P < .05). Xpert includes these seven types along with HPV 45 within three of the test's five channels and we defined these eight types as restricted genotyping . Full genotyping was defined as all 14 high‐risk types. Sensitivity estimates for full genotyping using LA were similar to that of restricted genotyping: 83.9% (full) vs 79.0% (restricted) in women without HIV and 93.0% (full) vs 88.9% (restricted) in women living with HIV. Specificity estimates improved for restricted vs full genotyping: 87.4% (full) vs 90.8% (restricted) in women without HIV and 63.7% (full) vs 71.4% (restricted) in women living with HIV. To optimize the performance of HPV testing for cervical cancer screening in high‐burden, under‐resourced settings like South Africa, only HPV 16, 18, 31, 33, 35, 45, 52, 58 could be included to define screen‐positive. We recommend the inclusion of HPV45 for its known link to adenocarcinoma.Human papillomavirus (HPV) testing is highly sensitive compared to cytology, with the trade‐off of being less specific. We investigated whether select combinations of HPV genotypes, ascertained by Linear Array (LA) and Xpert HPV (GX), can optimize sensitivity/specificity trade‐offs to detect high‐grade cervical intraepithelial neoplasia (CIN2+). In a study in Cape Town, South Africa, 586 women living without and 535 living with HIV, aged 30‐65 years, were recruited. Each woman underwent a pelvic exam to collect cervical samples (tested by LA and GX for 14 high‐risk HPV genotypes) and underwent colposcopy with histological sampling to determine CIN2+. In multivariable logistic regression of LA results, only HPV genotypes 16, 18, 31, 33, 35, 52, 58 were significantly associated with CIN2+ ( Human papillomavirus (HPV) testing is highly sensitive compared to cytology, with the trade‐off of being less specific. Among South African women, we have shown that restricting the definition of screen‐positive to women with HPV genotypes 16/18/45/31/33/35/52/58 substantially improves the specificity of screening while producing only minor reductions in sensitivity compared to inclusion of all 14 HPV genotypes conventionally included as high risk in HPV assays. Genotyping for specific HPV genotypes could provide risk stratification of HPV‐positive women by identifying HPV infections that are at highest risk of persisting and developing into invasive cervical cancer.In this study, we evaluated the impact on sensitivity and specificity for CIN2+ of restricting the number of HPV genotypes identified using the Roche Linear Array (LA) method that identifies each of the high‐risk HPV genotypes individually. We also compared the individual LA results to the grouped HPV typing inherent in the Xpert HPV (GX) assay.22.1We recruited women from two populations in Cape Town, South Africa. One was a community‐based clinic (screening population) located in Khayelitsha township which provides general women's health services to the local community. The other was women referred to a colposcopy clinic at Groote Schuur Hospital, a university teaching hospital, for evaluation of abnormal cervical cytology . Women aged 30‐65 years were eligible to participate if they: (a) had a known and documented HIV status, (b) were not previously treated for cervical disease, (c) did not have a hysterectomy, and (d) were not pregnant. The study aimed to recruit approximately equal numbers of women living with and without HIV. Seven hundred and fifteen women were recruited at the community‐based clinic and 406 from the referral colposcopy clinic .2.2This study was approved by institutional review boards at Columbia University (AAAO3652) and the University of Cape Town. Following written informed consent, each woman underwent a pelvic examination by a physician and had two liquid‐based cytology specimens  collected using a cytobrush and plastic spatula for each (Medscand).After the collection of the cervical samples, a colposcopic examination was performed. Multiple cervical biopsies were obtained of the most abnormal‐appearing areas of the cervix and/or an endocervical curettage (ECC) if there was no visible cervical lesion. If clinically indicated based on clinical protocol, a Loop Electrosurgical Excision Procedure (LEEP) was performed. Women from the screening population were told to return in 6 weeks after enrollment to receive their histology results. For those women recruited at the screening clinic, a second colposcopy was undertaken in those with HPV at baseline who had not had cervical intraepithelial neoplasia grade 2 or greater (CIN2+) detected at baseline. A third colposcopy was undertaken if the second colposcopy still failed to identify CIN2+. Random four‐quadrant biopsies were undertaken at these repeat colposcopies to reduce risks of missing disease.Histological samples collected via biopsy, LEEP, and/or ECC were first evaluated by a pathologist in Cape Town and then blindly reviewed by an expert pathologist in the US. Results were classified using the cervical intraepithelial neoplasia (CIN) classification system: within normal limits (WNL), CIN grade 1 (CIN1), CIN grade 2 (CIN2), CIN grade 3 (CIN3), and cancer.2.3One mL of the first cervical sample was tested at the Khayelitsha screening clinic using the Xpert HPV assay (Cepheid). The remaining 19 mL were stored and tested later in batches at the University of Cape Town using Linear Array (LA) (Roche Diagnostics). The second sample was stored for possible later testing.Xpert HPV (GX) is a clinic‐based, point‐of‐care HPV assay that tests for 14 high‐risk HPV types in five separate channels through real‐time PCR. Each single‐use Xpert HPV cartridge is prefilled with the necessary reagents, primers, and fluorescent probes to amplify the E6 and E7 regions of HPV DNA automatically on the GeneXpert System (Cepheid). Presence of HPV DNA that meets cycle thresholds is indicated by fluorescence in the following five channels: HPV16; HPV18, 45; HPV31, 33, 35, 52, 58; HPV51, 59; and HPV39, 56, 66, 68.The Roche Linear Array (LA) is a laboratory‐based assay that is not generally used in clinical practice but which enables the identification of 37 high‐risk HPV genotypes individually. PGMY09/11 primers are used to amplify the L1 region of the HPV genome using real‐time PCR. The resulting amplicons undergo reverse‐line blot hybridization to individually detect genotypes.2.4t‐tests for continuous variables and chi‐squared or Fisher's exact tests for categorical variables. Overall and type‐specific HPV prevalence was calculated using only data from the general population. Frequency tables were used to determine HPV distributions by age group, HIV status, and cervical disease status. Diagnostic agreement between GX and LA was compared using percent overall agreement and Cohen's kappa statistic. We used multivariable logistic regression to determine which HPV genotypes were significantly associated with CIN2+ and CIN3+ (P < .05). By study design, women with CIN2+ and CIN3+ identified in both the general and the referral populations were included whereas women with <CIN2 or <CIN3 only from the general population were included in the analyses. This design was selected to obtain a sufficiently large number of women with cervical disease with the resources available.Clinical and demographic information was summarized using descriptive statistics. Groups were compared using two‐sample 33.1P < .001), had fewer children (P < .001), were less educated (P = .001), and had more prior cytology screenings (P = .006) than women without HIV. Of the women living with HIV, 265 (79.82%) reported currently taking antiretroviral medications. There were no differences observed for tobacco use (P = .954) and employment status (P = .740) by HIV status.Seven hundred and fifteen women were recruited at the community‐based clinic in Khayelitsha and 406 women from the referral colposcopy clinic. Of the 1121 women, 1118 had valid GX and LA results. Baseline demographic and clinical characteristics of the women recruited at the community‐based clinic are summarized by HIV status in Table 3.23.2.1For both assays, HPV prevalence for any type was higher regardless of age group for women living with HIV (48.19% [42.82%‐53.57%)] by GX, 45.78% [40.42%‐51.14%)] by LA) compared to women without HIV (16.23% [12.53%‐19.93%)] by GX, 15.71% [12.06%‐19.36%] by LA)  was the most common followed by HPV 18, 45 (18.07%), HPV 39, 56, 66, 68 (10.84%), HPV 16 (10.54%), and HPV 51, 59 (6.63%). In women without HIV, the rankings were HPV 31, 33, 35, 52, 58 (8.12%) followed by HPV 39, 56, 66, 68 (3.66%); HPV 16 (3.40%); HPV 18, 45 (3.40%); and HPV 51, 59 (2.88%).Using LA, the three most prevalent individual HPV types for women living with HIV were HPV 45 (10.24%), HPV 16 (9.94%), and HPV 18 (7.23%)/HPV 58 (7.23%). For women without HIV, HPV 16 (3.40%), HPV 51 (2.88%), and HPV 35 (2.09%)/HPV 52 (2.09%) were most prevalent.3.2.3Of the women identified as HPV‐positive by GX N = 222) and LA (N = 212), multiple infections were more common among women living with HIV  compared to women without HIV  , with a strong kappa value of 0.85 . Of the 222 women identified as HPV positive by GX, 194 (87.39%) were also deemed positive according to LA. Similarly, 194 (91.51%) of the 212 women identified as HPV positive by LA were also positive by GX. When the HPV genotypes were grouped following the five channels of GX, four of the five channel groups had strong kappa values: 0.86  for HPV16; 0.89  for HPV 18,45; 0.83  for HPV 31,33,35,52,58; 0.85  for HPV51,59. The channel detecting HPV 39, 56, 66, 68 had only a moderate kappa value of 0.72  compared to LA detection of these same genotypes.Figure Figure 3.4P < .05) associated with CIN2+ in the multivariable analysis regardless of HIV status. For LA, only HPV types 16, 18, 31, 33, 35, 52, and 58 were significantly associated with CIN2+ in multivariable analysis. HPV 59, which was significantly associated with CIN2+ in univariable analysis, was no longer associated with CIN2+ once the above‐mentioned HPV types were included in the model. In sum, both GX and LA assays identified the same seven HPV genotypes. The exception was HPV45 which was not significant as an individual type in the LA analysis but was included along with HPV 18 in one channel on GX. For the purpose of further analysis, we included HPV45 (due to its known links to adenocarcinoma) along with the seven consistently identified genotypes, in our calculations of selected typing. Full typing was defined as all five channels for GX and the 14 individual high‐risk HPV types for LA.Table Results from the univariable and multivariable logistic regression analyses for CIN3+ were similar and are included as Table S1.3.5After selecting the genotypes that were significantly associated with CIN2+ from the multivariable logistic regression results, we calculated sensitivity and specificity comparing full vs restricted genotyping. For both assays, there was only a slight decrease in sensitivity for restricted vs full genotyping. For women living with HIV, the sensitivity to detect CIN2+ with full typing was 93.60% [88.85%‐96.76%] by GX and 93.02% [88.13%‐96.34%] by LA, which decreased to 90.70% [85.33%‐94.59%] by GX and 88.95% [83.29%‐93.22%] by LA for restricted genotyping. For women without HIV, the sensitivity to detect CIN2+ with full typing was 88.71% [81.78%‐93.69%] by GX and 83.87% [76.19%‐89.86%] by LA, which decreased to 87.10% [79.89%‐92.44%] by GX and 79.03% [70.81%‐85.85%] by LA for restricted genotyping . Nine of the 11 were reported as “no isolates” on LA, one was HPV 53 and one was IS39. In four women with CIN2+ who did not have one of the 14 genotypes detected by GX, but who did have them detected by LA, the HPV genotypes detected in LA were: 45; 16,83; 68,70; and 51, 53, 62, 71, 82, 83.Specificity estimates improved with restricted compared to full genotyping. Specificity for full genotyping among women living with HIV was 60.07% [54.00%‐65.93%] for GX and 63.74% [57.73%‐69.45%] for LA. Specificity increased to 67.77% [61.87%‐73.27%] by GX and 71.43% [65.67%‐76.71%] by LA for restricted genotyping. For women without HIV, specificity was 86.87% [82.93%‐90.19%] for GX and 87.43% [83.54%‐90.68%] for LA. Specificity increased to 89.66% [86.04%‐92.62%] by GX and 90.78% [87.30%‐93.56%] by LA for restricted genotyping  for full vs restricted vs 16, 18 genotyping were 23.73%, 29.79%, and 26.32% for women without HIV and 34.87%, 38.58% and 40.00% for women living with HIV.Sensitivity estimates were slightly higher and specificity estimates lower for detection of CIN3+ compared to CIN2+ but the benefits of restricted compared to full genotyping were similar (Table S2).4To optimize the clinical performance of HPV testing in screening programs, our results indicate that only HPV 16, 18, 45, 31, 33, 35, 52, and 58 should be tested for. Although HPV 45 was not significantly associated with our disease endpoint, CIN2+, we include this genotype since it is the third most commonly identified genotype in invasive cervical carcinoma.We examined whether the inclusion of only types 16 and 18 might be a simpler approach to HPV type selection. We found that restricting only to these types led to an unacceptable decline in sensitivity. Positive predictive value in the group with types 16 and 18 was not appreciably better than the PPV in the group of eight types that we selected.In our analysis, we have additionally shown that point‐of‐care HPV DNA testing is a robust option for primary cervical cancer screening in limited‐resource settings like South Africa. The point‐of‐care GX used in South Africa has the benefit of rapid same‐day results and minimal lab infrastructure/personnel. In this analysis we found it to have equivalent clinical performance characteristics as laboratory‐based LA.The GX platform is a pragmatic choice for South Africa given its current widespread usage across the country for tuberculosis testingThere are a few limitations to our study. First, we used disease endpoints (CIN2+/CIN3+) in our analysis but realize that not all of these cervical lesions will progress to cancer. Some researchers criticize the use of CIN2+ due to lack of reproducibility and the higher likelihood of these lesions regressing compared to CIN3+.The landscape of cervical cancer prevention is rapidly changing and on the horizon is primary screening with HPV DNA testing in resource‐constrained settings.LJ, RS, ZM, AW, RB, AT, JM, WYT, LD, and LK have no conflicts to declare. DP and SC declare that they receive salary, benefits, and equity from Danaher Corp. Cepheid is a wholly owned entity of Danaher Corporation. TW declares that he serves as a consultant in clinical trial design and as an expert pathologist for HPV vaccine and/or diagnostic trials for Becton, Dickinson and Company, BD Life Sciences – Diagnostic Systems, Roche, and Inovio Pharmaceuticals and as a speaker for Roche and Becton, Dickinson and Company, BD Life Sciences – Diagnostic Systems.LK, TW, and LD designed the study. RS, RB, and LD recruited the participants and did the clinical follow‐up. DP and SC provided technical assistance on human papillomavirus testing with Xpert. ZM and AW conducted human papillomavirus testing with Linear Array. LJ, LK, and WYT undertook the statistical analyses. LJ and LK wrote the first draft. JM, DP, and TW provided crucial input in data interpretation. All authors reviewed the content of the final version of the manuscript.Table S1‐S2Click here for additional data file.


The natural history of the CIN1 lesions is characterized by an elevated rate of spontaneous regression (80%), some authors recognize a capacity to progress to HSIL in 10% of cases, and other authors do not recognize the capacity of progression of LSIL (CIN1). This study was aimed to evaluate the incidence of progression to HSIL (CIN3) in women with a histological diagnosis of LSIL (CIN1). Furthermore, to this end, we studied the histological outcomes of cone specimens collected by the LEEP.  All the data were retrospectively analyzed. All participants underwent a follow-up of 4 years, during which each woman underwent an HPV test and genotyping, cervical cytological sampling, or biopsy every six months. The endpoint was the histological confirmation of CIN3 lesions in any moment during follow-up.  Progression to CIN3 occurred in 7 cases . Analyzing the histological exams of the cones of the 7 cases that progressed to CIN3, we found the coexistence of CIN1 and CIN3 lesions in all cases.  After 4 years of follow-up, only 1.5% (7/475) of the women with LSIL developed CIN3, all within the first two years of follow-up, and were immediately treated. The most likely explanations for “progression” from LSIL to HSIL are (1) actual progression, (2) underdiagnosis of HSIL on initial biopsy, (3) overdiagnosis of HSIL on follow-up biopsy/cone, and (4) CIN3 arose de novo. Analyzing the histological exams of the cones of the 7 cases that progressed to high-grade, we found the coexistence of CIN1 and CIN3 lesions in all cases. Some recent studies have shown that a viral genotype corresponds to different lesions in the same cervix; therefore, CIN1 coexisting with CIN3 does not always indicate progression of CIN1. Other authors have doubted the capacity of LSIL to progress. There is still much debate concerning the management of patients with LSIL CIN1) of the uterine cervix, defined as the transient expression of a self-limited human papillomavirus (HPV) infection  of the u, 3. The The natural history of the CIN1 lesions is characterized by an elevated rate of spontaneous regression 80%) , 6, some0% , 6, sIn light of these considerations, this study was aimed to evaluate the incidence of progression to HSIL (CIN3) in women with a histological diagnosis of LSIL (CIN1) and the correlation with the genotype as well as the type of viral infection, with six monthly follow-ups for 4 years. Furthermore, to this end, we studied the histological outcomes of cone specimens collected by the loop electrosurgical excision procedure (LEEP).We enrolled 751 women at the outpatient clinic of Colposcopy and Cervico-vaginal Pathology at the University of Catania, Italy, with a cytological diagnosis of LSIL (CIN1).The first participants were enrolled in March 2014.Patients' age was from 24 to 49 years, with an average of 29 ± 8.3 years and a median of 33 years.Positive at the cytological and histological exam for LSIL/CIN1Positive HPV testCompleted 4 years of FUInclusion criteria are as follows:Women were not enrolled in this study if they were pregnant, with a diagnosis of or under therapy for previous cervical dysplasia, immune-depressed, or with infections caused by human immunodeficiency virus (HIV-positive).All the data of the women in our database who met the inclusion criteria were retrospectively analyzed in an observational cohort study.Only 475 patients met the inclusion criteria; their clinical data was gathered regarding sexual and daily habits that could facilitate virus acquisition.All the participants underwent colposcopy guided biopsy of suspected areas after an anomalous cervical cytology examination and successive colposcopy, according to current guidelines , 4.All participants underwent a follow-up of 4 years, during which each woman underwent an HPV test and genotyping, cervical cytological sampling (Bethesda system), and all the participants with positive colposcopy or viral persistence underwent biopsy every six months. The endpoint was the histological confirmation of CIN3 lesions in any moment during follow-up.The women who showed progression to CIN3 underwent large loop excision of the transformation zone (LEEP).Histologic evaluation was performed with specimens collected by a colposcopy-directed biopsy and/or cone specimens collected by the loop excision procedure. Chronological data of follow-ups were compiled and analyzed for regression, persistence, and progression of the lesions over time. Regression is defined as CIN 1 lesions that return to normal cytology. Persistence is defined as CIN 1 lesions demonstrated by biopsy. Progression is defined as histologically confirmed CIN3. In this retrospective study, all the histological diagnoses were reviewed.The search for viral DNA (HPV-test) was carried out using PCR after the extraction of the cervical sample using Thin-prep. The automated DNA extraction was carried out on the NucliSenseasyMAG system  following the manufacturer's HPV 1.1 protocol. Amplification of HPV DNA was accomplished by HPV-HS Bio  nested polymerase chain reaction (PCR) for the detection of HPV-DNA sequences within the L1 ORF, according to the manufacturer's recommendations.HPV typing was carried out with specific probes for the most frequent HPV types . HPV typing allows the identification of 11 LR genotypes  and 18 HR genotypes . Samples that were positive by nested PCR but negative in reverse line blot for any of these types were considered as undetermined HPV. The cervical swab for the HPV test was taken from the endocervical canal and the transformation zone.We classified HPV infection in transient if the second genotyping shows a different genotype, persistent if the second sample shows the same genotype after 6 months or 1 year from the first, clearance if two consecutive samples of DNA HPV are negative at an interval of about 6 months after a positive sample. Viral clearance does not always coincide with regression of the lesion; thus, the women underwent HPV  and cytological samples or biopsy. After 2 negative cytological samples and a negative HPV test, women reenter screening .Moreover, a single HPV infection is characterized by the presence of a single genotype, multiple HPV infections from the concomitant presence of more genotypes; in this case, the causality is attributed based on the finding of the same HPV type of the previous samples.The study was conducted in accordance with the 1975 declaration of Helsinki. The investigations were conducted through the retrospective review of the medical database; the protocol was notified to the Catania1 Ethics Committee of the Catania Polyclinic, according to the AIFA law of 20 March 2008. Furthermore, the consent of the study participants was deemed unnecessary as the study only concerned the retrospective review of the medical database.χ2 test, and, when appropriate, Fisher's exact test was used to calculate the significance  of the difference between groups.Statistical analysis of the data was made using the software package SPSS 15.0 . Descriptive statistics were expressed as frequency, arithmetic mean, standard deviation (S.D.), and percentages. The analysis of the data was made using the At the histological examination, carried out after biopsy, 511/626 patients  had one low/grade precancerous lesion (LSIL/CIN 1); 115 (18.3%) patients had a negative histological diagnosis. The 511 CIN1 positive women were given an HPV test; of these, 36 samples (7%) were found to be inadequate; these patients were excluded from the study. Thus, the study group was composed of 475 women who were CIN1 positive at the histological exam and HPV positive and completed the four years of follow-up.Viral genotyping was positive for infections from a single HPV genotype for 180/475 patients (37.9%), and 295/475 patients (62.1%) were, instead, positive for multiple infections . We had All the participants (475 women) completed the follow-up period (48 months), and 1.5% (7/475) developed CIN3 and represented 11.5% (7/61) of the women who had persistent HPV infection.We found 61 (12.9%) persistent infections and 414 (87.1%) transient infections .Of the transient infections, 251 were multiple and 163 were single; in almost all of the cases, viral clearance occurred within 18 months and regression of the lesion within 24 months; in 9% of the cases, infection was transient from papillomavirus even if the CIN1 lesion disappeared. The rate of regression for histologically confirmed CIN1 lesions was always elevated. The rate at 12 months was 54,8% and at 24 months 35,9%. Overall transient infections histologically regressed in 100% of the cases within the 4 years of follow-up. In transient infections, both single and multiple, genotypes 16, 54, 58, and 31, were the most frequent.We had 61 cases of persistent HPV infection, 17 single infections, and 44 multiple infections, in our study. In this group, viral and histological lesion clearance occurred in 43 cases (33 multiple and 10 single), with an average persistence of 2.5 years, and progression to CIN3 in 7 cases (5 cases of single infection and 2 cases of multiple infection). Progression occurred in the first 24-36 months of follow-up .The most frequent genotypes in the persistent infection group were HPV 16, HPV 31, and HPV 33. To date, 12 cases have long-term persistence. Analyzing the histological exams of the cones of the 7 cases that progressed to CIN3, we found the coexistence of CIN1 and CIN3 lesions in all cases .The diagnosis and prevention of cervicocarcinoma are based on the presupposition of Richard  that preAn important role is played by a woman's immune system and by the presence of cofactors such as smoking, use of oral contraceptives, and genital herpes infections. Furthermore, it has been demonstrated that the natural history of the evolution of CIN depends on the type of HPV that infects the cervix  and the Some authors –14 have The cases of persistent HPV infection were 12.9% (61/475), 68,9% (42/61) of these cases reached clinical recovery with viral clearance, persistence had an average duration of 2.5 years, 19.6% (12/61) persist to date, without alterations of the histological state of CIN1, and 11.5% (7/61) of cases had progression; in 7 cases to CIN3, all these lesions occurred during the first 36 months of F0ll0w-up.Overall, 82.1% of the women with CIN1 regressed within 2 years, while 1.5% progressed to a high-grade lesion within 3 years.Moreover, 19.6% (12/61) of persistent infections were not able to eliminate the virus in 4 years.The fact that our study sample is made up of women already at baseline with low-grade lesion and positive for HPV infection does not allow us to know the time between the first detection of HPV infection and the first detection of cytological abnormalities. We can say that from the observation of the woman with LSIL to the onset of the high-grade lesion, the average of 36 months has elapsed. This finding is consistent with previous studies that reported the onset of HSIL within 3 years of the first detection of HPV infection. Even authors , 15 asseTransient HPV infections were clinically irrelevant with a high percentage of viral clearance and histological regression, while cases of viral persistence are those that clinicians have to be interested in: they are nearly all linked to a single genotype; in fact, cases of persistence of 2 or more genotypes are very rare. It is known that the persistence of specific viral genotypes is associated with a greater risk of developing high-grade lesions. In particular, persistent HPV 16 infection is known as the most significant prognostic factor in the progression of cervical lesions.In our precedent study , the preThe most represented genotypes in persistent infections with progression are HPV 16 and HPV 31, the most frequent in Italy and Europe , while HRecent data from the control arm of the FUTURE I study reported a persistence of 12 months or more, and the highest risk of progression to CIN2+ for HPV16 and HPV33 .The women with progression were, on average, 32 years old and had one single persistent infection with one of the following genotypes: HPV 16, HPV 31, or HPV 33. The low rate of subsequent CIN 3 suggests that follow-up time, rather than immediate ablation or resection of the transformation zone, is appropriate in these women. Excisional treatments are associated with physical, psychological, and obstetric morbidity  and can The most likely explanations for “progression” from LSIL to HSIL are (1) actual progression, (2) underdiagnosis of HSIL on initial biopsy, (3) overdiagnosis of HSIL on follow-up biopsy/cone, and (4) CIN3 arose de novo. There is a significant risk that an HSIL diagnosis following an LSIL biopsy is a diagnostic error and should be reviewed by an additional observer. Less likely is that the initial LSIL biopsy is an undercalled HSIL (CIN3). Analyzing the histological exams of the cones of the 7 cases that progressed to high-grade, we found the coexistence of CIN1 and CIN3 lesions in all cases.We asked ourselves if CIN1 is the same lesion for which the woman underwent follow-up and that it remained the same and thus did not progress, if CIN3 has always been present and not diagnosed or if it occurred “de novo” over about 6 months. Park  demonstrIn our cases, we had a rapid development of high-grade lesions; in fact, the cases of CIN3 and carcinoma occurred within 24-36 months of follow-up to which the women were undergoing for persistent HPV CIN1. Other authors have doubted the capacity of LSIL to progress .Today, it is accepted that CIN3 can arise de novo, rather than be the result of a continuum of CIN1. It is known that CIN1 is due to a viral cytopathic effect and not necessarily the onset of the progression to cancer. Lityens  show thaTherefore, from the analysis of the data, we can say that it is not the presence of CIN1 that determines the risk of a successive CIN3, but the diagnosis of persistent HPV 16, 31, 33, or 18 infection. This knowledge would avoid carrying out intensive follow-ups and unnecessary treatment  and the missing diagnosis at biopsy of CIN3, arose de novo, coexisting with CIN1.


At univariate analysis, HPV vaccination after LEEP (odds ratio (OR) = 0.4, p-value = 0.020) emerged as an independent protective factor. Choosing as an outcome of the analysis only recurrence as severe cervical lesions, the protective role of HPV vaccination after LEEP was found to be much more relevant with an odds ratio of 0.2 . Administration of an HPV vaccine after LEEP seems to reduce the risk of recurrence, thus suggesting that HPV vaccination has a role as an adjuvant treatment after LEEP.The aim of this study was to assess the role of a human papilloma virus (HPV) vaccine after loop electrosurgical excision procedure (LEEP) in reducing recurrent cervical dysplasia. A series of 503 women with cervical dysplasia received LEEP between January 2012 and October 2018. Of these patients, 379 were treated between January 2012 and June 2017, thus ensuring an adequate follow-up time. We made three attempts to establish telephone contact with each patient; 77 women did not respond and were excluded from the final study population, which consisted of 302 patients. One hundred eighty-two (60.7%) women were vaccinated with an HPV vaccine within 4 weeks of LEEP and 103 (34.3%) were followed up with but not vaccinated. Recurrence of cervical dysplasia requiring a further LEEP procedure occurred in 30 (10.5%) women, of whom 17 (16.5%) were not vaccinated and 13 (7.1%) were vaccinated ( Human papilloma virus (HPV) causes one of the most prevalent sexually transmitted infections, and it has been estimated that around 80% of women will be exposed to HPV during their lifetime . DespiteAt the beginning of the 20th century, cervical cancer represented the second most common incident cause of cancer-related deaths in the female population in the United States . The mortality rate decreased to 2.3 deaths per 100,000 females in 2019 ,6,7,8,9.If the introduction of pap smears represents the first milestone in the fight against HPV-related malignancies, the second milestone is certainly represented by the development of HPV vaccines ,6,7,8,9.Therefore, with the aim of clarifying whether HPV vaccination after LEEP may be associated with a reduced recurrence rate in women with cervical dysplasia, we conducted this single-institution observational retrospective study.We analyzed a series of women with a confirmed diagnosis of cervical dysplasia who underwent LEEP between January 2012 and October 2018 at the Obstetrics and Gynecology Unit of an Italian university hospital. Only women with ≥2 years of follow-up were enrolled on the assumption that ~90% of post-LEEP recurrences occur within the first 24 months after surgery ,16,17,18After January 2015, all women were counseled with regard to the potential benefits and risks of HPV vaccination and, after acceptance, received within 4 weeks after LEEP the first dose of an HPV vaccine, which was administered at the local general community hospital. Only the first three women who enrolled in the study received the bivalent vaccine; all other patients that were included in the study were administered the quadrivalent HPV vaccine.The clinical follow-up included a pap smear and a colposcopy every 6 months for the first 2 years after LEEP. Furthermore, in accordance with international guidelines, if margins were positive, then cytology and endometrial curettage at the 3-month evaluation were performed.All women who participated in the study were contacted by telephone and interviewed on oral contraceptive prescriptions, smoking habits, total number of sexual partners, number of pregnancies, concomitant drugs, and occurrence of infectious diseases.A treatment outcome failure was defined as a patient with ≥1 recurrence of cervical dysplasia after LEEP.HPV genotype at the time of relapse was assessed only in the group of vaccinated women on histological specimens obtained at the time of recurrence.According to Italian law on observational studies, ethical approval is not needed for the analysis of anonymous retrospective data. Institutional Review Board (IRB) approval was obtained (IRB code 2018-375).LEEP was performed under local anesthesia using wire loop electrodes with a diathermy apparatus set. A suture was placed at the 12 o’clock position of the specimen for orientation before fixation in 10% formalin. Cervical dysplasia grading and a histologic assessment of exocervical and endocervical margins of resection were carried out; in the case of involvement of surgical margins, clinical monitoring was performed, followed by surgical excision for persistent severe dysplasia. Furthermore, cone length and p16 expression were assessed in all specimens.t and Mann–Whitney tests were performed for parametric and nonparametric variables, respectively.An ad hoc electronic form was prepared to collect qualitative and quantitative variables from medical charts. Qualitative variables are described with absolute and relative (percentage) frequencies, whereas quantitative variables are summarized with means  or medians , depending on their parametric distribution. Comparisons between vaccinated and nonvaccinated patients were performed with a chi-squared or Fisher’s exact test for qualitative variables, whereas Student’s A logistic regression analysis was carried out to assess the relationship between recurrence rate and demographic, epidemiological, and clinical data.p-value less than 0.05 was considered to be statistically significant. All statistical analyses were performed with the statistical software STATA version 15 .A two-tailed A total of 503 women underwent LEEP due to cervical dysplasia during the study period. However, only 379 patients were followed-up with for ≥2 years. Seventy-seven (20.3%) women did not respond to our telephone calls and were excluded from the analysis .A total of 182 (63.8%) women received an HPV vaccination within 4 weeks after LEEP. The remaining 103 (36.2%) patients entered a routine follow-up program but did not receive an HPV vaccine .As described in p-value = 0.0004; Women administered an HPV vaccine after LEEP were 4 years younger when compared with nonvaccinated patients  .Disease recurrence occurred in 17 (16.5%) and 13 (7.1%) nonvaccinated and vaccinated women, respectively (= 0.010) . A statiHPV genotype at the time of relapse was retrieved in five women receiving vaccination after LEEP. In particular, two patients showed HPV 16 infection (both with HPV 16 at diagnosis), one woman was affected by HPV 33 , in one patient we detected HPV 51 (high-risk HPV at diagnosis), and in the remaining woman we observed HPV 31 infection (high-risk HPV at diagnosis).p-value = 0.020). The protective role of HPV vaccination after LEEP was found to be more relevant to protection against severe (CIN3/carcinoma in situ) cervical lesions .The successful implementation of HPV vaccination strategies has significantly reduced the incidence of cervical cancer . HoweverIn particular, contrasting results on the risk of recurrence have been described in women treated with LEEP for a cervical dysplasia ,14. In 2On the other hand, in 2016, a randomized clinical trial that involved 1711 women, each of whom either received or did not receive an HPV vaccine after surgical excision, did not confirm previously reported data. The authors concluded that vaccination does not protect against infections/lesions after treatment . TherefoIn this context, the results of our study should not be underestimated. In fact, we demonstrated in a series of more than 300 women a statistically significant reduction of recurrence rate in women receiving a quadrivalent HPV vaccination after LEEP when compared to women who did not receive a vaccine, thus confirming previous results . FurtherTherefore, while our results do not enable us to draw definitive conclusions, they do support the introduction of post-LEEP HPV vaccination in women receiving an excisional procedure due to cervical dysplasia, particularly in the case of severe lesions. It seems reasonable to hypothesize that, particularly considering that the results from randomized clinical trials did not demonstrate an increase in virus clearance after HPV vaccine administration , the benIt should be acknowledged that, in our study, the cohort of patients who received an HPV vaccine showed a statistically significantly lower age as compared to women who did not receive an HPV vaccine, thus potentially influencing the rate of recurrence that was observed in the two groups. However, the results of the univariate analysis, from which HPV quadrivalent vaccine administration after LEEP emerged as the only predictor of failure after LEEP , stronglThe small sample size in comparison with other international studies and the high drop-out rate in our cohort could limit the reliability of our findings. Furthermore, the lack of HPV testing with genotype characterization before and after LEEP meant that we could not clarify the role that HPV plays in HPV-positive patients after LEEP intervention. However, the scientific evidence from our observational study, which is characterized by homogeneous groups for the main confounding variables, could help us to evaluate the preventive/therapeutic role of HPV vaccines in surgically treated patients.In conclusion, the administration of an HPV vaccine after LEEP reduces the risk of recurrence, suggesting that HPV vaccines can effectively be used as an adjuvant treatment after an excisional procedure. Further controlled clinical trials are needed to better elucidate the efficacy of the currently available HPV vaccines in different epidemiological scenarios.


LGarcinia mangostana . or mangosteen fruit, has been applied in various biomedical products because of its minimal skin irritation, and prominent anti-inflammatory, antimicrobial and immune-modulating activities. Owing to its low water solubility, the particle formulations are necessary for the applications of α-mangostin in aqueous media. The particle formulations are usually prepared using surfactants and/or polymers, usually at a larger amount of these auxiliaries than the amount of α-mangostin itself. Here, we show the self-assembly of α-mangostin molecules into water-dispersible particles without a need of any polymers/surfactants. Investigations on chemical structure, crystallinity and thermal properties of the obtained α-mangostin particles, in comparison to the conventional α-mangostin crystalline solid, confirm no formation of the new compound during the particle formation and suggest changes in intermolecular interactions among α-mangostin molecules and significantly more hydroxyl functionality positioned at the particles' surface. The ability of the water suspension of the α-mangostin to inhibit the growth of Propionibacterium acnes, the acne-causing bacteria, is similar to that of the solution of the conventional α-mangostin in 5% dimethyl sulfoxide. Moreover, at 12.7 ppm in an aqueous environment of RAW 264.7 cell culture, α-mangostin suspension exhibits five times higher anti-inflammatory activity than the conventional α-mangostin solution, with the same acceptable cytotoxicity of less than 20% cell death.α-Mangostin, the extract from pericarp of  The pericarp of this fruit has been used as a medicinal agent by southeast Asians for centuries. During the last few decades, scientific researchers have reported that α-mangostin possesses various biological activities including anti-inflammatory -1-piperazineethanesulfonic acid , 1% v/v gentamicin and 10% v/v fetal bovine serum ), under a humidified atmosphere of 5% CO2.2.2.2.Amount of nitrite is an indicator of NO synthesis. Here, the amount of nitrite was measured in the culture medium by a colorimetric method using Griess reagent . In the 2.2.2.3.−1) and MTT solution . Then cells were incubated in a humidified atmosphere at 37°C for 4 h. After that, DMSO was added (200 µl well−1) to dissolve purple formazan. After completely dissolving, the absorbance was measured at 540 nm. Water was used as a control and complete media was used as a blank. Cell viability was calculated as follows:The evaluation of cell viability was based on the mitochondrial dehydrogenase activity in the living cells to reduce MTT into a purple formazan product which can be measured by UV absorption spectroscopy . Here, t3.ai–aiii). When the starting α-mangostin concentration reached 10% w/v, observable precipitates (not dispersible in water) started to appear. Higher concentration (30% w/v) led to more precipitates (bi). We explain these observations as follows. When water was slowly added to the ethanolic solution of α-mangostin, phase separation of α-mangostin occurred, as α-mangostin molecules are poorly soluble in water. Because the addition of water was slow and the concentration of α-mangostin was not too high, these molecules arranged themselves in such a way that the hydrophobic part was away from water and the hydrophilic part interacted with the added water. At higher starting α-mangostin concentrations, more α-mangostin molecules were surrounding each growing particle during the water addition, causing faster deposition of the molecules onto each growing particle, resulting in more α-mangostin molecules being integrated into each particle, thus a bigger sized particle was obtained. This faster deposition also resulted in less time for molecular orientation to take place for the depositing molecules. With less hydrophilic moieties properly facing out at the particles' surface, the particle's surface became more hydrophobic and resulted in the particle being even more attractive to α-mangostin molecules. These contribute to the random aggregation of α-mangostin molecules into large precipitates.By slowly introducing water into the ethaolic solution of α-mangostin, particulate formation was observed as the clear solution turned cloudy. The dry particles, obtained by removing ethanol from the suspension by vacuum and then subjecting the aqueous suspension to freeze drying, could disperse well in water. We systematically studied the effect of α-mangostin concentrations  on the particle formation. The result clearly showed that at the concentration range of 0.5–5.0% w/v of α-mangostin in the starting ethanolic solution, only water-dispersible particles were obtained, no settling down precipitate was observed. The diameter of the formed particles increased with increasing concentration of the starting α-mangostin in ethanol. The biggest particles that still showed good water dispensability possess diameters of less than 10 µm ai–aiii. ipitates bi. We exWe prepared α-mangostin particles at various final ethanol to water ratios , dried the obtained suspension, then analysed the morphology of the products. From the SEM images (psupason@chula.ac.th) with the diameters of 324.35 ± 71.45 and 467.97 ± 188.51 nm, for those made with propanol (MG-P) and butanol (MG-B), respectively. Other solvents gave precipitates.We investigated the effect of replacing ethanol with other water-miscible solvents. Among propanol, butanol, methanol, acetone and DMSO, only propanol and butanol gave water-dispersible particles of α-mangostin. SEM images indicate that these dry particles are spherical , which was significantly bigger than the size of the dry particles observed by SEM (312.17 ± 55.11 nm). The MG-P and MG-B suspensions in water, however, showed bimodal distributions . We speculated that the bimodal distribution was possibly a result of the breakup of large particles during the organic solvent evaporation at the end of the process. It was possible that the evaporation of propanol or butanol that had been trapped in the particles might cause particle destabilization and break-up. This did not happen when ethanol was used because ethanol is more hydrophilic and thus entrapment was probably not as significant. In addition, the boiling point of ethanol is the lowest among the three solvents; therefore, evaporation could take place easily. By contrast, the trapped propanol and butanol which possess higher boiling points probably built up vapour pressure inside the particles at higher temperatures where intermolecular bonds among α-mangostin were weakening, resulting in particle disintegration. Further study is needed to make a better understanding of the mechanism of the particle formation and their size distribution.The average zeta potential values of MG-E, MG-P and MG-B, in water, are −34.1 ± 0.72, −30.1 ± 0.21 and −32.9 ± 1.56 mV, respectively. These zeta potential values agree well with the observation that water dispersions of the three α-mangostin particles are stable.1H NMR spectrum. Indeed, no chemical transformation took place during the particle formation, the 1H NMR spectrum of the MG-E particles is identical to that of the original bulk α-mangostin . This also applies to the 1H-NMR spectra of MG-P and MG-B.To make sure that no chemical transformation took place on the α-mangostin structure during the preparation of the particles, we dissolved the obtained MG-E in DMSO-d6 and acquired their a). It should be noted here that, unlike 1H NMR that all intermolecular forces within the particles did not exist owing to the complete dissolution of the material before analysis, FT-IR analyses were performed directly with the material in their self-assembled particulate state. The O-H stretching vibration of the original α-mangostin shows up as the double broad peaks at 3412.2 and 3246.3 cm−1 whereas that of the three α-mangostin particles appears as a single broad peak at 3385.4 cm−1. The C-O stretching vibration of vinyl ether of the original α-mangostin appears as the double peaks at 1238.2 and 1222.8 cm−1 whereas that of the three α-mangostin particles appears as the single peak at 1226.8 cm−1. This indicates that the self-assembled α-mangostin molecules in the particles probably exist in more than one configuration. The stretching vibration of the C-O bond of the -C-O-H functionality appears as two sets of the triple peaks at 1199.1, 1182.4, 1169.2 cm−1 and 1094.9, 1075.1, 1049.9 cm−1 for the original α-mangostin, whereas that of the three mangostin particles appears as two sets of the double peaks at 1184.1, 1154.2 cm−1 and 1078.7, 1044.1 cm−1. In addition, vibrational absorptions from functionalities associated with the aromatic π-π bonding of the original raw material also differ from those of the three particles, i.e. C=C stretching of the aromatic ring in the original raw material absorbs at 1607.3 and 1581.4 cm−1, whereas that of the three particles absorbs at 1605.1 and 1580.4 cm−1, and the C=O stretching of the aromatic ketone in the original raw material absorbs at 1640.1 cm−1, whereas that of the three particles absorbs at 1643.4 cm−1. Because all the tested materials are pure α-mangostin with the same molecular structure, the differences in the FT-IR spectra imply different configuration settings of the molecules and different intermolecular interactions. The information indicates that H-bonding and π-π stacking among α-mangostin molecules in the original α-mangostin differ from those in the three α-mangostin particles.Interestingly, significant differences in the FT-IR spectrum of the original α-mangostin and those of the α-mangostin particles  could be clearly observed a. It shoθ at 16.1, 18.5, 19.2, 20.2, 21.0, 21.8, 22.7, 23.6, 24.4, 25.6, 26.3, 27.7, 28.5, 30.8 and 32.6° (figure 2b blue line). By contrast, the XRD patterns of MG-E, MG-P and MG-B  contain only a single broad peak, clearly indicating amorphous structure. These results confirm that the assembly of the α-mangostin molecules into water-dispersible α-mangostin particles results in different molecular packing from that of the original water-immiscible α-mangostin solid.The XRD pattern of the original α-mangostin reveals crystalline character with the 2c). The original α-mangostin, MG-E, MG-P and MG-B show the maximum decomposition temperature at 361.62, 347.53, 334.97 and 335.78°C, respectively. These results indicate different molecular packing of α-mangostin molecules in the four materials. The molecular packing of the α-mangostin particles is less tight compared to that of the original α-mangostin. In addition, intermolecular forces of the MG-P and MG-B  are a little weaker than that of the MG-E (particles prepared using ethanol as solvent). This lower thermal stability of the water-dispersible particles as compared to that of the original α-mangostin corresponds well to the disappearance of the crystallinity of particles observed in the above XRD patterns and the difference in the intermolecular interactions implied by their FT-IR spectra. All these observations also agree with our proposed model of solvent entrapment in the particles. The presence of solvent molecules at the inside of the particles during the assembling process would prevent crystalline packing inside the particles and decrease thermal stability.Thermograms of the original α-mangostin, MG-E, MG-P and MG-B are not identical c. The orNext we probed the surface of the particles with the XPS analysis. Although the C1 s spectra of the original α-mangostin and the MG-E . This result indicates that the α-mangostin colloidal particles in water possess the same anti-P. acne, as that of the original α-mangostin in organic solvent.Next, we tested the ability of the original α-mangostin and the MG-E to inhibit the growth of a) indicates that the anti-inflammatory activity of MG-E suspension is significantly higher than that of the original α-mangostin solution. The MTT assay indicates that the MG-E at concentrations up to 12.7 ppm is not toxic to the tested cells . To our surprise, at this highest non-toxic concentration of 12.7 ppm, the MG-E suspension was five times more potent in inhibiting the inflammatory reaction in the cells than the original α-mangostin solution. Thus, it can be concluded that the α-mangostin particles obtained from this self-assembly process possess better anti-inflammatory activity than the original α-mangostin, and this obtained anti-inflammatory activity was not owing to the cytotoxicity effect of α-mangostin. We speculate that the colloidal state of the material probably helps improve the efficiency of the uptake of the sample by RAW 264.7 cells. In other words, the MG-E colloids probably can get into the RAW 264.7 cells more effectively than α-mangostin in the true solution state.Using the RAW 264.7 cell line, we tested the anti-inflammatory activities, another well-known biological activity of α-mangostin, of the MG-E via the NO assay. The original α-mangostin was first dissolved in DMSO and then diluted with water (to 5% DMSO), whereas the MG-E was directly dispersed in water. Here, anti-inflammatory activity of the tested sample was determined as the ability of the sample to suppress the NO production, which was calculated from the amount of the nitrite accumulated in the culture supernatants. The % NO production a indicat4.P. acne at a similar level to that of the original α-mangostin solution (used with some DMSO to aid solubility). When tested by the NO assay in the RAW 265.7 cell line at a concentration that was not toxic to the cells, the aqueous suspension of the α-mangostin particles showed five times higher anti-inflammatory activity than the original α-mangostin in the solution state. We speculate that the α-mangostin particles probably could be more effectively taken up into the cells as compared to α-mangostin in the solution state.Here, we have shown that through the use of water and appropriate water-miscible solvents, self-assembly of the water-insoluble α-mangostin into the water-dispersible particles of pure α-mangostin could be induced. The prepared pure α-mangostin particles can be easily dispersed into a stable suspension in water with no need of any auxiliary agent. FT-IR, XRD spectra and thermograms indicate that molecular packing and intermolecular interactions of the α-mangostin molecules in the particles are different from those in the original solid. The original solid seems to possess stronger intermolecular attractions as witnessed through its better thermal stability. The alteration of the intermolecular forces in the prepared particles revolves around the aromatic hydroxyl groups and the aromatic π-π stacking as suggested from the FT-IR spectra. The difference in molecular packing of the prepared particles from that of the original solid is also reflected in the loss of crystallinity upon particle formation as shown in their XRD spectra. Water dispersibility of the particles could be explained at the molecular level through the observation, via XPS, of the significantly higher abundance of polar hydroxyl groups at the particle surface, as compared to that at the surface of the original solid. We speculate that the enthalpy compensation from the hydrogen bonding interactions between water molecules and these hydroxyl groups at the particle's surface is the driving force for the system to favour the particle formation regardless of the weaker intermolecular forces among α-mangostin molecules inside the particles. When tested by the disc diffusion assay, the aqueous suspension of the α-mangostin particles could inhibit the growth of


Nurses and midwives play important roles in educating the public on cervical cancer prevention strategies.This study sought to assess nurses’ and midwives’ knowledge of, attitudes towards, and acceptance of human papillomavirus (HPV) vaccination in relation to their background characteristics.A descriptive cross-sectional study using questionnaires was conducted with a convenience sample of 318 female nurses and midwives, ages 20 to 59, at the Korle-Bu Teaching Hospital in Ghana. The data were summarised using frequencies, percentages, chi-square tests, and Fisher’s exact tests.n = 132) of the participants had high levels of knowledge about cervical cancer risk factors, and 17.6% (n = 56) of the respondents had received at least one dose of the HPV vaccine. Reasons for receiving the HPV vaccination included advice from a colleague  and perceived threat of cervical cancer . Of the 262 respondents who had not been vaccinated, 24.45% (n = 78) strongly agreed and 28.0% (n = 89) agreed with the statement that there was limited information on HPV vaccination. Also, there were statistically significant associations between age , marital status , completed level of education , and duration of working at the hospital  and acceptance of HPV vaccination.The results indicated that 41.5% (This study demonstrated gaps in knowledge about cervical cancer risk factors and attitudes towards HPV vaccination, indicating the need for targeted measures to improve knowledge and attitudes. Also, measures to increase acceptance of HPV vaccination among nurses and midwives should consider their sociodemographic characteristics. HPV infections are most common in young adults, but it generally takes decades for high-risk HPV infections to develop into cervical cancer. HPV is a sexually transmitted infection and the causative agent of cervical cancer , 2. It iPrimary and secondary prevention strategies, including HPV vaccination, screening, and early treatment of precancerous lesions, are critical to decrease incidence and mortality. High-income countries have been successful in decreasing the morbidity and mortality of cervical cancer by 70% through effective cervical cancer screening interventions , 9.HPV vaccination is one of the preventive strategies for cervical cancer. Although some high-income countries have adopted two novel prophylactic vaccines and other secondary preventive methods  to deal The HPV Bivalent Vaccine Cervarix, manufactured by GlaxoSmithKline, can be administered in two to three doses based on the age of the person . In 2013Nurses’ and midwives’ knowledge of HPV vaccination is critical, as these professionals play essential roles in disease prevention and health promotion by educating the public through culturally-relevant health education and promotion strategies. A previous study involving nursing, medical, and dental students reported that participants with at least a moderate or high knowledge of cervical cancer had a greater chance of intending to receive HPV vaccination. Additionally, a positive attitude about HPV was associated with intention to screen . It is pDespite these factors that could impede HPV vaccination among health professionals, nurses and midwives are viewed as role models in matters relating to health , includiA descriptive cross-sectional study was conducted at the KBTH. The KBTH is a premier health facility located in the southern part of Ghana. It is the third-largest hospital in Africa and a major referral centre in Ghana. The facility has a bed capacity of approximately 2000 and 17 clinical and diagnostic departments. It also provides sophisticated and scientific investigative procedures and specialisation in various fields, such as neurosurgery, dentistry, eye/ear/nose and throat (ENT), renal surgery, orthopaedics, oncology, dermatology, cardiothoracic surgery, radiotherapy, paediatric surgery, reconstructive plastic surgery and burns. It has an average daily attendance of 1500 clients and 250 admissions. The choice of this teaching hospital was appropriate because it is a major referral point in the country and has a significant number of female nurses and midwives.The target population of this study consisted of female nurses and midwives at KBTH. Data from the KBTH records unit indicate that there are 1750 female nurses and midwives on staff . This stThe sample size required for the study was determined using Yamane’s 1998 formula for sample size determination .\documeWhere:n = sample sizeN= study populationα= margin of error, which is 0.05 with a significance level of 95%.Based on this formula, a sample size of 325 was anticipated for the study. We added 10% to cover any uncertainties . A totalA questionnaire was used as the data collection tool with items adapted from previous studies , 25. TheThe five reasons for accepting the HPV vaccination were as follows: advice from family or friends, advice from a colleague health worker, perceived threat of cervical cancer, media discussion on cervical cancer, and a friend or colleague who died from the disease. The items in this domain were on a four-point Likert scale: strongly agree, agree, disagree, and strongly disagree. This elicited multiple responses from the participants.The barriers to HPV vaccination domain consisted of 12 items: the vaccine is expensive, going for the HPV vaccine is a waste of time, the unavailability of vaccination sites, there is limited information on the HPV vaccine, I don’t know what the HPV vaccine is all about, there are no health education programmes to promote HPV vaccination, HPV vaccination is not necessary, I do not have money to take the vaccine, fear of adverse effects of the vaccine, risk of becoming infected with HPV, anticipation of family disapproval, and fear of experiencing pain during injections. These barrier items were rated on a four-point Likert scale: strongly agree, agree, disagree, and strongly disagree.The research instrument also covered respondents’ sociodemographic characteristics, including age, marital status, religion, completed level of education, and duration of working in the hospital.The instrument was pretested on 50 nurses and midwives in a nearby health facility. Ambiguities with some of the items were detected, and questions were reworded. For example, “Have you ever had cervical cancer vaccination”? was changed to “Have you received at least one dose of HPV vaccination”? to improve clarity. The instrument was shown to at least two experts to determine the adequacy of the items in measuring the intended concepts. Reliability for the items on the knowledge domain was assessed using Kuder-Richardson Formula 20. The coefficients of reliability for the Likert-type items were determined using Cronbach’s alpha. The following reliabilities were obtained for the various domains: knowledge = .806, reasons for accepting HPV vaccination = .711, and barriers = .824.Three registered nurses were trained to assist with the data collection. The data collection took approximately six weeks from September to October 2018. The questionnaires were administered to the participants after the purpose of the study had been explained to them and they had voluntarily agreed to participate in the study. Some of the participants completed the questionnaires in the nurses’ lounge after they had finished their shifts. Most participants completed their questionnaires off-site and returned them to the research assistants on their next shifts.Data were analysed using frequencies, percentages, chi-square and Fisher’s exact tests. Respondents with knowledge scores of 59% and below were categorised as having low knowledge about cervical cancer risk factors. Those with a score of 60 to 79% were considered to have moderate knowledge, while those with a knowledge score of 80% and above were considered to have high knowledge. These cut points were based on a previous study . With thn = 151) were aged 20 to 29 years. In relation to their marital status, 45.0% (n = 143) were married, while 43.7% (n = 139) had never married. In regard to religion, 82.7% (n = 263) were Christians, while 15.4% (n = 49) were Muslims. In connection with respondents’ level of education, 48.1% (n = 153) had a 3-year diploma, while 37.1% (n = 118) had a bachelor’s degree. The results further indicate that 37.1% (n = 118) had been working in the teaching hospital for one to five years, while 10.7% (n = 34) had been working in the facility for over 11 years.The results in Table n = 132) of the participants had high knowledge of the risk factors for cervical cancer, while 29.6% (n = 94) had low knowledge, as shown in Table n = 176) indicated that they had at least one Pap smear test, while 44.7% (n = 142) had not had a test. Additionally, only 17.6% (n = 56) had received at least one dose of HPV vaccination, while 82.4% (n = 262) had not received a dose.Regarding knowledge of cervical cancer risk factors, 41.5% n = 13 of the pn = 20) strongly agreed and 6.6% (n = 21) agreed with the statement that they received HPV vaccination because a health worker colleague recommended it. In addition, 6.0% (n = 19) strongly agreed and 5.7% (n = 18) agreed that they had the vaccination due to the perceived threat of cervical cancer.Table n = 78) strongly agreed and 28.0% (n = 89) agreed with the statement that there was limited information about HPV vaccination. Additionally, 21.7% (n = 69) strongly agreed and 23.9% (n = 76) agreed with the statement that the vaccine was expensive. Additionally, 10.1% (n = 32) and 22.3% (n = 71) strongly agreed and agreed, respectively, with the statement that fear of experiencing pain during injection was a barrier to seeking HPV vaccination. Fear of adverse effects of the vaccination was also cited, as 15.1% (n = 48) strongly agreed and 20.8% (n = 66) agreed to that assertion. However, 35.2% (n = 112) disagreed and 31.8% (n = 101) strongly disagreed with the assertion that anticipation of family disapproval was a barrier to HPV vaccination.In connection with the barriers to obtaining HPV vaccination, Table 2 = 23.746, p = 0.001), marital status , completed level of education , and duration of working at the hospital  in regard to acceptance of HPV vaccination. However, there was no statistically significant association between religion  and acceptance of HPV vaccination.Table The purpose of this study was to assess nurses’ and midwives’ knowledge of, attitudes towards, and acceptance of HPV vaccination in relation to their background characteristics. HPV vaccination is critical in the prevention of cervical cancer. Nurses and midwives play pivotal roles in health promotion and the prevention of diseases. Consequently, their level of knowledge and acceptance of HPV vaccination may have a direct impact on their own and their clients’ health promotion activities. The findings of this study indicate that less than half of the sample studied had a high level of knowledge about cervical cancer risk factors. A possible explanation could be that these health professionals may not have had a continuous professional development programme on cervical cancer prevention. It could also be assumed that their training curriculum did not adequately cover cervical cancer. The present finding is consistent with the findings of a cross-sectional study published in 2014; the study was conducted among midwives, students and patients in Serbia, and it found that their knowledge level was unsatisfactory . DespiteThe findings further showed that approximately half of the sample had at least one Pap smear. This is encouraging, as nurses and midwives who have had the test may be able to provide detailed information about it. Earlier studies conducted in Canada, Nigeria and Kenya affirmed the willingness of women to obtain a Pap smear, and the majority had done so –35. ThesDespite evidence that knowledge about cervical cancer could translate into prevention and screening , the accSeveral studies have reported low rates of HPV vaccination –40. In aFurthermore, although nurses and midwives are mandated by their codes of practice to perform their tasks without letting personal values interfere, it seems difficult if not impossible to detach some of these personal values from practice. Given the role of nurses and midwives in ensuring professionalism in dealing with clients to promote safe, effective and client-centred care outcomes , these pThe findings further suggest that some factors could potentially hinder nurses and midwives from participating in HPV vaccination, including limited information and lack of health education programmes to promote HPV vaccination. An earlier work cited knowledge gaps on the part of healthcare providers as a critical barrier in HPV vaccination, as it affects their ability to effectively engage in patient education . In GhanFurthermore, 45.6% of the participants viewed the cost of HPV vaccination as expensive. In Ghana, it costs GHC 250 the equivalent of approximately $50 (US) to obtain a dose of HPV vaccination, Cervarix, at the KBTH. Therefore, the three doses of Cervarix vaccine recommended will cost approximately $150 (US). The scope of the National Health Insurance Scheme does not cover HPV vaccination, which could discourage potential participants from obtaining the vaccine, especially if they cannot afford the out-of-pocket costs. Other empirical works have reported cost as a major barrier to accessing HPV vaccination , 44. EffFear of adverse effects of the vaccination was cited as a barrier by 35.9% of the nurses and midwives. It is worth mentioning that a previous study found nurses’ unwillingness to accept the vaccination to be associated with beliefs about the efficacy and safety of the vaccine . This hiAlso, some of the participants cited fear of experiencing pain during injections as a barrier to HPV vaccination. However, surprisingly, a survey conducted in North Carolina among adolescents showed that half of the sampled population complained of pain after receiving an injection, with 2% of the parents complaining their daughters experienced severe pain after the injection. The parents also reported that the pain their daughters experienced after receiving HPV vaccination was either comparable or less intense than the pain from other vaccines . This fiThe study also explored the sociodemographic factors associated with the acceptance of HPV vaccination. Age was found to be associated with HPV vaccination. Earlier works found young girls to have favourable attitudes towards HPV vaccination , 46. YouIn this study, level of education was statistically significantly associated with the acceptance of HPV vaccination. It is known that disparities exist in cervical cancer prevention strategies among the general population, especially across different levels of education . MaternaIn the same way, the duration of working in the hospital was associated with the acceptance of HPV vaccination. A possible explanation could be that those who had more work experience may have had more access to continuing education or professional development programmes on HPV vaccination, learned about HPV and cervical cancer on the job or nursed a patient with that condition. In a study conducted in the United Kingdom, experience and trust in doctors influenced vaccine acceptance .Generally, religion seems to have a great influence on decisions regarding appropriate health behaviour . For exaThe respondents were sampled by convenience, so there is the possibility of selection bias. Therefore, the results should be interpreted with caution. This sampling approach, however, was the best method, considering the nature of the study. Although nurses and midwives routinely give other vaccines, this study only focused on HPV vaccination. Therefore, the association between other vaccines and HPV vaccination was not assessed.This study demonstrated gaps in knowledge of cervical cancer risk factors and attitudes towards HPV vaccination, which requires targeted measures to improve knowledge and attitudes. Despite the low rates of acceptance of HPV vaccination among the sample studied, perceived threats of cervical cancer and recommendation of HPV vaccination by colleague nurses and midwives motivated some of the participants to receive the HPV vaccination. Moreover, the study identified critical barriers that hindered participants from seeking HPV vaccination, including fear of adverse effects of HPV vaccination, fear of experiencing pain during injections and cost of the vaccine. The provision of a comprehensive training programme on HPV vaccination and safety net intervention to cover the cost of the vaccine could reduce barriers and increase uptake. This study also highlighted the need for stakeholders to consider important sociodemographic factors in efforts to increase the acceptability of HPV vaccination.


Phalaenopsis represents an important cash crop worldwide. Abundant flower colors observed in Phalaenopsis orchids range from red-purple, purple, purple-violet, violet, and violet-blue. However, violet-blue orchids are less bred than are those of other colors. Anthocyanin, vacuolar pH and metal ions are three major factors influencing flower color. This study aimed to identify the factors causing the violet-blue color in Phalaenopsis flowers and to analyze whether delphinidin accumulation and blue pigmentation formation can be achieved by transient overexpression of heterologous F3’5’H in Phalaenopsis.Phalaenopsis flowers with red-purple, purple, purple-violet, and violet to violet-blue color, but no true-blue color and no delphinidin was detected. Concomitantly, the expression of PeF3’H  was high, but that of PhF3’5’H  was low or absent in various-colored Phalaenopsis flowers. Transient overexpression of DgF3’5’H (Delphinium grandiflorum) and PeMYB2 in a white Phalaenopsis cultivar resulted a 53.6% delphinidin accumulation and a novel blue color formation. In contrast, transient overexpression of both PhF3’5’H and PeMYB2 did not lead to delphinidin accumulation. Sequence analysis showed that the substrate recognition site 6 (SRS6) of PhF3’5’H was consistently different from DgF3’5’Hs at positions 5, 8 and 10. Prediction of molecular docking of the substrates showed a contrary binding direction of aromatic rings (B-ring) with the SRS6 domain of DgF3’5’H and PhF3’5’H. In addition, the pH values of violet-blue and purple Phalaenopsis flowers ranged from 5.33 to 5.54 and 4.77 to 5.04, respectively. Furthermore, the molar ratio of metal ions  to anthocyanin in violet-blue color Phalaenopsis was 190-, 49-, and 51-fold higher, respectively, than those in purple-color Phalaenopsis.Cyanidin-based anthocyanin was highly accumulated in Phalaenopsis and was concomitant with a high pH value and high molar ratio of Al3+, Ca2+ and Fe3+ to anthocyanin content. Enhanced expression of delphinidin is needed to produce true-blue Phalaenopsis.Cyanidin-based anthocyanin was detected in violet-blue color  Phalaenopsis hybrids are among the most popular orchids in flower markets because of their long-lived and spectacular flowers. One of the special agronomic traits for Phalaenopsis is their abundant flower color, including white, yellow and red-purple. For the red-purple series, the flower colors range from red-purple, purple, purple-violet, violet and violet-blue  without blue-, violet- or purple-flowered cultivars are due to the absence of the F3’5’H gene that encodes the key enzyme for delphinidin-based anthocyanin biosynthesis [Phalaenopsis flowers.Previous studies showed many commercial horticultural plants without blue hue flowers due to lack of F3’5’H. Molecular approaches have been used to create blue-color flowers in rose , 7 and cynthesis . The floynthesis . Thus, FInNHX1 and InNHX2 translate proteins responsible for transporting K+/Na+ into the vacuoles in morning glory, and produce weakly alkaline vacuoles at the flowering stage [+(K+)/H+ exchanger (NHX) drives a vacuolar pH increase during flower opening, causing a color shift from red to blue [ph 5 and ph 1, vacuolar acidification was reduced and caused purple-colored petunia [2+ and Fe3+ are essential for blue coloration in blue poppy flower [3+ and Al3+ may affect blue coloration in the blue cornflower Centaurea cyanus [Different levels of vacuolar pH could cause a color shift in flowers with the same anthocyanin compounds. Flowers with the same anthocyanin compounds can have a color change with diverse levels of vacuolar pH. For example, an increase in pH from 6.6 to 7.7 due to the polyacylation of peonidin glycoside cause blue coloration in the corolla of morning glory . In adding stage –13. Na+ to access whether delphinidin could accumulate. This study will set the groundwork for molecular breeding of Phalaenopsis cultivars with novel blue color pigmentation.Here, we aimed to study the mechanisms in the formation of violet-blue color in Phalaenopsis, anthocyanins were extracted and analyzed by using HPLC. Pure compounds of both cyanidin and delphinidin were used as standards  ‘S304’ and violet-blue P. Purple Martin, whereas delphinidin was highly detected in blue-color Delphinium grandiflorum    and PeMYB2 resulted in 53.6% delphinidin and 46.4% cyanidin accumulation  Fig. a. We premulation . Overexplor Fig. b. OverexThere are six substrate recognition sites (SRSs) near the heme group of F3’5’H; mutation of the individual amino acids at these sites might influence enzyme function –27.PhF3’5’H cannot produce delphinidin, we wondered about divergence in amino acids in its sequences. The deduced amino acid sequences of PhF3’5’H were analyzed. They contained three conserved regions including an AGTDTS cytochrome P450 conversed region, EXXR oxygen binding motif and FGAGRRICAG heme binding domain  residue at position 8 of SRS6, isolated from P. Purple Martin and P. hybrid ‘King Car’  residue at position 8 of SRS6, isolated from P. aphrodite, P. equestris, P. bellina, P. schilleriana, P. lueddemanniana, P. Purple Martin and P. violacea ‘Indigo Blue’  at position 8 of SRS6  on PhF3’5’H and DgF3’5’H were predicted by using SwissDock. Of the approximately 30 different conformations obtained in the substrate docking prediction with N, DHQ and E, the lowest energy conformation was selected as the most probable binding model. Prediction results showed that the aromatic rings (B-ring) of N, DHQ and E were oriented toward the N-terminal of SRS4 and SRS1 of PhF3’5’H Fig. d-f. In c+ concentrations among various-colored Phalaenopsis, the pH value was measured from flower extracts  ‘KS1226’, had pH values of 5.52 and 5.54, respectively .To confirm whether anthocyanins under various levels of acidification will present different colors, anthocyanins were extracted from purple-violet ili Fig. a and addili Fig. b. The anili Fig. b. In theili Fig. c. In addili Fig. d. This rues Fig. d. In facPhalaenopsis cultivars. Five metal ions including Mg2+, Al3+, Ca2+, Fe3+ and Zn2+ were analyzed in the purple color cultivar P. OX Honey ‘OX1372’, violet-blue color cultivar P. Kenneth Schubert, and dark-purple colored P. OX Firebird ‘OX1506 mutant’ by using ICP-MS , whereas that between the molar ratios of Mg2+ and Zn2+ to cyanidin and blue color formation was low .The molar ratios of AlPhalaenopsis orchids, cyanidin-based anthocyanin was highly accumulated in flowers from red-purple to violet-blue. However, violet-blue Phalaenopsis cultivars have higher pH in flower sap than do purple cultivars.Vacuolar pH is higher in blue flowers than red flowers in petunia, morning glory and hydrangea, but blue and red flowers have the same anthocyanin compound , 14, 28.3+ to delphinidin-based anthocyanin 39-fold higher in blue than red cells [3+ to delphinidin-based anthocyanin was 40-fold higher in blue than red flowers [3+ to cyanidin-based anthocyanin was 51-fold higher in blue than purple flowers [3+ is necessary to form the blue color in cyanidin-based flowers including cornflower and blue poppy [2+ is essential for producing metalloanthocyanin in several plants, including Commelina communis, Centaurea cyanus, Salvia patens, S. uliginosa and Nemophila menziesli [3+, Ca2+ and Fe3+ metal elements to cyanidin-based anthocyanin was 190-, 49-, and 51-fold higher, respectively, in violet-blue than purple Phalaenopsis.Previous studies showed that blue-red bicolor tulips had a molar ratio of Feed cells . In blue flowers . In blue flowers . Fe3+ isue poppy , 32, 33.enziesli . Our resThe large size of amino acids results in eliminating F3’5’H function. Substitution of Ser to Thr at position 8 of SRS6 can decrease 5′-hydroxylation activity . SimilarArabidopsis thaliana [In addition, results of substrate docking showed that the aromatic rings (B-ring) of N, DHQ and E were oriented toward the N-terminal of SRS4 and SRS1 of PhF3’5’H Fig. , whereasthaliana . PhF3’5’F3’H must be silenced for high delphinidin accumulation to lead to a blue hue because both F3’H and F3’5’H compete for the same substrates of N and DHK [F3’5’H-induced plants with and without endogenous F3’H silenced [F3’5’H and PhDFR, 94 and 100% delphinidin accumulation resulted in purple 82A and mauve 75A groups, respectively, in carnation [F3’5’H and PhDFR resulted in a 95% delphinidin accumulation and the violet group 85B [PeMYB2 and DgF3’5’H in the V3 tepal caused the highest delphinidin accumulation (53.6%), and the flower color shifted from white (NN155C) to violet-blue (91A). Even though relatively lower delphinidin accumulation was observed with overexpression of DgF3’5’H in Phalaenopsis, higher delphinidin accumulation and a bluer violet-blue resulted as compared with other overexpressed plants. This is the first report to show a novel blue hue created in white Phalaenopsis cultivars. Our findings suggest that with the addition of PeMYB2, DgF3’5’H was able to compete the substrates of N and DHK with endogenous F3’H in Phalaenopsis to accumulate the delphinidin compound. Silencing endogenous F3’H accompanied by DgF3’5’H-induced transgenic Phalaenopsis is required to produce high delphinidin accumulation and a true-blue color formation in Phalaenopsis.Endogenous  and DHK . For trasilenced . Previousilenced . In addiarnation , 7. In rF3’5’H and also PhDFR were introduced into host plants of rose and carnation. PhDFR strongly utilizes dihydromyricetin (DHM) but not DHK, thereby benefiting delphinidin production [F3’5’H was introduced, and endogenous F3’H was silenced. Knockdown of F3’H can increase delphinidin production [Different strategies of genetic regulation in the anthocyanin biosynthesis pathway have been used . Heteroloduction . In contoduction . Of noteoduction . These cMYB transcription factor, PeMYB2, and heterologous F3’5’H (DgF3’5’H) while knocking down the expression of endogenous F3’H. This can set the ground work for molecular breeding of Phalaenopsis cultivars with real blue pigment.In conclusion, we proved the initial assumption framework of how white-color orchids are converted to blue hue by overexpressing an anthocyanin regulating Phalaenopsis was caused by both reduced vacuolar acidification and enhanced molar ratio between Ca2+ and Fe3+ to cyanidin-based anthocyanin. Delphinidin did not accumulate in any colors of Phalaenopsis cultivars because of the altered amino acids and possibly adverse substrate binding direction, PhF3’5’H has little to no ability for delphinidin accumulation. On overexpressing both DgF3’5’H and PeMYB2, we observed a novel violet-blue color in the white Phalaenopsis cultivar, with 53.6% delphinidin and 46.3% cyanidin accumulation. This study will benefit the understanding of the regulatory mechanism of violet-blue color formation and set the groundwork for molecular breeding of Phalaenopsis cultivars with novel blue color pigmentation.The violet-blue color formation in Phalaenopsis cultivars with various colors including red-purple, purple, purple-violet, violet, violet-blue and white were collected from various local orchid nurseries  . Purple P. hybrid ‘King Car’ was collected from King Car Biotechnology Industrial Co. . Purple-violet P. Big Chili, white P. aphrodite subsp. formosana, and white P. Sogo Yukidian ‘V3’ were collected from Taiwan Sugar Corp. . Violet P. violacea ‘Indigo Blue’ was collected from Meidarland Orchids . Violet P. (Germaine Vincent x Samera ‘indigo’) ‘S304’ was collected from Keng-Liang Liu Orchid Nursery . Violet P. (Kenneth Schubert x Samera) ‘KS1226’ and Violet-blue P. Purple Martin were collected from Kung Sir Orchids . Violet-blue P. tau Chiang Sapphire was collected from Sunhope Garden Biotec . Violet-blue P. Kenneth Schubert was collected from Taida Horticultural Co. . The white P. Sogo Yukidian ‘V3’ and purple P. OX Honey ‘OX1372’ were used for transient overexpression analysis because their colors of tepals were beneficial for delphinidin accumulation and blue color. All plant materials were collected according to institutional guidelines and grown in the greenhouse at Nation Cheng Kung University  under natural light and at 23–27 °C. All plant materials used in this study including the Phalaenopsis cultivars and Delphinium grandiflorum are commercially available, not collected from the wild. All plant materials were kept in permissible green house at NCKU. Experimental research on plants comply with institutional, national, or international guidelines. The authenticity of these materials has been verified by each orchid nursery owners.For color analysis, Phalaenopsis  ‘S304’ and P. (Kenneth Schubert x Samera) ‘KS1226’ are classified in the violet 86A, N87A and N87B groups, respectively , a housekeeping gene, was used for normalization of qRT-PCR results [For each gene, primer pairs were designed within the gene-specific regions , with the following accession numbers: Campanula medium F3’5’H (D14590), Catharanthus roseus F3’5’H (AJ011862), Eustoma grandiflorum F3’5’H (D14589), Petunia hybrida F3’5’H (Z22544), Delphinium grandiflorum F3’5’H (AY856345), Vitis amurensis F3’5’H (FJ645756.1), Petunia hybrida F3’H (AF155332), Torenia hybrid cultivar F3’H (AB057673), Chrysanthemum morifolium F3’H (AB523844.1), Glycine max F3’H (AB191404), Narcissus tazetta F3’H (JX292106.1), Arachis hypogaea F3’H (JN572892.1), Antirrhinum majus FNSII (AB028151), Callistephus chinensis FNSII (AF188612), Arabidopsis thaliana AtAHA2 (AEE85731.1), Arabidopsis thaliana AtAHA4 (Q9SU58.2), Nicotiana plumbaginifolia NPPMA1 (AAA34094), Petunia x hybrida PhPH5 (DQ334807), Nicotiana plumbaginifolia NpPMA9 (AF156684), Populus trichocarpa PtAHA10-like (XP_002326625.1), Vitis vinifera VvAHA10-like (CBI35782), Oryza sativa OsAHA9 (AJ440220), Arabidopsis thaliana AtAHA10 (NP173169), Citrobacter sp. CsMgtA (ZP04559661), Escherichia coli EcMgtA (YP672334), Klebsiella pneumoniae KpMgtA (YP002917472), Petunia x hybrida PhPH1 (AHH24342.1), Vitis vinifera VvPH1-like (CBI41039), Ricinus communis RcPH1-like (XP002533565), Populus trichocarpa PtPH1like (XP002306511), Arabidopsis thaliana AtACA2 (NP195497) and Oryza sativa Os03g0616400 (NP001050661).Sequence alignment involved using Align X . Phylogenetic analysis trees were constructed with the neighbor-joining method and evaluated by bootstrap analysis with MEGA5 , with 10https://swissmodel.expasy.org/). The X-ray crystal of P450 1A2 (PDB ID: 2hi4.1) was used as the template [http://www.swissdock.ch/docking). Finally, UCSF Chimera was used to visualize the predicted binding model [The prediction of F3’5’H tertiary protein structures involved using SWISS-MODELL  35S promoter was used following Hsu et al., (2015) [XhoI, and then ligated to p1304NhXb. The recombinant constructs were bombarded into Agrobacterium tumefaciens EHA105 using electroproration. The engineered A. tumefaciens was grown at 28 °C overnight, refreshed culture to OD600 = 0.8–1, and then harvested by centrifugation. Cell pellets were resuspended in Murashige and Skoog medium supplied with 100 μM acetosyringone, and incubated at room temperature for 30 min. The bacterial suspensions were infiltrated into the base of sepals and petals of V3 flowers, and incubated at 25 °C with a 10 h light/14 h dark photoperiod. Five days post bombardment, the infiltrated flowers were photographed and then stored at − 80 °C for anthocyanin extraction. The transient overexpression assay was performed in five different plants for each experiment, and repeated three times independently.For transient overexpression of , (2015) . Briefly2HPO4) and 0.1 M citric acid (C6H8O7) at various pH values and measured by spectrophotometric analysis from OD 300–800 nm .The complete petals of flowers were directly ground, centrifuged at 13,000 rpm  for 15 min, then supernatant was immediately measured by using a pH electrode . In addition, the flower sap was diluted in 1 ml buffer solution containing 0.2 M sodium phosphate dibasic  at 400 watts and increased to 180 °C within 15 min and sustained for 15 min. After digestion, samples were diluted with 2% HNO3 for metal ion measurement by using ICP-MS analysis . High-performance microwave and ICP-MS were both performed at the Institute of Biomedical Engineering and Nanomedicine of National Health Research Institutes .Powders of colorful petals were dried by using a freeze dryer , then ground by using a homogenizer . Next, 100 mg of the dried sample was digested in 5 ml of 70% HNOThe correlation between the concentration of metal ion and violet blue color formation and pH value and violet blue color formation were analyzed statistically with Pearson’s correlation coefficient .Additional file 1: Flowers used in this study, and their definition of color code. Flower color of Phalaenopsis and Delphinium hybrid were assigned according to the Royal Horticultural Society Color Chart.Additional file 2: Flower color of various flowering development stages of violet-blue P. Kenneth Schubert and P. Purple Martin.(A) P. Kenneth Schubert in various flowering development stages range from flower opening (D) and after flower opening 5 days (D + 5), 10 days (D + 10), 15 days (D + 15), and 20 days (D + 20). (B) Flowers of P. Kenneth Schubert and, (C) the whole plant of P. Purple Martin. Scale bar is 2.5 cm.Additional file 3: Primers used in this study. List of primers for qRT PCR, gene isolation, 5′ and 3′ RACE.