The objective of this study was to discover the possible correlation between p16INK4A expression and the LR/HR-HPV infection in condyloma acuminate (CA) lesions. INK4A was detected by immunohistochemistry (IHC) staining. The positive interpretation was carried out by scoring which score 0 was negative, score 1 was sporadic, score 2 was focal, and score 3 was diffuses. The HPV genotypes were identified by reverse line blot, and 40 genotypes of HPV detected, including HR-HPV  and LR-HPV . This cross-sectional study was conducted during January-December 2017 on 33 CA patients. The expression of p16INK4A was significantly correlated with HR-HPV infection. Patients infected with HR-HPV had 0.644 times higher possibility to express p16INK4A gene compared to those infected with LR-HPV. LR-HPV genotypes detected in CA patients were HPVs 6, 11, 42, 61, 54, 81, 87, 89, and 90 and HR-HPV genotypes were HPVs 18, 26, 45, 51, 52, 67, 68B, 69, and 82. LR-HPV was found in 19/33 of patients and HR-HPV was in 14/33 of patients. The expression of p16INK4A in CA lesions was diffuse in15.2% of patients, was focal in 24.2% of patients , was sporadic in 39.4% of patients were, and was negative in 21.2% of patients . In LR-HPV group, there was no diffuse expression, focal expression was observed in 15.8%, sporadic in 47.4%, and negative in 36.8%, while in HR-HPV group, p16INK4A expression was detected in all lesions , in a way that its expression was diffuse in 35.7%, focal in 35.7%, and sporadic in 28.6%. The expression of p16INK4A expression by IHC can be used as a biomarker for HR-HPV infection diagnosis. IHC is a routine method in histopathological diagnosis, therefore the detection of p16 Condyloma acuminata (CA) is the most frequent sexually transmitted disease worldwide . It is also known as genital warts or anogenital warts. These lesions are usually single or multiple appearing in the anogenital region and causing symptoms of itching, vaginal discharge, and bleeding. CA can be flat or lobulated and appears as pearl-like, filiform, plaque eruption, or cauliflower projection . Its prevalence is increasing worldwide. The prevalence of CA in Italian female population during 2009-2010 was 3.8 cases per 1,000 women per year , in the UK population during 2010-2012 was 3.8% in males and 4.6% in females , and in the US population during 2013-2014 was 2.9% . A systematic review on the incident of anogenital warts showed that their prevalence ranged from 160 to 289 per 100,000 persons. New incident of anogenital warts ranged from 103 to 168 per 100,000 persons among males and 76 to 191 per 100,000 persons among females .The most common cause of CA is Human Papillomavirus (HPV). There are two groups of HPV, including high risk (HR) HPV and low risk (LR) HPV. HR-HPV are HPV genotypes of 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 61, 73, and 82, and LR-HPV are HPV genotypes of 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81 . LR-HPV causes CA, but a study in China showed that the infection of HR-HPV was also found in CA lesions. In some cases, CA is caused by the combination of LR and HR-HPV infections . Persistent infection of LR or HR-HPV is a risk factor for the transformation of epithelial to benign hyperplasia or premalignant lesions. HR-HPV is related to the occurrence of malignancy in women, as cervical cancer . Some literature considers HR-HPV as the main causative agent responsible for the cervical cancer . The detection of HPV genotype is very important to prevent, establish early diagnosis, and initiate the treatment in cervical cancer. Determination of LR or HR-HPV in anogenital warts can be used as a factor to predict the progression of lesions to benign or malignant lesions.INK4A protein. The expression of p16INK4A can also be considered as a marker of E7 gene activity . p16INK4A plays a role in cell cycle regulation and it is involved in the processes of apoptosis, angiogenesis, cell invasion. This activity may be associated with overexpression in cancer . The expression of p16INK4A is a marker to determine the prognosis of a malignancy caused by HPV infection , suggesting that p16INK4A can be a specific marker for HPV infection and may correlate with the type of HR-HPV or LR-HPV. The objective of this study was to analyze the correlation between p16INK4A expression and the LR-HPV or HR-HPV in CA lesions. This study identified the genotype of HPV and performed immunohistochemical analysis of p16INK4A staining of. The E7 HPV protein plays a role in the cell transformation process. It binds to important proteins, such as the pRB and cyclin A/CDK2 complex, inhibiting the interaction between Rb and E2F. The E7 protein of HR-HPV deactivates pRB, resulting in the accumulation of p16The samples collectionThis cross-sectional study was conducted at Outpatient Clinic of Department Dermatology and Venereology, Dr. Soetomo General Academic Hospital, Surabaya, Indonesia, from January 2017 to December 2017. The study was ethically approved by the Medical Ethic Research at Dr. Soetomo General Academic Hospital, Surabaya .All patients with CA, both male and female, who were willing to participate in the study, were included. Informed consent was obtained from the participants. Menstruating and pregnant women, those who suffered from an active pelvis and / or acute cervicitis, men or women with a diagnosis of HIV and AIDS, and those who were not willing to participate in the study were excluded.The specimens were 33 tissues of CA. Tissue from each patient was divided into 2 parts, one part for tissue processing into paraffin block preparations followed by histopathological diagnosis and immunohistochemistry (IHC) staining, while another for the examination of the HPV genotype. Histopathological diagnosis of CA and analysis of IHC staining was performed by a pathologist. Expression of p16INK4AINK4A Antibody (clone 1E12E10) IHC-plus™ LS-B5261 (LS Bio). The interpretation was positive if cells were stained in the nucleus or combined in the nucleus and cytoplasm. The assessment was carried out based on guidelines presented by Klaes et al. (2001) as follows:The expression of p16INK4A was detected by IHC staining using Anti-CDKN2A/ p16Score 0 (negative): if cells were stained positive <1% of all cellsScore 1 (sporadic): if cells were stained positive <5% of all cellsScore 2 : if cells wee stained positive <25% of all cellsScore 3 (diffuses): if cells were stained positive > 25% of all cells. Genotyping of HPVVirus extraction was carried out from CA tissues using the QIAamp DNA Mini Kit (Qiagen) kit and according to the manufacturer’s protocol. Genotyping of HPV was performed by Polymerase Chain Reaction (PCR), then followed by reverse line blot using the Ampliquality HPV type express v 3.0 kit . This kit could detect 40 genotypes of HPV, including HR-HPV were HPV 16, 18, 26, 31, 33,35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68 , 69, 73, and 82 and LR-HPV were HPV 6, 11, 40, 42, 43, 44, 54, 55, 61, 62, 64, 70, 71, 72, 81, 83, 84, 87, 89, and 90.Statistical analysisINK4A on CA patients with HR-HPV or LR-HPV groups infection was analyzed by Mann Whitney test (significant if p <0.05). The correlation between variables was analyzed by two-tailed Spearman’s rho (significant if p <0.05). The difference in the expression of p16Characteristics of patient This study was performed on 33 patients suffering from CA. Patients’ characteristic, such as sex, age, sexual partner, duration of the symptoms, medical history, presence of lesion in the partner, history of treatment, and shape, type, and location of the lesion were investigated. The patients consisted of 12 (36.4%) males and 21 (63.6%) females. The patients aged from 18 to 64 years. The highest frequency was allocated to the age group of 15-24 years old (51.5%). Regarding sexual partner, it was found that 30/33 of the patients (90.9%) were heterosexual (men having sex with women), 2/33 (6.1%) were bisexual (having sex with the same and different sex), and 1/33 (3.0%) were homosexual (having sex with the same sex). Regarding symptoms duration, the highest frequency was allocated to the duration of 1to 3 months. Mostly, the patients experienced CA for the first time, there was no lesion in their partner, and received TCA therapy, respectively. Considering the shape, type, and location of the lesion, it was revealed that the highest frequencis were allocated to papule shape, multiple type of lesions, and gland penis for male and labia majora for female . HPV genotype in CA lesions The genotype of HPV included the infection of HPV-HR and LR, both single and multiple infections. The single or multiple infections of HPV-LR was assumed as infection of LR- HPV. The multiple infection of LR-HPV with HR-HPV was assumed as infection of HR-HPV. The genotype of LR-HPV were HPV 6, 11, 42, 61, 54, 81, 87, 89, and 90, and of HR-HPV were 18, 26, 45, 51, 52, 67, 68B, 69, and 82. LR-HPV was found in 19/33 (57.6%) of the patients and HR-HPV was seen in 14/33 (42.4%) of the patients . The mosp16INK4A expression in CA lesionsINK4A in CA lesions showed that nucleus or the combination of nucleus and cytoplasm of cell was stained in brown color, indicating in sporadic, focal, or diffuse . Correlation between p16INK4A expression and LR-HPV and HR-HPV infections was moderate . The expression of p16 diffuse . The res diffuse . There wThe microscopic features of CA lesions based on haematoxylin eosin (HE) staining are parakeratosis, hyperkeratosis, hypergranulosis, basal cell hyperplasia, and koilocytic . In this study, CA lesions were seen as papules, cauli flowers, and flat-tapping papules. The microscopic features found in this study were hyperkeratosis, parakeratosis, papillomatosis, hypergranulosis, and hyperplasia of basal cell, as well as the koilocytes that usually is associated with HPV infection. The most common cause of CA is infection by HPV. There are more than 40 genotypes of HPV that can infect the anogenital area that are usually infected by LR-HPV including HPV 6 and HPV 11 in single infection, but most commonly co-infection with LR-HPV or HR-HPV . The results of this study showed that all the tissues taken from the patients, who were clinically diagnosed with CA, were positive for LR-HPV or HR-HPV infections, in single infection or multiple infection. Most of the patients were infected by LR-HPV, while 42.4% were infected by LR-HPV and co-infected with HR-HPV. In line with this study, a cross-sectional study in Kuwait on 156 patients with genital warts showed that 102/156 (65.4%) of the patients were infected by LR-HPV and 54/156 (34.6%) of the patients were infected by HR-HPV. About 88.4% of the patients in the aforementioned study had single infection and 11.6% had multiple infections . Another study in Spain showed that the frequency of LR-HPV infection was 63/138 (45.6%) in their patients and 71/138 (41.4%) of anogenital warts patients were infected by HR-HPV . The other study on 66 anogenital warts specimens showed that LR-HPV infected 42/66 (62.1%) which was dominated by HPV 6 (47%), and HPV-11 (13.6%), as well as HPV 18 and HPV 3 . We discovered that that beside LR-HPV, CA was also co-infected with HR-HPV that could develop to malignant cancer. Therefore, identification of HPV genotype can predict the risk of developing related diseases. In addition, determination of HPV genotype affects treatment management, patients’ follow-up, and prevention strategies The findings of this study revealed that 57.6% of CA patients were infected by LR-HPV and the rest were infected by both LR-HPV and HR-HPV infections. The most common HPV genotypes were HPV 11 and HPV 6 for LR-HPV, followed by HPV 18, HPV 51, and HPV 82 for HR-HPV. The incidence of CA and the progression of the disease can be prevented by vaccination. Recently, there are 3 commercial vaccines against HPV infection, namely Gardasil to prevent infections caused by HPV 6, 11, 16, and 18, Cervarix to prevent infections caused by HPV 16, and 18, and Gardasil 9 to prevent infections caused by HPV 6, 11, 61, 18, 31, 33, 45, 52, and 58 . A previous study on anogenital benign lesions showed that HPV genotype in CA patients was dominated by HPV 11 and HPV 6 among 13 female patients  and 12 male patients . In pre-cancerous lesion and cancerous lesion of uterine cervix, those were dominated by HPV 16 (62.68%), then followed by HPV 18 (20.9%), HPV 45 (5.97%), HPV 52 (5.97%), and HPV 67 (4.48%) . Vaccination programs is expected to reduce the incidence of these diseases, but there are some genotypes of HPV that cannot be targeted by the current vaccines. Therefore, strategies to prevent the incidence of CA or the progression of malignancy still need to be developed. INK4A feedback mechanism. HPV E7 induces degradation of pRB by an ubiquitin proteosome pathway that causes the loss feedback mechanism of p16INK4A and then leads to accumulation of p16INK4A, presenting as overexpression of p16INK4A . Oncoprotein E6 and E7 of HR-HPV play a role in cancer development. HR-HPV E6 mediates p53 inactivation by binding to the conserved domains of E6AP (E6-linked protein) to form the E6/ E6AP/p53 complex. This complex causes degradation of p53 by ubiquitination mechanism. HR-HPV E7 targets to degrade the retinoblastoma protein (pRB). In normal cells, when cells are prevented from entering the S phase, the pRB binds to the E2F family of transcription factor, so that the cell stops at the checkpoint of G1-S phase, activating cell cycles arrest. pRb, which is phosphorylated by cyclin D1/CDK 4/6 complex, causes E2F to be released and enter the nucleus. The cell enters the S phase which then starts the activation of gene transcription . Furthermore, phosphorylated pRB is a p16INK4A in this study was sporadic (39.4%), focal (24.2%), diffuse (15.2%), and negative (21.2%). Another study on p16INK4A expression in 24 CA specimens showed that overexpression of p16INK4A were sporadic in 11/24 of the specimens (45.8%), were focal in 7/24 of the specimens (29.2%), and were negative in 6/24 of the specimens (25%) . The expression of p16INK4A in cervix tended to increase from cervical normal epithelium to invasive cervical cancer  and cervical adenocarcinoma . The expression of p16INK4A in HR-HPV showed that all specimens were positive ranging from sporadic to diffuse. In LR-HPV were sporadic and focal, whereas 36.8% of specimens were not expressed in p16INK4A and none of specimens expressed in diffuse category. Other study on anal lesion with HR HPV infection showed that all specimens expressed p16INK4A ranging from sporadic to diffuse, whereas those infected with LR-HPV were mostly patchy . In oropharyngeal squamous cell carcinomas and tonsillar dysplasia that were positive for HPV 16, p16INK4A expression was also diffuse. However, in benign and pre-malignant lesions were positive for HPV 6 and HPV 11 showed variations from negative to strong positive . All cervical lesions infected with HR-HPV had a significant, strong, and diffuse p16INK4A expression, whereas those infected with LR-HPV showed mild p16INK4A expression (score 1) .The overexpression of p16INK4A significantly correlated with HR-HPV infection, in a way that CA lesions infected by HR-HPV had 0.644 times more chances to express p16INK4A compared to CA lesions infected by LR-HPV. As previous studies showed that p16INK4A expression was associated with HR-HPV infection in oropharyngeal squamous , mucosal squamous cell carcinomas of the head and neck , cervical squamous intraepithelial lesion , and invasive cervical and vaginal carcinomas . HR-HPV is integrated into the host cell genome, whereas LR-HPV prefers extra chromosome as episome, so that the expression of E6 and E7 oncoproteins are within the regulatory framework of E1 and E2 HPV . HR-HPVs, such as HPV 18, 33, HPV 51, HPV 58, and HPV 59, were found to be integrated, while HPVs 30, 35, 39, 44, 45, 53, 56, 59, 74 and 82 did not tend to be integrated, but to be in episome form . In this study, it was found that HR-HPVs 18, 51, and 82. As reported by Nkili-Meyong et al., (2019), HPV 18 was founded to be integrated in 55% of the positive HPV 18 specimen, HPV 51 was integrated in 25% of the positive HPV 51 specimen, and HPV 82 was founded in extra chromosome. This integration was associated with partial or total deletion of E1 and E2 genes, leading to the overexpression of E6 and E7 due to loss of feedback mechanism by E2 protein . In addition, the HR-HPV E7 oncoproteins have higher affinity to bind pRB than LR-HPV E7, increasing the accumulation of p16INK4A. Therefore, all specimens infected by HR-HPV in this study were positive for the expression of p16INK4A in sporadic, focal, and diffuse forms, while those infected by LR-HPV had sporadic and focal, but not diffuse expression. These findings highlighted that the expression of p16INK4A. p16INK4A expression was correlated with HR-HPV infection moderately. IHC is a routine method to perform the diagnostic of histopathological. Therefore, IHC of p16INK4A could be used as biomarker for HR-HPV infection to predict the malignancy development of CA lesions. In conclusion, this study found significant differences between CA specimens infected by LR-HPV  and HR-HPV  regarding the expression of p16Concepts, design, definition of intellectual content: Gondo Mastutik. Literature search: Gondo Mastutik, Afria Arista. Clinical studies: Gondo Mastutik, Dwi Murtiastutik, Trisniartami Setyaningrum. Experimental studies: Gondo Mastutik, Alphania Rahniayu, Afria Arista. Data acquisition: Nila Kurniasari, Alphania Rahniayu, Anny Setijo Rahaju. Data analysis: Gondo Mastutik, Alphania Rahniayu, Anny Setijo Rahaju.Statistical analysis: Gondo Mastutik, Nila Kurniasari, Anny Setijo Rahaju. Manuscript preparation and manuscript editing: Gondo Mastutik, Afria Arista, Anny Setijo Rahaju. Manuscript review: Gondo Mastutik, Alphania Rahniayu, Dwi Murtiastutik, Trisniartami Setyaningrum, Erna Sulistyani.


Preventive Services Task Force endorsement of HPV testing as a primary approach to CC screening offers an opportunity to explore self-sampling  as an alternative to clinician sampling.3 Self-sampled HPV testing with timely follow-up care has the potential to improve CC screening uptake particularly in low resource settings. With the goal to inform self-sampling interventions to improve HPV testing uptake among underscreened women, this study examines awareness and use of HPV testing and acceptability of self-sampling and differences by sociodemographic factors among CC screening-eligible women living in Minnesota.Recent US data reveal concerning declines in cervical cancer (CC) screening rates and persistent disparities in CC screening and related outcomes by sociodemographic factors.Data were from the omnibus 2019 Minnesota State Survey implemented by the Minnesota Center for Survey Research at the University of Minnesota (UMN) through telephone interviews between October 2019 and March 2020, using a simple random sample of Minnesota adult residents with a landline or cell phone . Telephone interviewing was abruptly terminated on March 16, 2020, when UMN suspended all on-campus work due to COVID-19. This study was approved by the UMN Institutional Review Board.CC screening-eligible respondents  self-reported whether they had heard of HPV testing for CC screening and whether they have had a HPV or Pap test. Respondents were also asked to compare self-sampling for HPV testing to Pap testing done by a clinician in terms of convenience, embarrassment, ease, and pain, and likelihood of following up abnormal results with further testing. Sociodemographic factors including race/ethnicity, education, marital status, household income, housing, and metropolitan area were measured.We report descriptive statistics for awareness of HPV testing as a screening option and CC screening history, and acceptance of self-sampling for HPV testing. We examined sociodemographic differences using chi-square test or Fisher’s exact test.p=.041) while women ages 21–29 or 40–59 less frequently had HPV testing (p=.002). Women without a college degree (versus college graduates) less frequently heard of or had HPV testing . Additionally, women ages 21–29, racial/ethnic minority (versus non-Hispanic white) women, and women who rent (versus own) their homes less frequently had Pap testing .Table p<.001), while women living in the largely rural greater Minnesota region (versus Twin Cities metropolitan area) more frequently rated self-sampling as more convenient and easier . Lastly, 94.8% of respondents reported that they would be likely to follow up with further testing upon receiving an abnormal result from the self-sampled HPV test.Table 5 suggesting interventions promoting HPV testing and self-sampling should focus on underserved populations to reduce existing CC disparities. These data can inform interventions to improve HPV testing uptake among underscreened women through self-sampling.Self-sampling for HPV testing was perceived as more convenient, less embarrassing, easier, and less painful than clinician sampling for Pap testing by most screening-eligible women we surveyed, especially women without a college degree and women living in the greater Minnesota region. Additionally, over 90% of women reported they would seek follow-up testing of an abnormal result from a self-sampled HPV test. These findings suggest that self-sampling has the potential to improve HPV testing uptake for women in medically underserved and rural communities. The disparities by age, education, race/ethnicity, and housing in HPV testing awareness and reported CC screening history confirm previous findings,Limitations of this research include the low response rate, which may introduce bias and limit the generalizability, and the small sample size, which limited statistical power for exploring interaction effects.


One of the main questions in the design of a trial is how many subjects should beassigned to each treatment condition. Previous research has shown that equalrandomization is not necessarily the best choice. We study the optimalallocation for a novel trial design, the sequential multiple assignmentrandomized trial, where subjects receive a sequence of treatments across variousstages. A subject's randomization probabilities to treatments in the next stagedepend on whether he or she responded to treatment in the current stage. Weconsider a prototypical sequential multiple assignment randomized trial designwith two stages. Within such a design, many pairwise comparisons of treatmentsequences can be made, and a multiple-objective optimal design strategy isproposed to consider all such comparisons simultaneously. The optimal design issought under either a fixed total sample size or a fixed budget. A Shiny App ismade available to find the optimal allocations and to evaluate the efficiency ofcompeting designs. As the optimal design depends on the response rates tofirst-stage treatments, maximin optimal design methodology is used to findrobust optimal designs. The proposed methodology is illustrated using asequential multiple assignment randomized trial example on weight lossmanagement. However, it may be preferable to allocate more participants to one arm thanto another, for instance, when variances and/or costs vary across the treatmentarms,15 or when outcomes arecategorical rather than quantitative.610 The derivation of the optimalallocation of units to treatment conditions has not only been done for individuallyrandomized trials, but also for more complex trial designs such ascluster-randomized trials,1116 and trials with partiallynested data.1719 From a statistical point ofview, it is more efficient to assign more subjects to the condition with the lowestcosts and highest variance. Other, more practical, reasons to use unequal allocationover equal allocation include resource constraints, administrative, political orethical concerns or when the aim is to gain experience from an intervention and tostudy its feasibility.20In many randomized controlled trials, participants are equally allocated tointervention arms. Such a design is consistent with the view of clinical equipoisethat must exist before the start of the trial.2124 An ATS individualizestreatments to subjects via decision rules that adjust the type, intensity, dosage ordelivery of a treatment and specify when, whether and how to proceed at certaincritical clinical decisions. For instance, those subjects for whom their assignedtreatment turns out to be beneficial may continue the same treatment, while thoseothers may be assigned to another treatment. The use of sequential treatments isoften necessary because of: (i) heterogeneous treatment outcomes across subjects,(ii) change in treatment goals over time, (iii) the need to balance potential risksand benefits or (iv) to reduce costs when intensive treatment is notnecessary.26 Also, the use of sequential treatments implies multipleclinical decisions to be taken throughout the course of the study. These clinicaldecisions are formalized through ATSs.The focus of these references is on trials where subjects are randomized to eitherone single treatment or a combination of treatments, but do not change theirassigned treatments during the course of the trial. This is a drawback since in realresearch practice some subjects may benefit more from one treatment and others morefrom another. Adaptive treatment strategies (ATSs), which are also called dynamictreatment regimens or adaptive interventions, are more flexible in the sense thatthey allow changing treatments over time.27 SMARTs are multi-stagerandomized trial designs that are used to inform on the development of multiple ATSsembedded in it. The use of SMART designs allows researchers to evaluate the timing,sequencing and adaptive selection of treatments by using randomization anddeveloping the best sequence(s) of treatments that lead to the optimal outcomes inthe long term. In SMARTs, participants are allowed to switch through multiplestages, where each stage corresponds to a clinical decision, and subjects may berandomized at each stage. Sequenced randomization ensures that at each decisionpoint the groups of participants assigned to the intervention options are balancedin terms of patient characteristics. This adds flexibility, allowing participants toremain on those treatments that are having an effect and giving the possibility toswitch away to patients being treated with less effective options. This has madeSMART designs appealing in a broad variety of health care, behavioural andpsychological settings.Based on the number of treatments and treatment switches, various competing ATSs maybe developed and they may be compared to one another in a so-called sequentialmultiple assignment randomized trial (SMART).2834 but did not study the optimalallocation of units to treatment sequences and the loss of efficiency of using equalrather than unequal allocation.Multiple ATSs are embedded in a SMART and the main question in the design phase of aSMART is how many subjects should be assigned to each ATS, and whether an unequalallocation is better than an equal allocation. Some recent papers studied therelation between sample size and power for SMART designs, in the setting of a cluster-randomized trial. In our contribution, we focuson individual randomization. We focus on sample sizes to be used when comparing twoATSs that start with different first-stage treatments. Four of such pairwisecomparisons can be made in their prototypical SMART design, and one comparison maybe of more importance than another. We therefore use multiple-objective optimaldesign methodology to consider all comparisons simultaneously, while taking intoaccount their relative importance. Multiple-objective optimal designs are useful when the study has multiple andconflicting objectives, such multiple pairwise comparisons of marginal means of ATSsin a SMART. It combines these objectives in one optimality criterion and tries toseek a design that is highly efficient for each of these criteria. We provide aShiny App to calculate the optimal allocation of units and to evaluate theefficiency of the design with equal allocation. We demonstrate our optimal designmethodology on the basis of a SMART example that compares two different treatments,nutrition (NUT) and physical activity (PHY), for weight loss management. Our focusis on SMARTs with a quantitative outcome with individual randomization. In otherwords, we do not focus on cluster-randomized SMARTs or other complex SMART designswith clustered data.The aim of this paper is to derive optimal allocations of units for a prototypicalSMART design. This is a two-stage design where all units are randomized to twotreatment conditions in the first stage. Those who respond to their assignedtreatment are not re-randomized in the second stage, while those who do not respondare re-randomized to two second-stage treatments. This design was considered earlierby NeCamp et al.The remainder of our contribution is organized as follows. Section ‘PrototypicalSMART design’ further discusses the prototypical SMART design and its embedded ATSs.Furthermore, this section introduces the example of weight loss management. Section‘Derivation of the optimal design’ derives the optimal allocation of units forstudies in which either the total sample size or the budget is fixed. In the lattercase, we consider the realistic situation where costs may vary across treatmentconditions. The optimal allocation turns out to depend on the subjects’probabilities to respond to their first-stage treatment. We therefore also focus onmaximin optimal designs that are robust to incorrect prior estimates of theseprobabilities. Furthermore, Section ‘Derivation of the optimal design’ introducesthe Shiny App that we developed for finding the optimal design. Section ‘A SMARTexample’ demonstrates our optimal design methodology on the basis of the weight lossexample. It shows how the optimal design is influenced by the costs per treatment,proportion of responders to first-stage treatments and the relative importance ofthe four pairwise comparisons. Section ‘Discussion’ summarizes our findings,discusses limitations of this contribution and gives directions for futureresearch. but using different notation). The observed covariates and treatmentassignment at stage k are denoted k. Within a SMART multiple ATSs are embedded; these aredenoted j. If the SMART has two stages, then k, a tailoring variableis measured which determines if a subject has responded to the treatment in thatstage or not. In other words, this variable determines which treatment the subjectis assigned to in the subsequent stage. At the end of the study  the continuous outcome variable Before we focus on the prototypical SMART, we rehearse some general ingredients forarbitrary SMART comparing first-stage intervention options; (ii) comparing second-stage interventionoptions; (iii) comparing two or more embedded ATSs in the study starting with thesame first-stage intervention option or (iv) comparing two or more embedded ATSs inthe study starting with different first-stage intervention options.Bariatric surgery is an effective treatment for obese patients to lose weight.Given its costs, potentially harmful side effects and the risk of death,patients in the Netherlands are only considered eligible if they can demonstratethey have previously attempted other means to lose weight. Two treatments are anincrease in PHY and a change in NUT.R refers to second-stage treatmentassigned to responders, while the superscript NR denotes second-stagetreatment assigned to non-responders. obsessive–compulsive disorder and chronic pain.The SMART design of this example is a simplification of the prototypical SMARTdesign in the sense that just two treatments are involved. Responders continuewith their first-stage treatment, while non-responders are randomized to theother treatment or a combination of both treatments. This specific SMART designwas previously used for, among others, the treatment of anxiety disorder,jth subject as measured at the end of stage 2, with For a given ATS  in the sense that we allow the proportions The weights follow from the fact that there is a structural imbalance betweenresponders and non-responders: the non-responders are re-randomized but theresponders are not. For instance, for ATS N is the totalsample size of the SMART and The weighted mean for the continuous primary outcome of interest for ATS given byE(Y¯di)=Following from , we obta∑jNd1Wj=γ1)p2=N∑jNd1Wj2plugging :7)VarVar(5)Using their respective subject-specific weights, formulae for the variance of theweighted mean We consider pairwise comparisons of ATSs that start with different first-stagetreatments. The expected difference in weighted means of two such ATSs Considering the ATSs embedded in our example, four possible pairwise comparisonsexist, with corresponding variances: (i)  For that reason, a multiple-objective optimal design is used, so that allof the four pairwise comparisons are taken into account simultaneously. We do soby using a weighted sum of the four objectives, where weights are to be chosenby the user. The use of weights allows placing more emphasis on the oneobjective than another, subject to the researcher's interests and the goals ofthe study. A constraint is put on the weights such that their sum is equal to 1.The optimal design problem becomes a multiple-objective optimal design problem.The aim is to minimize the optimality criterionThe optimal design N. This is a realistic scenario when studying treatmentsfor a rare disease or condition, but it can also be used when resourceconstraints allow recruiting a fixed number of subjects. It is assumed that apriori estimates of the response rates In this scenario, the optimal design is sought under a fixed total sample sizeN, but only onthe choice of the weights. In particular, The optimal design minimizes the objective in ; it is fN. A detailedderivation of the optimal design is given in the online supplement.The optimal randomization probability for the first-stage treatment A takes on amore complicated form:C for treating subjects should not exceed the budgetB. The costs are calculated asN thatcan be used decreases when the costs increase. This implies that a design is notonly determined by the proportions but also by the total sample size: In this scenario, we consider a budgetary constraint: the total costs to allow specification of intervals, rather than point estimates, of thetwo response rates.The optimal design depends on the response rates Define the parameter space for the response rates and the designspace For each possible combination of the two response rates in theparameter space, compute the locally optimal design For each design in This procedure yields the design which is most robust to amisspecification of the response rates and it can be used when working under afixed budget or under a fixed total sample size.The maximin optimal design . The following steps should be taken in such a power analysis: N into theequations of N has to be calculated from thebudget, costs and optimal proportions. This can be done on the basisof equations ,,Var identical for the chosen sets of weights,response rates and costs. As a result, the minimal RE of the balanced design asgiven in Tables 5 and 6 of Supplemental material is almost equal to that of the RE of thebalanced design in A comparison of Considering our example of a prototypical SMART design, we derived the optimal design 3Under a fixed budget, the optimal proportions are influenced also by the cost oftreatments, besides the aforementioned weights and response rates. When includingcost of treatments into account, the performance in terms of RE of the optimaldesign It is especially advised to use the optimal design rather than the balanced designwhen the second set of weights, It should be mentioned that the optimal designs are locally optimal, as they dependon the two unknown response rates We derived our optimal design under the assumption that outcomes of subjects in ATSsthat start with different first-stage treatments are independent of each other,resulting in a zero correlation between weighted mean outcomes of ATSs starting withdifferent first-stage treatments. There are situations in which this assumption maybe violated. Consider for instance the situation in our weight loss example wherejust a limited number of personal trainers is available. It may then occur, apersonal trainer trains subjects from ATSs starting with different first-stagetreatments. In such a case, the outcomes of subjects who have been trained by thesame personal trainer become dependent because of the trainer's skills, enthusiasm,experience, etc. In such a case, the assumption of independence is violated andhence our optimal design is not applicable. Such a problem can be easily solved byletting each personal trainer only train subjects from ATSs that start with the samefirst-stage treatment.55 Clustered data occur, amongothers, in cluster-randomized trials and multicentre trials. In such studies notonly the total number of subjects in each treatment sequence needs to be determined,but also the number of clusters and cluster size. The optimal design will depend on the intraclass correlation coefficient,which measures the degree of dependence of outcomes within the same cluster.One limitation of this study is that it does not take clustered data structures intoaccount, while such data may also occur in SMARTs.58 and online. It would be necessary to study optimal designs for such other types of SMARTdesigns.Another limitation of this study is that formulae and methodology only apply to theprototypical SMART designs in To our knowledge, this is the first paper that studies optimal allocation totreatments in SMARTs. Our Shiny App allows researchers in the fields of biomedical,health and social sciences to derive the optimal design for their SMART and tocalculate the efficiency of a balanced design. We hope that this paper will furthercontribute to the development and implementation of SMARTs.Click here for additional data file.Supplemental material, sj-docx-1-smm-10.1177_09622802211037066 for Optimalallocation to treatments in a sequential multiple assignment randomized trial byAndrea Morciano and Mirjam Moerbeek in Statistical Methods in MedicalResearch


N-terminal probrain natriuretic peptide (NT-ProBNP) combined with a simplified Geneva scale for the prognosis of acute pulmonary embolism (APE).  To explore the predictive value of mean platelet volume (MPV) and plasma n = 45) and a poor prognosis group (n = 23). The clinical data, laboratory clinical indexes, and simplified Geneva scale scores were recorded for the two groups. The risk factors of poor prognosis were analyzed by binary multivariate logistic regression analysis; the predictive ability of each index on the prognosis of patients with APE was analyzed by the ROC curve.  The clinical data of 68 patients with APE admitted to our hospital from October 2017 to October 2019 were collected. According to the prognosis, the patients were divided into a good prognosis group (P < 0.05). PLT, platelet distribution width (PDW), MPV, and plasma NT-ProBNP in the poor prognosis group were higher than those in the good prognosis group (P < 0.05). The simplified Geneva scale score of the poor prognosis group was higher than that of the good prognosis group (P < 0.05). PDW, MPV, plasma NT-ProBNP, and simplified Geneva scale were all independent risk factors for the poor prognosis of APE patients (P < 0.05). The AUC of MPV in predicting the prognosis of APE patients was 0.818 (95% CI: 0.712–0.925). When the optimal cutoff value was 0.571, the sensitivity was 77.1%, and the specificity was 80.0%. The AUC of plasma NT-ProBNP in predicting the prognosis of APE patients was 0.762 (95% CI: 0.634–0.891). When the optimal cutoff value was 0.475, the sensitivity was 71.5%, and the specificity was 76.0%. The AUC of the simplified Geneva scale in predicting the prognosis of APE patients was 0.749 (95% CI: 0.618–0.879). When the optimal cutoff value was 0.469, the sensitivity was 82.9%, and the specificity was 64.0%. The AUC of MPV and plasma NT-ProBNP combined with the simplified Geneva scale in predicting the prognosis of APE patients was 0.907 (95% CI: 0.826–0.988). When the optimal cutoff value was 0.726, the sensitivity was 88.6%, and the specificity was 84.0%.  The incidences of deep vein thrombosis, diabetes, and hyperlipidemia in the poor prognosis group were higher than those in the good prognosis group ( MPV, plasma NT-ProBNP, and simplified Geneva scale have a certain predictive value for the prognosis of APE. Compared with a single index, the combination of the three indexes has a significant improvement in predicting the prognosis of APE and has better clinical value. Acute pulmonary embolism (APE) refers to a variety of emboli that block the pulmonary artery and its branches, leading to pulmonary circulation disorders, resulting in a variety of pathological and physiological changes. APE is mainly manifested as sudden chest pain, dyspnea, hemoptysis, and other manifestations. It has a rapid onset and rapid development and has the characteristics of low specificity and poor prognosis , 2. In rN-terminal pro-brain natriuretic peptide (NT-ProBNP) is a metabolic substance in the process of myocardial cells producing BNP, which can reflect the state of the body's heart function. The higher the level of NT-ProBNP, the larger the area of the embolized blood vessel, the more severe the degree of right heart dysfunction and myocardial damage, the worse the prognosis of patients. NT-ProBNP has a slower clearance in the blood and a long half-life, and the concentration of NT-ProBNP in plasma is higher than that of BNP, and it has no biological activity. Its detection is not easy to be affected by drugs, and the determination results are more accurate and sensitive and have been applied in the diagnosis and prognosis of a variety of diseases [Mean platelet volume (MPV) is an indicator of platelet function, which can reflect the production and activation of platelets, and plays an important role in the diagnosis of acute pancreatitis, colorectal cancer, respiratory diseases, and other diseases –7. N-terdiseases . Studiesdiseases . The simdiseases .The objective of this study was to investigate the predictive value of MPV and plasma NT-ProBNP combined with a simplified Geneva scale in predicting the prognosis of APE.The clinical data of 68 patients with APE admitted to our hospital from October 2017 to October 2019 were collected. Among them, 39 were males, and 29 were females, with an average age of (56.23 ± 11.86) years.① Symptoms: the clinical manifestations were complex and diverse, with low specificity, most of which were dyspnea, chest pain, syncope, hemoptysis, and other symptoms. ② Signs: mainly the signs of the respiratory system and circulatory system, manifested as increased respiratory rate (>20 beats/min), increased heart rate (>90 beats/min), cyanosis, and hypotension. ③ Pulmonary artery CTA: the image conforms to the following manifestations: direct signs: (1) partial thromboembolism, the thrombus was free in the vascular lumen, and the surrounding contrast agent surrounds and presents a circular low-density shadow. (2) Complete thromboembolism, the distal blood vessels were not visible, and the official cavity was completely blocked into a cup or irregular club. (3) Annular mural thrombus, showing mural filling defect .The diagnostic criteria of APE were met. All patients were diagnosed by CT angiography, radionuclide pulmonary ventilation/perfusion scan, and pulmonary artery angiography. Onset time <24 h; age ≥18 years; and the medical history data were complete.The exclusion criteria were as follows: chronic thromboembolic pulmonary hypertension; anticoagulant or thrombolytic therapy had been performed before admission; heart, liver, and kidney insufficiency; severe respiratory infections; severe blood system disease; blood indicators that have not met the requirements; previous cerebral infarction; patients who had undergone coronary artery bypass grafting or stenting; pregnant and lactating women; mentally ill; and unable to follow visitors.In this study, clinical data of study subjects were retrospectively analyzed, and since there were no intervention activities for patients in this study, the process of signing informed consent could be exempted in this study.n = 45) and a poor prognosis group (n = 23).All patients were followed up for 1 month, and the prognosis of the patients after discharge from the hospital was recorded. Poor prognosis was defined as adverse events such as death (death due to various reasons), deterioration of the disease (increased severity of the disease), APE recurrence (after recovery and discharge and readmission to APE), mechanical ventilation, and tracheal intubation. According to the prognosis, the patients were divided into a good prognosis group , smoking history, drinking history, predisposing factors, and primary diseases of all patients were recorded.The laboratory clinical indexes of the patients were recorded 24 h after admission: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), platelet count (PLT), platelet distribution width (PDW), MPV, plasma NT-ProBNP, and so on.The simplified Geneva scale score was recorded for all patients, with a total of 9 indicators, a total score of 0–10 points. The higher the score, the greater the risk of APE. The specific scoring operation of this scale was carried out by nurses in our department . The incidences of deep vein thrombosis, diabetes, and hyperlipidemia in the poor prognosis group were higher than those in the good prognosis group . PLT, PDW, MPV, and plasma NT-ProBNP in the poor prognosis group were higher than those in the good prognosis group . When the optimal cutoff value was 0.571, the sensitivity was 77.1%, and the specificity was 80.0%; The AUC of plasma NT-ProBNP in predicting the prognosis of APE patients was 0.762 (95% CI 0.634–0.891). When the optimal cutoff value was 0.475, the sensitivity was 71.5%, and the specificity was 76.0%. The AUC of the simplified Geneva scale in predicting the prognosis of APE patients was 0.749 (95% CI 0.618–0.879). When the optimal cutoff value was 0.469, the sensitivity was 82.9%, and the specificity was 64.0%; The AUC of MPV, and plasma NT-ProBNP combined with simplified Geneva scale in predicting the prognosis of APE patients was 0.907 (95% CI 0.826–0.988). When the optimal cutoff value was 0.726, the sensitivity was 88.6%, and the specificity was 84.0% .As an acute and severe disease with frequent clinical occurrence, APE is characterized by atypical clinical manifestations, the rapid development of the disease, and poor prognosis; its prevalence of APE increases with age; and it has a higher risk of death . The stuThe results of this study showed that the incidence of deep vein thrombosis, diabetes, and hyperlipidemia in the poor prognosis group were higher than those in the good prognosis group. This suggested that deep vein thrombosis, diabetes, and hyperlipidemia can all affect the prognosis of APE patients. The possible reasons are: when deep vein thrombosis is formed, the thrombus can fall off and can travel along the bloodstream to the lung, causing the blood vessel to block, which in turn leads to an increased risk of APE recurrence and poor prognosis. In addition, the blood of patients with diabetes and hyperlipidemia is often in a state of hypercoagulation; the blood of patients is relatively viscous, prone to the formation of thrombosis, and can damage the blood vessel wall, leading to the thrombus falling off, thereby affecting the prognosis of APE patients .As a parameter indicating the volume and size of platelets, MPV can reflect the function of platelets and is closely related to the production of thrombus. Once suffering from APE, the patient's body will produce a variety of inflammatory factors, thereby destroying the endothelium of the pulmonary vessels, resulting in the production and activation of platelets, thereby increasing the level of MPV . Gunay'sIn addition, the ROC curve results showed that the AUC of MPV, plasma NT-ProBNP, and simplified Geneva scale in predicting the prognosis of APE patients were 0.818 (95% CI 0.712–0.925), 0.762 (95% CI 0.634–0.891), and 0.749 (95% CI 0.618–0.879), respectively, and the AUC of MPV and plasma NT-ProBNP combined with simplified Geneva scale in predicting the prognosis of APE patients was 0.907 (95% CI 0.826–0.988). The results suggested that MPV, plasma NT-ProBNP, and simplified Geneva scale have a certain predictive value for the prognosis of APE. Compared with a single index, the combination of the three indexes has a significant improvement in predicting the prognosis of APE, which has better clinical value.In summary, MPV, plasma NT-ProBNP, and simplified Geneva scale have a certain predictive value for the prognosis of APE. Compared with a single index, the combination of the three indexes has a significant improvement in predicting the prognosis of APE and has better clinical value. This study has the limitation of the small number of included cases, and the other influencing factors of patients were not comprehensively observed. Therefore, we need to collect a larger sample of cases for further verification in the future and exclude other factors for a comprehensive discussion of the prognosis.


Toxoplasma gondii infection has been reported in numerous species of marine mammals, some of them with fatal consequences. A serosurvey for T. gondii infection was conducted in pinnipeds from an oceanographic park in Portugal (n = 60); stranded pinnipeds on the Portuguese coast (n = 10); and pinnipeds captured in Lorenzensplate, Germany (n = 99). Sera from 169 pinnipeds were tested for the presence of antibodies to T. gondii by the modified agglutination test with a cut-off titre of 25. An overall seroprevalence of 8.9%  was observed. Antibody titres of 25, 50, 100, 1600 and ≥3200 were found in five (33.3%), two (13.3%), five (33.3%), one (6.7%) and two (13.3%) animals, respectively. Pinnipeds under human care had a seroprevalence of 20.0% (12/60), in contrast to 2.8% (3/109) in wild pinnipeds (p < 0.001). General results suggest a low exposure of wild pinnipeds to T. gondii, while the seroprevalence found in pinnipeds under human care highlights the importance of carrying out further studies. This is the first serological survey of T. gondii in pinnipeds in Portugal and the first infection report in South African fur seal . Toxoplasma gondii, which affects a wide range of homeothermic animals, including marine mammals. Despite the existence of several identified transmission routes, the exact mode of T. gondii transmission to marine mammals remains unknown [T. gondii. Some sources of oocyst contamination include freshwater outflows to the sea, surface runoffs and litter from domestic cats flushed down toilets [T. gondii does not multiply in poikilothermic animals, bivalve molluscs, sardines and anchovies can assimilate and concentrate oocysts [T. gondii transmission, although rare in pinnipeds, has been documented. Systemic toxoplasmosis was confirmed in a full-term stillborn Hawaiian monk seal (Neomonachus schauinslandi) pup and in aborted California sea lion foetuses  [T. gondii infections in marine mammals under human care and wild marine mammals are of clinical and public health importance [T. gondii contamination of the marine environment [Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular parasite  unknown . The pre toilets ,2. The i toilets . While T oocysts ,5,6,7. T oocysts . Transplrnianus) ,10. Certrnianus) . Thus, Tportance . Additioironment . T. gondii in pinnipeds worldwide [T. gondii infection in pinnipeds in Portugal. The purpose of this study was to perform a serological study on T. gondii infection in pinnipeds from the zoological collection of an oceanographic park , as well as in pinnipeds stranded on the Portuguese coast and then rehabilitated at a rehabilitation centre for marine species , and in pinnipeds captured shortly for health assessment in Lorenzensplate, Wadden Sea, Germany.While numerous serological studies have been performed to determine the seroprevalence of antibodies to orldwide , to the T. gondii were found in 15 (8.9%) of the 169 pinnipeds  (T. gondii were detected in five of the eight (62.5%) species tested, namely: Harbor seal (Phoca vitulina), California sea lion, South American sea lion (Otaria flavescens), South African fur seal  and hooded seal (Cystophora cristata).Antibodies to .1–14.2) . Titres T. gondii in pinnipeds according to the independent variables studied. A significantly different seroprevalence (p < 0.001) was observed in pinnipeds under human care  compared with wild pinnipeds . In addition, there were statistically significant differences (p < 0.001) in frequencies of antibodies between otariids  and phocids . Concerning diet, pinnipeds fed fresh and frozen fish had lower seropositivity  than pinnipeds fed only frozen fish  (p < 0.001). T. gondii was significantly different (p = 0.043) in pinnipeds under human care with clinical signs  compared with apparently healthy animals . On the other hand, the seroprevalence of T. gondii infection did not vary significantly in pinnipeds with respect to family (p = 0.107), sex (p = 0.508), age (p = 1.0), birthplace (p = 0.670), presence of felids around habitats, gestation (p = 0.340) and abortion (p = 1.0). The prevalence of antibodies to With respect to paired samples, i.e., from the same individual, there were 11 out of the 12 seropositive pinnipeds under human care: Six were seropositive for both samples and five seronegative in the oldest sample . p = 1.0), age (p = 0.421) and clinical status (p = 1.0).In wild pinnipeds, statistically significant differences were not detected between the prevalence of infection and sex  was lower than in surveys elsewhere [In the present study, the overall lsewhere . In the lsewhere ,19,20,21T. gondii detected in pinnipeds under human care was higher (20.0%) and significantly different from the one obtained in wild pinnipeds (2.8%), which may be explained by several factors, including the increased potential sources of infection in pinnipeds under human care. A likely source of infection is contamination of pool’s water source with sporulated oocysts, because there is a stray cat colony in the park, although managed through a veterinary care program that includes identification, vaccination, deworming and sterilization. In addition to direct exposure of water pool to feline faeces, pinnipeds could also be exposed to the oocysts mechanically transported through the trainer’s clothing or boots or even attending veterinarian, despite the use of specific footwear in areas where the animals are housed. The disinfection of swimming pool water in Zoomarine is carried out using chlorine and ozone treatments, but chlorination treatments (100 mg/L for 24 h) and ozone (6 mg/L for 12 min or 9.4 mg/L for 20 min) [T. gondii oocysts [T. gondii as a source of infection in seropositive animals cannot be ruled out. Arthropods and rodents may serve as mechanical vectors of T. gondii, but pest control is carried out regularly. The seroprevalence found in pinnipeds under human care in the present study (20.0%) was similar to the one found in South American sea lions from different Spanish zoological collections  [The prevalence of antibodies to  20 min) ,23 may n, 12.5%)  but cons, 12.5%)  and the , 12.5%) . T. gondii. Comparatively, antibodies to T. gondii were not detected in any of the 120 harbor seals captured in Svalbard, Norway [T. gondii in wild pinnipeds. These results may be explained by the low temperature of the waters that the seals included in this study inhabit, namely, the Wadden Sea and the North Atlantic Ocean, which may limit the survival of the oocysts [The seroprevalence in wild animals reported in the present study suggests a low frequency of exposure to , Norway , neither, Norway . In nort, Norway . Contrar, Norway  and the , Norway . In grey, Norway . Despite oocysts . Since t oocysts , the ser oocysts .T. gondii by indirect fluorescent antibody test . In New Zealand sea lions, only 6.0% (3/50) of the animals sampled were positive by latex agglutination test, using a cut-off titre of 32 [Because among marine mammals, otariids spend more time on land than phocids, given the anatomical particularities that distinguish them , it is ere of 32 .The results obtained in phocids in the present study are in agreement with studies carried out in wild grey seals (5.8%) from France  and in HConcerning diet, and contrary to our expectations, a higher seroprevalence was observed in individuals fed exclusively on frozen fish. Considering that Zoomarine’s pinniped collection diet is based on thawed fish, previously frozen and stored at −20 °C for at least 21 days, it would be expected that the seroprevalence would be lower in these animals. However, it is important to remember that oocysts can survive freezing [The higher seroprevalence obtained in pinnipeds under human care with clinical signs compared to that obtained in apparently healthy pinnipeds suggest that immunocompromised animals or animals with concomitant infections may be more prone to infection but serologic findings are only an aid to diagnosis of clinical toxoplasmosis .T. gondii were found in two harbor seals, two California sea lions, four South American sea lions and four South African fur seals. It should be noted that the lowest titre , was found in harbor seals and California sea lions, while South African fur seals had the highest titres . To the authors’ knowledge, this is the first study to report the presence of antibodies to T. gondii in South African fur seals. While seropositive South African fur seal with the highest titre (≥3200) had occasional clinical signs such as coughing, diarrhea and aggressiveness since its arrival at Zoomarine, some of which are observed in marine mammals with toxoplasmosis [Of the 60 pinnipeds under human care included in this study, antibodies to T. gondii in marine mammals is not frequent [Regarding paired samples, of 11 out of the 12 seropositive pinnipeds under human care, six were seropositive in both samples and five were seronegative in the oldest sample. These results suggest that in six animals the primary infection occurred in a place other than Zoomarine. However, the samples were not collected immediately after the animals entered the park and therefore it cannot be assumed that the infection did not happen at Zoomarine. On the contrary, five of those 11 seropositive animals tested negative in the old samples, suggesting that the infection occurred at Zoomarine. Of the 5 animals mentioned, one was the only seropositive pinniped born at Zoomarine. This pup probably acquired infection post-natally because its dam tested negative by MAT (data not shown). Additionally, vertical transmission of frequent ,33,35,36With respect to the antibody titre obtained in old and recent samples, we found that in three animals the titre decreased, in two it remained identical and in one it increased. The decrease in antibody titres in three animals over time is in agreement with the hypothesis that in general titres decrease over time . In the T. gondii is unknown, the natural resistance to this parasite may also explain the low seroprevalences obtained [The seropositive wild pinnipeds included two harbor seals (titres of 50 and ≥3200) captured in Lorenzensplate and one hooded seal (titre of 25) stranded on the south coast of Portugal. While the three species sampled have different eating habits, which may contribute to different exposure to the parasite, the results obtained may reflect permanency in contaminated coastal areas. Grey seals and hooded seals are species that spend more time out at sea, unlike e.g. harbor seals, thus reducing the likelihood of exposure to oocysts shed in coastal regions . The obsobtained .T. gondii infection and the lack of a protective vaccine, prevention of infection by T. gondii acquires special importance, particularly in animals under human care. Hygiene measures, such as washing hands of trainers and veterinarians before and after contact with pinnipeds, as well as before and after handling fish, might reduce T. gondii transmission. Cats should not be allowed in or near the holding facility of marine mammals, and other agents such as arthropods that can constitute a means of dissemination of oocysts should be controlled.Because there are no drugs that eliminate In the present study, a total of 169 pinnipeds were tested, among which 60 were pinnipeds under human care and 109 were wild individuals .Convenience-based sampling resulted in the collection of samples of seven different pinniped species from Mundo Aquático SA—Zoomarine Portugal, an oceanographic theme park located in the Algarve, south of mainland Portugal . The samWild pinniped samples included hooded and grey seals stranded on the Portuguese coast, between 2001 and 2014, and were obtained after the animals had been admitted to Porto d’Abrigo—the Rehabilitation Centre for Marine Species of Zoomarine Portugal. Samples from harbor seals captured in Lorenzensplate, in the Wadden Sea, Germany  were alsg for 5 min. After separation, the serum of each sample was transferred to properly identified tubes and was then stored at −20 °C until analysis.In otariids, the blood was collected from the interdigital veins of the pelvic flippers with a 23 G butterfly catheter. In phocids, blood was collected from the extradural vein with 20 G needles or hind flippers (for details see ). In botWhenever possible, epidemiological data were collected for each animal, namely: Sampling date, provenance (under human care/wild), family (Otariidae/Phocidae), species, sex , age (subadult/adult), diet (fresh and/or frozen fish/frozen fish), clinical status at the time of blood collection , birthplace (Zoomarine/other location), presence of felids around habitats (absent/present), pregnancy (non-pregnant/pregnant) and abortion (none miscarriage/≥1 miscarriage). Data on the control of arthropods and rodents were also recorded. Clinical manifestations included gastrointestinal , respiratory  or neurological  signs.T. gondii with a MAT commercial kit  according to the manufacturer’s instructions. Sera were assayed at a two-fold serial dilution from 1:25 to 1:3200. Positive and negative controls were supplied with the kit and included in each testing plate.Serum samples were tested for specific IgG antibodies to T. gondii in animals [T. gondii in experimentally [The results obtained were expressed as an antibody titre, i.e., the reciprocal of the highest dilution at which agglutination  was still visible after 5–18 h incubation at room temperature. A cut-off titre of 25 was chosen based on previous studies on pinnipeds ,18,19,20mentally  and natumentally ,24,26,34mentally ,43.T. gondii and explanatory variables  were analysed using the chi-square or Fisher’s exact tests. The exact binomial test was used to calculate CI for the proportions, with a 95% confidence level. A p < 0.05 was defined as statistically significant.Statistical analysis was performed using the IBM SPSS 26.0 program for Windows. Association between the prevalence of antibodies to T. gondii. It is noteworthy that infection in captive animals was higher than in wild animals, probably related to exposure to waters contaminated with oocysts excreted by cats. Prevention of access of felids to the enclosure where pinnipeds are housed and contiguous areas, efficient rodent and arthropod control programs, a diet based on previously frozen thawed fish and the use of proper hygiene practices are some of the measures that can minimize the risk of exposure to this parasite. In conclusion, the results of the present study document that pinnipeds under human care and wild pinnipeds in Portugal are exposed to


Thirty‐one percent of CIN1 and 26% of ≥CIN2 were attributed to HPV51, HPV53, HPV56, and HPV68. Multivariate analysis revealed that HPV53, HPV81, age, menopause, cervical transformation area and involved glands were independent risk factors for ≥CIN2 group compared to the CIN1 group (p < 0.05). Additionally, among the 14 non‐9‐valent vaccine covered HPV subtypes, the infection rates of HPV53, 56, 51, and 68 were higher in this study. In conclusion, our study demonstrated the distribution and pathogenic risk of non‐9‐valent vaccine covered HPV subtypes in cervical lesions. These findings might supply a foundation for optimizing cervical cancer prevention in the post‐vaccine era.Human papillomavirus (HPV) infection is the main cause of female precancerous lesions and cervical cancer. The development and application of HPV prophylactic vaccines have been recognized as a major effective intervention for the control of cervical lesions. However, the infection rate and clinical characters of non‐9‐valent vaccine covered HPV subtypes are still worth studying. In this retrospective study, we included patients diagnosed and treated in the Department of Gynecology of Shanghai General Hospital between January 2017 and February 2021. The clinical features of non‐9‐valent vaccine covered HPV subtypes were explored in 2179 patients who have normal results, 338 patients with cervical intraepithelial neoplasia 1 (CIN1), and 153 patients with ≥CIN2. Univariate analysis showed that compared to the normal cervix group, age ≥50, pregnancy ≥5, delivery ≥3, menopause, no condom use, and cervical transformation zone type III were risk factors for CIN1 or ≥CIN2 ( We identified the most non‐9‐valent vaccine covered carcinogenic HPV types, so as to provides a basis for further optimizing screening methods and eliminating cervical cancer in the post‐vaccine era. VIVIANE, FUTURE III, and other studies have confirmed the preventive effect of HPV vaccine on cervical cancer,In this retrospective study, we studied the non‐9‐valent vaccine covered HPV subtypes in cervical lesions, analyzed their distribution and the clinical characteristics of these HPV‐infected patients. We also identified the most non‐9‐valent vaccine covered carcinogenic HPV types and the risk factors of CIN patients.22.1The clinical data of 7400 patients treated at the Department of Gynecology of Shanghai General Hospital from January 2017 to February 2021 were analyzed in this retrospective study. All cases provided cervical histopathological results and pre‐treatment HPV typing results. According to the inclusion or exclusion criteria, 2670 patients with non‐9‐valent vaccines covered HPV subtype infections were selected for analysis. The patients were divided into normal group (2179 cases), CIN1 group (338 cases), and ≥CIN2 (including CIN2 and CIN3 and carcinoma in situ) group (153 cases). The HPV typing data, histopathological data, and age were collected from each group.The inclusion criteria were as follows: (1) cervical HPV and cytology sampling qualified; (2) colposcopy was performed in the gynecological clinic of our hospital; (3) HPV classification, ThinPrep cytologic test (TCT), Pathological results of colposcopy biopsy, and baseline data were obtained. Exclusion criteria were as follows: (1) previous hysterectomy or conization; (2) women infected with any type of the 9‐valent vaccine covered HPV; (3) other malignant tumors.2.2The PCR‐reverse dot‐hybridization technology was used for HPV detection using a PCR‐RDB HPV genotyping assay ,2.3Cytological classification of the samples was performed by two experienced pathologists based on the Bethesda System Standard 2001 in a double‐blinded manner.2.4The diagnosis of cervical diseases in all patients was based on cervical histopathology. The cervical tissue is obtained from colposcopy biopsy or surgical submission specimens. The histopathological results of the highest level of the cervix were viewed as the final diagnosis of the disease. The specimens were processed using the standard histopathological methods and evaluated by at least two pathologists. All colposcopy operations were performed by qualified colposcopy specialists at our Center. The pathology results were classified as normal, CIN1, CIN2, CIN3, and carcinoma in situ.2.5p < 0.05 indicate a statistical difference significantly. All statistical analyses were performed using software package of social science statistical software version 26 .The clinical characteristics and multiple HPV infections of the patients were tested by chi‐square test. For samples with multiple HPV infections, the infection of one HPV subtype contributed to the occurrence of the disease as a partial attribution. The prevalence rate of HPV genotypes and the attribution rate of cervical lesions are based on the literature,33.1p < 0.05) , and ≥CIN2 group had the highest number of pregnancies and parities, with an average of 2.99 and 1.52, respectively. The univariate analysis was used to compare the clinical variables. Compared to the normal group, age ≥50 years, pregnancy ≥5 times, parity ≥3 times, menopause, no condom use, cervical transformation zone type III, and HPV‐negative were risk factors for CIN1 or ≥CIN2  in polymorphic infections. In CIN1 group, the infection rates of HPV53, 56 and 51 were higher: 14.3%, 14.2%, and 12.4%, respectively. However, among the patients with multi‐type HPV infection, the infection rates of HPV35 and 81 were second only to HPV53, accounting for 4.14%. In patients with ≥CIN2, the infection rates of HPV56 (13.1%), 51 (11.8%) and 53 (10.5) were higher, and the subtypes with high monotype HPV infection rates were 51 (8.5%), 68 (5.2%), and 53 and 56 (4.6%). However, in polymorphic infections, the infection rate of HPV35 and 39 was higher than that of HPV53 , followed by HPV51 (9.5%) and 56 (6.2%). The ≥CIN2 grade is mainly attributed to HPV51 (9.2%), followed by HPV68 (5.9%) and 56 (5.6%). Among all CINs, the attribution rates of HPV51, 53, and 56 were 18.8%, 34.3%, and 46.2%, respectively. Meanwhile, HPV51, 53, 56, and 68 accounted for the majority of cervical lesions in each group, and the cumulative attribution rates were 31.5% in CIN1 and 26.0% in ≥CIN2 . Compared to the normal group, the HPV infection covered by high‐risk non‐9‐valent vaccine was a risk factor for ≥CIN2 (p = 0.027)  and lowest in ≥CIN2 group (37.9%). The infection rate of two subtypes of HPV increased with the aggravation of cervical lesions, and the infection rate of patients with ≥CIN2 was the highest (22.9%), while triple and quadruple infections were most common in the CIN1 group with infection rates of 5.0% and 1.2%, respectively. Furthermore, the infection rate of HPV subtypes covered by high‐risk non‐9‐valent vaccine was higher than that covered by low‐risk non‐9‐valent vaccine among all three groups of patients. Conversely, the infection rate of HPV subtypes covered by low‐risk non‐9‐valent vaccine decreased gradually. Consecutively, two subtypes of HPV infections were found to be risk factors for ≥CIN2 compared to the normal and CIN1 groups . Cervical transformation zone was an also critical risk factor (OR = 4.1). In patients with TCT ≥ atypical squamous cells of unknown significance (ASC‐US), HPV35, HPV42, HPV83, age, menopause, and cervical transformation zone were independent risk factors for CIN (Table p < 0.05)  Table .3.6Finally, we analyzed age‐stratified HPV distribution of the subjects. Among the individuals <25 years old, the infection rate of HPV51 was the highest (25.5%). Among the patients aged 25–34, 35–44, 45–54, and 55–64 years, the HPV subtype with the highest infection rate was HPV53. Among the patients ≥65 years old, the infection rates of HPV51 and 53 were the highest at 17.1%  group and 68.4% in the HSIL group.The attribution rate of HPV51 in CIN1 (9.5%) and ≥CIN2 (9.2%) in current study was higher than that of the findings of Chan et al.,Diverse from the results of the previous study,The current study found that the higher infection rates of HPV subtypes not covered with 9‐valent vaccine in different age groups were HPV51, 56, and 53. A previous study found that the most common non‐9‐valent vaccine coverage HPV genotypes in women <50‐years‐old were HPV59, 39, and 56, while in elderly women >50 years old, the most common HPV subtypes were 56 and 68,Since the current study showed that some non‐9‐valent vaccine covered HPV subtypes was vigilant for the risk of cervical lesions, we still need to focus on the cervical precancerous lesions and cervical cancer caused by non‐9‐valent vaccines HPV subtypes (including HPV‐negative). In the case of women who have been vaccinated, we should also strengthen the universal education and pay attention to the non‐9‐valent vaccine covered HPV subtypes, such as HPV35, 51, 53, 56, 81, and 83. In addition, with the change in social population structure and increased aging, for elderly women, if there is low‐risk HPV (such as 81 and 83) infection, it still needs to be considered in combination with specific clinical conditions.Nevertheless, there are some deficiencies in the present study. First, the retrospective study is easily affected by the selection offset. Second, we lack data on the other risk factors for patients with non‐9‐vaccine covered HPV subtypes, including smoking volume, economic status, educational level, number of sexual partners, family history, etc.In conclusion, our study analyzed the infection rate of non‐9‐valent vaccines covered HPV subtypes in cervical lesions. The clinical characteristics and risk factors of CIN patients with non‐9‐valent vaccines covered HPV subtypes were also explored. The results suggest that sufficient follow‐up of future studies with a larger sample size can further assess the carcinogenic risk of non‐9‐valent vaccines HPV covered subtypes, which might be helpful to optimize the screening and prevention strategies of cervical cancer in the post‐vaccine era.The study is approved by the Ethics Committee of the Shanghai General Hospital (reference 2021SQ263). As all analyses were performed on pseudonymized and already collected data, written consent from participants was not required.The authors have no potential conflict  to disclose in terms of this manuscript.S Wu and J Zhang conceived and designed the whole project and drafted the manuscript. M Ma, J Zhu, and Y Yang analyzed the data and carried out data interpretations. X Wang and Y Jin helped data discussion. M Ma, J Zhu, and Y Yang wrote the manuscript. J Zhang revised the manuscript. All authors have read and approved the final copy.Table S1Click here for additional data file.Table S2Click here for additional data file.


The fluorescent dye indocyanine green (ICG) has emerged as a promising tracer for intraoperative detection of sentinel lymph nodes (SLNs) in early‐stage cervical cancer. Although researchers suggest the SLN detection of ICG is equal to the more conventional combined approach of a radiotracer and blue dye, no consensus has been reached.99mTc) with blue dye.We aimed to assess the differences in overall and bilateral SLN detection rates with ICG versus the combined approach, the radiotracer technetium‐99m  with ICG versus 73.5%  with 99mTc with blue dye. This resulted in a significant and clinically relevant risk difference of 16.6% . With sensitivity analysis, the risk difference of the bilateral detection rate maintained in favor of ICG but was no longer significant .From 118 studies retrieved seven studies  were included, encompassing 589 patients. No significant differences were found in the pooled overall SLN detection rate of ICG versus 99mTc with blue dye in patients with early‐stage cervical cancer. However, in adherence with the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) guidelines, the quality of evidence is too low to provide strong recommendations and directly omit the combined approach of 99mTc with blue dye.ICG appears to provide higher bilateral SLN detection rates compared to  The radiotracer enables preoperative imaging with SPECT‐CT, aiding in a rapid SLN identification with intraoperative detection of the radioactive signal through tissue.Currently, two predominant methods for detecting SLNs in cervical cancer are in use. The first is the more conventional combination of the radiotracer technetium‐99m nanocolloid  and logistics of ICG (which does not require injection in a controlled environment), will favor it over With this systematic review and meta‐analysis, we aim to assess the differences in SLN detection between ICG and the combined approach of 99mTc with blue dye in early‐stage cervical cancer patients.22.1MEDLINE via PubMed from inception (1946) to January 1, 2021Embase from inception (1947) to January 1, 2021The Cochrane Central Register of Controlled Trials  in the Cochrane LibraryWe conducted a systematic review and meta‐analysis following the Preferred Reporting Items for Systematic reviews and Meta‐Analyses (PRISMA) guideline.The search query combined synonyms, abbreviations, and alternative spellings for “cervical cancer,” “sentinel node biopsy,” “ICG,” and “technetium‐99m,” based on prior systematic reviews and input from a reference librarian. When the search strategy identified a conference abstract, we searched MEDLINE for an associated full‐text article by the same authors. We checked the reference lists of both the included studies and other systematic reviews on a similar topic for potentially relevant references.2.299mTc with blue dye in patients with stage I‐II cervical cancer were included to ensure equal patient populations and surgical setting. We excluded studies that did not contain original data, conference abstracts, case reports, and editorials. The references of all included studies were cross‐checked for possible additional literature. Two reviewers  independently selected eligible studies by first screening title and abstract, and subsequently reviewing full texts to determine their eligibility. Divergent opinions were resolved by consensus discussion. Any remaining disagreements were resolved by a third reviewer (C.G.).The search results were limited to English, Dutch, French, and German language. Peer‐reviewed studies reporting on a comparison of the overall and/or bilateral SLN detection rate of ICG versus 2.3The methodological quality of all included studies was appraised independently by two reviewers  using the validated ROBINS‐I tool.The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) guidelines were used to grade the overall quality of evidence and strength of recommendations per outcome measure.Differences in the quality assessment were discussed to reach a consensus between the two aforementioned reviewers. Remaining disagreements were resolved by a third reviewer (C.G.).2.499mTc with blue dye); (e) type of surgical approach; (f) patient and tumor characteristics ; (g) technical details on the use of ICG ; (h) technical details on the use of 99mTc with blue dye ; (i) median/mean number of SLNs resected per patient per modality; (j) intra‐operative SLN detection rates for each modality; (k) false negatives per modality ; (l) histopathological assessment; and (m) adverse events of each modality. Studies reporting data on other malignancies in addition to cervical cancer were included and only the data on cervical cancer were extracted. Disagreements in data extraction were resolved by discussion between the two reviewers; any remaining disagreements were resolved by the third reviewer (C.G.). We contacted individual authors of studies when further clarification was desired.We developed a data extraction sheet before collecting the data. For each study, the two reviewers  independently collected the following data: (a) author and publication details; (b) study design; (c) study population; (d) sample size per modality . This can be an indication of an incorrectly identified SLN. An alternate definition of false negatives is missing a tumor‐positive SLN because it is not detected by one modality (“non‐detection”) but was detected by another modality. This definition can only be applied in cross‐sectional studies (with intrapatient comparison) and will be referred to as “false‐negative mapping.”The primary outcomes were overall and bilateral SLN detection rates per modality for ICG only or the combination of P < .05. When  overlap in the patients of two or more of the included studies occurred, possibly over‐ or underestimating the pooled outcome, a sensitivity analysis was performed to assess the effect of excluding overlapping studies.All analyses were performed using the statistical software R, version 4.0.0  in conjunction with the “meta” package, version 4.12‐0, created by G. Schwarzer. The overall and bilateral detection rates, with 95% confidence intervals (95%CI), were calculated from the included studies. Using a random‐effects meta‐analytical model, wherein studies are weighed based on their inverse variance , we calculated the pooled risk differences of the primary outcomes for both modalities. Corresponding forest plots were created. We created funnel plots, wherein standard errors are plotted against the risk differences, to visually assess the risk of selective reporting , with formal significance testing (linear regression test) only when more than 10 studies were included. Statistical significance was set at 33.199mTc with blue dye . The references cited in the included eight articles were cross‐checked and did not yield any additional eligible studies.Figure Using the ROBINS‐I tool, the overall risk of bias was judged as “serious” in six studies and “moderate” in one study. One study had two domains judged as a “critical” risk, automatically leading to an overall “critical” risk of bias and exclusion from the analysis Figure .30 Com3.2The remaining seven studies included 589 patients Table . Six stu99mTc with blue dye, and 33 patients (5.6%) received ICG in adjunct to 99mTc with blue dye . In the 33 patients receiving both modalities, the SLNs were identified with ICG first, followed by identification with 99mTc and blue dye. In this cross‐sectional study deviations from the intended protocol existed as in nine (out of 33) patients no blue dye was administered due to unclear reasons.99mTc with blue dye group” received blue dye due to unclear reasons  of ICG injection was fairly equal, but the method of blue dye and particularly ed Table . Three o3.399mTc with blue dye showed a pooled proportion of 98.7% (95%CI 96.5‐100.0%) and 95.6% (95%CI 92.1‐99.2%), respectively   . The overall detection rate of ICG and y Figure . No sign) Figure . The poo) Figure , with a ) Figure . Visual ) Figure .99mTc with blue dye did not differ substantially: 98.4% (95%CI 95.2‐100.0%) and 96.0% (95%CI 93.0‐98.9%), respectively  , with a risk difference of 13.2% .Due to partial overlap of the patient populations from five studiesy Figure . Nor did) Figure . Althougp Figure , with a6)Figure .Table 3.499mTc with blue dye).99mTc with blue dye group.99mTc with blue dye (intrapatient comparison), with tumor‐positive non‐SLNs in final pathology.Six studies presented information on false negative SLNs Table . Three s99mTc with blue dye resulted in unilateral SLN detection and consequently missed a tumor‐positive SLN on the contralateral side, which was identified by ICG. In none of the cases, any ICG negative but 99mTc or blue dye positive SLN showed tumor infiltration. Due to the limited data, the false negatives (using both definitions) could not be reliably pooled. None of the included studies reported adverse events of ICG, 99mTc or blue dye.False‐negative mapping  occurred in one case (out of 33 patients).3.5I2 > 50%) and potential of publication bias.The quality of evidence for both the outcome measures overall and bilateral detection rate was scored as very low due to the inclusion of mainly observational cohort studies , of which several studies with a high risk of bias . In addition, the pooled proportion of bilateral detection showed a relatively high heterogeneity (499mTc with blue dye), by including nearly 600 patients in a meta‐analysis. Our results showed the overall detection rate of both modalities was high and not significantly different. A clinically relevant and significant risk difference in bilateral detection rate was observed of almost 17% in favor of ICG. Due to the limited data provided in the included studies, it was not possible to accurately compare the safety  of both modalities. The risk of bias in the included studies was substantial due to the (mainly) retrospective cohort study designs and the inability to blind the surgeons to the detection of different tracers in the cross‐sectional study. Therefore, we were not able to strongly recommend one approach for detecting SLNs over the other.With this systematic review, we assessed the clinical evidence of seven low‐quality studies, all comparing the SLN detection of the two modalities , not painful since it is intraoperatively injected and logistically less challenging since the injection does not require a radiation‐safe environment. Rare adverse events have been reported with ICG99mTc nanocolloid.The underlying hypothesis of a higher bilateral SLN detection with ICG versus the conventional tracers, especially 99mTc).99mTc linked to ICG could overcome these issues.99mTc, which also reduces the risk of detecting second echelon nodes.Nevertheless, ICG to has its disadvantages. The FILM trial, comparing ICG with blue dye only, reported a higher occurrence of presumed SLNs with a bright signal on NIR fluorescence imaging that was not confirmed to be nodes on final pathology but only lymphatic trunks or adipose tissue.99mTc with blue dye than what we found in our meta‐analysis. In the multi‐institutional prospective study on SLN procedure in early‐stage cervical cancer, the SENTICOL I study, a 97.8% overall detection rate and 76.5% bilateral detection rate were reported for SLN mapping with 99mTc with blue dye.99mTc with blue dye found in this meta‐analysis was substantially lower, it is comparable to the bilateral detection rate of 72% reported in a review by Tax et al.99mTc with blue dye or do the studies included in this meta‐analysis achieve sub‐optimal results with the combined approach? One of the factors that may explain the difference in detection rates, and provide an answer to this, is the learning curve effect.Recent literature reported higher SLN detection rates from using 99mTc with blue dye, to achieve >90%  SLN detection, has been established in endometrial cancer at 30 cases per surgeon.99mTc with blue dye. In this meta‐analysis most studies started their SLN procedure with 99mTc with blue dye before switching to ICG, potentially favoring the latter. Additionally, researchers suggest NIR fluorescence imaging has a steep learning curve, since most surgeons are trained in operating while using a monitor (in laparoscopic surgery), and practiced in the use of an additional probe.Previous literature has suggested that a learning curve of the SLN procedure exists. The learning curve of SLN mapping with 99mTc with blue dye have been described. Balaya et al showed tumor size >2 cm, BMI above 30 kg/m2, and age above 70 years yielded lower bilateral SLN detection rates.99mTc with blue dye group (100% (33/33) versus 64% (21/33), respectively; P = .001), while no significant difference existed in patients with a tumor size ≤2 cm (97% (34/35) versus 86% (37/43), respectively).Other risk factors for failed SLN detection of A strength of this review is its sole focus on cervical cancer, as previous reports on SLN mapping mixed both cervical and endometrial cancer patients.99mTc with blue dye (without comparison group) often reported higher bilateral detection rates than the studies included in this systematic review. Secondly, there were certain case mix and methodological differences between the studies that may have influenced the detection rates. The surgical modality in the selected studies differed from robot‐assisted laparoscopy, conventional laparoscopy, and laparotomy, all with different ICG‐NIR fluorescence platforms. Finally, the partial overlap in populations that existed between some studies could have led to overestimation of the risk differences. This effect is likely limited in view of our results from the sensitivity analysis. Leaving all or some of these studies in or out of the primary analysis could have resulted in selection bias from a review standpoint.Our systematic review has limitations. Firstly, we could not rule out publication bias, which reflects the increased likelihood of a study being published when the study has a positive result, occurred. The funnel plots showed no convincing indication of larger studies with lower SE's reporting smaller benefits of ICG compared to smaller studies but, with only seven studies included, no formal significance testing could be reliably performed. As mentioned before, studies on the SLN detection rate of 599mTc and blue dye. Given the advantages of ICG, these promising results could potentially lead to a widely adopted shift from 99mTc with blue dye to the use of ICG only. However, in adherence with the GRADE approach, the quality of evidence is too low to provide strong recommendations and directly omit the combined approach of a radiotracer with blue dye. Larger prospective studies —preferably with the bilateral detection rate and false‐negative mapping as endpoints— are needed to further substantiate the superiority of ICG.In early‐stage cervical cancer patients, the use of ICG appears to result in higher bilateral SLN detection compared to the more conventional combination of Ilse Baeten: Conceptualization; formal analysis; investigation; methodology; visualization; writing‐original draft. Jacob Hoogendam: Conceptualization; formal analysis; investigation; methodology; visualization; writing‐original draft. Bernadette Jeremiasse: Validation; writing‐review & editing. Arthur Braat: Validation; writing‐review & editing. Wouter Veldhuis: Validation; writing‐review & editing. Geertuida Jonges: Validation; writing‐review & editing. Ina Jürgenliemk‐Schulz: Validation; writing‐review & editing. Carla van Gils: Methodology; validation; writing‐review & editing. Ronald Zweemer: Conceptualization; supervision; validation; writing‐review & editing. Cornelis Gerestein: Conceptualization; investigation; methodology; project administration; validation; writing‐review & editing.R.Z. is a proctor for robot‐assisted surgery in gynecological oncology on behalf of Intuitive Surgical Inc. C.v.G. reports grants and other from Bayer Pharma, all outside the submitted work. The other authors declare no conflicts of interest.Not applicable.Appendix S1. Systematic search stringsClick here for additional data file.Figure S1. Quality assessment using the ROBINS‐I toolClick here for additional data file.Figure S2. Intraoperative pictures of robot‐assisted SLN procedure with ICG on the left A, and right pelvic site B. White light inspection (upper) and NIR fluorescence light inspection (lower)Click here for additional data file.Figure S3. Pooled overall SLN detection of ICG A, and 99mTc with blue dye BClick here for additional data file.Figure S4. Pooled bilateral SLN detection of ICG A, and 99mTc with blue dye BClick here for additional data file.Figure S5. Pooled overall SLN detection of ICG A, and 99mTc with blue dye B, in the sensitivity analysisClick here for additional data file.Figure S6. Pooled bilateral SLN detection of ICG A, and 99mTc with blue dye B, in the sensitivity analysisClick here for additional data file.Table S1. Detailed study characteristicsClick here for additional data file.


Since then, several agencies including the WHO and the American Cancer Society have echoed the use of HPV testing as the preferred primary screening method beginning at age 25 years, with cotesting or cytology testing alone recommended only if primary HPV testing was unavailable. Currently, only two tests, Cobas  and Onclarity , have received US Food and Drug Administration approval as primary HPV tests and five others as cotests, including Digene's Hybrid Capture II assay (hc2).2Unlike cytology-based screening, molecular detection of HPV collected from cervicovaginal swabs is less influenced by targeted sampling from the transformation zone of the cervix or by specimen adequacy and cell morphology. This has opened the possibility for patients to collect their own HPV samples without undergoing an examination that many perceive as embarrassing. The ability to collect one's own specimens lends a form of empowerment and participation in one's health that is difficult to achieve when asked to undress and be examined in an unfamiliar environment. There is growing evidence that self-collection has benefits that include anxiety reduction, less pain and discomfort, and lower-cost screening opportunities whether implemented through door-to-door kit distributions or in a private room at a health facility.In underserved and hard-to-reach populations, self-collection additionally has been posited to increase access to screening with the potential to decrease social inequities that often pose a barrier to cervical cancer screening.3 demonstrated that sensitivity for self-collected HPV was similar to provider-collected HPV detection when analyzed with polymerase chain reaction but demonstrated lower sensitivity for cervical intraepithelial neoplasia (CIN) 2 with hybrid capture 2 technology on which careHPV is based. This has led to questioning whether using careHPV for cervical cancer screening with self-collected specimens is the appropriate approach.2 Given the additional infrastructure and costs for polymerase chain reaction, careHPV is a more cost-effective platform for use in countries with low resources. There is however limited data comparing self- versus provider-collected HPV detection using careHPV. Head-to-head comparison using paired sampling of provider- and self-collected specimens of Katanga et al4 is therefore important in demonstrating good concordance despite lower positivity rates in self-collected (14%) versus provider-collected (19%) specimens.A meta-analysis by Arbyn et alcareHPV is superior (69.6% and 71.3% for detecting CIN 2 and 3 lesions with careHPV versus 21%-73.6% for CIN2+ lesions with VIA). To increase sensitivity of the test, a cutoff of 1.0 has been recommended for vaginal careHPV detection, to improve its sensitivity to levels reached with provider-collected specimens.6careHPV has been widely used in cervical cancer screening programs in developing countries and is an accepted platform providing access to screening in hard-to-reach and rural populations. Results can be obtained within 4 hours, but for cost efficiency, runs must be performed in batches of 90 wells often delaying results until at least the following day.Compared with visual inspection with acetic acid, the most common and cost-effective method of screening in low- and middle-income countries (LMICs), sensitivity of vaginal careHPV self-collection has high acceptability, and scalability, it comes with some challenges that must be overcome if it is to live up to its WHO prequalified status as an in vitro diagnostics for use in LMICs. There is a steep learning curve for technicians who have never used similar equipment, and on a personal level, two refresher visits by company representative were needed for technicians to feel secure running batches successfully with a minimal number of discarded wells.Although careHPV costs must be reduced. It is incumbent upon companies that develop these platforms to ensure that the cost of these tests is more affordable, $2-$3 US dollars, so that women in the most remote villages of the world can have access to HPV screening.The desktop unit is inexpensive and sturdy but given the cost of the reagents and kits, which must be continuously replenished from one single vendor on the African continent, the downstream cost to patients runs between $5 and $7 US dollars per person, which is prohibitive for most women in LMICs. To further scale up HPV screening for cervical cancer in unscreened populations and fulfill the initial target of the 90/70/90 strategy to eliminate cervical cancer,