity of the condition, the advancement must, so far, be reliant on retrospective and prospective cohort studies.


According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 10 Human papillomaviruses (HPVs), a family of viruses with double-stranded circular DNA, are responsible for various tumorous lesions, ranging from warts to cancers of the cervix, anus, and head and neck . In partE1, E2, E4, E5, E6, and E7), two late genes (L1 and L2), and the long control region (LCR). HPV-6 and -11 detected in LP are categorized as low-risk HPV according to their association with cancer. HPV initially infects basal cells in the mucosal and cutaneous epithelium through membrane disruption [E1 and E2) and the packaging genes (L1 and L2). The rest of the genes  are highly diverse and their involvement is observed in the cell cycle, escape from immune surveillance, and viral release [The approximately 8-kbp HPV genome is divided into three sections: six early genes  assay for most of the early mRNAs. We also established a monoclonal antibody to immunohistochemically localize E4 in HPV-6 LP . The resreatment .In the present study, given that HPV-11 causes more severe disease than HPV-6 , we furtSpecimens were collected from the following patients at our hospital between 2005 and 2021: 28 patients with LP, 10 with laryngeal cancer, 5 who underwent tumor-free vocal cord removal during pharyngolaryngoesophagectomy, 5 with chronic tonsillitis, 5 with oropharyngeal cancer, and 5 with tongue cancer, 4 with nasoseptal exophytic papilloma, and 1 each with paranasal exophytic papilloma, inverted papilloma with squamous cell carcinoma, nasopharyngeal papilloma, and oropharyngeal squamous papilloma. The HPV genotypes of the oropharyngeal and laryngeal cancer samples were analyzed in our previous studies ,14. All DNA was extracted from fresh-frozen samples and subjected to PCR using the degenerated consensus primer sets that were designed to amplify the L1 region, as in our previous studies ,15. We eE6, E7, E1, E2, E4, E5a, E5b, L2, and L1 mRNAs, as described in our previous studies [E6 copy number/ng cellular DNA. Details of the methods are provided in the Total RNA was extracted from fresh-frozen papilloma samples and subjected to quantitative real-time PCR to measure the absolute levels of  studies . More prBiotinylated DNA probes were prepared and used to perform ISH of HPV DNA, as described in our previous study  and the E6, E2, E4, and E5b genes of HPV-11 . PCR was performed under the following conditions: 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, and finally 72 °C for 5 min. The PCR product was amplified with HPV-11 E1^E4-NdeI-F and HPV-11 E1^E4-BamHI-XhoI-R primers was digested with NdeI and XhoI, and then subcloned into the NdeI/XhoI restriction sites of the pET22b (+) vector , resulting in the pET22b (+) 1× HPV-11 E1^E4 vector. Then, the PCR fragment amplified with the HPV-11 E1^E4-BglII-F and HPV-11 E1^E4-BamHI-XhoI-R primers was digested with BglII and XhoI and subcloned into the BamHI and XhoI restriction sites of the pET22b (+) 1× HPV-11 E1^E4 vector, resulting in the pET22b (+) 2× HPV-11 E1^E4 vector. Finally, the above PCR fragment digested with BglII and XhoI was subcloned again into the BamHI and XhoI restriction sites of the pET22b (+) 2× HPV-11 E1^E4 vector, resulting in the pET22b (+) 3× HPV-11 E1^E4 vector. As the pET22b (+) vector contains a His-tag (6 histidine residues) at the C-terminal region of the multiple-cloning site, the pET22b (+) 3× HPV-11 E1^E4 vector was expected to express a 31.8-kDa protein (three tandem fused HPV-11 E1^E4 plus the His-tag). The pET22b (+) 3×-HPV-11 E1^E4 vector was transformed into E. coli BL21 (DE3). The transformed E. coli were cultured for 15 h at 25 °C in Luria Broth medium containing 100 mg/mL ampicillin and 1 mM isopropyl β-D-1-thiogalactopyranoside. After centrifugation at 20,000× g for 5 min, the pellet was suspended in ice-cold PBS and sonicated for 30 min. After centrifugation at 20,000× g for 10 min, the pellet was suspended in ice-cold PBS, including 6 M guanidine hydrochloride, and sonicated for 30 min. Then, dithiothreitol was added to the lysate , mixed, and placed on ice for 1 h. After centrifugation at 20,000× g for 5 min, the supernatant was collected and purified with a His-trap column as described previously [To produce recombinant HPV-11 E1^E4 protein in eviously . To remoA specific pathogen-free rabbit was immunized with the above 3× HPV-11 E1^E4 protein . On day 0, initial immunization was administered with 400 μg of the protein combined with Freund’s complete adjuvant. Subsequent immunization was administered with Freund’s incomplete adjuvant, including 200 μg of the protein, at 2-week intervals . Antiserum was obtained by bleeding the rabbit on day 49.Antibodies against HPV-11 E1^E4 and HPV-6 E1^E4 were evaluated by Western blotting, as in our previous study [FFPE samples from 66 patients were used to evaluate the sensitivity and specificity of the newly developed anti-HPV-11 E1^E4 antibody and anti-HPV-6 E1^E4 antibody in immunohistochemistry, using the same process as in our previous study .Viral mRNA levels in terms of the percentage of viral mRNA expression were analyzed by the Kruskal–Wallis test. Statistically significant differences in the mRNA levels of the nine viral genes were detected using the Dwass–Steel–Critchlow–Fligner multiple comparison test. The correlations between viral load and viral mRNA levels were analyzed by Spearman’s rank-ordered test. Comparisons of viral load between positive and negative immunohistochemistry staining of HPV-6-infected papilloma  with the anti-HPV-6 E1^E4 antibody were performed by the Mann–Whitney U-test. This study was conducted with the approval of the Institutional Review Board of the University of Ryukyus (project identification code 35) and conformed to the principles of the Declaration of Helsinki and its later amendments. Written informed consent was obtained from all participants prior to sample collection.5 copies/ng DNA  than those of E6, E7, E1, E2, L2, and L1 . Indeed, E4, E5a, and E5b accounted for 88% of the total expression of the nine viral mRNAs . There was no significant correlation between viral DNA load and the mRNA levels of E6, E7, E1, E2, E4, E5a, E5b, L2, and L1.The expression levels of the nine HPV-11 virus mRNAs were ascertained in 10 samples from three patients with LP, one with nasoseptal papilloma, and one with nasopharyngeal papilloma . As showE4 and E5b mRNAs were markedly expressed and located in the cytoplasm of the upper third cell layer of LP . Meanwhile, signals were not observed in LP, normal vocal cord, chronic tonsillitis, and cancerous lesions in the laryngeal, oropharyngeal, and tongue samples without HPV-6 , as well as functional “accessory” genes  in HPV-11-infected LP [E4 was the most highly expressed mRNA, followed by E5b and E5a in LP, which stood in stark contrast to the much lower expression of E6, E7, E1, E2, L2, and L1. We further investigated the intraepithelial distribution of E4 and E5b transcripts in LP, finding that E4 and E5b mRNAs were expressed in similar superficial epithelial layers. The DNA and RNA levels of HPV-11 genes and immunolocalization of E4 protein in HPV-11 resembled those of HPV-6 [In addition, we examined viral load, in situ localization of viral DNA, and viral gene expression, including that of the “core” genes directly associated with viral DNA replication ), the following three promoters have been identified in the HPV-11 genome: E6 promoter (nt 90), E7 promoter (nt 264), and E1 promoter (nts 674–714). The PaVE database also shows 19 polycistronic messages transcribed by these promoters, among which E5a and E5b are the most encoded genes (n = 18), followed by E4 , E2 (n = 7), E7 (n = 4), E6 (n = 3), L1 (n = 2), and E1 and L2 (n = 1). The expression of each viral gene examined in this study was in good agreement with the number of genes encoded by the message (E5a and E5b are always transcribed simultaneously, but the level of E5b expression was markedly different from that of E5a, with E5b showing a similar level of expression as E4 in this study. In our previous study, HPV-6 mRNA expression of E5a and E5b differed greatly in LP samples, consistent with the results of the present study. Although the precise reason for the observed differences in E5a and E5b mRNA expression is not clear, the splicing of E5a and E5b mRNAs might occur differently in different LPs. Further study is needed to clarify how mRNA splicing occurs in low-risk HPV.The E4 protein encoded by HPV-16 DNA appears to be able to bind keratin as well as reorganize the keratin network of the cell, indicating possible involvement in the release and transmission of the virus . Meanwhi message b. AccordThe life cycle of the virus synchronizes with the program of epithelial differentiation, possibly through the action of differentially expressed cellular transcription factors binding to the LCR throughout multiple layers of epithelia . Viral p


We develop and study a theory of optimal transport for vector measures. We resolve in the negative a conjecture of Klartag, that given a vector measure on Euclidean space with total mass zero, the mass of any transport set is again zero. We provide a counterexample to the conjecture. We generalise the Kantorovich–Rubinstein duality to the vector measures setting. Employing the generalisation, we answer the conjecture in the affirmative provided there exists an optimal transport with absolutely continuous marginals of its total variation. In this note we develop theory of optimal transport of vector measures. Let us first briefly describe the topic of classical optimal transport.X, d), how to transfer one distribution onto the other in an optimal way. The criterion of optimality was to minimise the average transported distance. Since then the topic has been developed extensively and much of this development has been done recently. We refer the reader to the books of Villani (see [In 1781 Gaspard Monge (see ) asked tani  that we deal with is as follows:The precise formulation of the optimal transport problem for an mentclass2pt{minimmentclass2pt{minimThe conjecture of Klartag (ii)(iii)(iv)u is an optimal potential for Moreover, if the above conditions hold, thenLet Assume that (iii) holds. Observe thatSuppose now that (i) holds. Clearlyconcentrated on a subset We say that a measure Assume that The assertion is thatsee e.g. ) any 1-LLet us first provide an affirmative answer to the conjecture of Klartag, under the provision of the existence of optimal transport with absolutely continuous marginals of its total variation.A leaf u that is 1-Lipschitz with respect to Euclidean norms are closed and convex sets. Two distinct leaves may intersect at most by their relative boundaries.It is proven in  that leatransport set associated with u if it enjoys the following property: if u and Let u is a transport set.Let us remark that a Borel set B(u) the set of all points u such that B(u) is of Lebesgue measure zero.We shall denote by mentclasspt{minimaSuppose that u be an optimal potential for A associated with u: (i)(ii)(iii)u is an optimal potential of Assume that A is a transport set, by the definition of B(u),u is an optimal potential. Also By [ose that  holds trWe shall now provide necessary tools for the aforementioned counterexample to the conjecture of Klartag.In fact we shall provide a more general theorem for which we shall consider locally uniformly closed subsets subsets X and converge weakly* to Let X, we haveX,By the assumption, for any continuous and bounded map Below we shall denote by u be an optimal potential of A be the union of all leaves of dimension at least one. Then A is Borel measurable. Suppose that there exists an optimal transport u is of Let A is Borel measurable. Suppose that there exists an optimal transport u is of Observe thatIn the theorem below we shall provide a counterexample to the conjecture of Klartag.Assume that In particular, there is no optimal transport Choose any entclass1pt{minimah is 1-Lipschitz. By the Kirszbraun theorem we may assume that h is defined on the entire space. Moreover forh is an optimal potential and Suppose that points By Theorem u is a maximal, with respect to the order induced by inclusion, set A that enjoys the property that if u, then for any The proof of the following theorem is based on the same idea as the proof of Theorem A of (i)(ii)P denotes a projection onto a complement W of V.for any absolutely continuous Suppose that the norms are Euclidean. Then, if any Let Suppose that f is isometric,f that satisfies (m. This follows by the Hahn–Banach theorem. As v we infer that u is affine. This proves part of i).By the assumption,entclass1pt{minimau is affine then there exists a subspace u. Here P denotes a projection onto a complement W of V. Indeed, let V is not a leaf of u. Then there exists u is affine, it is also an isometry on V is a leaf of u. Thus ii) is proven.If V and any V intersects at most m of the balls We shall now provide an example of a vector measure V are the only leaves of an affine map. This holds true, since any point in V and the leaves of a map foliate To prove the last part of the theorem, it is enough to prove that the translates of mentclasspt{minima


People experiencing homelessness during the 2017–2018 California wildfires faced significant risks of disruption. Homeless service organizations (HSOs) are an essential safety net for this population. To learn about how HSOs performed during the wildfires, this study interviewed U.S. Department of Veterans Affairs (VA) staff overseeing HSOs providing transitional housing under the VA's Grant and Per Diem (GPD) program to Veterans experiencing homelessness. We employed a comparative case study approach exploring GPD organizations' disaster response actions, including evacuating Veterans from wildfire‐affected areas or taking in disaster‐displaced Veterans. This article presents three themes in the GPD organizations' disaster response: (1) Organizations benefitted from close collaboration and communication with the VA during the disaster, creating a safety net to ensure Veterans' well‐being and enact rapid re‐housing to prevent homelessness; (2) Organization staff performed heroically under stressful disaster conditions; and (3) Organizations benefitted from the written disaster plans that VA requires them to create, but were not as well‐prepared for wildfires as they had been for earthquakes. As emergent threats such as the COVID‐19 pandemic, wildfires, and a very active 2020 hurricane season amplify the importance of mitigating risks, comprehensive disaster planning is needed to ensure the safety and support of people experiencing homelessness. From October 8–14, 2017, the 37,000‐acre Tubbs Fire and the 57,000‐acre Nuns Fire tore through Sonoma and Napa Counties, home to California's famous wine country. Strong winds carried the Tubbs Fire from Napa into Sonoma County before reaching parts of eastern Santa Rosa . Traveling at an incredible speed which left firefighters no time to issue comprehensive warnings, the Tubbs Fire jumped the US‐101 freeway, destroying 2900 homes, killing 44 people, forcing 90,000 people to evacuate, and exacerbating the demand for housing in areas already reeling from an affordable housing crisis. Unlike many wildfires whose destruction is mainly felt in sparsely populated areas on the edge of urban‐wildland areas, the Tubbs Fire wrought most of its damage within the city of Santa Rosa, causing $1.2 billion in damage and forcing a hospital to evacuate  in Butte County was almost completely obliterated, as the fire destroyed 14,000 homes and killed 86 people  Homeless Programs constitute a vital part of the VA's long‐term effort to end homelessness among Veterans. The VA Grant and Per Diem (VA GPD) program provides funding to non‐VA, community based HSOs to provide transitional housing and other services to Veterans experiencing homelessness. Veterans can stay up to 2 years to help them achieve permanent housing , led by a “GPD liaison” who monitors the Veterans' progress. The GPD inspection covers a wide scope of areas, including life safety, security, sanitation, and clinical services. In 2013, VA GPD leaders added a new regulation requiring all GPD grantees to have a written disaster plan that is coordinated with emergency managers for their local jurisdiction  impacts to the GPD programs; (2) how the VA and the GPD programs collaborated; and (3) how well the GPD programs' disaster preparedness efforts addressed these disasters. The VA Greater Los Angeles Health Care System Institutional Review Board approved this study. Interviews were digitally audio‐recorded with the respondents' permission and transcribed.Interview transcripts were iteratively analyzed using Atlas.ti  qualitative analysis software. Interviews were coded using both an inductive grounded theory approach of identifying themes from the data that were most salient to VA staff perspectives as well as the three areas of deductive domains governing the interview guide  with the Grant Per Diem program. She went over to the house and physically…said—'you guys have to go'.”There was some confusion in the communications, as the GPD program leadership informed the VA that the Veterans had already gone to the evacuation center, while VA staff contacted the Veterans directly and learned that they were still in the house, with no intention to evacuate. When the GPD program director learned that the Veterans were reluctant to leave, she and other staff immediately traveled to the house and coordinated the evacuation. The GPD program had help from the house monitor, a former firefighter, who ultimately agreed that it would be safer to evacuate. Four of the Veterans went to the evacuation center in their own cars, while one refused to leave and remained at the house due to an alcohol dependence issue.“they really did not want to go but once they were there, they seemed to get comfortable fairly quickly…. I would say there was some camaraderie about the evacuation center.”VA staff reported that Veterans became comfortable once they arrived at the evacuation shelter, finding a sense of community there despite their initial reluctance:“all of the Veterans that were in the house at that time, they were able to find permanent housing…. And we were totally prepared that these Veterans would need to stay in Grant Per Diem past their time that they were supposed to have discharged. So we didn't have that problem with them, so we were very, very lucky.”The evacuated Veterans were given the option of relocating to another GPD site 25 miles away, but all opted to remain at the disaster shelter, saying that they expected to be able to safely return to the house very quickly. The first 2 days in the shelter, the house monitor helped the GPD and VA staff keep track of the Veterans for the daily evacuation status report, verifying that those evacuated were still at the shelter, and going to the house daily to check on the remaining Veteran. On the third day, the house monitor successfully found permanent housing out of the area and left the shelter. The remaining Veterans were in the shelter for 5 days. Despite the Tubbs Fire's devastating impact to the housing market, six out of the seven Veterans enrolled in the program during the fires ultimately found permanent housing before the end of their 2‐year limit in the GPD program:“the (organization) staff actually went to the house and confirmed who was leaving the house. So that was all monitored by the (organization) staff, with constant coordination. You know, they let me know every step of the way what was going on, and I just want to commend them, because several of their staff lost their homes.Despite the communication and other challenges, VA staff praised the GPD staff's efforts in coordinating the evacuation and collaborating with the VA to track the Veterans daily during their stay at the shelter. They noted that the GPD staff were juggling these responsibilities during a stressful and chaotic time, with air quality problems and uncertainty about whether phone lines or freeway access would be compromised at any moment. While they were evacuating the Veterans and reporting to the VA, GPD staff were also monitoring their own evacuation situation and whether their own homes had been lost:This close collaborative relationship between the VA liaison and the GPD led to Veterans voluntarily evacuating, with monitoring from GPD staff. The next case study examines another GPD, where staff did not encounter resistance to evacuation, but had to coordinate Veterans' relocation quite closely with multiple VA liaisons.3.2This program, which first became a GPD grantee in 2004, had 8–10 Veterans, who evacuated for 5 days to another GPD facility 97 miles away in Sacramento that was operated by the same umbrella organization. While the Santa Rosa program was a house where Veterans lived without on‐site staff, the Sacramento facility had been operating a 42‐bed full‐service GPD program since 1995 with on‐site staff. GPD program leadership reached out to VA regional staff on October 8, when the Tubbs Fir e began, to inform them of the plan to evacuate as a precaution, due to the fire's unpredictable spread. The next day, these Veterans were transported to the Sacramento facility by the GPD staff, remaining there for 5 days until the fire had been contained. Recognizing the difficulties inherent in finding housing in an area where so much of the housing stock was destroyed by fire, the VA offered Veterans the option of enrolling in the Sacramento GPD program, but all of them were anxious to return home and ultimately opted to return to the Santa Rosa program after the evacuations were over.“…So [we]had to bring out—I think it was (VA) Law Enforcement and Facilities Management to take a look at how it was set up. How the bunks were being placed, making sure we had no blockages to fire egresses, to make sure there weren't too many people in a room, and if I remember correctly, we got lucky. It was only one gender. So I didn't have to worry about female versus male safety stuff….it was all males. So once—I think when the PD and the fire people came through, we just had to….Move the cots around, just to make sure there was no blockages to any fire egresses.”The evacuation of Veterans from the Santa Rosa to Sacramento locations required some coordination with the VA because it involved Veterans being physically moved from the San Francisco VAMC catchment area to the Northern California VAMC catchment areas. Because two different VAMC GPD programs coordinated the movement of Veterans, staff at both VAMCs, as well as the VA's regional staff, had to be involved and kept informed of the evacuation. The two VAMCs' staff decided not to officially transfer the Veterans' enrollment between the GPD programs during the evacuation, as that would have also required them to transfer the per diem funding amount to the new site for that time. However, not being officially enrolled in the Sacramento GPD program meant that these Veterans were not assigned rooms and beds within that program. Instead, the GPD evacuated Veterans were all placed in a single congregate room with cots at the Sacramento GPD facility. While the VA did not need to do a comprehensive inspection of the Sacramento facility, they had to bring in staff to inspect the congregate living space to ensure that the arrangement was safe and met basic standards:The Sacramento VAMC staff also enrolled the Veterans in their health care system during the evacuation so their access to health care could continue uninterrupted.“the impression I had was they were glad to be out of the area in case it caught on fire…I felt like they felt safe and they were trying to feel a part of, and I know like the case managers made sure they reached out. And that particular campus has a really nice cafeteria where all the food and stuff is made so all that stuff was made available to them. I think a couple of them kind of were anxious to get back in the area and they were… asking for answers when stuff would clear and they could go back.”VA staff overseeing the GPD programs in both regions reported that the transfer went smoothly. Once the Veterans were evacuated to Sacramento, Santa Rosa GPD program staff went to the new site daily during the evacuation to ensure their continued care and reported to the VA about the Veterans' daily evacuation status and any needs that arose. VA staff reported that, other than ensuring that the GPD program was supported and received their per diem funds, they did not have to do very much to ensure a successful disaster collaboration with the GPD. While evacuated Veterans were anxious, they were happy to be away from the danger of the fire and largely felt comfortable at the Sacramento facility, where they had access to staff support:The close relationship between GPD and VA ensured that inspections required for relocation to an alternate facility went smoothly and that Veterans were comfortable in the new location. The next case illustrates how the VA and GPD coordinated to take in disaster‐displaced Veterans who were not enrolled in GPD programs.3.3In disasters and other situations where the need arises, the VA and its GPD grantees collaborate to open vacant beds in GPD programs to any Veterans in need, regardless of whether they were previously enrolled in any VA homeless programs. During both the 2017 Tubbs Fire and the 2018 Camp Fire, Veterans living in private residences subsidized by HUD‐VASH vouchers and Veterans who presented as homeless in disaster shelters were offered vacant beds in GPD transitional housing programs, enabling them to access a plethora of supportive services designed to assist them in finding permanent housing. However, this essential role requires a significant amount of disaster coordination and communication between the VA and its GPD grantees. It requires that GPD grantees be prepared and agile to step into this role during and after disasters, even if they are not otherwise affected.During regional disasters, this collaboration requires VA GPD program staff to work across VAMC catchment areas, obtaining bed vacancy information from their respective GPD grantees and coordinating placement with other VA Homeless programs with Veterans in need. Often, the VISN network coordinator will be involved in this coordination as well, particularly when disaster‐affected Veterans within one VAMC catchment area must be transferred to GPD programs in another VAMC's jurisdiction. VA GPD liaisons and supervisors all reported that their grantee organizations performed extremely well as VA partners when asked about their ability to absorb Veterans.“And then the GPDs, they are always so great. They always make sure the Vet has—you know, whatever…we had three Veterans who were in HUD‐VASH apartments and their apartments burned down…they spent one night in a hotel and (GPD organization) took them the next day.”Many GPD organizations quickly stepped up to absorb these Veterans in need and did their best to ensure that displaced Veterans received needed services. As one VA liaison reported:“we did get one and the thing that was amazing about it…as soon as I emailed my supervisor and the HUD‐VASH Supervisor and said ‘Hey, I got this guy who decided to relocate from Paradise', they instantly gave him a HUD‐VASH voucher.”When the 2018 Camp Fire devasted the City of Paradise in Butte County, the geographically closest GPD organizations were located 88 miles away in the Sacramento area. Sacramento GPDs were able to take in Veterans who were displaced by the Camp Fire because they were only at 90% capacity and had plenty of room. One VA GPD staff member recounted the story of a Camp Fire survivor who was experiencing homelessness, and was placed in a Sacramento‐area GPD program after being identified as a Veteran at a disaster shelter:After the massive displacement caused by the wildfires, other VA Homeless Programs such as Supportive Services for Veteran Families and HUD‐VASH also responded quickly and proactively, expediting grants and housing vouchers to assist affected Veterans. Speaking less than a year after the fire, a GPD staff member reported that this Veteran now had access to VA health care within walking distance for the first time and was actively working toward finding permanent housing using his HUD‐VASH voucher. The GPD program was a vital resource that gave him a transitional residence, enabling him to access critical supports such as health care and a stable address to bolster his efforts to find permanent housing.“They really weren't that interested in an environment that sort of had rules and boundaries and so it's been hard to really help those Veterans…I think…these were Veterans that were just kind of living on their own in Paradise, on the edge of being homeless, but not really engaging with the community that much.”VA GPD staff also noted some challenges involved with efforts to place wildfire survivor Veterans into GPD programs. Some Veteran survivors of the 2017 Tubbs Fire sought to enter GPD programs in neighboring Marin County through this program, but VA staff were unable place them in GPD housing due to lack of capacity. VA GPD staff noted that Marin County GPD programs quickly ran out of available beds after the fires, since they were also absorbing Veterans from local disaster shelters. VA GPD staff also noted that some of the new Veterans who entered GPD programs after being displaced in the wildfires experienced challenges adapting to structured living in the GPD setting.Undoubtedly, moving into a new area after surviving a stressful and chaotic disaster and having to adapt to a new environment with institutional rules can be a distressing experience. VA staff reported that over time, these Veterans eventually adjusted to the GPD environment and the problems were quickly resolved. In offering vacant program slots to new participants, GPD organizations play a vital role in disaster response and recovery for disaster‐affected Veterans, serving as an essential lifeline for homelessness prevention and rapid rehousing.4These case studies underscore how community‐based organizations (CBOs) providing preexisting services to marginalized populations play a crucial role in disaster response and recovery. Not only did they ensure the safety and well‐being of Veteran participants living in these VA‐funded housing programs, but they also partnered with VA to take in additional Veterans of varying degrees of homelessness status who were displaced by the wildfires. The literature notes that CBOs typically lend their vital skill sets to assist in community disaster response and recovery, often taking on additional roles, such as housing displaced individuals  or hearing about how a similar organization struggled during a disaster  can ensure that their member organizations all have a set of common disaster plan elements. All organizations can connect with peer service provider non‐profits through networks like Voluntary Organizations Active in Disasters.The emergency management literature underscores the importance of coordinated collaborative relationships between the government and nonprofit sectors for disaster response Kapucu, . IdeallyThird, the 2017–2018 wildfires generated an awareness—nationally and locally—of unanticipated gaps in the required GPD disaster plans, as several VA staff noted. With the increase in human development in the wildland‐urban interface, these experiences and risks were somewhat new to these organizations (Brenkert‐Smith et al., Finally, the lessons learned from the 2017–2018 disaster experience contributed to the VA's adoption of a disaster protocol to provide all HSOs with disaster information for Veterans to follow, and to check on all Veterans participating in VA homeless programs in a disaster. It also facilitated policies to improve the VA's situational awareness about impacts to Veterans experiencing homelessness by formally integrating VA homeless program staff into the VA's emergency management roles and responsibilities (VA Homeless Programs Emergency Management Plan draft, This study has limitations that should be acknowledged. First, it is a multisite cross‐sectional examination of VA‐funded HSOs during a specific disaster in Northern California, and its findings may not necessarily be applicable to other disasters in different settings. Second, this study was only able to interview VA homeless program staff, so the perspectives of the GPD organizations or Veterans may yield different insights. However, the GPD Liaisons interviewed in this study were closely involved with the GPD organizations and called Veterans daily to check on their well‐being. Hence, they should be able to provide a faithful accounting of what they heard directly from GPD staff and Veterans. Future research should expand interviews to include GPD staff or Veterans. In addition, the close coordination structure between the VA and its GPD grantees may limit these findings' applicability to nonprofit organizations that lack such a deeply engaged funder to provide a back‐up form of support for clients during disasters. Finally, GPD grantees differ from other HSOs in regard to having a disaster plan mandate, further limiting applicability. We cannot assume that all HSOs will have disaster plans. However, it has been found that organizations providing housing to populations experiencing homelessness were among the best prepared and were more likely to have disaster plans than food‐provider organizations (Ritchie et al, 6The experience of the 2017–2018 Northern California wildfires underscores the essential role of residential HSOs and other service providers in ensuring the safety and support of people experiencing homelessness during the chaos and confusion of disasters. Climate experts predict that the ferocity of these two fire seasons is due to become increasingly common in the Western U.S. in future years (Karklis et al., https://publons.com/publon/10.1002/jcop.22653The peer review history for this article is available at The authors declare that there are no conflict of interests.


Acanthamoeba spp. can cause amoebic keratitis (AK). Chlorhexidine is effective for AK treatment as monotherapy, but with a relative failure on drug bioavailability in the deep corneal stroma. The combination of chlorhexidine and propamidine isethionate is recommended in the current AK treatment. However, the effectiveness of treatment depends on the parasite and virulence strains. This study aims to determine the potential of Garcinia mangostana pericarp extract and α-mangostin against Acanthamoeba triangularis, as well as the combination with chlorhexidine in the treatment of Acanthamoeba infection. The minimal inhibitory concentrations (MICs) of the extract and α-mangostin were assessed in trophozoites with 0.25 and 0.5 mg/mL, for cysts with 4 and 1 mg/mL, respectively. The MIC of the extract and α-mangostin inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. The extract and α-mangostin combined with chlorhexidine demonstrated good synergism, resulting in a reduction of 1/4–1/16 of the MIC. The SEM results showed that Acanthamoeba cells treated with a single drug and its combination caused damage to the cell membrane and irregular cell shapes. A good combination displayed by the extract or α-mangostin and chlorhexidine, described for the first time. Therefore, this approach is promising as an alternative method for the management of Acanthamoeba infection in the future. Acanthamoeba is an opportunistic amoeba distributed in diverse natural habitats1. This organism have two main forms: the trophozoite, an invasive stage; and cyst, a highly resistant stage in a very harsh conditions2. Based on the size, shape and features of cysts, Acanthamoeba spp. have been divided into three groups . However, Acanthamoeba spp. has been classified into 22 different genotypes (T1–T22 Genotype) based on molecular technique, which used 18S rRNA gene sequencing5. Among them, T4 genotyge is the most isolated in clinical and environmental samples, followed by genotypes T3 and T5. In addition, the T4 genotype is the most virulent because it has a significant potential for binding to host cells than other genotypes6.Acanthamoeba spp. are the causative agents of amoebic keratitis (AK) and granulomatous amoebic encephalitis (GAE). AK can cause permanent loss of vision7. The rate of infectious keratitis is becoming alarming in recent times, a problem that may be related to a sudden increase in the population of contact lens wearers2 Similarly, Acanthamoeba encysts penetrated deeply into the corneal stroma8 as such, the cyst wall becomes impervious to existing drugs, and this becomes a drawback for further studies in the areas of drug formulations and designed for this organism.Garcinia mangostana Linn. is generally known as mangosteen and belongs to the family Clusiaceae. It is a tropical tree, widely distributed in Southeast Asia9. The pericarps of this fruit are commonly used in traditional medicine to treat several diseases which are non-toxic and safe to use11. Major compound in the mangosteen pericarps is xanthone group, particularly α-mangostin, which exhibited antibacterial activity12, antifungal activity13, antioxidant activity14 anti-cancer activity17, anti-inflammatory activity19, antiparasitic activity22.Acanthamoeba activity of G. mangostana extract to date. Hence, our study sought to investigate the effective concentration of the G. mangostana extract and α-mangostin on the growth inhibition of Acanthamoeba spp. and to demonstrate its synergistic effects combined with chlorhexidine on anti-Acanthamoeba activity.To the best of our knowledge, we discover that there is no single report on the anti-Acanthamoeba sp. WU19001 from our previous study aligned using CLUSTAL W and revealed the highest variation, as shown in Fig. Acanthamoeba triangularis KX232518.1 (99.74% similarity). The sequence homology search for the 35 Acanthamoeba spp. in the National Center for Biotechnology Information (NCBI) database showed WU19001 formed Acanthamoeba genotype T4 cluster  susceptible to the extract and α-mangostin at all concentrations when compared to control. The extract and α-mangostin showed higher susceptibility against Acanthamoeba trophozoites than cysts.   Fig. a,b.FigurAcanthamoeba cysts decreased to 2.3 × 105 and 0.8 × 105 cells/mL when treated with MIC concentration of the extract and α-mangostin, respectively. We observed the re-growth which reached to the number of viable cells of 1.4 × 105 and 2.2 × 105 cells/mL in the presence of the extract and α-mangostin at 72 h of incubation. However, the treated cysts showed a decrease in viable cells when compared with the control that reached up to 5.8 × 105 cells/mL at 72 h of incubation  was observed in the combination of chlorhexidine and the extract. Chlorhexidine at 0.002 mg/mL was synergistic when combined with α-mangostin at 0.032, 0.062 and 0.125 mg/mL   after 24 h of treatment  Fig. a. For α-mL) Fig. b. The efent Fig. c. To redA. triangularis infection with herb-drug based combination strategy. Also, we highlight the potential of the anti-Acanthamoeba activity of G. mangostana which also known as the “queen of fruits”.The herb-drug interaction may impact to the potential health-promoting effect of increasing drug efficacy or decreasing common adverse effect. As such, we present in this study an effective treatment for G. mangostana which have been presented in several parts of plant such as pericarp, whole fruits, leaves and bark6. The pericarp of mangosteen fruit are the most abundant of xanthones α-mangostin 23. In addition, there are various components of other xanthones, such as β-mangostin, gartanin, 8-deoxygartanin, garcinones A, B, C, D and E, mangostinone, 9-hydroxycalabaxanthone, and isomangostin24. Nevertheless, previous studies have reported that the concentration of α-mangostin correlates with the biological activity of mangosteen extract10. Interestingly, the compound α-mangostin from G. mangostana has ever been reported its property against other parasites like Plasmodium falciparum22.In previous literature, xanthones is a major phytochemical compound from G. mangostana extract and α-mangostin on anti-parasitic activity25, but the effect on Acanthamoeba spp. has so far never been explored. Our study therefore demonstrated for the first time of G. mangostana extract from the pericarp and α-mangostin exhibit anti-Acanthamoeba activity against A. triangularis trophozoites and cysts. A. triangularis WU19001 was a selected strain parasite used in this study due to its characteristic property belong to group II (genotype T4). Group 2 Acanthamoeba species are pathogenic in nature, typically having double-walled with a wrinkled ectocyst and stellate polygonal, triangular, or oval endocyst26. A. triangularis was originally isolated from human faeces27 and currently discovered from environmental water souces, as reported in this study. In 2008, the pathogenic strain of A. triangularis has firstly reported in a clinically confirmed case of Acanthamoeba keratitis in Korea28. Due to climate change and global warming, it is postulated that A. triangularis would be more likely to be associated to clinical scenario in times to come as it is widely contaminated in the environment.Some studies have reported the effects of Acanthamoeba infection seems impossible due to the high resistance of the cysts to anti-Acanthamoeba drugs. Therefore, we also investigated the effective concentration of G. mangostana extract and α-mangostin against A. triangualris trophozoite and cyst stages. Our results showed that the extract and α-mangostin can inhibit A. triangularis in PYG medium. It is noteworthy that the number of Acanthamoeba trophozoites and cysts were significantly reduced (p < 0.05) in the treatment with MIC concentrations of the extract and α-mangostin. They were found to have the greatest growth-inhibitor for trophozoites at 72 h. For the cystic form, they survived on 72 h  as a result of the destruction of the ectocyst walls after the treatment given with G. mangostana extract, α-mangostin, chlorhexidine and in combination.The mode of action considered in this study was confirmed by scanning electron microscopy (SEM), as shown in Figs. 50 values 39 μM (0.016 mg/mL). Our preliminary finding is however surprisingly different from a previous study reported that α-mangostin was non-toxic to nontumorigenic human pancreatic duct epithelial cells, at a dose as high as 40 μM36. In addition, xanthones in G. mangostana pericarp proved to be non-toxic to mice in vivo when administered orally at a dose of 100 mg/kg of body weight/day for 7 days37. Since α-mangostin is gaining more popular to be used against infectious diseases, therefore, it strongly suggests for further comprehensive studies such as chemical structure modification and structure–activity relationship as well as nanotechnology to evaluate the mechanism of action of these compounds against Acanthamoeba spp., before any conclusion could be made.For a pure compound, α-mangostin exhibited toxicity to Vero cells at ICG. mangostana from the pericarp and α-mangostin possess anti-Acanthamoeba activities against A. triangularis trophozoite and cyst stages. Moreover, the present study focuses on the novel combination for treatment of Acanthamoeba infection. The combination of G. mangostana extract and α-mangostin with chlorhexidine generated synergistic effects which increased effectively for the treatment of Acantrhamoeba infection.Besed on the results obtained from this study, ethanolic extract of G. mangostana pericarp powder was soaked in 200 mL of ethanol for 7 days. The extract was filtered through Whatman No. 1  using a vacuum and pressure pump. The solution was evaporated to dryness under reduced pressure using a rotary evaporator to obtain G. mangostana extract. The 200 mg of extract was subjected to column chromatography using silica gel as a stationary phase and eluted with 20% acetone in 750 mL of hexane. The collected fractions were chromatographed on a silica gel thin-layer chromatography compared with the authentic compound, α-mangostin. Fractions with one spot of α-mangostin on chromatogram were combined. The solvent was removed to give 30 mg yellow solid of α-mangostin. The extract and α-mangostin were dissolved in 99.5% DMSO and stored at − 20 °C until use.The 50 g of dry A. triangularis WU19001, a strain from the recreational reservoir at Walailak University, Nakhon Si Thammarat-Thailand, was used in this study38. The parasite was grown in PYG medium . The trophozoites were observed after 72 h of incubation at room temperature and were cultured in this medium for 1 week The 90% mature cysts were obtained PYG medium. The cysts were harvested when the cultures were incubated for at least 1 week without addition fresh medium. The parasite reproduced exponentially until they reached the maximum level of 1 × 106 cells/mL, after which a reduced nutrients led to encystation (cyst formation) due to unfavorable conditions for the parasite’s growth especially into trophozoite stage. At the end, all fully homogenic inoculum of mature cysts were successfully harvested. Trophozoites and cysts were centrifuged at 4000 rpm for 5 min and re-suspended in fresh PYG thereafter. For counting, 50 μL of cell suspension was mixed with 50 μL of trypan blue. Viability was investigated under the inverted microscope based on the principle of the dye can cross the membrane of dead cells with blue color, but not intact membrane of viable cells with colorless appearances.G. mangostana extract and α-mangostin was determined using the microtiter broth dilution method38. The extract and α-mangostin were diluted to give a final concentration of 4, 2, 1, 0.5, 0.25, 0.125, 0.062 mg/mL in a 96-well microplate. Then 100 µL of 2 × 105 cells/mL of trophozoites and cysts were inoculated into each well. Chlorhexidine and 1% DMSO were included as a positive and negative control, respectively. The plates were incubated at room temperature for 24 h. The viability of parasites was calculated as follows: % viability = (mean of the viable parasite/control) × 100. The MIC value was defined as the lowest concentration that inhibited > 90% of viable growth when compared with the control.The minimum inhibitory concentration (MIC) for A. triangularis of G. mangostana extract and α-mangostin was carried out following the procedure that previously described1 with modifications. The trophozoites and cysts (2 × 105 cells/mL) were incubated with the extract and α-mangostin in the MIC, 0.5 × MIC and 0.25 × MIC, except for untreated control tubes, which had only PYG medium and incubated at room temperature for 72 h. At 24 h intervals, the viability of parasite was determined by staining with 0.2% trypan blue.In the present study, the growth inhibition on 39 was used to evaluate the interaction between G. mangostana extract/α-mangostin and chlorhexidine against A. triangularis. Subsequently, the microdilution assay was performed in a 96-well plate with a final volume of 200 µL. The extract, α-mangostin and chlorhexidine were diluted with PYG to obtain 4 times to their final concentrations of 1/16 MIC, 1/8 MIC, 1/4 MIC, 1/2 MIC and MIC. A total of 100 µL of the extract + chlorhexidine or α-mangostin + chlorhexidine were prepared in 96-well plate and added 100 µL of parasite suspension containing 2 × 105 cells/mL into the wells. The plates were incubated at room temperature for 24 h. The viability of parasite was defined as the lowest concentration that inhibited > 90% of growth when compared to the negative control. The assessment of the results was defined as the Fractional Inhibitory Concentration Index (FICI), which was calculated using the following:The checkerboard methodThe combination was considered synergistic for FICI ≤ 0.5, additive for 0.5 < FICI ≤ 1, indifferent for 1 < FICI < 4, and antagonistic for FICI ≥ 4, according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) definition.A. triangularis were treated with chlorhexidine, G. mangostana extract, α-mangostin and in combinations. After incubation, cells were collected by centrifugation at 4000 rpm for 5 min and re-suspended in phosphate buffer saline (PBS). Cells in 1% DMSO were used as negative controls. Samples were fixed with 2.5% glutaraldehyde overnight. The samples were further dehydrated with a series of graded alcohol , mounted on aluminum stubs, and allowed to dry using a critical point dryer. Samples were then coated with gold particles and the morphology of A. triangularis trophozoites and cysts after treatment was subsequently examined under SEM  at the Center for Scientific and Technological Equipment, Walailak University, Nakhon Si Thammarat, Thailand38.Trophozoites and cysts of G. mangostana extract, α-mangostin, chlorhexidine and combination sets were evaluated using the Vero cell line (8200F270602). Cells were cultured in Dulbecco's Modified Eagle's (DMEM) medium  supplemented with 10% FBS and 1% antibiotic containing penicillin G of 100 units/mL, streptomycin of 100 μg/mL. The culture was incubated at 37 °C in a humidified atmosphere and 5% CO2. After the cells reached 90% confluence, detachment was performed using trypsin ethylene diamine tetraacetic acid (EDTA), incubated at 37 °C in 5% CO2. Single cells at a density of 1.5 × 104 cells/100 μL were seeded into each well of a 96-well polystyrene plate and allowed to attach for 24 h. Then, 100 μL of the extract and α-mangostin, previously prepared at multiple concentrations, chlorhexidine and combination sets were gently added. After incubation for 24 h, the cytotoxic effects were determined using the MTT assay. The absorbance was measured using a microplate reader  at 570 nm. The percent survival was calculated using the following equation:The cytotoxic effects of 40.ABt and ABu denote the absorbance values of treated and untreated cells, respectivelyp < 0.05 was considered statistically significant.The experiments were performed in triplicate. All data were recorded and entered using the statistical package software . The data were expressed as mean ± SD. Statistical analysis was analyzed by the two-tailed unpaired Student’s t-test. In all analyzes, Supplementary Information.