Although penile cancer (PC) is uncommon in developed countries, it is widespread in developing countries. The state of Maranhão  has the highest global incidence recorded for PC, and, despite its socioeconomic vulnerability, it has been attributed to human papillomavirus (HPV) infection. This study aimed to determine the histopathological features, the prevalence of HPV infection, and the immunohistochemical profile of PC in Maranhão.INK4a, p53, and ki-67.A retrospective cohort of 200 PC cases were evaluated. HPV detection was performed using nested-PCR followed by direct sequencing for genotyping. Immunohistochemistry (IHC) was performed using monoclonal antibodies anti-p16p = 0.001). IHC revealed frequent p16INK4a overexpression (26.0%) associated with basaloid (p < 0.001) and high-grade tumors (p = 0.008). Interestingly, p16 appears not to be a better prognostic factor in our disease-free survival analysis, as previously reported. We also demonstrated high ki-67 and p53 expression in a subset of cases, which was related to worse prognostic factors such as high-grade tumors, angiolymphatic and perineural invasion, and lymph node metastasis. We found a significant impact of high ki-67  and p53  expression on decreasing patients’ survival, as well as grade, pT, stage, pattern, and depth of invasion .Our data revealed a delay of 17 months in diagnosis, a high rate of penile amputation (96.5%), and HPV infection (80.5%) in patients from Maranhão (Molecular detection). We demonstrated the high rate of HPV in PC also by histopathological and IHC analysis. Most patients presented koilocytosis (75.5%), which was associated with those reporting more than 10 different sexual partners during their lifetime (Our data reaffirmed the high incidence of HPV infection in PC cases from Maranhão and offer new insights into potential factors that may contribute to the high PC incidence in the region. We highlighted the possible association of HPV with worse clinical prognosis factors, differently from what was observed in other regions. Furthermore, our IHC analysis reinforces p16, ki-67, and p53 expression as important diagnosis and/or prognosis biomarkers, potentially used in the clinical setting in emerging countries such as Brazil.The online version contains supplementary material available at 10.1186/s12885-022-10127-z. Penile cancer (PC) is an uncommon neoplasm in developed countries. However, it has a high incidence in developing countries, mainly in Asia, Africa, and South America , 2. WhenNevertheless, some risk factors have been proposed for PCs. The disease appears to be more prevalent in older men living in rural zones and those with low education levels . PC has According to data from the International Agency for Research on Cancer (IARC), the age-standardized rate (ASR) of PC in the United States is 0.5/100.000 men [In contrast, the highest incidence was reported in Uganda, Africa . According to the IARC, Brazil is ranked thirteenth worldwide for incidence of PC, with a rate of 1.6/100.000 men [Since the prevalence of PC appears to vary considerably between certain regions of Brazil, local studies are essential to better understand the reality of PC in the country. These studies reveal the North and Northeast of Brazil as the regions with the highest incidence of the disease, where according to Korkes et al. 2020, between 2006 and 2018 the incidence of PC in these regions was 5.4 per 100,000 men [In northeastern Brazil, the state of Maranhão has attracted the attention of the scientific community owing to its high prevalence of PC . In 2018Our research group and others have aimed to better understand PC features in Maranhão. Over the past five years, these studies have drawn attention to a clinical, epidemiological, and etiological profile distinct from that observed in other studies carried out in Brazil and worldwide, with marked heterogeneity of histological subtypes and high prevalence of HPV infection , 9–13.Therefore, the present study proposes a panoramic analysis of PC in Maranhão, describing the clinical, histopathological, HPV status, and immunohistochemical (IHC) features for diagnostic and/or prognostic importance markers.This was a cross-sectional, experimental, and retrospective study. A survey of cases diagnosed with PC between 2006 and 2020 was carried out in two reference hospitals  and Hospital do Câncer Aldenora Bello (HCAB)), both located in São Luís, Maranhão, Brazil. Only patients 18 years of age or older with clinical and histopathological PC diagnosis who underwent surgery, and who had tissues available for histological review were included in the study. Patients with clinical and histopathological diagnoses of PC performed under other services and with materials in inadequate conditions for analysis were excluded.A total of 236 cases of PC (2006–2020) were identified; however, only 200 cases met the inclusion criteria. The study involved a review of clinical and histopathological findings, HPV detection and genotyping, and protein expression profiling by IHC. All clinical data described were collected from medical records. Histopathological and IHC analyzes were performed at the Laboratory of Immunofluorescence and Electronic Microscopy (LIME/HU-UFMA), and molecular assays were performed at the Clinical Research Center (CEPEC) of the HU-UFMA, São Luís, Maranhão, Brazil, and in the Nucleus of Research in Oncology (NPO), at the Federal University of Pará (UFPA), Belém, Pará, Brazil.Both for histopathological and molecular analysis we used formalin-fixed paraffin-embedded (FFPE) tumor tissue, which was stored under appropriate conditions in the archives of the pathology service of each hospital included in the study.n = 200) were subjected to hematoxylin and eosin (H&E) staining to confirm the histological diagnosis and classification of tumors according to the criteria of the classification of malignant tumors (TNM) in the American Joint Committee on Cancer (AJCC), 8th edition. All tumors were independently evaluated by three pathologists . In cases of disagreement, the criteria were reviewed a second time.Specimens  tissue, three 10 µm cuts of each paraffin-embedded tissue were made in a histological microtome and transferred to a 2.0 ml microtube. Then, 1.0 µL of xylol was added to the tube, vortexed, and incubated in a thermoblock for 5 min at 50 °C to remove excess paraffin. The samples were centrifuged at 14,000 rpm for 2 min, and the supernatant was discarded. The xylol-washing process was repeated twice. Then 1.0 mL of absolute ethanol was added to each sample, centrifuged again at 14,000 rpm for 2 min, the supernatant was discarded, and the ethanol wash was repeated two more times. Samples were then incubated at 37ºC for 40 min to dry all alcohol in the sample. DNA isolation was done with the QIAamp DNA FFPE Tissue Kit , according to the manufacturer’s instructions. Each sample was diluted in 50 µL of RNase-free TE, pH 8.0, and quantified using a NanoDropOne® spectrophotometer , with concentrations expressed in ng/µL, and purity analysis with measures 260/280 (between 1.8 and 2.0) and 260/230 (above one). DNA samples were immediately used for molecular detection of HPV or stored at -20 °C.DNA extraction resulted in only 113 samples within the expected quality standards, so HPV detection was performed only in these cases. Each sample was subjected to PCR (Polymerase Chain Reaction) for the β-globin endogenous gene as second quality control. All samples were β-globin positive. HPV detection was performed by nested-PCR. In the first PCR, a set of generic primers called PGMY09/11 was used, which produced a 450 bp amplicon of the L1-HPV capsid region . In the http://blast.ncbi.nlm.nih.gov/Blast.cgi).For HPV genotyping, the second PCR amplicons of HPV-positive cases were sequenced in a single direction using the GP5 + primer. Automatic sequencing was performed on the ABI PRISM 3500XL Genetic Analyzer, using the BigDye™ Terminator v3.1 Cycle Sequencing kit (Applied Biosystems™). The sequences obtained were uploaded to the MEGA 6.0 and compared with those from the GeneBank/NCBI database using the Basic Local Alignment Search Tool (BLAST) for genotyping (n = 27) were excluded.IHC analysis was performed on 3 µm-thick sections from FFPE tumor tissue from 173 patients, using the EnVision FLEX Mini Kit High pH System . Antigen retrieval was performed using PT Link equipment (Agilent Technologies) following the manufacturer's instructions. Slides were next washed in Dako Wash Buffer, blocking of endogenous peroxidase was done for 5 min, primary monoclonal antibody for each evaluated marker was applied for 60 min, polymer application (20 min), and staining in chromogen 3,3’ diaminobenzidine (DAB) for 10 min, counterstaining with Harris hematoxylin (5 min), and analysis under an optical microscope. Table INK4a, the Cubilla et al. (2011) criteria were used, which proposed the following categories: (0) complete absence of p16INK4a staining in all tumor cells; (1) spotty, patchy, and discontinuous staining in some of the tumor cells; (2) more extensive but discontinuous staining pattern with small clusters of positive tumor cells; (3) cytoplasmic or nuclear staining in all tumor cells, except in hyperkeratotic or parakeratotic areas when present [INK4a.Immunostaining was measured semi-quantitatively by two pathologists independently (GEBS and SPCM), and when in disagreement, the slides were reviewed together for consensus. For p16 present . Only caFor p53, cases with < 20% of tumor cells stained were considered negative, 20-50% were classified as positive ( +), and > 50% of tumor cells were positive (+ +) . For ki-For DFS analysis, only cases with clear information in the medical record  were included. Only 134 cases met the inclusion criteria. We defined the time of diagnosis as the beginning of follow-up and noted the events of recurrence and/or lymph node metastasis during the analysis period. We considered recurrence when the tumor returned after the first surgical treatment and a period during which it could not be detected.p < 0.05 were considered significant.Data were tabulated using Excel (Microsoft Office 2019) and statistically analyzed using the SPSS® software v. 22 . The association analysis between clinical, histopathological, immunohistochemical, and HPV infection variables was performed using bivariate tests (chi-square test or Fisher's exact test). The Kaplan–Meier method was used to estimate the DFS, and the log-rank test was applied to compare the survival curves. For all analyses, results with In this study, we evaluated 200 patients with clinical and pathological diagnoses of PC treated in the state of Maranhão, Brazil, between 2006 and 2020. Of these, 40 (20.0%) were admitted to the HU-UFMA and 160 (80.0%) were admitted to the HCAB. Figure There was a high frequency of patients with risky sexual behavior, with 54.1% reporting more than 10 different sexual partners during their lifetime (mostly having sex without condoms), and 58.7% practicing zoophilia . Among those who reported a history of sexually transmitted infections (STIs), gonorrhea was the most frequent (67.6%). Only one patient tested positive for the human immunodeficiency virus (HIV). Among those who reported phimosis, 43.0% underwent circumcision in adulthood, usually preceded by surgical treatment of the tumor.Surgical treatment was performed in all cases, mainly partial penis amputation (72.5%). The main affected topography was the glans (91.9%), either individually (44.4%), or simultaneously with the foreskin and/or coronal sulcus (27.3%) or penile body (20.2%). The tumor size ranged from 0.6 cm to > 10.0 cm, the majority being between 2.0 and 5.0 cm (61.9%). Tumors were unifocal in 89.9% of cases. An ulcerated aspect occurred in 30.8%, or ulcer-vegetating in 25.8%, and with an expansive pattern of growth in 75.3%.In H&E analysis, all tumors were squamous cell carcinomas (SCC), poorly differentiated in 49.0% of cases, and mainly of the usual (39.5%) and warty (29.0%) subtypes. The presence of carcinoma in situ associated with invasive lesion was observed in 82.0% of cases, and lichen sclerosus in 27.3%. Angiolymphatic and perineural involvement was present in 35.5% and 36.5% of cases, respectively, and sarcomatoid dedifferentiation in 17.1% of cases.We also evaluated the thickness and depth of tumor invasion. We considered “thickness” as the measure between the largest point on the tumor surface and the deepest point of the infiltration front and as “depth” the distance between the basement membrane and the deepest point of infiltration. Slight majorities had a thickness > 5.0 cm (61.0%) and depth < 5.0 cm 52.4%). According to the TNM classification, these tumors were mainly in stage pT3 (48.0%), pNx (92.5%), and pMx (100.0%). Pathological stage IIb at diagnosis was seen in 44.0% of cases. The complete characterization of the histopathological parameters is presented in Table .4%. AccoThe main complaints reported were the appearance of a nodule/tumor in the penis (46.5%); ulcerated wound (21.0%), secretion (12.3%), nodule with presence of secretion (7.9%), dysuria and/or oliguria (7.0%), and other symptoms, such as a burning sensation, discomfort in the penile region, or itching (5.3%). These patients took an average of 17 months to seek medical help from the onset of symptoms  and usually with locally advanced tumors .p = 0.001), warty tumors (p < 0.0001), HPV-associated histological subtypes (p < 0.0001), and p53 negativity (p = 0.007).Additionally, we identified histological alterations suggestive of HPV infection (koilocytosis) in 78.5% of cases Fig. . The prePhysical examination of palpable lymph nodes was performed in 102 of the 200 cases evaluated . Of these, 61 (59.8%) had palpable lymph nodes unilaterally or bilaterally, 47 (46.1%) at admission, and 14 13.7%) after tumor amputation. 74 cases underwent inguinal lymphadenectomy, which was performed unilaterally in 32 cases (43.2%) and bilaterally in 42 cases (56.8%). The presence of lymph node metastasis was confirmed in the histopathological analysis in 47 cases (63.5%), and 13 (27.6%) were simultaneous with primary tumor amputation/diagnosis. Extracapsular involvement was observed in 37 (78.7%) of the 74 patients with lymph node metastasis. The lymph node involvement data are presented in Table .7% afterIn Fig. p = 0.042) and the presence of carcinoma in situ associated (p = 0.029), while the absence of HPV was associated with tumors in advanced stages (pT3-pT4) (p = 0.042).HPV positivity was associated with tumors located in the glans  overexpressed p16INK4a and 74.0% (128/173) were negative. The p16INK4a overexpression  were associated with basaloid (p < 0.0001), poorly differentiated (p = 0.008), and high proliferative tumors . Furthermore, the absence of p16INK4a overexpression was associated with tumors without angiolymphatic invasion (p = 0.031). The full description of the statistical analyses for koilocytosis, molecular detection of HPV, and p16INK4a expression is described in the supplementary material , presence of carcinoma in situ (p = 0.0.41), HPV-associated histology (p < 0.0001), absence of perineural invasion (p = 0.036), koilocytosis (p = 0.002), and tumors with an expansive growth pattern (p = 0.002). In contrast, p53 positivity was associated with poorly differentiated tumors (p < 0.0001), pT3-pT4 tumors (p = 0.031), stage II disease (p = 0.020), and lymph node metastasis (p = 0.046).For p53, 55.0% of the cases were negative (95/173), and 45.0% were positive. Among the positive cases, 56.4% were ( +) (44/78) and 43.6% were (+ +) (34/78). The absence of p53 expression was associated with warty tumors (p < 0.0001), poorly differentiated tumors (p < 0.0001), presence of angiolymphatic , and perineural invasion , pT3-pT4 tumors , stage III-IV disease , and lymph node metastasis .For ki-67, 26.6% of the cases had high expression (46/173), and 73.4% had low expression (127/173). When analyzing only the hotspot zones, 57.8% of tumors showed high expression (100/173), and 42.2% exhibited low ki-67 expression (73/173). The high expression of ki-67, either global or in hotspot, was associated with basaloid tumors (p = 0.017), with an absence of sarcomatoid transformation , an expansive invasion pattern , small tumors , negativity for p53 , and with no p16INK4a overexpression .In contrast, low ki-67 expression was associated with warty tumors  of the 200 cases evaluated. Approximately 66 patients (33.0%) were excluded because they did not have enough information in their medical records for follow-up analysis or were lost to follow-up. The follow-up time ranged from 1.0 to 114 months (mean 20.96 + 21.32). By the end, 53 cases (53/134) had local recurrence and/or lymph node metastasis (39.5%), and 81 cases (81/134) had no evidence of the disease after surgical treatment (60.5%).INK4a), and p53 and ki-67 protein expression. In Fig. p = 0.056) and age on the DFS of these patients (log-rank p = 0.106), however, we did not observe a statistically significant difference. In Fig. INK4a, p53, and ki67 , where we observed a statistically significant difference only with p53 (log-rank p = 0.032) and ki-67 expression .We performed the Kaplan–Meier test to estimate the DFS and the log-rank test to compare the survival curves according to clinical, histopathological, HPV infection parameters . The southern region seems to account for the lowest percentage of cases and this may be associated with the evasion of these patients to cities in neighboring states, such as Piauí , or to the southern region of Brazil (looking for better therapeutics approaches). The large migration of PC patients from poor regions to underdeveloped areas is commonly reported in Brazil , suggestSocioeconomic vulnerability seems to be one of the main risk factors for PC . In the Exploring the profile of these patients, we observed that most of them reported poor genital hygiene habits, an important and known risk factor for PC . Proper In addition to the level of education, phimosis also seems to contribute to inadequate cleaning of the penis due to the difficulty or inability in exposing the glans. This condition was described in 70.5% of the cases evaluated in our study and is frequently reported in PC patients. In general, the prevalence of phimosis in PC is approximately 25% to 75%,  while itFurthermore, we observed a high frequency of risky sexual behavior (54.1%), which can significantly contribute to the acquisition of sexually transmitted infections, such as HPV , 37. A hUpon evaluating the primary symptoms of these patients, we found that the medical records mainly described the appearance of a nodule/tumor in the penis (46.5%) or a foul-smelling ulcerative wound (21.0%). These symptoms are consistent with the main manifestations of the disease, which include changes in skin thickness or color, a wound not having healed in four weeks, rash, bleeding from the penis or under the foreskin, and a smelly odor . Vieira As the penis is an external organ, the PC symptoms are classic and easily recognizable. However, we noted an average delay of 17 months between the onset of symptoms and seeking medical care, similar to what was previously described in Maranhão by Vieira et al. (2020), who described a delay of 18.9 months . These fIn this context, access to basic health, especially in rural areas, is scarce, making it difficult to carry out follow-up and early diagnosis of several diseases, including PC. All these factors contribute to an advanced stage at diagnosis, a high rate of metastasis, limitations in the therapeutic approach, and a low overall survival rate. Population-based analyses from Europe and the United States have shown that, unlike other types of tumors, tumor-specific survival rates for PC have not shown any improvement since 1990 , 44.Our findings reinforce a recurrent social-economic profile of patients with PC , 45, 46 The high prevalence of HPV-positive tumors reported in the last five years has been one of the most notable findings in Maranhão. The first study on the detection and genotyping of HPV in local cases was conducted by De Sousa et al. (2015)  and MartAll these studies carried out in the region showed a frequency of HPV-positive tumors above the global average of approximately 46.9—50.8% –51, and In DFS analysis, we did not observe any difference in survival curves according to the HPV status. In contrast, in a cohort from Maranhão, Martins et al. (2018) found better DFS in HPV-positive PC patients than in HPV-negative PC patients ; this reHistopathological analysis revealed morphological alterations suggestive of HPV infection (koilocytosis) in 78.5% of the evaluated cases. This corroborates previous findings in Maranhão, where koilocytosis was observed in 74.5% of the PC analyzed . The preKoifman et al. (2011) observed koilocytosis in 88.4% of PC cases evaluated, with high specificity (90%), but low sensitivity in detecting the virus . De-PaulThe presence of koilocytosis is well accepted as pathognomonic evidence for possible the presence of HPV, and has been widely used in screening for cases of viral infection . HoweverTo better understand aspects related to HPV infection, we searched the literature for data on the global prevalence of the virus in the general population. In Brazil, the prevalence of HPV in individuals without clinical symptoms appears to vary from 30 to 75%, depending on the region studied . InteresThe high prevalence of healthy individuals infected with HPV in Maranhão has been attributed to factors such as early initiation of sexual activity  and the high number of sexual partners . In the INK4a overexpression, only one case was not positive for any of these three parameters. Interestingly, this was the first case report of pseudoangiosarcomatous squamous cell carcinoma on the penis, an uncommon and highly aggressive variant of squamous cell carcinoma [Knowledge of the regional prevalence of HPV infection is important for understanding the local etiology of PC, as the incidence of HPV-associated tumors seems to be proportional to the prevalence of viral infection in healthy individuals . In our arcinoma .INK4a protein expression analysis, which is widely used as an indirect marker of HR-HPV infection and has been associated with basaloid and high-grade tumors [INK4a overexpression does not exclude infection by LR-HPV or even the absence of HPV infection [In addition to koilocyte and HPV molecular detection, we performed p16e tumors , 68, 69.nfection , 70.INK4a overexpression in 26.0% of PC cases. In the present study, all basaloid tumors showed p16INK4a overexpression, reinforcing its secondary use as a tool in the differential diagnosis of this histological subtype [INK4a overexpression was associated with the absence of angiolymphatic invasion.We found p16 subtype , 17, 70.INK4a overexpression is closely correlated with the presence of the HR-HPV genotype in PC, as observed in cervical carcinoma and other HPV-related tumors [INK4a overexpression and HR-HPV infection was observed. We suggest that limitations in HPV detection methods may explain these findings, such as: (1) we used DNA samples obtained from FFPE tissue, unlike Martins et al. (2018), who used fresh tumor samples; and (2) viral genotyping was not done for all positive cases.According to Cubilla et al. (2011), p16d tumors , 70. HowINK4a overexpression was low compared to that described by Martins et al. (2018) in Maranhão, where 40% of the cases had overexpression of p16INK4a [INK4a might underlie this difference. Martins et al. (2018) described four basaloid tumors in a total of 55 cases, while here we had eight basaloid tumors in approximately 173 cases.Despite the high frequency of cases with koilocytes and HPV-positivity, the frequency of p16p16INK4a . We specINK4a expression. However, patients with p16INK4aoverexpression lived on average less (30.1 months) when compared to patients without p16INK4a overexpression (46.8 months). These findings do not corroborate with previous studies where PC men with HPV or p16INK4a overexpression generally have a significantly more favorable DFS compared to those without HPV or p16INK4a overexpression [INK4a overexpression was significantly associated with and was an independent factor for better DFS in PC patients  [We also did not observe a statistically significant difference in DFS with the level of p16pression . Further< 0.001) .INK4a overexpression according to the following criteria: (1) basaloid tumors were excluded; and only (2) HR-HPV positive cases; (3) usual subtype tumors; (4) warty subtype tumors; and (5) stage I-II and III-IV tumors. However, in all analyses, there was no significant difference in DFS, and the mean survival time of the p16INK4a overexpressing group remained lower than that of no overexpressed p16INK4a.Given these controversial data, we performed DFS analysis for p16INK4a expression, making it difficult to compare their findings in Maranhão with those observed in the present study [INK4a overexpression (log-rank p = 0.753) or molecular HPV positivity (log-rank p = 0.979) [INK4a overexpression in PC patients from these Brazilian regions.Unfortunately, Martins et al. (2018) did not describe survival data based on p16nt study . However= 0.979) . Accordi= 0.979) . Our datFor histopathological classification, all the evaluated tumors were classified as squamous cell carcinoma (SCC), the most common type of PC . AccordiIn the present study, most cases were the usual subtype (39.5%), followed by the warty (29.8%). These frequencies differ from other studies in the area, such as that carried out by Cubilla et al. (2001), where out of 61 tumors evaluated, 59% corresponded to the usual subtype and only 10% corresponded to warty tumors . In 2010In 2018, Martins et al. observed that 47.3% of the PC cases evaluated in Maranhão were usual and 29.1% were warty subtypes , all mixThe high frequency of warty tumors in the present study and in others carried out in Maranhão, as well as the prevalence of mixed tumors with HPV subtypes associated, differs drastically from that observed in other studies, and supports the high prevalence of HPV-associated PC in the region , 77.When we evaluated the p53 expression profile, negative cases were more prevalent (55.0%) than positive cases (45.0%). The statistical analysis revealed that p53 positivity was associated with worse prognostic factors such as high-grade tumors (G3), pT3-pT4, those in stage II, and those with lymph node metastasis, corroborating previous studies with PC and other tumor types –81.Previously, all poorly differentiated PCs analyzed by Marinescu et al. (2016) were p53 positive , and LopAccording to the DGIdb database, the TP53 gene is the target of 193 therapeutic drugs , many ofIn a study conducted by Jacob et al. (2019), most patients with metastatic PC had mutations in TP53 and were HPV-negative  This sugIn contrast to what we observed for p53 positivity, the absence of p53 expression was associated with warty tumors, presence of carcinoma in situ, tumors with HPV-associated histology, absence of perineural invasion, koilocytosis, and expansive growth pattern. These findings corroborate the main pathway for HPV action, where the viral oncoprotein E6 binds to the p53 protein and inhibits its tumor suppressor function . In addiMost tumors analyzed in the present study had a low global expression of ki-67 (73.4%), but high expression in hotspot zones (57.8%). Here, we observed an association between high ki-67 expression and basaloid and high-grade tumors, presence of angiolymphatic and perineural invasion, pT3-pT4 tumors, stage III-IV, and lymph node metastasis. The association between high expression of ki-67 and worse prognostic factors has been previously described in PC , 88, 89.Ki-67 analysis has been used as a good parameter to assess the proliferative index and aggressiveness of numerous tumors , 91. In p < 0.0005), but they did not observe a relationship with advanced staging or lymph node metastasis, unlike our findings [A strong association between high ki-67 expression and poorly differentiated tumors has been described previously in PC . Berdjisfindings .The association between high ki-67 expression and angiolymphatic and perineural invasion is another important finding of our study and was previously described for PC. Angiolymphatic and perineural invasions are known to be associated with a worse prognosis, and its detection is sometimes influenced by the observer (pathologist) bias, suggesting that IHC for ki-67 can refine the diagnosis and prompt one to search for neural or vascular invasion in the histopathological analysis , 93.Protzel et al. (2007) and Zhu et al. (2007) described a strong association between high ki-67 expression and lymph node metastasis in PC , 88, andDespite the association with worse prognostic factors and lymph node metastasis, we did not observe a statistically significant difference between the DFS rate according to the ki-67 expression profile, similar to the study published by Stankiewicz et al. (2012) . We hypoPC is a neglected, heterogeneous, aggressive, and deadly disease that is typically not diagnosed early. There is a high incidence of PC in the Maranhão state, and little is known about the local etiological profile of these tumors. Most patients are socioeconomically vulnerable and come from the state's countryside. There are few centers for treating the disease in the region, most of which are located in the capital, São Luís, making early diagnosis and monitoring of these patients challenging.Here, we discussed the impact of the geographic isolation of the state capital on the displacement of these patients, suggesting that the actual incidence of the disease is more significant than that described previously . Our finAccording to Ornellas & Ornellas (2018), when PC is considered globally, only a tiny proportion is described as HPV-associated. Therefore, increased men's education and prevention strategies such as condom use, hygienic measures, smoking cessation, and avoidance of chronic inflammatory states can considerably impact the pathogenesis of precancerous lesions of the penis . In MaraINK4a overexpression appear to be related to decreased disease-free survival and what is its impact on overall and cancer-specific survival in PC patients? To answer these questions we need to explore the correlation between CDKN2a (p16INK4a) copy number, mRNA expression, methylation profile, and protein expression (IHC).Our data discuss HPV infection in a multi-methodological way, integrating histological, molecular, and immunohistochemical aspects and bringing new insights into the role of HPV on histological presentation and clinical behavior of these tumors. An interesting question involves the histological and prognostic variation observed in HPV-associated tumors. For example, why do warty and basaloid tumors, both HPV-associated, have widely different prognoses? Why does p16INK4a can reveal the real status of HPV infection in these patients, whether it is active and impacting tumor progression or part of the HPV-positive cases are just a background due to the high rate of HPV infection in the general population, especially in Maranhão (as discussed).Another important approach that we are currently investigating is the biological profile of viral oncoproteins (E6 and E7), both by gene (mRNA) and protein expression. These data linked to the characterization of p16The joint analysis of this information can help to better characterize each of the cited markers and their real role in the patient's prognosis. The perspective of our group has been, given the vast majority of HPV-positive cases, to investigate the differences within this group. We are currently performing all these analyzes on a prospective cohort of 60 cases from Maranhão, involving histological , genomic , and metagenomic tools to unravel these questions. All these approaches ongoing rely on the expertise of geneticists, physicians, pathologists, and oncologists with experience in PC to perform an integrative analysis between genomic (multi-omics) and the clinical aspects of PC. We hope with this approach to also elucidate the role of changes in p53 in PC carcinogenesis, evaluating somatic mutations and gene expression patterns.INK4a, p53, and ki-67 expression have clinical relevance and seem to impact the DFS of these patients. The test used to assess these protein expression profiles (IHC) is offered free of charge by the Brazilian Unified Health System (SUS) and can be used in histopathological routines to help diagnostic and prognostic classification of these patients.At the time, the associations found between p16Despite the difficulties inherent in the PC study, the present study strongly points to a clinical-histopathological and etiological profile different from that observed in other studies in Brazil and worldwide. It highlights the importance of local and collaborative research aimed at understanding these tumors' etiological aspects, behavior, and progression, especially in places with high incidences, such as Brazil. We emphasize that, due to limitations in current protocols for the management of PC patients, early diagnosis becomes even more decisive for better therapeutic conduct. This reality is expected to change since recently, the Brazilian consensus statement for managing PC patients in low- and middle-income countries has been proposed .In the present study, we presented important clinical-histopathological and pathophysiological features of PC. However, some limitations may reduce the clinical impact of the data obtained and must be considered. First, the retrospective aspect of the study makes it difficult to have reliable control over the variables evaluated, creating gaps in the data. Second, the use of DNA from FFPE tumor samples may increase HPV-PCR false negatives and the absence of genotyping data for all HPV-positive tumors limits the stratification of cases according to the HPV genotype. Third, this study had many censored patients in DFS analysis due to loss of follow-up and there is no data regarding cancer-specific and/or overall survival. Despite this, the data obtained from a large Brazilian cohort of PC present a wealth of information that significantly contributes to a better understanding of this neglected disease. All limitations may be further explored in a future prospective study, as discussed.INK4a overexpression can be used as a marker for basaloid tumors, an HR-HPV-related histological subtype. The IHC is easily accessible in poorer regions such as Brazil, reinforcing its applicability in clinical practice.Our study obtained important clinical and histopathological data for the characterization of the epidemiological profile of PC in Maranhão, Brazil. Our IHC findings revealed important parameters with prognostic values, including ki-67 and p53 expression associated with lymph node metastasis, grade, pT, stage, pattern, and depth of invasion, as well as ki-67 and p53 expression associated with decreased survival of PC patients. Additionally, p16Our data reaffirmed the high incidence of human papillomavirus (HPV) infection in PC and highlighted its possible association with worse clinical prognosis factors in the cohort evaluated, differently from what was observed in other regions. The association between HPV histological changes and men with risky sexual behavior draws attention to the urgency of effective public policies to vaccinate men to prevent the disease. This is especially important in Brazil, where there is a high rate of amputations as the first treatment option. Finally, our integrative approach offers new insights into possible factors that together contribute to Maranhão having the highest global incidence of PC ever recorded and highlights the importance of studies that aim to understand the pathophysiological behavior of HPV-related PCs better.Additional file 1: Supplementary material (S1) Table 1. Primers used for HPV detection in penile cancer samples Table 2. PCR mix preparation and thermocycling conditions.Additional file 2: Supplementary material (S2) Table 1. Statistical data for koilocytosis, molecular detection of HPV, and p16INK4a expression analysis in penile cancer.Additional file 3: Supplementary material (S3) Table 1. Statistical data for p53 and ki-67 protein expression analysis in penile cancer.


Head and neck cancers (HNSCCs), especially in the advanced stages, are predominantly treated by radiochemotherapy, including cisplatin. The cure rates are clearly higher for HPV-positive HNSCCs when compared to HPV-negative HNSCCs. For both entities, this treatment is accompanied by serious adverse reactions, mainly due to cisplatin administration. We reported earlier that for both HPV-positive and negative HNSCC cells, the effect of radiotherapy was strongly enhanced when pretreated using the dual PI3K/mTOR inhibitor NVP-BEZ235 (BEZ235). The current study shows that for HPV-positive cells, BEZ235 will strongly enhance the effect of cisplatin alone. More important, preincubation with BEZ235 was found to alter the purely additive effect normally seen when cisplatin is combined with radiation into a strong synergistic enhancement. This tri-modal combination might allow for the enhancement of the effect of radiochemotherapy, even with reduced cisplatin.The standard of care for advanced head and neck cancers (HNSCCs) is radiochemotherapy, including cisplatin. This treatment results in a cure rate of approximately 85% for oropharyngeal HPV-positive HNSCCs, in contrast to only 50% for HPV-negative HNSCCs, and is accompanied by severe side effects for both entities. Therefore, innovative treatment modalities are required, resulting in a better outcome for HPV-negative HNSCCs, and lowering the adverse effects for both entities. The effect of the dual PI3K/mTOR inhibitor NVP-BEZ235 on a combined treatment with cisplatin and radiation was studied in six HPV-negative and six HPV-positive HNSCC cell lines. Cisplatin alone was slightly more effective in HPV-positive cells. This could be attributed to a defect in homologous recombination, as demonstrated by depleting RAD51. Solely for HPV-positive cells, pretreatment with BEZ235 resulted in enhanced cisplatin sensitivity. For the combination of cisplatin and radiation, additive effects were observed. However, when pretreated with BEZ235, this combination changed into a synergistic interaction, with a slightly stronger enhancement for HPV-positive cells. This increase could be attributed to a diminished degree of DSB repair in G1, as visualized via the detection of γH2AX/53BP1 foci. BEZ235 can be used to enhance the effect of combined treatment with cisplatin and radiation in both HPV-negative and -positive HNSCCs. Advanced HNSCCs are mainly treated with radiotherapy, combined with concurrent cisplatin-based chemotherapy ,2. For HThe combination of cisplatin and radiation was established at the beginning of this century by several large trials showing that the overall survival of HNSCC patients was enhanced by 8 to 11% when compared to radiation alone ,6,7. FurCisplatin is a planar molecule that needs to be activated within the cell, which is then able to bind to the DNA, causing a variety of DNA crosslinks leading either to intra-strand 90%) or inter-strand crosslinks  0% or int,11,12. WHowever, it was noted that RCT with cisplatin is accompanied by severe side effects ,6. ThereIt is generally assumed that therapy might be improved for HNSCC when using molecular targeting. In this respect, the PI3K/Akt/mTOR pathway is regarded as being an optimal target, since it is often mutated in HPV pos. and HPV neg. HNSCCs ,25,26,27The dual inhibitor NVP-BEZ235  can obtain a strong suppression of the dual PI3K/mTOR pathway . When coIt was recently reported by us  that BEZIt was tested for HPV neg. and pos. HNSCC cells whether BEZ235 could also be used to enhance the effect of cisplatin alone, but most of all, the effect of the combined treatment with cisplatin and radiation. These experiments were performed with six HPV neg., as well as six HPV pos. HNSCC cell lines, to cover the huge molecular heterogeneity generally seen for this entity . It was 2 atmosphere and routinely tested for mycoplasma contamination .Immunofluorescence analysis was performed as previously described . Brieflyt-test was used to check for statistical significance, with a significance level p < 0.05. All statistical analysis and graphics were made using GraphPad Prism version 9.0 .If not stated otherwise, all experiments were performed in triplicates and repeated at least three times. Data are presented as the mean values ± standard error of the mean (SEM). A Student’s p value of p = 0.119 and p = 0.037 when UD-SCC-2 was excluded.HPV pos. cells are defective in DSB repair performed by HR, as demonstrated by the lack of formation of RAD51 foci after X-irradiation . In ordep = 0.001).It was recently shown by us that a pretreatment with BEZ235, which is a potent dual inhibitor of the PI3K/mTOR signaling pathway, resulted in an enhanced degree of radiosensitivity for both HPV neg. and pos. HNSCC cells, and that this was due to an inhibited NHEJ . It was The clear enhancement of cisplatin seen for HPV pos. cells after pretreatment with BEZ235 suggests that this might also result from defective HR. This assumption was tested by suppressing HR in three HPV neg. cells using, again, KD of RAD51. It was of special interest to test whether BEZ235 can also be used to enhance the combined effect of cisplatin and radiation. p = 0.0001 vs. HPV neg. p = 0.03).As demonstrated previously, the increase in cell killing achieved when BEZ235 was added prior to irradiation could be attributed to an enhanced number of residual DSBs, as detected by the co-staining of γH2AX/53BP1 foci . This asIn nuclei with hp-signals, the co-localization of γH2AX and 53BP1 foci was less than 20% d. In conCisplatin alone only resulted in a moderate increase in the number of foci counted in G1 cells c. These Head and neck cancers (HNSCCs), especially in the advanced stage, are treated by radiochemotherapy, including cisplatin, but with a poor outcome for HPV neg. HNSCCs. Moreover, this treatment is often accompanied by severe side effects primarily resulting from cisplatin administration. Therefore, the aim of this study was to test whether BEZ235, which is an effective dual inhibitor of the PI3K/mTOR signaling pathway, can be used to enhance the effect of cisplatin alone, but especially when combined with radiation.The study was performed with six HPV neg. and six HPV pos. HNSCC cell lines to cover the molecular heterogeneity that is well-known for this entity . The celOverall, cisplatin sensitivity was characterized by a huge degree of variation, with HPV pos. cells being slightly more sensitive when compared to HPV neg. cells, albeit without reaching significance. However, when the data was analyzed together with data from other reports using a similar protocol ,54, an oIt is shown here for the first time that the higher cisplatin sensitivity of HPV pos. cells might in part result from their defective HR. This defect was recently verified for HPV pos. cells via a lack of formation of RAD51 foci after irradiation , which iHR is involved in the repair of cisplatin-induced DSBs ,10,11,12Concerning the heterogeneous cisplatin sensitivity, there is great interest in establishing robust markers for prediction ,64. So fWe also found that cisplatin sensitivity is strongly enhanced in HPV pos. but not HPV neg. cells, when pretreated with BEZ235. This difference in enhancement could likewise be attributed to the defect of HR in HPV pos. cells. When HR was also depressed in HPV neg. cells via KD of RAD51, a similar degree of enhancement of cisplatin by BEZ235 was obtained, as for HPV pos. cells.It was recently shown by us that at a concentration of 50 nM, BEZ235 NHEJ is suppressed in HNSCC cells, while HR is not affected . NHEJ isThe enhancement of cisplatin sensitivity seen for the HPV neg. cell line UM-SCC-11b when pretreated with BEZ235 does not result from a defective HR. It was previously shown that HR is functional in UM-SCC-11b, as demonstrated by an active formation of RAD51 foci after irradiation, and by its response to the CDK inhibitor roscovitine . SurprisThe most important observation made here is that BEZ235 can be used to enhance the combined effect of cisplatin and radiation in HNSCC cells. In contrast, no such enhancement was seen for HNSCC cells, when cisplatin and radiation were combined with the multi-kinase inhibitor sorafenib . For HNSThe enhanced cell killing effect of the combined treatment with cisplatin and radiation when using a pretreatment with BEZ235 was seen for both HPV neg. and pos. cells, with a slightly stronger effect for the latter. This enhancement is considered partly to result from a diminished degree of DSB repair in G1, as demonstrated for two cell lines by the correlation between the decrease in survival and the increase in residual DSBs, as detected in G1 cells by co-staining with γH2AX and 53BP1. Since BEZ235 inhibits NHEJ and also induces a G1-arrest, as shown previously , more ciOverall, our data indicate that BEZ235 might be used as an excellent tool to enhance the combined effect of cisplatin and radiation. This enhancement might even allow for a reduction in the amount of cisplatin given to the patient, thereby reducing the side effects, but still reaching an excellent response. Unfortunately, BEZ235 has slipped from focus because of the negative results obtained when it is given as monotherapy (NCT01856101). This is a big drawback, because other PI3K inhibitors such as BKM120 and GDC0980 appear to be less efficient . For the


To explore the utility of the scoring system for screening and early warning of cervical cancer based on big data analysis.A total of 420 women undergoing physical examination in Shenyang from January 2021 to January 2022 were screened by convenient sampling as the study subjects. All females accepted the human papilloma virus (HPV) tests and thin-prep cytology test (TCT), a Rating Questionnaire for Screening and Early Warning of Cervical Cancer was developed, and a warning threshold was derived according to the scores of the questionnaire and the goodness of fit for the results of HPV+TCT tests. The patients were graded according to the threshold, and corresponding intervention strategies for patients of different grades were developed.p < 0.05), and TCT results showed that there was a significant difference in the number of patients with CIN I before and after intervention (p < 0.05).Among the 420 people undergoing physical examination, 92 (21.90%) obtained scores ≥8 points, and 328 (78.10%) obtained scores < 8 points; in diagnosing cervical cancer, the Rating Questionnaire for Screening and Early Warning of Cervical Cancer had an AUC value of 0.848, specificity of 97.22%, and sensitivity of 86.46%; after scientific intervention, HPV test results showed a significant decrease in both high-risk positive cases and low-risk positive cases (The scoring system for screening and early warning of cervical cancer based on big data analysis presents certain clinical value in the clinical screening of cervical cancer, which can further improve the screening coverage, is of great significance for the diagnosis and treatment of disease, and helps physician implement hierarchical diagnosis and treatment quickly and precisely. Cervical cancer is a malignant tumor disease that endangers women's health . InvestiA total of 420 women undergoing physical examination in Shenyang from January 2021 to January 2022 were screened by convenient sampling as the study subjects. The study met the World Medical Association Declaration of Helsinki (2013)  and reviInclusion criteria: (1) Women with history of sexual life; (2) those with good understanding and could objectively and accurately fill in the questionnaire; and (3) those who voluntarily joined the study. Exclusion criteria: (1) Those who had cervical cancer and operation history; (2) those who were unable to fill in the questionnaire due to other reasons; and (3) those who were unwilling to join the study.By reviewing a large number of published works on the epidemiology of cervical cancer and related influencing factors, the first version of Rating Questionnaire for Screening and Early Warning of Cervical Cancer was developed, which included the following sections. (1) The general data of the study subjects, such as their age, place of residence, occupation, educational degree, marital status, and the paying method of medical treatment;(2) Sexual habits, such as age at first sexual activity, number of sexual partners, and whether condoms were used at the time of intercourse;(3) Personal situation, such as number of times of miscarriages, number of times of pregnancies, presence or absence of an intrauterine contraceptive device (IUD), and presence or absence of immunodeficiency diseases; and(4) Relevant symptoms or vital signs, such as presence or absence of cervical contact bleeding, cervical erosion, or cervical polyps.In addition to the personal information of the study subjects, a total of 20 items were scored, and the maximum score was 20 points. The scoring method was as follows: 1 point was assigned to each question , and higher scores indicated higher risk of illness of the study subjects.A Delphi Consultation Questionnaire was developed according to the corresponding general format. After 2 rounds of expert consultation and pre-survey to 30 females, relevant data were organized and the indicators were perfected in combination with the experts' suggestions, which then concluded that the Cronbach's α coefficient and split-half coefficient of the questionnaire were, respectively, 0.796 and 0.691, the validity index of each item level was ≥0.945, and the content validity index of the scale level was 0.950.The HPV special sampler was inserted to the orificium externum isthmus of the subjects, rotated for 5 turns, and then slowly taken out and put into the HPV specimen bottle containing preserving liquid. The HPV subtyping genes chip was used to detect 26 types of HPV genes , and all operations were conducted according to the specifications of the apparatus and reagent.in situ (CIS); and squamous cell carcinoma (SCC).The sampler tip was inserted into the cervical canal and slowly rotated along the axis for 5 turns, the sampling brush tip was removed and put into the cell preservation solution, the specimen underwent standardized processing and was made into a thin-layer cell smear and fixed with 95% alcohol, and then Papanicolaou stain was performed. For diagnostic gradation of cytology, the Bethesda system (TBS) was adopted, to be specific, TCT negative, referring to no CLN was seen ; TCT abnormalities, such as a typical squamous cells of undetermined significance (ASC-US); low-grade squamous intraepithelial lesion (LSIL), which was graded as weak CIN (CIN I); high-grade squamous intraepithelial lesion (HSIL), which was graded as moderate and expressed CIN (CIN II and CIN III) and carcinoma Each section of the investigation was under strict quality control, in the stage of designing, the scientificity and feasibility of the scheme and questionnaire applied were verified; before investigation, the researchers were trained with uniform guidelines, and then they provided guidance and correction on any queries and errors made by the study subjects when filling in the questionnaire; after the completion of the questionnaire, 2 personnel were assigned to collect and organize the questionnaires, Epidata3.1 was used for data entry, SPSS20.0 was used for statistical analysis, and an early warning threshold of 8 points for the questionnaire was derived by comparing the analytic findings and gynecological examination results.According to patients' HPV test and TCT results and the scores of the Rating Questionnaire for Screening and Early Warning of Cervical Cancer, the threshold was specified as 8 points, and detailed intervention measures were developed according to the threshold.Detailed hierarchical intervention measures were developed according to the threshold. (1) Below threshold: For those who were free from infection and without prior HPV testing, testing was recommended for 3 consecutive years, and if the results were negative, testing could then be done every 2–3 years; (2) Above threshold: this group was high-risk HPV infected individuals, who should visit gynecological clinics according to the TCT result, and the effectiveness of intervention measures should be analyzed by comparing the results of TCT and HPV test before and after the intervention.The experimental data were statistically analyzed and processed by the software SPSS26.0, the diagnostic efficacy of the Rating Questionnaire for Screening and Early Warning of Cervical Cancer on cervical cancer was analyzed by the area under the Receiver Operating Characteristic (ROC) curve.See Among the 420 female undergoing physical examination, 92 (21.90%) obtained scores ≥ 8 points, and 328 (78.10%) obtained scores < 8 points.See See p < 0.05), and TCT results showed that there was a significant difference in the number of patients with CIN I before and after intervention (p < 0.05), and no significant differences in other types were observed (p > 0.05). See After scientific intervention, HPV test results showed a significant decrease in both high-risk positive cases and low-risk positive cases , and TCT results denoted that the number of patients with CINI was greatly reduced, which illustrated that the implementation of scientific control measures could lower the risk of cervical cancer to a certain extent. Studies have confirmed , through a process of PCR target preference, target amplification, automated real-time detection, and selective amplification, and then identify the corresponding HPV replication with greater precision than second-generation hybrid capture assay. Currently, TCT is the more advanced and mature screening technique for cervical cancer, but it has certain false-negative rate and false-positive rate for low-grade cervical lesions, and the concordance rate increases with higher cytological grade. Scientific means of clinical screening can screen out people at high risk of cervical cancer, reduce the follow-up interval, facilitate effective monitoring of cervical cytological alterations and early detection of cervical lesions, and TCT combined with HPV screening is beneficial to the triage management of cervical lesions, realizing early detection and treatment of the disease. In this study, more detailed prevention and treatment strategies were developed by typing the obtained examination findings. The findings showed that after intervention, both high-risk positive cases and low-risk positive cases were significantly reduced according to the HPV test results  Strengthen the infrastructure construction of the health system, invest corresponding financial, material, and human resources, strengthen the training efforts of primary care workers, and establish a complete system for AI-assisted diagnosis; (2) based on the domestic conditions, clarify the age and screening interval of subjects, identify high-risk individuals, encourage those of proper ages to actively participate in screening, establish a scientific surveillance and evaluation system, and encourage the population of the right age to get an HPV vaccination to gradually expand vaccination coverage; and (3) scientifically develop appropriate screening programs and strategies for the Chinese people to reduce the incidence of cervical cancer . DespiteThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Ethics Committee of General Hospital of Northern Theater. PLA. The patients/participants provided their written informed consent to participate in this study.DH, BY, and MS: conception and design and data analysis and interpretation. All authors: administrative support, provision of study materials or patients, collection and assembly of data, manuscript writing, and final approval of manuscript.This research was funded by Feasibility study on cervical cancer screening early warning scoring system for female physical examination population in Shenyang, Grant No. 20-205-4-088.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction and Background: Both human papillomavirus (HPV) and the human immunodeficiency virus (HIV) are sexually transmitted. High-risk (HR) HPV types are a causal factor in cervical cancer. Persistent HPV infection in this subset of immunocompromised women results in faster disease progression. The study determined the prevalence of HPV genotypes in cervicovaginal secretions of HIV seropositive women and the correlation with CD4 counts and cytology.Method: One hundred, non-pregnant, HIV-positive women of 18 years of age and above were enrolled in this cross-sectional study following approval by the institutional ethical committee. A written consent, questionnaire, followed by sample collection including a Papanicolaou (Pap) smear for cytology was undertaken. Cervicovaginal secretion samples were collected in the Digene® specimen transport medium (STM) . HPV genotyping was carried out with PCR amplification of a 65-base pair (bp) fragment in the L1 region of the HPV genome using the short PCR fragment (SPF10) primers followed by reverse hybridization by line probe assay (LPA) using the INNOLiPA HPV Genotyping Extra kit . Quantitation of HPV-16 and−18 viral loads (VLs) was done by real-time PCR. Results of Pap smear cytology were correlated with CD4 counts and HPV-16 and−18 VLs.Results: Mean age of the subjects was 34.9 years ± 7.2 years . HPV was detected in 62 of 93 (66.6%) samples. Twenty (32.25%) of these 62 samples harbored a single HPV genotype. Multiple genotypes (more than two) were detected in 38 (61.3%) samples. HPV-16 was the commonest genotype detected in 26 (27.9%) of all samples and 41.9% of HPV positive samples. Pap smear cytology was reported for 93 women included in the study. Women who had normal cytology were reported as negative for intraepithelial malignancy or lesion , two women had a high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion , atypical squamous cells of undetermined significance . Those smears with inadequate material were reported as scant (n = 6). The median CD4 count was 363/cu.mm (range 39–787) in HPV-positive women compared to 423/cu.mm (range 141–996) in those HPV-negative women. Quantitation of HPV-16 and−18 VL was done in duplicate for samples positive by PCR reverse hybridization (INNOLiPA). Of these 20 samples (65%), 12 samples were positive by real-time PCR. The normalized HPV-16 VL ranged between 18 and 240,000 copies/cell. The normalized HPV-18 VL in cervical samples ranged between ~24 and 60,000 copies/cell.Conclusion: HIV-positive women may be infected with multiple genotypes other than HPV-16 and−18. This may have implications on the vaccines available currently which target few specific genotypes only. Studies are required to determine the predictive role of HR HPV genotypes, in significant copy numbers especially in HIV seropositive women. It would be clinically relevant if the HPV VLs, cervical cytology, and CD4 counts are considered into cervical cancer screening programs for triage and follow-up of these women. To achieve the goal of eliminating cervical cancer by 2030, there is a need for a multipronged approach involving a commitment to screen, test, and treat reliably . A “PoinOne hundred consecutive, non-pregnant, HIV-positive women >18 years of age and reporting to the antiretroviral therapy (ART) clinic, Department of Obstetrics and Gynaecology and Department of Microbiology, of the All India Institute of Medical Sciences (AIIMS), New Delhi, India, were enrolled in this cross-sectional study. This study was conducted for 4 years following approval by the ethical committee. The inclusion criteria for including women in the study were HIV-positive women, aged 18–59 years, and who were willing to participate in the study. They were included irrespective of their treatment and CD4 counts. CD4 counts were done in the lab using a fluorescence-activated cell sorter (FACS) count analyzer . Women were excluded from the study if they were pregnant, hysterectomized, and had undergone a procedure on the cervix, or they were diagnosed with neoplastic lesions of the cervix. Written consent was taken from each participant, and a questionnaire was filled by a face-to-face interview. Details of their education, socioeconomic status, treatment history, most recent CD4 counts were noted.® cervical brush was used for collecting cells from the endocervix, it was rotated 3–5 times under direct vision and placed in the Digene specimen collection tube, containing 1 ml of the specimen transport medium . Pap smear slides for cytology were put in a fixative and sent to the cytopathologist for reporting. Doubtful or undetermined smears were reviewed independently by two cytopathologists. The cytopathologists were blinded to the HPV molecular assay results. The collection tubes with the cervical samples for HPV DNA testing were transported on ice to the laboratory and stored at −70°C until further processing.Consent was obtained, and the sample was collected after explaining the procedure to the women. Universal precautions were followed during the collection of cervicovaginal secretions. Cervicovaginal samples were collected for conventional cytology for Pap stain followed by samples for HPV DNA genotyping. The DigeneThe specimen in the STM was centrifuged and aliquoted for HPV DNA extraction. Detection and genotyping of HPV were done using the INNOLiPA HPV Genotyping test kit , a line probe assay (LPA) based on the principle of reverse hybridization , 14. Thi2), and 2.3 μl of enzyme mix (AmpliTaq Gold DNA polymerase) reagents provided in the INNOLiPA HPV Genotyping Extra assay . The PCR reaction was a 50 μl reaction performed in the thermal cycler as follows: decontamination at 37°C for 10 min, denaturation at 94°C for 9 min, 45 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 45 s, and extension at 72°C for 45 s. The biotinylated amplified HPV DNA PCR products were denatured and hybridized to oligonucleotide probes immobilized on a nitrocellulose membrane. After washing thoroughly, streptavidin-conjugated alkaline phosphatase was added, which bound to any biotinylated hybrid formed. A colorimetric reaction gave a purple/brown precipitate indicating a positive band. A specific pattern of bands as specified by the interpretive chart provided by the manufacturers was used to find out the HPV type, and results were read visually. A sample was considered positive if one of the defined type-specific banding patterns along with one of the HPV control lines was positive.Human papillomavirus genotyping was carried out by INNOLiPA HPV Genotyping Extra kit, . The kit detects 28 HPV types, such as low-risk (LR) and high-risk (HR) HPV types—HPV-6LR, −11LR,−16HR, −18HR,−26pHR, −31HR, −33HR, −35HR, −39HR, −40LR, −43LR, −44LR, −45HR, −51HR, −52HR, −53pHR, −54, −56HR, −58HR, −59HR, −66PHR, −68HR, −70LR, −69/71, −73HR, and −82HR. The sequence variation within the SPF-10 primers allows the recognition of these different HPV genotypes  were searched for suitable primer and probe-targeted sites. The basic local alignment search tool (BLAST) was used to select nucleotides spanning 231–434 bp. Primers were designed using the Invitrogen Perfect Primer site which successfully amplified a 301 bp product of the E6 gene of HPV-16. For HPV-18 primer, target sites spanned nucleotides 667–803, and 251 bp product of the E7 gene was successfully amplified by the custom-designed primers and 538 bp product of GAPDH. These amplicons were successfully cloned into a TOPO vector using the TOPO TA cloning kit . Escherichia coli (E. coli) cells were transformed, and the presence of the insert was confirmed by the blue/white screening of the transformed colonies grown overnight on the Luria Bertani (LB) agar. The plasmid was extracted, and the concentration of the constructed cloned plasmid was measured by spectrophotometry. On measuring, the plasmid DNA concentration for HPV-16 was 50.0 ng/μl, HPV-18 plasmid DNA concentration was 187.0 ng/μl, and constructed cloned GAPDH plasmid DNA concentration was 140.0 ng/μl. The 10-fold serial dilutions of the constructed plasmid containing the target gene were prepared, from 107 to 101 copies/μl, which corresponded to the cycle threshold (Ct) range of 18.3–41.95 on real-time PCR. The Ct values of HPV-16,−18, and GAPDH standards measured in duplicate are provided in the Quantification of HPV-16 and−18 VLs was done for samples that were positive for these by LPA using the TaqMan assay performed on the ABI Prism 7500 [Applied Biosciences ] and the Step One Plus platform. The targeted genome segments were the E6 and E7 regions of the HPV genome. Amplification was performed for HPV-16,−18, and one internal control (housekeeping gene) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using primers and probes designed in-house by using published references (The cloned plasmid containing the target gene (with known copy number) was used to make the standard curve for the TaqMan absolute quantification assay. Once the standard curves at different dilutions were prepared for HPV-16, HPV-18, and GAPDH in-house, and assay standardized, the samples were run along with these standards.Viral loads were detected by amplifying a 223 bp fragment of the E6 region of the HPV-16 genome and a 137 bp fragment of the E7 region for HPV-18. Real-time quantitative PCR was performed by adding 5 μl of extracted DNA to the TaqMan master mix, with 800 nM forward and reverse primers, and a 200 nM labeled probe, in a final volume of 25 μl. For HPV 16 PCR, the forward primer used was 5′ ATGACTTTGCTTTTCGGGAT 3′, reverse primer used was 5′CTTTGCTTTTCTTCAGGACA3′, and probe primer used was 5′ACGGTTTGTTGTATTGCTGTTCTA3′ (FAM-TAMRA). Thermal cycling conditions were as described previously . The volume was made up to 25 μl. The thermal cycling conditions were as follows: an initial hold at 50°C for 2 min followed by pre-PCR denaturation step at 95°C for 12 min, and then 50 cycles of 95°C for 15 s, and 55°C for 30 s. The results were expressed as GAPDH copies per cell  software v25 and STATA version 17 . Descriptive statistics, such as mean and range, were calculated for continuous variables. Frequency and percent values were computed for qualitative variables. A One hundred HIV-1 positive women were enrolled; none of these women was HIV-2 positive. The mean age of the subjects was 34.9 years ± 7.2 years . The demographic details have been published elsewhere . SamplesHuman papillomavirus DNA of any genotype was detected in 62 (66.6%) of the 93 samples, from 58 of which, the HPV detected could be typed, while four samples were untypeable (HPV-X). In all, 20 (32.25%) of these 62 samples harbored a single genotype, and multiple genotypes were detected in 38 (61.3 %) samples. HR HPV genotypes were present in 37 (39.8%) of the 93 samples tested, comprising 59.7% of the 62 HPV DNA positive samples by PCR-hybridization.Either HPV-16 and/or HPV-18 was present in 30 (32.3%) of 93 samples and 48.4% of the 62 HPV DNA positive samples. HPV-16 was detected in 26 (27.9%) of all samples and 41.9% of the HPV positive samples, while HPV-18 was detected in 5 (5.4%) of all samples and 8.1% of the HPV positive samples. One sample had both HPV-16 and HPV-18 on PCR hybridization.* (n = 26) and HPV-52 (n = 17). The other genotypes detected in decreasing order were as follows: HPV-74, −56*, −44, −45*, −69/71, −18*, −51*, −11, −59*, −66pHR, −6, −58*, −68*, −35*, −43, −40, −54, −31*, −39*, −53pHR, −73pHR, −26pHR, −61, −81, −33*, and −42.  HPV positive; Group III, HPV-16 positive; Group IV, HPV-16, and HPV-18 positive.The patients who were positive for HPV on reverse hybridization were divided into four groups for analysis. The four groups were as follows: After excluding six samples of the 93 samples reported as scant by the cytopathologist, results for 87 samples were available. Sixty-two (71.36%) women had normal cytology.n = 2), LSIL (n = 11), and ASCUS (n = 12). HPV-16 and HPV-18 positive samples were associated with a higher degree of cytological abnormalities at ASCUS threshold as compared with LR HPV, all HR HPV taken together, or HPV negative samples  and ASCUS (25%), in LSIL, the representation of HPV-16 was much higher (72.7%). There were only two HSIL cases in this study, one of which was positive for HPV-16, and the other had HPV-73 as the solitary HPV type. The association between Pap smear abnormality and HPV DNA positivity by PCR hybridization, in the four groups, was analyzed . There wOf the 26 cervical samples positive for HPV-16 by PCR reverse hybridization (INNOLiPA), real-time PCR was performed for 20 samples (in duplicate), of which the housekeeping gene GAPDH could not be amplified in two samples. The results of 18 samples are shown in The normalized HPV-18 VL in cervical samples was in a range of ~24–60,000 copies/ cell.n = 93) ranged from 39 to 1,056 cells/mm3 . Pap smear cytology results were correlated with the CD4 category. Distribution in different CD4 categories, as per the Centre for Disease Control (CDC) classification, along with the cytology in each category is shown in 3 as compared to those with CD4 counts >200 (22.89%).The CD4 count of the study group  was 363 cells/mm3, range , while in the HPV-negative subset it was higher at 423 cells/mm3, range (141–883), though the difference was not significant. Infection with HPV types other than HPV-16 and HPV-18 was associated with the lowest median CD4 count (168 cells/mm3) range (138–890).It was noted that the median CD4 count in the HPV-positive subset , but the difference was not significant (p = 0.273) p-value < 0.05 statistically significant.Distribution in different CD4 categories, as per the CDC classification, revealed HPV positivity was higher  in those with CD4 counts < 200 cells/mm3 was 0.264 times lower than those with CD4 < 200 cells/mm3. The 95% CI was 0.03–2.29 and was not significant.The logistic regression analysis was done, the odds ratio (OR) of having a positive hybridization in women with CD4 > 200 cells/mmp = 0.273); Group II, high-risk (HR) HPV positive (p = 0.62); Group III, HPV-16 positive (p = 0.999); Group IV, HPV-16 and HPV-18 positive (p = 0.896).Similarly, a Pap positivity had 0.360 odds of having a positive hybridization as compared with a negative Pap. The 95% CI was 0.108–1.19 and was not significant. A two-predictor logistic model was fitted to the data to test if CD4 and pap predicted the outcome. However, they were not found significantly related to the outcome. The patients who were positive for HPV on reverse hybridization were divided into four groups for analysis. Group I, any HPV positive (p = 0.011), for HPV-16 (p = 0.006), and most significantly, for HPV-16 and/or 18 , but not when the sample was positive for any HPV type (Group I).The nonparametric Pearson's Chi-square test showed the association between the categorical variables that the Pap smear cytology was differently distributed in samples harboring HRHPV-16 and−18 when compared with the samples, which were negative for any cytological abnormality. The Chi-square value was 48.24, and the Likelihood ratio of 29.61 was significant.Table 6. The standard curve for HPV-16 VL quantitation is shown in The viral load of HRHPV-16 along with CD4 counts and cytology is shown in A statistically significant correlation was seen in HPV-16 VL and the presence of abnormal Pap cytology .3  and LSIL. CD4 counts of the HPV-18 positive women were in the range of 112–533 cells/mm3 .n = 62) of the 93 samples. The prevalence has been reported differently by studies from all over the world. This difference is due to socioeconomic standards, region, cultural practices, and risk behavior , followed by HPV-74. Two samples were reported as HSIL in our study, one harbored HPV-16 and−52, and the other had a single HR HPV-73. The causal role of these other  genotypes needs to be investigated.A high prevalence of other HRHPV  genotypes, such as HPV-52 and HPV-58, has been reported in WLHIV . Some st3  women were HPV positive and “carcinogenic” HRHPV types were present in 35.3% (98/278). Half the women had multiple HPV genotypes, while multiple HRHPV genotypes were present in (27.8%) of women with “carcinogenic” HPV . Overallp = 0.011) than for HPV-16 alone (p = 0.006), and for HPV-16 and/or 18 (p < 0.001). The prevalence of abnormal Pap smear in WLHIV has been reported to be five times higher than the general population in which the rate of abnormal Pap smear is 5–6%.On comparing HPV genotypes and cervical cytology, we observed that the cytological abnormalities were higher with HPV positivity for HRHPV types (n = 62) of women had a normal Pap smear cytology. Abnormal cytology was seen in 24.2% of HIV-positive women. It has been reported to be 8–38% in previous studies. A meta-analysis for HPV-positive women (with HIV status unspecified), also showed a pronounced increase in HPV-16 positivity across cytological grades; from normal/ASCUS/ CIN1 (20–28%), through CIN2/HSIL (40/47%), to CIN3/ICC (58/63%)  .p-values ranging from 0.001 to 0.043, which was most pronounced in the HPV-negative group. Since a control group of women without HIV was not included in this study, comparisons were drawn with available data for this group from studies done at the same institute. In a meta-analysis from south Asia, the overall HPV positivity in women with normal cytology was 9–12% and increased to 52 and 76%, respectively, in those with LSIL and HSIL (A study was done at the same institute as this study reported HPV positivity as 7.6, 42.3, and 87.5% in HIV-negative women with normal cytology, LSIL, and HSIL . In thisand HSIL .Quantitative detection of HPV VL for HPV-16 and HPV-18 using real-time PCR was done. HPV VL was normalized to GAPDH for comparability across runs and samples. It was calculated as the ratio between the number of HPV copies, and the number of cells that are present. Normalizing allowed for correction of the VL for the number of target cells/DNA to even out variations due to sampling . Since HThe limitations of our study were the small sample size, which was not large enough to conclude. Studies on a larger cohort would be required to establish the correlation to decide the clinical VL cutoff . ProspecFuture studies are required to define the optimum frequency for screening intervals in HIV-positive women. In low-resource settings, screening strategies need to be organized based on the available resources. Incorporating self-sampling and POC tests for screening will need to be evaluated and developed into affordable and effective technologies in years to come.This study has detected HPV-16 as the commonest genotype in North Indian HIV seropositive women. Genotyping may help in risk stratification of HR types in HPV-associated cervical carcinogenesis. Monitoring of prevalent HPV types will become even more necessary with the widespread use of vaccines. Circulating HPV types may evolve or change as the population is vaccinated, prompting a change in the vaccine-targeted genotypes as well. Epidemiologically, it has implications for the development of newer HPV vaccines. HIV-positive women should be recommended cervical screening by Pap smear where facilities exist. Women should be actively screened by pap smears and HPV-specific molecular tests. Clinically relevant cutoffs of normalized HPV-16, 18 VLs in a high-risk subset of HIV-positive women may be incorporated into cancer screening programs. These in the future would be helpful to guide triage, follow-up, or treatment policies.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by All India Institute of Medical Sciences, New Delhi. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In-hospital pediatric cardiopulmonary arrest is rare. With more than 50% of patients not surviving to discharge following cardiopulmonary arrest, it is important that health-care providers (HCPs) respond appropriately to deteriorating patients. Our study evaluated the performance of basic life support skills using non-acute HCPs during pediatric inpatient resuscitation events. We conducted a retrospective chart review of all code blue team (CBT) activations in non-acute care areas of a tertiary care children’s hospital from 2008 to 2017. The main outcomes were frequency of life support algorithmic assessments and interventions  performed by non-acute HCPs prior to the arrival of CBT. CBT activation and outcome data were summarized descriptively. Logistic regression was used to assess for an association of outcomes with the presence of established leadership. A total of 60 CBT activations were retrieved, 48 of which had data available on isolated non-acute HCP performance. Most children (93%) survived to discharge. Critical action performance review revealed that an airway, breathing and pulse assessment was documented to have occurred in 33%, 69% and 29% of cases, respectively. A full primary assessment was documented in 6% of cases. The presence of established leadership was associated with the performance of a partial ABC assessment. Our results suggest that resuscitation performance of pediatric inpatient non-acute HCPs often does not adhere to standard life support guidelines. These results highlight the need to reconsider the current approaches used for non-acute HCP resuscitation training. In-hospital pediatric cardiopulmonary arrest (IHCA) occurring outside of acute care settings is an uncommon but potentially devastating event . While sPrior to the arrival of critical care support, non-acute care providers on in-patient pediatric wards must provide basic life supports, such as chest compressions and pulse checks, when patients deteriorate. Available data from simulation studies have shown that non-acute pediatric HCPs, including both nurses and physicians, frequently deviate from these andother actions outlined in standard life support algorithms ,10. ResuWhile this data comes from the simulation literature, it has been suggested in some studies that there is a correlation between resuscitation performance in simulation and in real-life settings. To our This was a retrospective observational study at an academic tertiary care pediatric hospital in Canada. The project was deemed not to require review by the hospital research ethics board. Code blue activations between 1 January 2008 and 30 June 2017 were identified and reviewed for the current study. Code blue activations are expected for cardiac or respiratory arrest, life-threatening emergencies , and intubations required outside a critical care area. Non-acute care providers (such as ward nurses) are required to maintain certification in American Heart Association (AHA) basic pediatric life support training and receive other acute-care education taught by educators (nurse or respiratory therapist) specific to their area of work. Pediatric resident trainees are required to be certified in both AHA Basic Life Support (BLS) and AHA Pediatric Advanced Life Support (PALS) and are provided certification and re-certification by the training program as needed. The hospital supports a simulation program, which provides regular mock code blue experiences in addition to the didactic small-group sessions provided by nursing educators on a variety of topics throughout the year. Members of the institutional code blue team include senior pediatric resident, anesthesia staff physician or resident, respiratory therapist, two critical-care trained nurses, pharmacist, pediatric intensive care physician or fellow, and a security officer. Our hospital has 166 inpatient beds with over 6000 admissions annually and a Medical Emergency Team (MET) that is a subset of the code blue team  available 24/7. The MET members receive regular training and exposure to critically ill patients, and the MET is typically requested for deteriorating patients in an effort to avoid ICU admission and code blue events.To focus on non-acute HCP actions, all activations of the code blue system for patients outside acute care areas  during the study period were reviewed. All code blue activations were included, even if it was subsequently determined that the incident was not life-threatening, defined as the absence of significant cardiac or respiratory compromise. Thiswas done because the actions of non-acute HCPs were still considered relevant, presuming their impression of the situation was of a significantly deteriorating patient. Code blue events that were activated by, or occurred in the presence of the MET, were excluded from the final dataset, as MET presence confounds the outcome of interest .The main outcomes of interest were the assessments and interventions  performed by non-acute HCPs prior to the arrival of the code blue team as documented in the patient chart (Table S1). These critical actions were adapted from those actions recommended in pediatric BLS and assessed in simulation studies ,10,12,13Code blue event characteristics and demographic information were collected to describe the patient and provider samples. Secondary outcomes  were also collected. Additionally, a secondary analysis associating critical action performance with the presence of established leadership was done. Leadership was considered ‘established’ if documentation listed one participant as directing the actions of other participants during the resuscitation prior to the arrival of the code blue team.Given the anticipated limitations of retrospective chart data, two forms of concurrent criterion validity data were also collected for comparison. First, critical action performance data by the code blue team was collected for the final 18 months of the study analysis period. These data were seen as relatively high quality as the code blue team uses a standardized template with a dedicated pre-assigned team member to document during resuscitations. Second, a convenience sample of MET nurses, ward nurses, RRTs, and physicians all with a history of involvement with at leastone code blue event were asked, ‘Imagine you were reviewing the last 10 code blue activations on the wards at [the hospital]. In the few minutes before the code team arrives, in how many of those 10 cases would you estimate the ward team has done the following: assess the airway for patency, assess breathing effort/effectiveness, assess circulation with a pulse check, assess all 3 of the above?’All code blue activations have been prospectively tracked and recorded at the hospital since 2007 with clear identifying data since 2008. The study dataset consisted of all code blue activations occurring within the hospital but outside of intensive care units or emergency department settings. The hospital charts were reviewed and event demographic data as well as the outcome measures listed above were extracted using a standard case report template. Outcomes of interest were extracted from the earliest documentation that described actions leading up to and including the activation of the code blue until the arrival of the code blue team.All data were de-identified and transferred to a REDCap database ,15. The Associations between the establishment of leadership and the performance of critical actions were measured using odds ratios (OR) estimated via logistic regression, together with 95% confidence intervals. In one instance, Firth logistic regression was performed due to difficulties encountered with convergence of the logistic regression algorithm, due to a zero cell count. All analyses were performed using R version 3.6.3.There were 60 code blue activations identified during the study period. Two charts were unavailable due to ongoing legal proceedings and were therefore not included in the analysis. Fifty-eight code blue activations were included in the analysisThirty-five percent (19/55) of code blue activations were for cardiopulmonary arrest, and more than half of activations  were for patients in respiratory distress or failure. Three code blue activations did not document the reason for the activation. The MET was activated prior to a code blue activation in 35% (20/57) of cases, with one instance where it was unclear from the documentation whether the MET was activated prior to the code blue activation. The MET itself activated the code blue in 17% (10/58) of cases for the purpose of intubation and mobilizing resources. Registered nurses (RNs) were the HCPmost likely to be present prior to code blue activation  .Table 1Overall, 93% (54/58) of children survived to discharge from hospital with only 1 patient dying prior to transfer to PICU. There were no instances of defibrillation . Of the As previously described, the 10 activations where the MET was present at the time of code blue activation were removed from the analysis of non-acute HCP performance. Forty-eight code blue activations were therefore analyzed for non-acute HCP critical action performance.An airway assessment was documented in 33% (16/48) of cases, a respiratory assessment was documented in 69% (33/48) of cases, and a pulse assessment was documented in 29% (14/48) of cases. A full ‘ABC’ assessment was documented in 6% (3/48) of cases . There wEstablishment of leadership was documented in 40% (19/48) of code blue activations prior to the code blueteam arrival . EstabliTwenty providers  were interviewed and provided their perceptions of critical action performance frequency prior to code blue team arrival. Also, critical action performance by thecode blue team was extracted from nine events that occurred in the final 18-months of the study period. Both sets of data and the main study findings are shown in The goal of this study was to determine whether pediatric non-acute HCP performance during real-life significant patient deterioration was consistent with standard life support guidelines. While by no means definitive, our data lend further credibility to simulation-based findings that non-acute HCPs do not consistently complete critical life support assessment and intervention actions. Documentation of a full ‘ABC’ assessment was rare, present in 6% (3/48) of cases prior to the code blue team arrival. A pulse check was documented in less than 30% (14/48) of cases, whereas respiratory status was most likely to be assessed (33/48 [69%]). In addition to the challenges with performing full ‘ABC’ assessments, we also found deviations from life support guidelines for BLS interventions. Airway maneuvers, including positioning and suctioning, were each documented in less than half of cases despite respiratory concerns being the most common reason for code blueactivation. Together, these results support the concern that provider performance is inconsistent with established life support guidelines in the real-life acute situations and suggest that current education models may be inadequate.Our results lend credibility to previous simulation studies that show errors in BLS skills by both physicians and nurses during mock codes ,10,17,18Our study supports the notion that novel educational approaches in resuscitation training are needed, as the potential for improved patient outcomes with better training is possible. Recent Building on our data and existing literature, including current recommendations, an effective training program should include principles of deliberate practice, be context-specific or compatible, occur at regular intervals, and be interprofessional in nature with a focus on nurses. Our insOur study also suggests that in addition to developing new training modalities for resuscitation-specific knowledge and skills, there may be utility infocusing on improving leadership skills. Simulation studies have suggested that leadership training can lead to improved and sustained cardiopulmonary resuscitation performance . LeadersOne of the major challenges faced in our study was the variability and quality of documentation. While our institution has standardized documentation for the code blue team with a dedicated real-time documenter, there is no standard documentation prior to the arrival of the code blue team. Documentation was noted to be variable in content and in individuals performing the documenting. We were unable to reliably identify which non-acute HCP performed critical actions when they occurred, which may have been useful data. The limitations of resuscitation documentation are not unique to our study, however, as this is a recognized problem that occurs even with critical care trained HCPs using standardized documentation templates . UltimatAcknowledging this possibility, in our opinion, three sources of data lend credibility to the validity of the findings. First, the results are reasonably similar to published findings from controlled simulation environments with challenges in adhering to basic life support algorithms ,10,17,18Our study suggests that the performance of non-acute pediatric HCPs, including both nurses and physicians, often deviates from established resuscitation guidelines when managing a deteriorating patient. The findings support the notion that these providers require a different educational approach to develop and maintain the relevant resuscitation skills they would be expected to perform in their clinical environment. Our study also supports ensuring particular attention to training ward nurses, as they are by far the most common provider type present at the time of a code blue activation.Click here for additional data file.


In Malaysia, the HPV immunization program has been introduced since 2010 as part of the national immunization plan for female students aged 13 years old. It was a very successful immunization program with good responses from students and parents until the start of COVID-19 pandemic in 2020. The COVID-19 pandemic caused the schools to be closed and resulted about 225000 female students aged 13 years old either missed their vaccination or have incomplete doses of HPV vaccination in 2020 and 2021. This could possibly lead to an increase in cases of cervical cancer and genital warts in the upcoming years. Hence, a wellorganized catch-up HPV vaccination program is vital in ensuring the aim of achieving zero HPV-related infections in the future. WHO has set a global goal to eliminate cervical cancer by 2030, and one of the targets is for 90 percent of girls aged 9 to 15 to be fully vaccinated against HPV (The Human Papillomavirus (HPV) vaccination was introduced by the World Health Organization (WHO) in 2006. Countless countries have integrated this vaccination into their national vaccination program in their effort to make HPV vaccination their primary strategy in the prevention and eradication of cervical cancer. Since then, three vaccines has been approved for global use by WHO, which are bivalent HPV-16/18 vaccine  with help from Ministry of Education (MOE), focused on vaccinating school girls aged 13 years old through school-based health service package. The school-based health package was designed to covered school-based health screening and services for students from primary school Standard 1 (age 7) till secondary school From 3 (age 15) . The HPVTo date, the national HPV Immunization Program has been implemented in 2,960 schools registered under MOE in urban and rural areas . To ensuAmong the challenges faced during the initial phase (2010–2011) was the lack of parental consent for their children to be given HPV vaccination. Two main contributing factors were identified: (1) concern of ingredients used to manufacture HPV vaccine and (2) poor knowledge of HPV vaccine against cervical cancer prevention , 8. A loDuring the sustained implementation phase 2012–2019), the national HPV immunization program was very successful. The HPV Immunization Program aim to vaccinate about 250,000 school girls aged 13 years old every year. It was observed that about 98% of parents/guardians consented for their children to be given HPV vaccine from 2012 to 2016 , the nat.Globally, the pandemic has left a huge impact in multiple sectors. According to WHO and the United Nations Children's Fund (UNICEF), the COVID-19 pandemic has caused 23 million children and 1.6 million girls globally to miss out on scheduled vaccinations and HPV vaccinations respectively in 2020 . In MalaThe economic impact caused by the COVID-19 pandemic had forced government to redistribute the national budget to strengthen the economic recovery rather than improving cervical cancer screening program. The total cost of the national HPV Immunization Program was estimated to be RM322.2 million where the cost of purchasing HPV vaccine was RM 319.2 million and the remaining RM 3 million was used in covering aspects of health education, training, and logistics of the involved personnel . It alsoThe long-term void caused by the HPV immunization program would possibly lead to an increase in cases of cervical cancer and genital warts in the upcoming years. A model-based analysis conducted in the United States revealed that the HPV vaccine coverage reached as low as 23% of the previous year's rate during the COVID-19 pandemic in 2020. This resulted in 51% cases of genital warts, 59% CIN 1 cases, and 55% CIN 2/3 of cases were projected to occur within the first 25 years in the current status quo of dropped vaccination rates due to the COVID-19 pandemic . Under tAs the national HPV immunization program has been disrupted by the pandemic, there is a need to put in place catch-up vaccination programs to ensure the closure of the large gaps created by COVID-19. A model-based study conducted in Japan showed that over a 100-year time horizon, there will be earlier and greater reductions in cervical cancer incidence through catch-up HPV vaccination programs where up to 484,248 cases of cervical cancer will be avoided over a 100-year span from 2021 to 2121 . FurtherThere are many aspects that need to be taken into account before the implementation of this program. From the parents' and individual point of view, they would query about the presence of interaction between HPV vaccination and other vaccinations particularly COVID-19 vaccination. There will be possibilities of vaccine hesitancy because they will be worried about the severity of side effects and adverse effects from the interaction between the HPV vaccination and COVID-19 vaccination. Besides that, the school administration plays a huge role in the implementation of this program. Before the implementation of the HPV catch-up program, the willingness of the schools to allocate time in the midst of catching up back on the missed curriculum that was caused by the multiple lockdowns carried out nationwide needs to be thoroughly assessed. The school will also need to arrange and limit the number of students taking the vaccination daily in line with the adherence to the rules and regulations put in place for the COVID-19 pandemic. The school's administration needs to set up with a system to successfully implement the catch-up HPV immunization program in their respective schools. This will surely pose a great challenge to the teachers to effectively arrange the timetable to give space for the students to receive their HPV vaccination. There should be collaborative efforts between MOH and MOE to effectively carry out the administration of HPV vaccination. MOH will need to provide adequate personal protective equipment and allocate healthcare workers to be sent to schools to carry out the administration of HPV vaccinations. MOH could also use this opportunity to overcome the lack of awareness among the public regarding the importance of HPV vaccination. The current pandemic will provide potential fertile grounds for overcoming doubts, suspicious, rumors, and conspiracy theories regarding vaccination in general among the members of the community . In concSR contributed to initial drafting. NK and CT performed first revision. MS completed the final revision of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.