Millions of deaths worldwide are a result of sepsis  and septic shock syndromes which originate from microbial infections and cause a dysregulated host immune response. These diseases share both clinical and immunological patterns that involve a plethora of biomarkers that can be quantified and used to explain the severity level of the disease. Therefore, we hypothesize that the severity of sepsis and septic shock in patients is a function of the concentration of biomarkers of patients.In our work, we quantified data from 30 biomarkers with direct immune function. We used distinct Feature Selection algorithms to isolate biomarkers to be fed into machine learning algorithms, whose mapping of the decision process would allow us to propose an early diagnostic tool.We isolated two biomarkers, i.e., Programmed Death Ligand-1 and Myeloperoxidase, that were flagged by the interpretation of an Artificial Neural Network. The upregulation of both biomarkers was indicated as contributing to increase the severity level in sepsis  and septic shock patients.In conclusion, we built a function considering biomarker concentrations to explain severity among sepsis, sepsis COVID, and septic shock patients. The rules of this function include biomarkers with known medical, biological, and immunological activity, favoring the development of an early diagnosis system based in knowledge extracted from artificial intelligence. They caThe fact that sepsis  and its severe subset  meet the criteria for sepsis-3 definition allows the employment of biomarkers as an early diagnostic tool . We seleThe inflammatory pathway of the diseases explored here leaves traces behind that might be employed in profiling the severity of the diseases themselves , 7. ManyIt has been stated  that a dIn this paper, we hypothesize that the severity of sepsis  and septic shock patients is a function of the concentration of biomarkers. For that, we aim to use feature selection algorithms that will isolate biomarkers as candidates for distinguishing the severity of multi-organ failure in sepsis, sepsis COVID, and septic shock patients. With subsets of the selected biomarkers, we aim to evaluate these subsets through interpretable Artificial Neural Networks, so the biomarker concentration that defines the severity of patients with sepsis, sepsis COVID, and septic shock can be used as an early diagnostic tool.22.1All the samples used in this study were obtained from a critically ill cohort of Intensive Care Unit (ICU) sepsis, sepsis COVID, and septic shock patients at St James’s Hospital in Dublin, Ireland. Institutional Research Board approval was granted by the SJH/TUH Joint Research Ethics Committee and The Health Research Consent Declaration Committee (HRCDC) under the register number REC: 2020-05 List 17 and project ID 0428. Biological samples, clinical findings, and laboratory data were collected at days 0, 3, and 14 after presentation of severe infection to monitor the progression and sepsis-induced immune-paralysis state at different stages of the disease. Sample collection took place from September 2020 to March 2021. Sequential Organ Failure Assessment (SOFA) score was obtained on admission to the ICU and at the matching collection timepoints for samples. The clinical variables for white blood cell (WBC) count (worst record of day 0), neutrophils (day 0), positive culture, and up to five comorbidities were attributed to each patient.2.2The concentration of biomarkers with potential altered functions in sepsis, sepsis COVID, and septic shock  patientsThe plasma concentration of both sIL6R and sGP130 was evaluated and quantified with Enzyme-Linked Immunosorbent Assay (ELISA) kits . These biomarkers were selected due to their crucial role in the IL-6 inflammatory pathway . Finally2.3Each patient in our cohort was assigned a Sequential Organ Failure Assessment (SOFA) score. For classification purposes, we binarized the SOFA score into two groups, i.e., High Degree Multi-Organ Failure (HDMOF) and Low Degree Multi-Organ Failure (LDMOF). We employed a cut-off value of 8, as reported by  to binar2.4All statistical procedures were performed using R. We used the Shapiro-Wilk test to find data distribution. To compare averages between groups, the Kruskal-Wallis test, t test, and ANOVA test were used through the Rstatix package (version 0.7.0). The Kaplan-Meier method under the survival R package (version 3.3-1) was used for calculating survival probabilities for each group . The level of significance was set to 0.05.2.5https://github.com/gustavsganzerla/covid-biomarker/blob/main/XAI/feature_selection.R and https://github.com/gustavsganzerla/covid-biomarker/blob/main/XAI/information_gain.py, respectively.To systematically choose biomarkers that are associated with high and low MOF, a Feature Selection (FS) step was applied, and the outcomes were benchmarked. The FS algorithms chosen for this study are representatives of three distinct classes of these methods. For a wrapper algorithm, we selected Boruta and applied the parameters specified in ; the fil2.6https://github.com/gustavsganzerla/covid-biomarker/blob/main/XAI/classific.R.We selected the algorithms Support Vector Machines (SVM), Random Forest (RF), Classification and Regression Trees (CART), K-Nearest Neighbors (KNN), and deep learning Artificial Neural Networks (ANN) to classify patients’ severity with immune biomarkers as input. For that, we performed a 10-fold cross-validation process on the input data. The test dataset encompassed 20% of the whole data. The first five classification algorithms were implemented in R through the Caret package (version 6.0-9) and their code is available at tf.keras.metrics. The architecture of the ANN has input layers that vary according to the set of biomarkers entered. Next, the model has two fully connected layers with 10 hidden neurons each. We also increased the epochs of the ANN until the error (binary cross entropy) kept dropping. The scripts containing the ANN simulation are available at https://github.com/gustavsganzerla/covid-biomarker/blob/main/XAI/SHAP-ANN.The ANN approach was developed in Python using the Tensorflow library (version 2.8.0). A sigmoid function was used in the output layer to return a probability of a given patient having MOF or not. The outcome was binarized through a confusion matrix built under the default 0.5 decision threshold applied in the outcome of the output neuron. The classes obtained were categorized as follows: True Positives (TPs), True Negatives (TNs), False Positive (FPs), and False Negatives (FNs). The ANN performance was evaluated in terms of accuracy, AUC, FPs, FNs, TPs, TNs, all of which can be found in the module 2.7We used Shapley Additive Explanations (SHAP)  to provikernel.explainer method in the SHAP module. As our classifier is not a tree-based algorithm, the Kernel SHAP is applied. Kernel SHAP will measure the contribution of each input feature to the outcome of the model, it consists of five steps:The SHAP approach is defined as a solid theoretical foundation that may be used to explain any predictive model locally and globally. From this, we employed the Sample all possible coalitions  in the dataset: KZ’ is obtained with the application of KZ’ to the predictive model.The prediction of each KZ’ is computed by the SHAP kernel.The weight of each The model is fitted.i.ϕReturn the SHAP coefficients In conclusion, A flowchart describing the data analytical process employed in this study is available in 33.1We provide p=0.051); however, the low p value indicates a trend among the three groups’ survivability rate, placing septic shock as the highest mortality rate after 28 days. When a 90-days analysis was considered (p = 0.044), where the septic shock group presented the lowest survival probability and sepsis the highest.We also employed a survival analysis by days 28 and 90 Figure 2Finally, to validate the binarization we performed, described in , we sele3.2i) PDL1, IL15, IL6, VCAM, IL1ra, IL1b, IL10, and CCL2 in Boruta; ii) MPO, VCAM, IL1b, VEGFC, IL17a, GMCSF, ANG2, CCL2, IL12, GRANB, and SGP130 in LR; and iii) PDL1, GRANB, IL15, ICAM, and IL1ra in IG.To select a subset of biomarkers that explain the target variables , we performed a feature selection process. We chose three algorithms of distinct classes, namely Boruta, LR, and IG. Each application returned a different subset of biomarkers with varied lengths. We show in 3.3We assessed the classification feasibility with five different machine learning algorithms, i.e., SVM, RF, CART, KNN, and ANN. We fed the classification models with the input variables identified by each one of our three FS algorithms Table 2.i) PDL1, IL15, IL6, VCAM, IL1ra, IL10, IL1b, IL4, and ii) MPO, VCAM, IL1b, VEGFC, IL17a, GMCSF, ANG2, CCL2, IL12, GRANB, and SGP130.The results displayed in 3.4To interpret the decision pattern of each ANN model, a SHAP approach was applied to the training dataset. Since the ANN trained/tested with the biomarkers obtained by the Information Gain approach did not produce satisfactory classification metrics, we opted not to interpret this erroneous classification model. For both Boruta and Lasso regression, we analyzed the SHAP value for each input biomarker. We targeted the LDMOF class out of our train dataset and checked the contribution of each biomarker in predicting the class. First, in Next, in 3.5i) the three diseases of our cohort  and ii) the two severity levels our binarization considered (LDMOF and HDMOF) (p = 0.007) was found only in the distinction of the MPO concentration among HDMOF and LDMOF patients (p = 0.045) in using MPO as a distinguisher of sepsis and sepsis COVID  to look for statistical differences between biomarker concentration and d HDMOF) Figure 64In our study, we could explain the severity of sepsis, sepsis COVID, and septic shock patients as a function of an unbalanced concentration of biomarkers. The input parameters of our function are subsets of biomarkers that explain a dysregulated host immune response. Moreover, we could isolate both MPO and PDL1 as the key contributors to the function.The output parameters of our function are high and low degree of multi-organ failure  of subgroups of patients that all meet sepsis-3 criteria, clinically placing the patients into a wider group, converging with past evidence . Our finNext we showed that the WBC count increased with severity. Neutrophils, the most abundant WBC, were higher in the severe manifestations of the diseases we assessed. Moreover, the WBC and neutrophil count was lower in COVID sepsis, matching previous references of neutrophils being one of the most responsive cells toward bacterial infection . We arguin-silico models.Statistically, no significant differences were found that distinguished our patients based on their severity . We trieAfter selecting optimal input biomarkers, we applied them in a classification system that uses this immunological information to predict the severity trajectory of critically ill patients. For that, our model, when fed with distinct subsets of biomarkers, could predict patients’ severity. In fact, biomarkers have themselves been proposed as good predictors of a plethora of medical conditions –36; on tWe interpret our ANN model by linking the concentration of biomarkers in explaining LDMOF. First, Multiple Organ Dysfunction and even death have been reported to be associated with increased levels of VCAM-1 in adults and neonates diagnosed with sepsis –40. SimiWe spotted four biomarkers that are negatively correlated with LDMOF, granting them a negative effect on a patient’s favorable outcome. First, low concentrations of IL17a have been reported as a good predictor for mortality in sepsis caused by distinct pathogens , 45. NexThe mapping of the XAI model provided meaningful information on the course of dysregulated immune responses and also converged with clinical interpretations regarding the neutrophil count of the disease models . A compelling example is the pro-inflammatory function our model attributed to MPO. This enzyme is primarily produced by the granulocytes of neutrophils. The overexpression of MPO generates harmful chemicals that have a detrimental effect on organ inflammation . Our modIn-silico models are an efficient paradigm of experimentation. Compelling examples are found in big data-based applications that have been assisting in several areas in the medical sciences, such as predicting heart attacks  and septic shock patients. After interpreting the results both clinically and immunologically, we found that there is solid medical and biological evidence for why the upregulation of PDL1 and MPO is a major driver of severity. To this extent, we posit that data mining routines such as the one we proposed be used to identify the biomarkers that can function as part of an early diagnosis system.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by Health Research Consent Declaration Committee (HRCDC) under the register REC: 2020-05 List 17 and project ID 0428. The patients/participants provided their written informed consent to participate in this study.GM, DK, IM-L, AG and AO designed the study. GM worked on the machine learning approach. GM prepared the figures. AO linked the machine’s decision pattern with immunological activity. AO, AA-R, and DK performed the immunoassays. AG, RC, and IM-L managed the clinical aspect of the study. GM, AO, AA-R prepared the draft version. All authors contributed to the article and approved the submitted version.


To evaluate the effects of a single ingestion of bovine lactoferrin (bLF) on oral and throat conditions under a low-humidity environment. A randomized, double-blind, 2-sequence, 2-treatment, and 2-period placebo-controlled crossover trial was conducted. Healthy adult subjects orally ingested bLF dissolved in water, or placebo water, followed by exposure to low humidity ) for 2 h. The primary endpoint was subjective oral and throat discomfort assessed by a visual analog scale (VAS), which positively correlated with the discomfort. Secondary endpoints were unstimulated whole salivary flow rate (UWSFR) and salivary immunoglobulin A (IgA) secretion rate. Overall, 40 subjects were randomly assigned to two sequences (20 each) and 34 were analyzed. The VAS values for oral and throat discomfort in the bLF treatment were significantly lower than in the placebo treatment, whereas UWSFR and IgA secretion rates were comparable between the two treatments. Adverse drug reactions were not observed. Subjective oral and throat discomfort associated with low humidity is suppressed by a single ingestion of bLF. Our findings demonstrate the novel use of bLF in a clinical situation that leverages its unique characteristics. Lactoferrin (LF) is an iron-binding glycoprotein present in bodily fluids such as mammalian breast milk, saliva, tears, and nasal secretions, exhibiting various biological functions ,2,3. TheThe amount of moisture present in the mouth is determined by the equilibrium between salivation and saliva evaporation. Loss of moisture in the mouth due to reduced salivation and/or excessive saliva evaporation can cause xerostomia . PatientThis study was conducted in accordance with the current version of the Declaration of Helsinki  and the This randomized, double-blind, two-sequence, two-treatment, and two-period placebo-controlled crossover trial was conducted at Tamagawa University  between September and October 2022. The alleviation of oral and throat discomforts was expected to be derived from the direct effects of bLF, mucin, and water complexes on oral and throat mucosa. Therefore, we considered that carryover effects were negligible and chose a crossover design with a 1-week washout period. The test had four periods: recruitment, assessment of eligibility, and randomization (1 month); treatment period 1 (1 day); washout (1 week); and treatment period 2 (1 day). The participants were randomly assigned (1:1) to ingest bLF in treatment period 1, followed by a placebo in treatment period 2 (Sequence A), or placebo in treatment period 1, followed by bLF in treatment period 2 (Sequence B). The intervention was conducted once during each treatment period.The participants were healthy adults from Tamagawa University. The inclusion criteria was being aged between 18–65 years. The exclusion criteria were as follows: (1) salivary flow rate less than 0.1 mL/min (diagnostic criteria for xerostomia); (2) disease in the teeth, mouth, pharynx, larynx, esophagus, or respiratory system; (3) consumption of functional foods or use of oral care products that influence the oral environment during the test period; (4) mouth breather; (5) burden on the mouth, pharynx, larynx, esophagus, and respiratory system; (6) smoker; (7) heavy drinker; (8) irregular wake-up time and bedtime; (9) serious disease in the liver, kidney, heart, lung, gastrointestinal tract, blood, endocrine system, and metabolic system; chronic disease; or serious medical history of these; (10) serious food allergy, such as milk or drug allergies, or a serious medical history of these conditions; (11) pregnant, under lactation, or expected to be pregnant during the course of the study; and (12) determined as ineligible for participation in this study, judging from the principal researcher/doctor’s opinions on the findings of their background. The background information confirmed by the principal researcher/doctor included recent hospital records, medication status, subjective symptoms, and basic demographics such as sex, age, body weight, and height. In determining the sample size, guided by an estimation of α error = 0.05, β error = 0.8, δ = 14.0, and σ = 30.0, the sample size was computed as 39 using EZR (version 1.40) leading us to set the target sample size as 40, accounting for the logistical aspects of the experimental procedure.® , cow’s milk-derived bLF concentrate, and it contained 96.3 mg of bLF (96.3% purity). bLF was dissolved in water before oral ingestion. We conducted a preliminary test to determine the optimal dose at which the effects of bLF could be expected and found that bLF was still retained in saliva at 28.8 and 1.3 µg/mL 1 and 2 h, respectively, after a single ingestion of 100 mg of MLF-1®. Therefore, we set the dose as above. The placebo was water used to dissolve MLF-1®. Prepared test foods were introduced into opaque bottles to make them indistinguishable. Then, the allocation manager or their substitute wrote the participants’ numbers on the bottle according to the copy of the randomization list. Moreover, the allocation manager confirmed that the test foods were indistinguishable in appearance, taste, and smell. The trial staff provided the packages to the participants based on the assigned numbers. All the staff in this trial, besides the allocation manager or their substitute, were blinded until the key code was broken.The participants were randomly allocated to sequence A or B by a third-party allocation manager. A randomization list was generated using a stratified method with sex as the stratified factor. The original randomization list was sealed until the key code was broken. The allocation manager or their substitute prepared the test foods. The test foods were bLF and the placebo. The bLF was 100 mg of MLF-1The participants fasted for 2 h before the start of the treatment periods. Before commencement, a thorough health assessment was conducted on the participants, ensuring the absence of any subjective symptoms affecting the oral and throat regions, as well as the absence of fever (<37.5 °C). Following this confirmation, the participants drank water to wash their mouths and spent an hour within a general hydrothermal environment ) to acclimatize. Subsequently, baseline evaluations were performed. Then, they orally ingested the test food and were exposed to low humidity  representative of the indoor environment during winter in Japan ,16. TheyThe primary endpoints were subjective oral and throat discomfort due to low humidity. Total oral and throat discomforts were assessed using the VAS, as described in previous studies ,21. Spect-test. Differences in the frequency of adverse events among the treatments were evaluated using the McNemar test. Demographic data were analyzed using the chi-square test for nominal variables and the t-test for continuous variables. The correlation between the change in the UWSFR and the VAS score was analyzed using Pearson’s correlation coefficient. Statistical analysis was performed using JMP version 14.0  and the level of statistical significance was set at p < 0.05 .Statistical analyses were conducted per the protocol set. The primary and secondary endpoints, VAS value of discomfort, UWSFR, and salivary IgA secretion rate were analyzed using a linear mixed model in which the treatment, period, and baseline values were fixed effects and participant identity was a variable effect. The analysis was conducted in accordance with a closed testing procedure to avoid multiple comparisons. The carryover effect was analyzed using a model in which the interaction between treatment and time was added to the fixed effects of the main analysis model. The differences between the treatments at baseline and between each time point and baseline for each treatment were analyzed using a paired The participants were recruited from 7 to 22 September 2022. In total, 40 healthy adults were enrolled; 20 were assigned to sequence A (bLF first) and the others were assigned to sequence B (placebo first). Overall, 3 participants dropped out due to consent withdrawal and 37 completed the test. In total, 3 other participants were excluded for meeting the exclusion criteria (UWSFR < 0.1 mL/min), leaving 34 for the per-protocol analysis . The demp > 0.10). Regarding specific oral and throat discomforts, the VAS score at the end of the test denoting “Difficulty in swallowing due to dryness”, “Dryness of the throat”, “Dryness of the lips”, and “Level of thirst” were significantly lower in the bLF treatment group than in the placebo treatment group  was negatively correlated with the VAS scores at 2 h regarding total oral and throat discomfort . Side effects or severe adverse events were evaluated in all the participants who ingested the test food. There was one adverse event, namely abdominal pain, after placebo treatment (1/37) and no adverse event after bLF treatment (0/36). No significant difference was observed between the two treatments. The principal doctor denied a causal relationship between ingesting the test food and the adverse event.In a low-humidity environment, oral and throat discomfort increased with time and a single ingestion of bLF before exposure to the environment yielded a notable alleviation of the discomfort in comparison to the placebo. Although the UWSFR following bLF treatment consistently demonstrated higher values across each time point in contrast to the placebo, statistical analysis did not reveal any significant disparity between the two treatments. In addition, it is worth noting that the augmentation in UWSFR was more pronounced at earlier time points subsequent to bLF treatment than after the placebo treatment. For the inclusion of participants, a physician conducted a comprehensive assessment, ensuring their overall health status. For those with a UWSFR less than 0.1 mL/min, the diagnostic criteria for xerostomia were excluded . MoreoveIn this study, we employed the VAS, which was initially developed for diagnosing xerostomia, to meticulously assess distinct instances of oral and throat discomfort. It is worth noting this VAS has been validated and found to exhibit a negative correlation with salivary flow rates . AdditioThe cause of discomfort due to the low-humidity environment was most likely owing to the lack of moisture in the mouth, similar to xerostomia. Therefore, the mechanism by which bLF suppresses the discomfort may involve the maintenance of moisture in the mouth. Our findings suggest three possible mechanisms involved in this maintenance. The first revolves around the possibility of increasing the salivary flow rate. The UWSFR at each time point was higher following the bLF treatment compared to the placebo treatment, albeit without achieving statistical significance. In addition, UWSFR significantly increased over shorter time intervals subsequent to bLF treatment, as opposed to the placebo treatment. These facts support the possibility of an increased UWSFR following bLF treatment. Psychological tension decreases salivation  and a prThe salivary IgA secretion rate is associated with mucosal immunity and protection against foreign substances. Our expectation was that under the conditions of a low humidity environment, alongside the UWSFR, the secretion rate would also decrease. Furthermore, we postulated that bLF administration might mitigate this decline by maintaining the salivary output. However, our observations revealed that these rates remained comparable to those observed in the placebo treatment group. The conclusions drawn from this study point towards a direct influence of bLF on oral and throat mucosa. Consequently, the mechanisms underpinning bLF’s effects are expected to be operational regardless of its delivery form, encompassing various consumable formats such as chewable and liquid products, except for completely insoluble food forms, such as enteric-coated products. In our pilot test, the concentration of bLF in saliva decreased below a few µg/mL in 2 h after its ingestion. Thus, even low concentrations of bLF may effectively suppress oral and throat discomfort caused by low humidity.In this study, we found that the ingestion of bLF suppressed subjective oral and throat discomfort due to a low-humidity environment. In addition, we discussed the possibility that bLF functions via the following mechanisms: modulating the UWSFR, saliva evaporation, and salivary wettability. However, this study had some limitations. Firstly, this method to assess discomfort is subjective because no objective method is generally used for the assessment. Therefore, the results of the subjective evaluation may have been subject to bias. Secondly, the experimental environment itself engendered physiological tension in some participants, potentially influencing the measurement outcomes. Thirdly, we did not evaluate the effects of other bLF-containing food forms than aqueous bLF solution. And finally, we could not measure the bLF concentration in the saliva after ingestion of bLF to confirm whether ingested bLF remained in the mouth. In our forthcoming studies, we intend to address these limitations and conduct a clinical trial to obtain more reliable results. Intake of bLF is beneficial for oral hygiene due to its antibacterial activity. However, knowledge regarding the other uses of bLF for oral health is limited. Based on our findings, we propose a novel use of bLF for oral and throat health that alleviates oral and throat discomfort in drying conditions as well as the possibility of its use as in easily consumable food products.


Leishmania infantum is distributed worldwide and affects humans and domestic and wild mammals. In Europe, specifically in the Mediterranean basin, leishmaniasis is endemic due to the concurrence of the phlebotomine vectors and reservoir mammals, including carnivorous wildlife species and other less studied wild species. In this article, spleen, skin, and eye or oral swabs taken from 134 wild mammals admitted to five wildlife recovery centers in Spain were used. PCR employing fragments of the Repeat region, ITS1, and SSUrRNA were used for detection, and positive samples were processed for sequencing. L. infantum was detected in three out of the nine species analyzed, including European hedgehog, European badger, and red squirrel, with percentages ranging from 11.53 to 35.71%, depending on the species. Most of the species showed higher percentages of positivity in spleen samples than in skin samples. A small number of animals from the remaining six species tested negative, including Algerian hedgehog, stone marten, least weasel, garden dormouse, western polecat, and Egyptian mongoose. Hedgehogs and badgers are good candidates for consideration as epidemiological sentinels and pose a higher risk as potential reservoirs of leishmaniasis based on their percentage of infection and wide distribution.Zoonotic leishmaniasis caused by  Leishmania. The Genus includes more than 20 zoonotic species with a worldwide distribution. Leishmania infantum is the most frequently reported species in the Mediterranean basin, causing a life threatening disease in humans and animals [Phlebotomus have been described as capable of transmitting L. infantum in southern Europe, with the most common being P. perniciosus and P. ariasi, which have a wide distribution in countries such as Italy, Portugal, France, and Spain [Leishmaniasis is a zoonotic disease caused by a protozoan parasite of the genus  animals ,2,3. Cut animals . In the  animals . The mai animals ,6. Aboutnd Spain ,8. Humannd Spain . The rolnd Spain .Vulpes vulpes), jackals (Canis aureus), and wolves (Canis lupus), have been extensively studied due to their similarity to dogs, but other species, such as European hedgehog (Erinaceus europaeus), European badger (Meles meles), common bat (Pipistrellus pipistrellus), rodents , red squirrel (Sciurus vulgaris), European and American minks [Martes martes), among others, have tested positive for L. infantum via both molecular and/or serological methods [In Europe, wild carnivores, like foxes , 26 red squirrels (Sciurus vulgaris), and 14 European badgers (Meles meles). The minimum number of animals included in each sample to detect the disease in these three species was calculated by employing previously described percentages of infection for each species: n = 11 for European badger (26% of infections) [n = 11 for red squirrel (25% of infections) [n = 8 for European hedgehog (34.4% of infections) [Samples were obtained, at convenience, from wildlife recovery centers in Spain. In total, 123 animals from 3 different mammal species were studied, including 83 European hedgehogs (ections) , n = 11 ections) , and n =ections) . The onlections) .n = 11) of other six species were included in the study: Egyptian mongoose (Herpestes ichneumon), Algerian hedgehog , stone marten (Martes foina), least weasel , garden dormouse (Elyomis quercinus), and western polecat (Mustela putorius). In addition, a small number , “La Granja de El Saler” , Mini-zoo , “El Chaparrillo” , and CERI “Iberian birds of prey Studies Center” . The areas of origin of the animals were the regions of Madrid, Castilla–La Mancha, Castilla y León, and the Valencian Community, Spain . All aniL. infantum promastigotes in culture and DNase free water were employed as positive and negative controls, respectively, in each batch.DNA was isolated from the samples using 10 (spleen) or 20 mg (ear skin or liver) of tissue sample based on the kit manufacturer’s recommendations. The Thermo Scientific GeneJet Genomic DNA Purification kit  was employed, and the manufacturer’s protocol was followed, except for DNA elution, which was carried out in 60 µL of elution buffer, instead of 100 µL. Sample derived from L. infantum using three different targets of DNA: Repeat region, SSUrRNA, and ITS1 [L. infantum promastigotes DNA) and negative (DNase free water) controls were employed in each reaction. For all PCR reactions, an initial step at 95 °C for 5 min was used to activate the enzyme, and a last step at 72 °C for 10 min was added for the elongation step. The intermediate steps, as well as other features, are described elsewhere [® Safe DNA gel stain  and visualized under UV light. Positive samples displayed a band on the gel related to the expected size of each target, including positive controls.DNA isolated from each sample was used in the detection of and ITS1 ,19,20. Tand ITS1 . In the and ITS1 . Finallylsewhere . Electro® Terminator v3.1 Cycle Sequencing Kit , following the manufacturer’s protocol. The obtained sequences were manually checked and aligned with Molecular Evolutionary Genetics Analysis (MEGA X) software and FinchTV 1.4.0 software . The consensus sequences of each positive sample were then compared with other available sequences using the BLAST tool  [All positive samples were cleaned and sequenced by a commercial company  using DNA Engine Tetrad 2 Peltier Thermal Cycler (BIO-RAD) and ABI BigDyeMD, USA) . All obtLeishmania species obtained from different authors available at GenBank. The Repeat region was only employed for detection and estimation of the percentage of homology.Phylogenetic trees were constructed using sequences available from GenBank, together with sequences obtained in this study that were of sufficient quality and length. Only trees for SSUrRNA and ITS1 regions are shown, since there were enough sequences from other Trypanosoma cruzi was included as an out-group reference. Sequences of the SSUrRNA region with a minimum length of 320 nucleotides were included, and a sequence of Trypanosoma cruzi was included as an out-group reference. In addition, sequences of L. infantum and other species of Leishmania derived from different sources were included to verify similarity. The evolutionary history was inferred using the Maximum Likelihood method based on the Tamura–Nei model [Sequences of the ITS1 region had a minimum length of 290 nucleotides, and a sequence of ei model . The treei model . BootstrLeishmania spp. reported in wild animals [® Inc., the Landmark, San Francisco, CA, USA) was employed to observe the overlaps between the two maps.The maps were created by taking as reference the map of prevalence of  animals  and the  animals . The AutA descriptive statistical analysis with absolute (n) and relative frequency of infection (%) was carried out, and a confidence level of 95% was estimated by employing the free online tool WinEpi (Working in Epidemiology) . L. infantum in at least one sample (spleen or skin) and one PCR target  tested positive for R target . PositivR target . The higThe area of leishmaniosis reported in humans and animals includes most of the Iberian Peninsula, which is in coincidence with the distribution of some of the species that tested positive in this study, namely European badger and European hedgehog, while red squirrel displayed a smaller area of distribution . The pron = 20)  .Considering all samples, the Repeat Region and the SSUrRNA PCRs displayed similar sensitivity, since 13 and 9 samples were positive, respectively. In total, 4.03% (5/124) and 7.08% (8/113) of positive samples were obtained in spleen and skin, respectively, when employing the Repeat region compared to 6.45% (8/124) and 0.89% (1/113) of positive samples rendered via the SSUrRNA PCR. The European badger displayed a higher number of positive samples when employing the SSUrRNA PCR of the spleen, while higher number of positives were obtained via the Repeat region PCR using the skin in European hedgehogs. The ITS1 tested positive in 4/12 and 1/9 samples in spleen and skin, respectively.All samples positive to PCRs were sequenced and submitted to GenBank, although faint reactions did not render a legible sequence. The accession numbers are as follows: OP594494–OP594504 for sequences of the Repeat region; OP588913–OP588918 for sequences of the SSUrRNA; OP588937 and OP588938 for samples positive at ITS1 PCR. L. infantum, with percentages of homology being 99–100%.A total of 6 sequences of the SSU target, 11 of the Repeat region, and 2 of the ITS1 target were successfully aligned in both directions and subjected to BLAST analysis. The rest of PCR positive samples rendered short sequences, since the signal was low. All of the samples corresponded to Phylogenetic trees were constructed by employing sequences from the two selected targets after cutting the sequences of the primers employed via the PCRs . The RepL. infantum sequence derived from a human in Italy, while the other sequence obtained from the skin of a European hedgehog (19/5763) was aligned in a slightly more distant position. The two sequences of the ITS region were employed to construct the phylogenetic tree with 290 bp. Although ITS1 sequences were grouped very close together in the phylogenetic tree, a slight difference was found between both sequences in this study. One sequence obtained from the spleen of a European hedgehog (18/4608) was closer to a L. infantum. Most of them had already been studied previously for the presence of L. infantum, but either a different target (kDNA) was employed in some of the studies [In this study, a high number of samples derived from wild animals were analyzed to determine the presence of  studies  or a smaThe results obtained for the European badger, with 35.71% of animals testing positive, are in line with other studies conducted in endemic or peri-endemic areas of Spain and Italy, where results ranged from 26 to 53.3%. In both cases, spleen samples were analyzed , with different DNA targets, ITS2, and kDNA ,25. ThisAtelerix and Paraechinus [Erinaceus europaeus [L. infantum, with 14.29% of samples being positive at the spleen or ear skin. These results are slightly lower than those obtained in the studies carried out by Muñoz-Madrid et al. [L. infantum detection was kDNA, which could have influenced this difference in results. kDNA is one of the most widely used targets in the detection of L. infantum, albeit with highly variable results, ranging from studies with no positive samples to studies with remarkably high percentages of positivity [In the case of hedgehogs, most studies focused on species of the genera aechinus ,28,29, wuropaeus ,30. In od et al.   compared to other tissue samples, such as skin (n = 9/113) (9.52% vs. 7.96%) (Leishmania [The results obtained in this study showed a higher percentage of positives using spleen samples (. 7.96%) . This reishmania ,34,36,37ishmania .L. infantum in this study and other studies is the skin, but the results are highly variable. This issue could be due to the difficulty of detecting these parasites in the absence of lesions. In the case of the animals studied here, none presented lesions compatible with L. infantum, which might be one reason that L. infantum is detected in skin samples less frequently than in the spleen. However, this observation was not true in hedgehogs, which displayed a higher percentage of positivity in skin (10.93% vs. 6.67%). Another explanation is that the distribution of the parasite depends on the analyzed species. Our findings agree with other studies, in which hedgehogs show similar or even higher percentages of positivity in skin than in spleen samples [Another tissue sample frequently used for the detection of  samples ,39. It would be interesting to analyze a larger number of less invasive samples, such as eye and oral swabs, which, in this study, have not yielded positive results, but encouraging results can be found in the literature, mainly in dogs and hedgehogs ,38. The L. infantum infection with the distribution of the parasite in the Iberian Peninsula, we considered that the species with the highest risk of being potential reservoirs are badgers and hedgehogs. Also, we would like to highlight that the information provided in this article is relevant to developing conservation strategies and management of wildlife populations. Finally, the research of zoonotic diseases in wildlife species make them sentinels that can be used to explore the epidemiological situation of endemic or emerging diseases.Considering the overlapping distribution of wild species positive for L. infantum in nine wild species of mammals, three of which tested positive for the parasite. The European badger was the species with the highest percentage of infection (35.71%). Lower percentages were found in red squirrel (11.53%) and European hedgehog (14.29%). Considering the distribution of the wild species analyzed and the parasite in the Iberian Peninsula, European hedgehogs and European badger could pose a higher risk of being potential reservoirs of leishmaniasis. Wild animals could act as sentinels to evaluate the epidemic status and predict emerging situations. The present study analyzed the occurrence of


Human papillomavirus (HPV) infection is associated with precancerous lesions and cancer of the genital tract both in women and men. The high incidence of cervical cancer worldwide focused the research on this infection mainly in women and to a lesser extent in men. In this review, we summarized epidemiological, immunological, and diagnostic data associated with HPV and cancer in men. We presented an overview of the main characteristics of HPV and infection in men that are associated with different types of cancer but also associated with male infertility. Men are considered important vectors of HPV transmission to women; therefore, identifying the sexual and social behavioral risk factors associated with HPV infection in men is critical to understand the etiology of the disease. It is also essential to describe how the immune response develops in men during HPV infection or when vaccinated, since this knowledge could help to control the viral transmission to women, decreasing the incidence of cervical cancer, but also could reduce other HPV-associated cancers among men who have sex with men (MSM). Finally, we summarized the methods used over time to detect and genotype HPV genomes, as well as some diagnostic tests that use cellular and viral biomarkers that were identified in HPV-related cancers. According to the World Health Organization, approximately 630 million people are infected with human papillomavirus (HPV) worldwide, with an estimated prevalence of 9–13% . In 2018Papillomaviridae family, and there are five genera  with more than 100 genotypes [Waf1 and p27Kip1 [+ T cells. Regarding MHC-II proteins, E5 prevents the activation of these molecules by inhibiting the degradation of the invariant chain, resulting in its intracellular accumulation and unable to load processed peptides for antigen presentation [The HPVs are members of the enotypes ,5. The Henotypes . These venotypes  to ensurenotypes  and the enotypes . The E7 entation . E5 can entation . E6 and entation .After infection, the HPV genomes are established as episomes. In this stage, the oncoproteins E6 and E7 are produced and regulated by the E2 protein through binding to the long control region (LCR) of HPV. During a productive infection that can lead to tissue lesions, the virus is maintained at a high number of episomes that depend on the cells’ differentiation program. Later on, during the progression from high-grade squamous intraepithelial lesions (HSIL) to cancer, the HPV genome generally is integrated into the cellular genome. This integration, where the circular DNA is disrupted between the E1 and E2 viral genes, leads to the loss of E2 expression and then the upregulated production of E6 and E7 oncoproteins. During the viral genome insertion, the expression of E5 is also lost, which initially functions in maintaining cell proliferation through the EGFR. Altogether, these events ultimately lead to a transformed phenotype .In another context, HPV research is mainly focused on women to study the development of cervical precancerous lesions and cancer, how to prevent these, and the application of ablative therapies and novel immunotherapies that aid in its control . A stratFurthermore, the immune response against HPV infection in men was not very well described. For this reason, the knowledge of how HPV infection develops in men and how the immune response is generated against it are questions that must be answered to help to control the infections in MSW (who could act as transmission vectors between women). However, also, the MSM group is a vulnerable fraction of the population that can be infected with HPV, which, together with HIV infection, increases the risk of developing lesions and cancer in different anatomical sites.This review aims to describe what is known about HPV infection in men and their sexual partners, the associated lesions, the immune response generated against the infection, and the vaccines, as well as laboratory tests to detect HPV infection in men.HPV is the most frequently acquired sexually transmitted infection (STI) worldwide and affects both women and men . Based oAccording to some reports, 50–80% of young men and women who are sexually active will acquire a genital HPV infection by having unprotected sexual intercourse with a person infected with at least one type of HPV at some point in their lives . Comparep = 0.007) and higher sperm DNA fragmentation (SDF) values (p = 0.003) than those HPV-negative [p < 0.001), vitality (p < 0.001), and normal sperm morphology (p < 0.05) as compared to the group of HPV-negative cases [The diagnosis of HPV infection in men mainly occurs when the lesions are apparent. Additionally, uncircumcised men present HPV infection in many genital sites and this was associated with infertility . It was negative . A studyve cases . HPV doeve cases . Anotherve cases . On the ve cases .For these reasons, it is important to develop specific diagnostic tests that allow early detection of HPV infection in men.In recent years, the interest in understanding the burden of HPV infection increased predominantly among heterosexual men . HoweverThe transmission rates of HPV infection are not easy to quantify for various reasons, one of which is that many couples are not monogamous. However, Bogaards and colleagues generated a transmission model and found an estimated transmission probability of 0.8 for HPV16 and 0.93 for HPV18 among heterosexual partners . AdditioSeveral research groups analyzed the infection of different HPV genotypes between individuals of a couple to establish if the individuals share the same HPV genotypes and how transmissibility could occur. In these studies, a variable number of couples participate, but concordances only can be obtained from couples where the HPV status and the genotype or genotypes are known for both partners . These sIn this sense, Nicolau and colleagues showed a complete concordance of the HPV group (HR or LR) in 36.7% of matched couples with an HPV positivity of 70% in men . In contOn the other hand, the prevalence of complete concordance for the same viral type in heterosexual couples was variable among studies ranging from 2.27% to 36.70%. Surprisingly, the sexual partners share only partial HPV genotypes or none at all, as opposed to having the same HPV types, as shown by the low positive concordance in most of these studies  21,34,3,40,41,42HPV is a common infection of the anal canal, and the prevalence varies according to sexual behavior. The highest prevalence (47.2%) was among men who have sex with men (MSM), and the lowest prevalence (12.2%) was among men who have sex with women (MSW) , except The principal risk factors associated with HPV infection in men include present and past sexual behavior, cigarette smoking, and lack of circumcision . HoweverHPV infections are associated with the development of cutaneous, anogenital, and head and neck lesions in men ,48. The In several studies carried out in MSM, MSW, and men who have sex with men and women (MSMW) and showing different types of lesions, genotypes of HR-HPV and LR-HPV were detected . The mosIn the case of normal anal samples, HPV types 6, 11, 16, and 18 were the more frequently found (10% to 14%) . HoweverOn the other hand, samples from normal penis showed a high prevalence of HR-HPV types 16, 51 59, 66 (5–8%), in condyloma lesions the LR types 6 and 11 30–49%), while in penile intraepithelial neoplasia (PIN), HGSIL and penile cancer, the HPV16 was the most prevalent  –49%, whi,61,62. Since HPV types were found in the mucosal epithelia, some authors expanded the search for these types in the oral cavity. They found that in the normal oral cavity of men, HPV16 showed a prevalence of 1% overall , but in One of the most common clinical manifestations of HPV infection is genital warts , also knGlobally, in 2018, anal cancer accounted for about 48,541 new cases and 19,129 deaths. In men, the incidence of anal cancer was 20,196 new cases, and 9,618 deaths, accounting for 0.21% of all new cancer cases . In the p = 0.011) with a high prevalence of HPV16 (35%) and HPV18 (19%) in HIV-positive and a low prevalence in HIV-negative for HPV16 (13%) and HPV18 (5%) in all MSM. Also, the prevalence of HPV16 in anal cancer was two times higher in HIV-positive than in HIV-negative MSM [Anal squamous cell carcinoma (ASCC) is the most common type of anal cancer. In men, 100% of ASCC is attributed to persistent infections by HR-HPVs , with HPtive MSM .On the other hand, a prospective cohort study of HIV-negative MSM found that anal HPV infection in this group is associated with a high risk of HIV acquisition. Thus, the increasing number of HPV types found in HIV-negative men that seroconverted to HIV-positive could reflect a higher number of anal lesions that facilitate HIV acquisition; nevertheless, there are few studies of the participation of HPV in HIV infection acquisition . In thisThe prevalence of infection with any HPV type in the anus in MSM is 1.5 times higher in HIV-positive than in HIV-negative men 79% vs. 47%). A similar trend was observed with MSW, although the prevalence was 3.6 times higher in HIV-positive than in HIV-negative individuals (43% vs. 12%) [% vs. 47%p = 0.022), which could be considered a protective effect [Several risk factors are associated with the development of anal cancer; among them are genital or rectal warts, persistent infections with HR-HPVs, HIV-positive, chronic immunosuppression, regular receptive anal intercourse, chronic hemorrhoids, syphilis, multiple sexual partners, gender, age, and smoking , also bee effect .p < 0.003). These data suggest that these molecules can be used as potential novel targets to predict the development of anal lesions HPV-associated in the future [To search for biomarkers associated with anal cancer, Walker and colleagues (2009) performed an anal cancer study to evaluate molecules that were reported as predictive biomarkers of favorable prognosis or potential targets in different types of cancers ,83,84. Te future ,83,84.p = 0.006). Interestingly, a PD-L1 expression ≥ 10% (TPS category 3 and higher) was significantly more often detected in men (50%) than in women (19.4%). TPS refers to a classification of “tumor proportional score” from 1 to 5. These results indicate that PD-L1 expression can be a prognostic factor for survival, although it is irrespective of the HPV status [Wessely and colleagues also analyzed women and men with anal cancer for expression of programmed death ligand-1 (PD-L1), whose role is to mediate anti-tumor immune responses. This study demonstrated that 64.3% of HPV-positive tumors and 54.5% of HPV-negative tumors express PD-L1, and those patients that express this molecule had better overall survival than patients with PD-L1 negative tumors  and in a cell line derived from it (IC2 cell line). They demonstrated that HPV16 DNA integrates into the chromosome band 8q24.21 at the MYC locus and observed MYC amplification (8–12 copies) in both the IC2 tumor and in the cell line, and in the case of the latter, MYC was overexpressed (3.4-fold) . A similIn a systematic review, Olsen and colleagues performed a meta-analysis evaluating HPV transcriptional activity and the correlation with the overexpression of p16. The researchers estimated the pooled prevalence of HPV DNA from different studies  and the pooled p16 positivity in penile cancer and penile intraepithelial neoplasia (PIN) worldwide. They observed that in penile cancer, the HPV DNA prevalence varies from 11.6% to 100%, but the pooled HPV DNA prevalence was 50.8%, and among patients with PIN, this was 79.8%. In HPV-positive penile cancers, the most common HPV type was HPV16, and the pooled prevalence between penile cancer (68.3%) and PIN (69.8%) were similar. However, the pooled prevalence of p16 positivity was much higher in HPV-positive (79.6%) than in HPV-negative (18.5%) penile cancer. Meanwhile, the overall prevalence of pooled p16 positivity in penile cancer (41.6%) and PIN (49.5%) were similar . These rp = 0.004) [Head and neck squamous cell carcinoma (HNSCC) is a global burden. In men, HNSCC is the fifth most common cancer worldwide. In 2018, this type of cancer accounted for 666,037 new cases and 340,750 deaths in men. This type of cancer is three-fold higher in men than in females worldwide , and app= 0.004) . In addi= 0.004) . The HPV= 0.004) . HPV16 i= 0.004) . It was = 0.004) .On the other hand, HPV-associated viral gene expression was evaluated in HNSCCs. Parfenov and colleagues found a higher expression of E2, E4, and E5 than of E6/E7 when HPV is not integrated into the cellular genome ,100. Onchttp://cbioportal.org accessed on 14 April 2023) with data from HPV-positive HNSCCs of The Cancer Genome Atlas [Cellular gene alterations were also significantly studied in HPV-positive and negative HNSCC and performed with samples from women and men. The gene alterations reported include gene expression alterations, mutations, deletions, copy number amplification (CNA), and fusions . Some peme Atlas ,103.Genes exhibiting increased expression in HPV-positive HNSCC tumors are associated with cellular events such as cell cycle, proliferation, migration, differentiation, apoptosis, DNA replication and repair, innate immune response, and tumor-suppressing . In thisp = 0.03) and also identified that high E5 levels are associated with recurrence-free survival (p = 0.02), although high EGFR levels correlate with decreased recurrence-free and overall survival . Furthermore, a comparison of recurrence-free survival between high levels of E5 and EGFR was inversely proportional [Another important gene implicated in proliferation and differentiation is the EGFR which is altered by mutations, CNA, and deletions ,103,104.ortional . Several tumor suppressor genes present mutations, deletions, and CNA. P53 mutations were found in various tumor cells, causing inactivation and loss of tumor suppressor protein functions, affecting targets such as the cell cycle inhibitor p21, DNA-repair proteins POLK and GADD45, pro-apoptotic proteins BAX and the proliferation-related protein P48 ,112. On A minor proportion of HPV-positive tumors from the TCGA data present 14% of recurrent deletions and 8% of truncating mutations of the gene of TNF receptor-associated factor 3 (TRAF3); this inactivation could allow HPV to evade innate and interferon responses. Therefore, some of these genes can be considered candidate therapeutic targets . In addiIt was also observed that some genes exhibit down-regulated expression in HPV-positive HNSCC like NAP1L2, which codifies for a protein implicated in nucleosome assembly, and KIRREL, which codifies for the NAPH1 protein that participates in cell-to-cell adhesion  101,102,106.Despite the diverse alterations in key cellular genes observed in HPV-positive HNSCCs, more detailed research is needed to establish the sequence of the events by which these alterations occur after HPV infection and that finally allow the development of this type of cancer.The role of immune responses, cellular and humoral, is crucial to determine whether an HPV infection progress to precancerous lesions or cancer. Several factors minimize the exposure of HPV to the immune system since the virus does not infect or multiply in antigen-presenting cells located in the epithelium, does not lyse keratinocytes, and does not cause viremia . SimilarFrom those studies, it was identified that the cells involved in the innate immune response against HPV are natural killer (NK) cells, which are generally present in virus-associated lesions and preneoplasic lesions. However, it was observed that there is resistance of the cells of cancerous lesions to be attacked by NKs, which at the same time present a decreased and restricted activity of immunostimulatory cytokines ,114. Other cells involved in the response against HPV are T lymphocytes, especially cytotoxic T lymphocytes (CTLs), which were attributed to the elimination of virus-infected cells ,116 and HPV infections are more frequent in the MSM population due to the higher prevalence of men with HIV compared to those from the MSW population. In the MSM-HIV positive group, there is an increment in the prevalence of HPV as well as in the number of types detected in the anal canal when the CD4+ T cell counts decrease, which suggests that CD4+ T cells play an important role in the control of HPV infection in this group; hence, broad surveillance is highly recommended ,122. WheTo closely determine the CD4+ T-cell response in the MSM group with anal lesions, peptides derived from HPV16 antigens were used, and they identified that 59.7% and 29.9% of the individuals presented a specific CD4+ T-cell response against E6 and E7, respectively, whereas E6- and E7-specific CD8+ T-cell responses were both around 19%. In this study, the researchers demonstrated that E6-specific CD4+ T-cell response is associated with HSIL regression .A meta-analysis carried out by Litwin and colleagues (2020) to evaluate infiltrating T-Cell markers in cervical carcinogenesis demonstrated that there were lower levels of CD3+, CD4+, and CD8+ cells in uterine cervical lesions than in cancer as compared to normal epithelium. Also, it was observed that the presence of FoxP3+ and CD25+ regulatory tumor-infiltrated T-cells was higher in persistent and precancerous lesions than in uterine cervical cancer. Regulatory T-cells are considered inhibitory of the immune response, so improved outcomes with lower regulatory T-cell levels. Successful evasion of the immune response is required for HPV infections, where infiltrating  T-cells are reduced and regulatory  tumor-infiltrated T-cells increase, allowing cervical disease progression . HoweverOn the other hand, when looking at the humoral immune response against HPV, this is mainly genotype-specific and is directed to the structural viral capsid protein L1, and those are important for virus neutralization . AntibodIn the case of the humoral immune response in men, it was observed that higher levels of serum antibodies against virus-like particles (VLPs) from HPV6 and 16 were detected in MSM having anal HPV infection with or without genital coinfection than MSM having only genital HPV infection. Also, this study demonstrated a strong association between seropositivity to HPV6 and the detection of HPV6 DNA in the anal canal among MSW and MSM. However, seropositivity to HPV16 and the presence of HPV16 DNA in the anal canal were associated only with the MSM group . Little is known about the antibody response against HPV early proteins in men, but one of those studies showed that in a high-risk men population, a high prevalence of E4 antibodies was present in the urethra and associated with the presence of HPV16 DNA, while the presence of E7 antibodies was high when the HPV16 infection was present in the penis . AnotherA vaccine derived from recombinant technology is the prophylactic HPV vaccine, which was shown to prevent human cancer development . Three phttps://www.cdc.gov/vaccines/hcp/vis/what-is-new.html, accessed on 14 April 2023).The third vaccine approved was the nonavalent HPV vaccine (9vHPV) (Merck & Co. Inc) in 2014, which contains the same four types and includes five HR-HPV types 31, 33, 45, 52, and 58. The 9vHPV was originally approved for girls 9–26 years old, and in 2018, the FDA extended the age of approval to be used in women and men from 27 to 45 years old . GardasiThe benefit of the HPV vaccine in reducing female cancer was well studied, and the mathematical models for cervical cancer indicate that the effectiveness of the vaccine in men is low and without much benefit in reducing the disease and is considered not cost-effective. However, men develop other HPV-associated cancers, such as anal, oropharyngeal, and from oral cavity, which are not necessarily covered by herd protection, especially the MSM population that remains positive for HPV-associated diseases . There were some studies that addressed the effectiveness of the HPV vaccine in different men populations. In this sense, Hillman and colleagues, in a randomized placebo-controlled, double-blind study, evaluated the immunogenicity of the 4vHPV vaccine in men in a three-dose scheme  and reported that ≥97.4% of MSW and MSM aged 16 to 26 years seroconverted for all types of HPV present in the vaccine at month 7 . HoweverTo analyze antibody titers in different studies, we used the geometric mean titers (GMT) values reported in milli-Merck units per milliliter (mMU/mL), and compared HPV serostatus at month 7 of men and women who received three doses of the 4vHPV vaccine. In Another cohort study in men aged 27 to 45 years old investigated whether the immunogenicity produced by the 4vHPV vaccine  was similar to that generated in young men (16–26 years). The researchers found that men at month 7 were 100% seropositive for all the HPV types contained in the vaccine. The GMT values (0 and 7 months) for each HPV type in the vaccine were: HPV6 (30–420 mMU/mL), HPV11 (40–517 mMU/mL), HPV16 (40–2229 mMU/mL), and HPV18 (16–300 mMU/mL), where the higher titers were for HPV16 . Thus, tGiuliano and colleagues, working with the same study group of young men aged 16–26 years, observed an efficacy of the vaccine of 65.5% against lesions related to HPV 6, 11, 16, or 18 types in the intention-to-treat population. And in the per-protocol population, an efficacy of 90.4% was observed against external genital lesions development related to HPV 6, 11, 16, or 18 types. The condyloma acuminata and all PIN lesions presented an incidence reduction of 89.4% and 100%, respectively. Although the point efficacy estimates for the boys and men in this study were numerically lower than those for girls and women in previous studies, the confidence intervals overlapped, suggesting that vaccine efficacy may be similar for the two sexes .In another study, Castellsagué and colleagues analyzed the immunogenicity of the 9vHPV vaccine in the group aged 16 to 26 years old with a three-dose regimen in women, MSW, and MSM. At month 7, the authors found a seroconversion of >99% for each of the HPV types included in the vaccine in all participants . The type-specific antibody response for HPV types of the 9vHPV vaccine was lower in MSM than in women or MSW, with a GMT ratio ranging from 0.70 to 0.89 (GMT MSM/GMT women) and 0.59 to 0.75 (GMT MSM/GMT MSW), respectively. In comparison with the type-specific antibody response in women and MSW with a GMT ratio ranging from 0.78 to 0.91 (GMT women/GMT MSW). This study did not present vaccine efficacy against the development of lesions in men .Recently, Drolet and colleagues realized a systematic review and meta-analysis of the population-level impact of vaccination on girls, women, boys, and men against HPV infections and anogenital wart diagnoses. They found, in boys aged 15–19 years and men aged 20–24 years, a 48% and 32% significant reduction in anogenital warts diagnoses, respectively. However, according to the multiple cohort vaccination and high routine vaccination coverage after 5–8 years of HPV vaccination, the percentage of diagnoses varies in boys younger than 20 years from 88% to 1% in countries with single-cohort vaccination and low routine vaccination coverage. Therefore, since many were infected with the vaccine HPV types, the effectiveness of the two-dose vaccine regimen in individuals older than 18 years should be considered, but until now, a three-dose schedule is recommended .The few studies on the immunogenicity of the HPV vaccine in men indicate that vaccination against HPV in men will help prevent the development of genital warts and various types of cancer, such as anal, penile, and head and neck. In addition, the vaccination of boys and men against HPV will also protect their sexual partners from developing lesions associated with HPV infection.https://www.cdc.gov/std/hpv/stdfact-hpv-and-men.htm, accessed on 29 April 2023). However, it is important to start looking for some specific tests, as more studies showed that men also develop HPV-associated malignancies, and herd immunity is not enough to protect them. These studies used some of the different tests that were approved to detect the presence of HPV in women, but those tests have to be standardized according to the type and the body site from which the samples are taken.There is no approved diagnostic test to detect HPV infection in men. The US Centers for Diseases Control and Prevention (CDC) does not recommend screening tests for HPV infection in men in the United States  for oncogenic HPV types. They found that MY09/11 consensus and type-specific PCR primers present sensitivity of 87.9% and 98.3%, respectively, and a specificity of 38.7% vs. 76.1% to detect biopsy-proven CIN2+ . Based oIn a cross-sectional study involving women and men, tissue samples from genital warts were screened for HPV infection using a qPCR assay containing the MY11/GP6 and HVP2/B5 primers to detect and quantitate HPV in genital warts. By using this test, the authors reported a prevalence in men of 33.3% and 66.7% of oncogenic and non-oncogenic HPV types, respectively . In compIn other studies, exfoliated cells, flat lesions, and cancer samples were taken from the penis to detect HPV DNA by using different tests such as reverse line blot, in situ hybridization (ISH), and PCR with different sets of primers. In the different studies, the prevalence reported for exfoliated cells was low for LR-HPV (<30%), this increased when flat lesions were present (43%), but they were not present in penile cancer. However, the prevalence of HR-HPVs in the penis was very high in exfoliated cells samples (40% to 71%), and this prevalence decreased in flat lesions and cancer   129,145,147,148.When samples of the distal urethra in men were taken to detect HPV DNA by reverse line blot hybridization assay or multiple HPV genotyping-Luminex system, many a very different prevalence was observed. The reverse line blot showed a prevalence for LR-HPV of 40% versus 10.8% prevalence detected by the multiple-Luminex, and the prevalence behaved similarly when looking for the HR-HPV types   129,149149. Subsequently, a modified version of PGMY09 and PGMY11 primers was used for PCR in some studies because they showed a higher detection rate of HPV infection than MY09 and MY11 primers. In addition, genotyping was performed by xMAP technology with specific HPV-L1 probes. This PCR test was used in samples of the anal canal in MSM and MSW groups and HIV-positive and -negative men. By using this new PCR, the prevalence in the anal canal for LR-HPV was low for the MSW group (5.4%) and increased in the MSM group (20% to 26%), and it did not seem to be higher in HIV-positive men (19.7%). The prevalence of HR-HPV in the anal canal was low for the MSW group (6.8%) and increased in the MSM group HIV-positive (65.3%)  150,151151. In gIn all these studies, when they looked for the prevalence of type-specific HPV16 and HPV18 was similar independent of the test or the set of primers used. For instance, in warts, penis, or urethra, the prevalence of HPV16 was between 6.9% and 12.9% (except for one study that did not find this HPV type), and for HPV18, the prevalence was from 3.2% to 7.2%. However, in the canal anal, the prevalence contrasts considerably especially in the MSM group. In this case, the lower prevalence of HPV16 was 2.2% in the MSW group, but it went up to 23% prevalence in the MSM group and to 33.7% in those HIV-positive. A similar trend in the prevalence was observed for HPV18, although those were, in general, lower than for HPV16   43,77,1,149,150.On the other hand, when looking at another type of HPV-associated cancer, in the case of HNSCC patients, it is essential to determine the HPV status. According to expert consensus opinions, the College of American Pathologists recommended routine HPV testing as a critical parameter for evaluating the clinical outcome of patients . The curp = 0.02). In comparison, medium/high E6 VL showed a slightly better clinical outcome, although this was not statistically significant (p = 0.67) [Another novel technic is the droplet digital PCR (ddPCR), which recently was used to determine the VL of E6 and L1 genes in OPC samples from women and men. Stevenson and colleagues, using this technique, found that a medium/high L1 VL was associated with a better clinical outcome, and it was statistically significant ( = 0.67) . The decision to use a specific test for the detection of HPV DNA in men is still not clear, since there are different tests in different formats, which were evaluated in the population; however, there is still no conclusive scientific evidence that allows us to determine which of all the diagnostic systems developed so far can give a more accurate diagnosis and prognosis for the disease. Therefore, a systematic analysis of the different HPV DNA detection tests used in the various population studies worldwide must be carried out and will depend on the reproducibility of the data in the different populations, as well as the sensitivity and specificity of the tests to select the most suitable one, as well as decide on the algorithm to follow in terms of the combination of biomarkers and the treatment to follow with the patients.Research about the relationship between HPV infection on cervical cancer is widely carried out due to the high incidence of this cancer worldwide. Nevertheless, these studies focused on men were carried on to a lesser extent because research becomes more complex due to a reduced incidence of all cancers attributable to HPV in men  . AdditioDetection of HPV in men is important as, usually, this group is asymptomatic for the infection. The diagnosis of HPV infection in men only occurs when there are apparent lesions or because of infertility problems; otherwise, men do not have a routinary test such as the Papanicolaou to detect precancerous lesions HPV-associated. Therefore, a selection of an HPV DNA detection test in combination with surrogate biomarkers should be implemented for men for early detection of HPV-associated anogenital lesions.Men’s vaccination is still under review, as herd immunity carried on from vaccinated women should protect them. However, the MSM group is at high risk of HPV-associated cancers, as the herd immunity from the women group does not function for them. Additionally, vaccination coverage worldwide is still too low to gain this type of immune protection. Then, it is relevant to consider this MSM group in the vaccination models, as this will help to prevent the development of various types of HPV-associated cancers, such as anal, penile, and head and neck. In addition, the vaccination of boys and men against HPV will also protect their sexual partners and avoid the spread of the HPV infection.


Aedes, Haemagogus and Sabethes genera were collected from six Brazilian states  and grouped into 246 pools, which were tested for YFV using RT-qPCR. We detected 20 positive pools from Minas Gerais, 5 from Goiás, and 1 from Bahia, including 12 of Hg. janthinomys and 5 of Ae. albopictus. This is the first description of natural YFV infection in this species and warns of the likelihood of urban YFV re-emergence with Ae. albopictus as a potential bridge vector. Three YFV sequences from Hg. janthinomys from Goiás and one from Minas Gerais, as well as one from Ae. albopictus from Minas Gerais were clustered within the 2016–2018 outbreak clade, indicating YFV spread from Midwest and its infection in a main and likely novel bridging vector species. Entomo-virological surveillance is critical for YFV monitoring in Brazil, which could highlight the need to strengthen YFV surveillance, vaccination coverage, and vector control measures.The largest outbreak of sylvatic yellow fever virus (YFV) in eight decades was recorded in Brazil between 2016–2018. Besides human and NHP surveillance, the entomo-virological approach is considered as a complementary tool. For this study, a total of 2904 mosquitoes of the  Orthoflavivirus flavi, the prototype virus of the Orthoflavivirus genus and Flaviviridae family [Aedes aegypti mosquitoes, the main vector, and humans. In the sylvatic cycle, Haemagogus and Sabethes genera mosquitoes act as vectors and non-human primates (NHP) as amplifier hosts, with humans occasionally serving as unintentional hosts [The yellow fever virus (YFV), e family , is an eal hosts .Ae. aegypti in Brazil until the 1960s, the virus remained endemic in forest areas of Northern and Midwestern Brazil , and is occasionally responsible for epizootics and human cases of yellow fever (YF) [Despite the absence of urban cases due to the brief eradication of ver (YF) ,3.The overgrowing human action in natural landscapes provokes an imbalance in ecosystem dynamics, exposing hosts, vectors, and known and unknown viruses to humans. In late 2016, the largest YF outbreak in Brazil in eight decades began in the countryside of Minas Gerais state and swiftly spread to other states of the Southeast Region, which is the most populated in the country and is within the Atlantic Forest biome. Until then, this Region, which registered 1865 human cases and 744 total deaths between 2016 and 2018, was not included under the Brazilian YFV vaccination program [Since NHPs are used as sentinels in sylvatic YFV surveillance due to their high susceptibility to the virus, the occurrence of epizootics implies viral circulation. It warns of the urgency to strengthen prevention and control measures, such as improving vaccination coverage in the area. The late detection and response could end in a severe outbreak, as was recently registered in Brazilian Atlantic Forest ,6,7,8.Entomo-virological surveillance can also be used as an important tool for the early detection of viral circulation and to find an epidemiological link to epizootics and human cases in investigation. Whereas entomological collection is focused on main and potential vectors of YFV in a specific region, viral detection in mosquitoes helps to foresee the escalation of an outbreak and could define the possible vectors implicated in YFV transmission ,8,9,10.Aedes, Haemagogus, and Sabethes genera mosquitoes, grouped into 246 pools and collected by the entomo-virological survey of the Brazilian Ministry of Health in six Brazilian states between 2016 and 2017.The possibility of urban YFV re-emergence in Brazil is feasible due to the YFV’s current dispersion over almost the entire country. In this scenario, sylvatic vectors are closer to urban areas, and opportunistic mosquitoes with vectorial potential could act as bridge vectors between the two transmission cycles ,12, reinThe mosquito samples used in this study came from the YFV entomo-virological survey of the Brazilian Ministry of Health. They were collected in six states  from four of the five Brazilian Regions between January 2016 and April 2017.Mosquitoes were collected by human attraction using hand nets, a polyester net bag of 30 cm in diameter with a 30 cm aluminum handle commonly used by our entomological surveillance team. The number of collectors ranged from two to four persons, depending on the municipality. Collections were done in the morning, preferably during the hours of highest sylvatic YFV vector density, between 9 am and 4 pm, in wild outdoor environments.The collected mosquitoes were transferred by oral suction to the identified cryotubes which were stored in liquid nitrogen at −196 °C and transported to the Department of Arbovirology and Hemorrhagic Fevers of the Evandro Chagas Institute prior to taxonomic identification and further analysis.On a −20 °C refrigerated table and using a stereo microscope Stemi 2000-C , mosquitoes were morphologically identified using dichotomous keys ,16,17,18Based on an adapted protocol , each pog for 10 min and 200 µL of supernatant was used for RNA extraction, which was performed with the Maxwell® 16 Viral Total Nucleic Acid Purification Kit  in the Maxwell® 16 System (Promega) instrument. Alternatively, the QIAamp viral RNA Kit (Qiagen) was used. Since these are mosquito samples, we used the Escherichia coli bacteriophage MS2 as a noncompetitive internal control RNA, which was added in a 2 µL volume in each sample.Pools were centrifuged at 13,000× ® Probe RT-PCR (Qiagen) (Thermo Fisher Scientific) and specific primers and a probe for the YFV 5′ untranslated region [The assay was performed using the QuantiTectd region . The 25 For the noncompetitive internal control RNA detection, the 25 µL reaction had the same composition as the YFV assay, but with the following set of primers and probe: MS2 forward , MS2 reverse , and MS2 probe  .In a 7500 Fast Real-Time PCR system (Thermo Fisher Scientific), the RT-qPCR assays were performed under the following cycling conditions: an initial RT step at 50 °C for 30 min, a denaturation step at 95 °C for 2 min, 45 cycles of 15 s at 95 °C and a final extension step of 1 min at 60 °C. Each sample was analyzed in duplicate and considered as positive when the average cycle threshold (Ct) value was less than 37 for both assays. The assay was validated by positive (YFV-infected mice brain tissue) and negative (nuclease-free water) controls.Aedes albopictus mosquitoes, was sequenced using the MinION® sequencing device  by the Oswaldo Cruz Foundation (FIOCRUZ). The genome assembly was also performed by FIOCRUZ following the methodology described in Giovanetti et al. [In a joint initiative, the AR843690 sample, a pool of i et al. .Other samples were prepared for sequencing by synthesizing first and second strands of complementary DNA, which were obtained with the cDNA Synthesis System Kit  and 400 µM Roche random primer. Agencourt AMPure XP Reagent Kit  magnetic beads were used for cDNA purification and Nextera XT DNA Library Preparation Kit  for cDNA library preparation, with 1 ng of cDNA input. Quantification of cDNA was assessed using Qubit 2.0 Fluorometer (Thermo Fisher Scientific), and the fragments’ size range was evaluated using a 2100 Bioanalyzer Instrument . Sequencing was performed on the MiniSeq platform (Illumina) using the MiniSeq High Output Kit (300 cycles) based on 150 bp paired-end technology.−3 e-value threshold. The contigs were inspected with MEGAN6 [Genome assembly was carried out through a de novo methodology using IDBA-UD v.1.1.3   and SPAdh MEGAN6  to identh MEGAN6 , contigsh MEGAN6 . The fivPhylogenetic inference with maximum likelihood (ML) analysis with 1000 bootstrap iterations  was perfAedes, Haemagogus, and Sabethes genera were collected between January 2016 and April 2017  and Pará (47 pools/954 specimens). Although Minas Gerais was the state with more grouped pools (63 pools), Pará had the most specimens collected during this study, with samples dating only from 2017. On the other hand, Mato Grosso was the state with the fewest mosquitoes sampled, which were only from the Aedes genus. Goiás was the only state with mosquitoes sampled in both years.Mosquitoes of the Haemagogus genus were organized into 50 pools with 620 specimens. Goiás was the only state from which Haemagogus mosquitoes were collected in 2016 (seven pools/128 specimens), and most of those collected in 2017 were from Minas Gerais (32 pools/272 specimens). From the Sabethes genus, 196 specimens were collected and grouped into 41 pools. Following a similar pattern as the Haemagogus genus, in 2016, Sabethes mosquitoes were collected only in Goiás (13 pools/88 specimens) and mostly in Minas Gerais in 2017 (16 pools/57 specimens). Goiás was the only state with no Sabethes mosquitoes collected in 2017. A description of each pool from 2016 and 2017 is available in Mosquitoes from the The 246 samples were tested for YFV and noncompetitive internal control RNA by RT-qPCR. The YFV amplicons were detected (positive) in 26 samples . The MS2From the 26 positive pools, 5 had mosquitoes collected in 2016, all from Jandaia (Goiás—Cerrado), and were sampled over a two-day interval. The other 21 samples were from 2017, and only 1 was sampled in Bahia state (Cocos—Cerrado), while all the others were captured in 10 different municipalities from Minas Gerais state (Atlantic Forest), sampled over a 10-day interval .Hg. janthinomys and one Sa. glaucodaemon, and their Ct value in duplicate ranged from 18.19/18.35 to 31.05/31.49. The only positive pool from Cocos, a Hg. janthinomys pool, had specimens collected during three days in March 2017 and a Ct value in duplicate of 28.62/28.64.The five positive samples from Jandaia consist of four pools of Ae. albopictus from Ituêta) to 35.46/35.75 . The 20 samples consisted of seven pools of Hg. janthinomys, five of Ae. albopictus, two of Ae. argyrothorax, two of Ae. scapularis, two of Ae. serratus, one of Hg. leucocelaenus, and one of Aedes sp. Between those samples, there were nine with only one to two specimens pooled, including two pools of Ae. albopictus, two of Ae. argyrothorax, and two of Ae. serratus.Minas Gerais was the state with the most positive samples, with twenty pools collected in 2017, mainly from Alvarenga, with five positive pools, and Caratinga, and Ituêta, with three positive pools each. Their Ct value in duplicate ranged from 23.82/23.93 (pool of Hg. janthinomys from Jandaia (Goiás), one from Hg. janthinomys from Alvarenga (Minas Gerais), and one from Ae. albopictus from Ituêta (Minas Gerais). Genomes were deposited in GenBank under the following accession numbers: OQ932914 (AR831907), OQ932915 (AR831908), OQ932916 (AR831909), MH329655 (AR843690), and MF370530 (AR843721).Five of the 26 positive samples met the quality control standards and were submitted to whole genome sequencing. The quality control standards are influenced by viral load, which is not usually high in mosquito samples. Three sequences were from The five new genome sequences belonged to the South America I clade. The two sequences from Minas Gerais were close to other sequences from the recent Brazilian outbreak and the three sequences from Goiás grouped together with another viral strain from the state of Goiás, collected in 2015 . InformaIn the last eight decades, YFV has been only maintained in its sylvatic cycle in Brazil and occasionally re-emerged in North, Midwest, and Southern Regions, indicating its constant tendency to spread to the Southeast, which was observed in the recent 2016–2018 outbreak ,35,36,37Haemagogus and Sabethes genera are considered the primary and secondary vectors of sylvatic YFV, respectively, and have a wide geographical distribution in Brazil [Haemagogus mosquitoes represent 21.35% of the 2904 collected mosquitoes and 20.3% of the 246 pools. On the other hand, mosquitoes of the Sabethes genus represent 6.75% of the 2904 collected mosquitoes and 16.6% of the 246 pools. Most mosquitoes of these genera are from Goiás and Minas Gerais, and were collected in 2016 and 2017, respectively. In addition, half of the 26 positive samples are from mosquitoes of the Haemagogus genus—and from these, only one is Hg. leucocelaenus—and another one is from the Sabethes genus, a pool of Sa. glaucodaemon from Jandaia. In the Americas, forest-living mosquitoes of the n Brazil ,8,10. InHg. janthinomys species has been identified as the primary vector of sylvatic YFV [Hg. leucocelaenus, a common secondary vector defined as a primary vector in forests in Southern Brazil in the absence of Hg. janthinomys [Haemagogus-positive pools from the Atlantic Forest support the vector presence and its participation in the maintenance of the 2016–2018 outbreak [In Brazil, the atic YFV ,8,10. Thatic YFV ,39. Hg. thinomys ,35, was Hg. janthinomys pool from Cocos, in the Bahia Cerrado, on the border with Northwestern Minas Gerais, corroborates the YFV spread from Minas Gerais to Bahia and the feasibility of YFV cases occurring in the region, which has a low vaccination coverage in humans, and the presence of main vectors and susceptible NHP [The positive ible NHP .Sabethes genus in general are considered secondary vectors of YFV and could present a potential role in the transmission of YFV in the absence or in low density of primary vectors. However, studies on the detection of YFV in Sa. glaucodaemon species and its role in the sylvatic YFV transmission cycle are scarce [Hg. janthinomys from the same municipality, it is unlikely that Sa. glaucodaemon participates in the active transmission of the virus there.Mosquitoes of the e scarce ,8. Due tAedes mosquitoes represent the majority of samples analyzed in this study: they comprised 71.9% of the 2904 collected mosquitoes and 63% of the 246 pools. This genus comprises species of great importance in public health, such as Ae. aegypti and Ae. albopictus. Ae. aegypti was the first vector discovered to be related to a virus (YFV) [Ae. albopictus has been detected and has spread in Brazil, and today more than 1000 municipalities have reported its presence in the peridomicile and adjacent natural or modified environments. Coincidentally, the highest infestation indices for Ae. albopictus in Brazil are reported mainly in the Southeast Region, where the outbreak occurred [us (YFV) ,41, reproccurred . These soccurred ,43.Aedes species from the Atlantic Forest: Ae. scapularis [Ae. serratus [Ae. argyrothorax; however, the real importance of YFV spillover in these three species in maintaining YFV in nature remains to be determined. Entomo-virological surveillance and experimental studies on vectorial competence could certainly contribute to enlightening their role in sylvatic YFV maintenance. The YFV was previously described in two of the positive apularis ,10,44, aserratus . Indeed,Ae. albopictus has been incriminated as a potential vector for YFV transmission [Based on experimental studies, smission ,43,45. Ssmission , there wAe. albopictus samples among the Minas Gerais pools, no subsequent detection of naturally infected Ae. albopictus in the region was made, thus the species was not implicated as a vector involved in the recent Brazilian YFV outbreak, during which reported cases were associated with the sylvatic transmission cycle [For a mosquito species to be considered as a potential vector for arbovirus transmission, it is necessary to combine criteria of all of the physiological and ecological factors of vector, host, pathogen, and environment that determine the vector status of a given arthropod population. The criteria are: (a) isolation of a specific virus from specimens collected in nature; (b) demonstration of infection in the mosquito following experimental feeding on a viremic host or virus suspension; (c) demonstration of transmission of virus by bite to a vertebrate host or demonstration of transmission through excretion of the virus in salivary fluids; and (d) field evidence confirming association of the mosquito species with the vertebrate population in which the virus infection is occurring ,48. Evenon cycle .Ae. albopictus was experimentally confirmed, and its biological characteristics, including the easy movement between both sylvatic and peri-urban environments associated with cases in humans and NHP, and its geographic distribution in Brazil, reinforce the concern about the risk of YFV re-urbanization in the country [Although it was not related to the outbreak, the vector competence of  country ,43,45.Ae. albopictus pools in this study suggests that this mosquito species could play the role of a bridge vector linking the sylvatic YFV to the urban cycle and establishing an intermediary transmission cycle, as documented in Africa with Ae. simpsoni and other Aedes mosquito species [The natural YFV infection detected in  species ,12, once species .In the phylogenetic analysis, the five sequences obtained in this study are clade-related to others from the 2016–2018 outbreak within South America I genotype of YFV ,50,51,52Hg. janthinomys and two other sequences obtained from NHP, all in 2017 from the Espírito Santo state. The proximity of Minas Gerais municipalities to Espírito Santo could explain their closeness due to virus spread, even with the multiple viral exchanges observed through the deposited sequences during the outbreak, which actually justifies the existence of several sub-clades with the 2017–2018 sequences in the phylogenetic analysis [The other two sequences are from geographically close Minas Gerais municipalities and clustered within the outbreak clade, close to a sequence which was obtained from a analysis .Entomo-virological surveillance has proven to be a crucial strategy in YFV surveillance in Brazil, linking and confirming data of the virus’ active transmission to NHP and humans, as well as shedding light on the involvement of unusual or potential vectors in the maintenance of YFV, which may pave the way for new studies. Ae. aegypti and Ae. albopictus.Analysis of main and potential vectors helps to understand their participation in the spread and maintenance of sylvatic YFV and the possibility of an oncoming re-emergence of urban YFV in Brazil. Furthermore, it could highlight the urgency to strengthen the monitoring of syndromic surveillance and NHP deaths, vaccination coverage, and vector control measures, including