BackgroundPyogenic skin infection (pyoderma) is a bacterial infection of the skin and its appendages. Primary pyoderma is caused by the direct invasion of healthy skin, whereas secondary pyoderma originates in diseased skin as superimposed conditions, such as scabies, pediculosis, wounds, insect bites, and eczema. This study aimed to identify the clinical patterns and risk factors of pyoderma in a pediatric population and to isolate various causative bacteria and determine their susceptibility patterns.MethodologyA prospective study was performed at the Medical College and Hospital, Kolkata, India, for one year (from August 2016 to July 2017), which included all children younger than 12 years with pyoderma attending the outpatient dermatology department . Sterile cotton swabs were used to aseptically collect exudates or pus from lesions and anterior nares, which were used for culture, identification, and antibiotic susceptibility testing of the causative organisms.ResultsStaphylococcus aureus, 18 (11.18%) were coagulase-negative staphylococci, 16 (9.94%) were Streptococcus pyogenes, and 1 (0.62%) was Pseudomonas aeruginosa. All staphylococci were susceptible to vancomycin and linezolid.During the study period, a total of 182 patients were included, 121 (66.48%) of whom had primary pyoderma and 61 (33.52%) of whom had secondary pyoderma. Of the 182 patients, 161 showed bacterial growth on culture: 126 (78.26%) were ConclusionsS. aureus, although the prevalence of methicillin-resistant S. aureus was low in this hospital. Proper identification and antibiogram are required for managing these cases.The most common cause of pyoderma in the pediatric age group is  Staphylococcus and Streptococcus [Pyoderma is defined as a purulent infection of the skin and constitutes one of the most common clinical conditions encountered by dermatologists. Pyoderma is classified as primary or secondary, with the former accounting for infection of healthy skin and the latter occurring from pre-existing skin disease . Primarytococcus .The spectrum of causative pyoderma agents and their antibiotic susceptibility patterns are constantly changing. Increasing antibiotic resistance hinders the effective management of such cases. More potent drugs should not be used for susceptible bacteria because they can lead to the spread of antibiotic resistance. For the successful treatment of pyoderma, detailed knowledge of its clinical patterns, its causative bacteria, and its contemporary antibiotic susceptibility pattern is needed.Staphylococcus.This study aimed to identify the clinical pattern and risk factors of pyoderma in the pediatric population and to isolate, identify, and determine the antibiotic susceptibility pattern of the various causative bacteria in primary and secondary pyoderma. This study also determined the nasal carriage of A prospective, cross-sectional study was performed at the Medical College and Hospital, Kolkata, a tertiary care center in Eastern India, for a period of one year (August 2016 to July 2017), after obtaining ethical clearance from the Institutional Ethics Committee of Medical College Kolkata (MC/KOL/IEC/NON-SPON/104/10-2015 dated 13-10-2015). All children 12 years of age or younger who were treatment-naïve and presented with pyogenic skin infection at the outpatient dermatology department were included in the study. Patients who had previously taken antibiotics for the disease concerned, those who were older than 12 years of age, or who had other infections  were excluded from this study.After obtaining informed consent from the patients’ parents, a detailed history regarding the patients’ age, sex, living conditions, lesion morphology, illness duration, disease onset and progression, ingestion of any drugs, and associated metabolic disorders was recorded, and a thorough clinical examination was performed. Sterile cotton swabs were used to aseptically collect exudates or pus from the lesions. Two samples were taken from the patients’ lesions and two from their anterior nares. In the case of intact pustular lesions, the pustule was ruptured with a sterile needle and material was collected on two sterile swabs. One swab from each set (pus and anterior nares) was used for Gram staining of the smear, and the other was immediately inoculated on blood agar, MacConkey’s agar, and brain heart infusion broth. The inoculated media were incubated at 37°C and kept for 48 hours, with readings taken after 24 and 48 hours as per the standard guidelines.Staphylococcus isolates were tested with cefoxitin (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), erythromycin (15 µg), clindamycin (2 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), amikacin (30 µg), gentamicin (10 µg), doxycycline (30 µg), and linezolid (30 µg) using the disc diffusion method and with vancomycin and teicoplanin using the E-strip method. To determine inducible clindamycin resistance, a D-test was performed with erythromycin and clindamycin discs. All data were entered into an Excel spreadsheet  and analyzed.Organism growth was identified by standard microbiological techniques, including Gram staining, relevant biochemical reactions, and the VITEK 2 Compact instrument . Antibiotic susceptibility testing was performed on Mueller-Hinton agar plates using the Kirby-Bauer disc diffusion method and was interpreted according to the Clinical and Laboratory Standards Institute guidelines (2016 version) . The StaDuring the one-year study period (from August 2016 to July 2017), a total of 182 patients were included. The children were aged from two days to 12 years. There were 13 (7.15%) children younger than one year, 68 (37.36%) were one to four years of age, 52 (28.57%) were five to eight years of age, and 49 (26.92%) were nine to twelve years of age . Of the 182 patients, 97 (53.29%) were male, 85 (46.71%) were female, 119 (65.38%) lived in an urban area, and 63 (34.62%) lived in a rural area.A total of 121 (66.48%) patients had primary pyoderma, and 61 (33.52%) had secondary pyoderma. Among the primary pyoderma cases, impetigo contagiosa (34.07%) was the most common type, followed by folliculitis (20.33%), including both superficial and deep folliculitis, whereas among the secondary pyoderma cases, scabies with secondary infection (14.84%) was more common than eczema with secondary infection (11.54%). Pain, fever, and discharge were the most common clinical features observed in 118 (64.83%) cases, followed by itching and fever in 18 (9.89%) cases. The lesion distribution was more common in the head and neck region in 61 (33.52%) cases, followed by the lower limbs in 36 (19.78%) cases. Pustules were the most common type of lesion observed in 112 (61.54%) cases, followed by crusting and erosion in 30 (16.48%) cases  were coagulase-negative staphylococci, 16 (9.94%) were S. pyogenes, and 1 (0.62%) was P. aeruginosa. The most common clinical condition was impetigo contagiosa  showed bacterial growth in culture whereas 21 (11.54%) had no growth after a 48-hour incubation period. Of these 161 cases, 126 (78.26%) were S. epidermidis (seven), S. lugdunensis (six), S. haemolyticus (two), S. hominis (two), and S. saprophyticus (one). All staphylococci were susceptible to vancomycin, teicoplanin, and linezolid. There were 17 (13.5%) cases of methicillin-resistant S. aureus (MRSA). The staphylococci were also most susceptible to fluoroquinolones and doxycycline  patients were colonized with it, of whom 64 (50.79%) were colonized with methicillin-sensitive S. aureus and nine (7.14%) were colonized with MRSA.Of the 126 cultures of This study, performed in a tertiary care center in Eastern India over a period of one year, included 182 cases of pyoderma in a pediatric population who fulfilled the inclusion criteria. Most 68, 37.36%) of the children were one to four years of age, with 52 (28.57%) in the five-to-eight-year age group. The preponderance of preschool-aged children might be because these children are exposed to unhygienic conditions. Hazarika et al. observed that pyoderma was more frequent in the one-to-five-year age group (52%) [8, 37.36%Most patients presented during the hot and humid months of May (15.52%), June (11.8%), July (13.04%), August (9.93%), and September (10.56%). We also observed a higher prevalence of primary pyoderma in the hot and humid months of May (13.66%), June (11.18%), July (9.93%), August (8.69%), and September (6.83%), whereas secondary pyoderma was more common in the winter months of December (6.84%) and January (6.83%). A study by Banerjee et al. observed that impetigo and furunculosis were more common during the rainy season, whereas scabies was more common during the winter .Primary pyoderma 66.48%) was more common than secondary pyoderma 33.52%). Among the primary pyoderma cases, impetigo contagiosa (34.07%) was the most common, followed by folliculitis (20.33%). Among the secondary pyoderma cases, scabies with secondary infection (14.84%) was more common than eczema with secondary infection (11.54%). In a similar study performed by Singh et al. in Rajasthan, 61% of the cases were primary pyoderma; among these, furuncle (27.2%) was the most common entity, followed by folliculitis (13.2%) and impetigo 9.4%), in contrast to our study, in which impetigo was the most common. Infectious eczematoid dermatitis (12.4%) and infected scabies (6.8%) were the two most common entities among secondary pyoderma [% was mor.4%, in c3.52%. AmS. aureus, or both [S. aureus growth was observed in 126 (78.26%) samples, followed by coagulase-negative staphylococci , S. pyogenes , and one sample showing P. aeruginosa growth (0.62%). Most of the recent studies in India have found that S. aureus is the most common cause of pyoderma in children. This finding has also been demonstrated by Hulmani et al. in Davangere (82/92 of positive cultures were S. aureus) [Pyoderma, impetigo, and impetigo contagiosa are terms used synonymously to describe discrete purulent lesions that are primary skin infections and are extremely prevalent in many parts of the world. In the large majority of cases, pyoderma is caused by β-hemolytic streptococci,  or both . Of the s 16, 9.9%, and on aureus) .S. aureus isolates in our study, 17 (13.5%) were MRSA. In a study performed in Kerala by Rani et al., the most common cause of pyoderma was S. aureus, with 13% being MRSA [S. aureus was completely susceptible to vancomycin, teicoplanin, and linezolid, similar to the findings of Sengupta et al. [Of the 126 tudy, 17 .5% were tudy, 17 .5% were S. aureus infections in Asian countries varies markedly from <5% to >35% [S. aureus cultures grown from the pyoderma samples of our patients, 73 (57.94%) patients were colonized with the bacteria, nine (7.14%) of whom were colonized with MRSA. Nasal colonization with S. aureus was observed in 59.7% of the patients in a study performed in Bengaluru [S. aureus nasal colonization was 52.3% and that of MRSA was 3.89% [It has been found that the proportion of MRSA among community-associated  to >35% . Of the engaluru . A commuas 3.89% .One limitation of this study was that a molecular characterization of the resistant bacteria was not performed. Another limitation was that most of the patients did not return for follow-up, possibly because they were cured.S. aureus, although the prevalence of MRSA was low, and they were all susceptible to vancomycin, teicoplanin, and linezolid. A thorough understanding of the clinical patterns of pyoderma, its causative agents, and its antimicrobial susceptibility patterns is essential for managing cases and preventing the spread of antimicrobial resistance.This study showed the pattern of pyoderma in pediatric age groups in a tertiary care center in Eastern India. More patients had primary pyoderma in the form of pustules, with the most common location being the head and neck region. Pyoderma cases were more prevalent in the hot and humid months of May, June, July, and August. The most common cause of pyoderma was


Salmonella, widely distributed in food-producing animals, has been considered the first priority for inclusion in the AMR surveillance program by the World Health Organization (WHO). Recent advances in rapid and affordable whole-genome sequencing (WGS) techniques lead to the emergence of WGS as a one-stop test to predict the antimicrobial susceptibility. Since the variation of sequencing and minimum inhibitory concentration (MIC) measurement methods could result in different results, this study aimed to develop WGS-based random forest models for predicting MIC values of 24 drugs using data generated from the same laboratories in Taiwan. The WGS data have been transformed as a feature vector of 10-mers for machine learning. Based on rigorous validation and independent tests, a good performance was obtained with an average mean absolute error (MAE) less than 1 for both validation and independent test. Feature selection was then applied to identify top-ranked 10-mers that can further improve the prediction performance. For surveillance purposes, the genome sequence-based machine learning methods could be utilized to monitor the difference between predicted and experimental MIC, where a large difference might be worthy of investigation on the emerging genomic determinants.Antimicrobial resistance (AMR) is a global health issue and surveillance of AMR can be useful for understanding AMR trends and planning intervention strategies. The online version contains supplementary material available at 10.1186/s13567-023-01141-5. Salmonella, belonging to the Enterobacteriaceae family, is a gram-negative rods bacillus. They are widely distributed in animals and are prevalent in food-producing animals, including cattle, porcine, and poultry  using the independent test dataset.Additional file 11. Prediction performance of ResFinder using the independent test dataset.


In(Ga)N epitaxial layers were grown on on-axis and off-axis (0001) sapphire substrates with an about 1100 nm-thick GaN buffer layer stack using organometallic chemical vapor deposition at 600 °C. The In(Ga)N layers consisted of a thin (~10–25 nm) continuous layer of small conical pyramids in which large conical pyramids with an approximate height of 50–80 nm were randomly distributed. The large pyramids were grown above the edge-type dislocations which originated in the GaN buffer; the dislocations did not penetrate the large, isolated pyramids. The large pyramids were well crystallized and relaxed with a small quantity of defects, such as dislocations, preferentially located at the contact zones of adjacent pyramids. The low temperature (6.5 K) photoluminescence spectra showed one clear maximum at 853 meV with a full width at half maximum (FWHM) of 75 meV and 859 meV with a FWHM of 80 meV for the off-axis and on-axis samples, respectively. Recent advances in the epitaxial growth of indium nitride layers led to the correction of the optical InN bandgap to 0.65 eV (previously expected to be 1.9 eV)  and open1-xGaxN were the chemical vapor deposition using organometallic precursors (MOCVD or MOVPE)  orientation). This corresponds to the basic idea that the presence of oriented steps in the GaN buffer layer served as nucleation centres for the growth of In(Ga)N pyramids and could diminish their twisting. However, the steps with different local density and height could emphasize the In(Ga)N pyramid tilting.The lack or presence of threading dislocations in the large pyramids was observed by TEM. The large pyramids were grown on the top of edge-type threading dislocations originating in the GaN layer a,b, but g.b = 0 is satisfied. There, g is the operating reflection and b is the dislocation Burgers vector [The edge-character of dislocations was confirmed by observing the contrast of the strained lattice in the vicinity of a dislocation. The contrast is missing  if sample is oriented in such a way that the invisibility criterion for dislocations s vector . Table 2Although the edge-type threading dislocations did not penetrate the large, isolated pyramids, the situation was different for pyramids that merged into larger clusters. xGa1-xN alloy. The PL maxima were at 853 meV with a FWHM of 75 meV and at 859 meV with a FWHM of 80 meV for the off-axis and on-axis samples, respectively. The on-axis sample exhibited a lower intensity of PL radiation.1-xGaxN layers was found -grown samples, leading to much narrower band-tails.The PL parameters are very similar to the published results on the relation between PL spectra shapes and composition of Inn of 30% . Tuna etn of 83% . They peIn 1-xGaxN pyramids and randomly distributed large In1-xGaxN pyramids with density of ~1 × 109 cm−2. In both samples, symmetric (2θ/ω) XRD scans showed wide continuous double maxima related to In(Ga)N materials with different Ga molar fractions because the pyramids are relaxed, so the changes of c-parameter could not be caused by different strains. The calculated Ga incorporation into the InN lattice was higher for the off-axis sample, which can be ascribed to the smaller average height of the large pyramids if one expects that the Ga incorporation in In1-xGaxN volume decreased with the increasing distance from the In1-xGaxN/GaN interface indicated by the double maxima found in the XRD results and also by the bent side walls of the large pyramids. However, the accuracy of Ga incorporation estimation is low due to weak and wide maxima of measured diffractions.The presented analyses suggest that the microstructure of both samples—on-axis and off-axis—was very similar with some interesting differences. As a result of self-assembled growth both samples consisted of a continuous thin layer of small InIt seems that presence of oriented cracks in GaN template grown on off-axis substrate indicating different mechanism of strain accommodation in GaN in this case did not influence significantly growth and properties of large In(Ga)N pyramids.1-xGaxN deposition.The TEM observations showed that large pyramids are grown over the edge threading dislocation. Their relationship could be explained by mechanism of self-assembled growth. As we have mentioned in the introduction, small island formation appeared very soon at the beginning of the growth to reduce the compressive strain energy due to the high misfit to the substrate. The islands were preferentially created on threading dislocations or surface steps—zones with locally decreased compression strain where larger In atoms can be preferentially located ,32,33,34The large pyramids were well crystallized and mainly free of dislocations. The strain was relieved by the creation of 60° misfit dislocation networks on the island/GaN interfaces as it is obvious for hetero-epitaxial systems with a high misfit , thus thWe need to point out that the densities of dislocations calculated from FWHM of ω-scans are only a theoretic expression of tilting and twisting misorientation of epitaxially grown pyramids here. The basic necessary conditions of homogeneous distribution of threading dislocations in the layer and the fact that the lattice misorientations are realized only (or mainly) by this threading dislocation system are not fulfilled. We used this concept because it is widely used, sometimes without any control if the supposed conditions are satisfied. Thus it could be sometimes confusing.1-xGaxN lattice quality and the PL spectra.The strongly coalesced small pyramids with the higher molar fraction of gallium were much more defective as the large ones. A possible reason that the PL spectra did not show any maxima splitting caused by the In(Ga)N crystals with two different Ga molar fractions was the presence of large quantity of recombination centres in the thin layer consisting of the small pyramids, effectively suppressing their PL emission. In such case, the PL emission from the on-axis sample should be stronger than that from the off-axis sample because of the higher total volume of on-axis large pyramids (the on-axis large pyramids had the slightly average higher density and height). Surprisingly, our observation was the opposite and the reason is not quite clear. The explanation needs further investigation, i.e., a correlation between the microstructure and In3 and trimethylindium (TMIn) as the respective N and In precursors we deposited In(Ga)N layers with unintentionally incorporated Ga in InN lattice. The In(Ga)N layers consisted of a thin (~10–25 nm) continuous layer of small conical pyramids in which large conical pyramids with the approximate height of 50–80 nm were randomly distributed. The Ga incorporation were inhomogeneous, the Ga ratio were higher in the thin layer of small pyramids, and further, the Ga incorporation gradient was observed in large pyramids. The large pyramids were grown above the edge-type dislocations which originated in the GaN buffer; the dislocations did not penetrate the large, isolated pyramids. The large pyramids were well crystallized and relaxed with only a small quantity of defects, such as dislocations located at the contact zones of coalesced pyramids. The low temperature (6.5 K) photoluminescence spectra showed one clear maximum at 853 meV with a full width at half maximum (FWHM) of 75 meV and 859 meV with a FWHM of 80 meV for the off-axis and on-axis samples, respectively. It can be explained by suppressing PL emission from the thin layer consisting of the small pyramids due to presence of large quantity of recombination centres and by recombination kinetics in an inhomogeneous material with fluctuating band-gaps. Excited electrons and holes relax preferentially to the positions in the sample with the narrowest band-gap, which could be very similar in the both samples.Using only NH


Despite some controversy, increases or decreases, respectively, of brain dopamine activity induced by drugs or that occur physiologically, usually improves or worsens, respectively, sexual activity. These findings suggest that an altered central dopaminergic tone plays a role in mental pathologies characterized by aberrant sexual behavior, and that pro-erectile D4 receptor agonists may be considered a new strategy for the treatment of erectile dysfunction in men.Early and recent studies show that dopamine through its neuronal systems and receptor subtypes plays different roles in the control of male sexual behavior. These studies show that (i) the mesolimbic/mesocortical dopaminergic system plays a key role in the preparatory phase of sexual behavior, e.g., in sexual arousal, motivation and reward, whereas the nigrostriatal system controls the sensory-motor coordination necessary for copulation, (ii) the incertohypothalamic system is involved in the consummatory aspects of sexual behavior (penile erection and copulation), but evidence for its role in sexual motivation is also available, (iii) the pro-sexual effects of dopamine occur in concert with neural systems interconnecting the hypothalamus and preoptic area with the spinal cord, ventral tegmental area and other limbic brain areas and (iv) D Therenaline ,9. Togetrenaline ,11, and renaline ,18,19,20renaline ). Among renaline ,23,24, mrenaline ,26), thorenaline ,16,27,28renaline ,30,31 (T1 and D5) and D2  receptor families, has led in recent years to the synthesis of molecules that act selectively as agonists or antagonists of these dopamine receptor subtypes. This has reopened the research aimed at defining the exact role of the different dopamine receptor subtypes, mainly of the D2 family , present in the areas of the brain controlling the different aspects of sexual behavior , from sexual motivation to erectile function and copulatory behavior  to copulatory behavior with a receptive female, in the species most studied so far, e.g., rats, but also other animal species including men. The aim is to show first to what extent the results of the recent studies confirm or confute the old theories suggesting that dopamine facilitates sexual behavior, and second, to present an updated description of the lastest discoveries and identify new possible strategies for the therapy of erectile dysfunction and/or other related sexual dysfunctions by modifying the activity of dopamine at the central level. The studies selected for review were chosen from Pubmed and Google Scholar medlines done with search terms such as dopamine; dopamine agonists and antagonists; erectile function; and male sexual behavior in different animal species, from rat to man only, on the basis of the presence of experiments aimed at studying the sexual role of dopamine in Penile erection is produced by a complex neural central and peripheral interaction which induces muscle and vascular changes in the erectile tissues of the male genital apparatus . This interaction is also complicated by humoral and endocrine influences, mediated mainly by testosterone and its metabolites, which occur at the central and peripheral levels 5-Tyr(Me)2-Orn8-vasotocin, Pen1,Phe(Me)2,Thr4,Orn8-oxytocin and d(CH2)5-Tyr(Me)2-Arg8-vasopressin given intracerebroventricularly, but not in the PVN, with a potency parallel to their rank order in blocking oxytocinergic receptors . T. T255]. 2 and D4 subtype ..296].The above differences in extracellular dopamine in the nucleus accumbens dialysate found in DAT-KO, HET and WT rats took place in concomitance with modifications in sexual activity, as shown by the different number of non-contact penile erection episodes seen when the female rat was inaccessible and the changes in mount, intromission and ejaculation frequencies recorded during copulation in the three rat sub-lines. Accordingly, (i) HET rats displayed more non-contact penile erections than DAT-KO and WT rats as well; (ii) HET and, to a lesser extent, DAT-KO rats displayed more mounts than WT rats; and (iii) DAT-KO rats displayed more ejaculations (higher ejaculation frequency) than WT and, to a lesser extent, HET rats. In addition, in line with the findings of the last two tests done before microdialysis: (i) DAT-KO and HET rats show lower mount, intromission and ejaculation latencies than WT rats; (ii) HET rats show higher and DAT-KO rats lower mount frequency than WT rats; and (iii) DAT-KO rats show lower intromission frequency and higher ejaculation frequency than WT and HET rats, but to a lesser extent . TogetheThe studies reviewed above show that central dopaminergic systems play a main role in both the anticipatory/appetitive  and/or consummatory  phases of male sexual behavior, although with different relevance in these two phases among them. In fact, although it is commonly accepted that mesolimbic/mesocortical dopaminergic neurons are those playing the main role in the anticipatory components of male sexual behavior ,250,257,The main involvement of mesolimbic/mesocortical dopaminergic neurons in the anticipatory/appetitive phases of copulatory behavior and in sexual arousal/motivation as well was confirmed by biochemical studies showing that dopamine neurotransmission  is activated more in the nucleus accumbens compared to the striatum during the anticipatory phase of copulatory behavior of sexually naive male rats with a sexually receptive, but not non-receptive female rat or in sexually experienced male rats with a receptive female rat ,306,307.Altogether, the studies reviewed above confirm and extend the results of earlier studies that support the hypothesis that the mesolimbic dopaminergic system plays a main role in the anticipatory/motivational phase, whereas the nigrostriatal dopaminergic system supplies the sensory-motor coordination necessary for the consummation of the behavior. In addition, these studies also show that the incertohypothalamic dopaminergic neurons are involved in sexual motivation, possibly by interacting with mesolimbic dopaminergic neurons. Finally, the results of these studies provide evidence that copulatory behavior is also rewarding and that its rewarding effects are mediated by increased dopamine release in the nucleus accumbens, as with that of other rewarding stimuli, i.e., feeding, drinking and self-stimulation ,308,309.The studies reviewed in this work confirm and extend the results of earlier studies suggesting that dopamine exerts a facilitatory role in erectile function and sexual behavior in male rats. Since in male rats mixed dopamine and D2-like receptor agonists induce penile erection , and among the main effects of these compounds on copulation are either a decrease in mount and intromission latency and an increase in ejaculation frequency, dopamine was assumed to improve both sexual motivation and sexual performance in male rats. Accordingly, among the copulatory parameters measured , changes in mount latency only are considered to reflect modifications in sexual motivation , whereas the other parameters provide only insights on the consummatory phase of the behavior, or on the rate at which copulation takes place. As recalled above , the fac2)5Tyr(Me)2-Orn8-vasotocin given intracerebroventricularly [The role of dopamine in both sexual arousal and motivation and in sexual performance was definitively confirmed when extracellular dopamine and its metabolites were found increased in the dialysate from the nucleus accumbens ,142,143 icularly  or into icularly . Togetheicularly . Thus, ticularly ,311,312)icularly ,313,314)icularly .More recent studies on this matter show that the extracellular dopamine increase in the nucleus accumbens and medial prefrontal cortex is correlated to the dopaminergic tone of the male rats, which determines their pattern of copulatory performance. Accordingly, rat sub-lines with a grater dopaminergic tone than their control counterparts obtained by selective breeding protocols over multiple generations for showing opposite specific behavioral tracts , a specific spontaneous behavioral response (LY versus HY rats) or by genetic mutations (DAT-KO versus HET and WT rats) show improved erectile function and copulatory behavior and specific patterns of copulation that occur in parallel with changes in dopamine activity in the nucleus accumbens and in the medial prefrontal cortex when compared to their lower dopaminergic tone counterparts ,296,316.The fact that rat sub-lines psychogenetically selected for showing specific (often opposite and contrasting) traits or even to show a spontaneous behavior or not, over multiple generations, show similar differences in sexual behavior and that a different dopaminergic tone present in these rat sub-lines, which resembles that of DAT-KO rats versus HET and WT rats, plays a main role in these differences deserves some comment.First, it is feasible that the behavioral differences between the above rat sub-lines are related to genetic and environmental factors, which concur with modifications in sexual behavior, related to alterations in the function of brain neurotransmitters .This raises the possibility that, depending on the behavioral criteria on which the psychogenetic selection is based, phenotypes may be generated that are strongly related to each other not only in behavioral traits but also in sharing central and/or peripheral neural and neuroendocrine substrates of their different behavioral patterns. This makes psychogenetically selected animal sub-lines important animal models to study associations between neurotransmitters function and behavior, including sexual behavior in physiological conditions or in the presence of brain lesions, mental illness or after the administration of psychoactive drugs ,316. In Second, it is also likely that associations between dopamine function and alterations in copulatory behavior observed in these rat sub-lines, also take place in humans. Although it is difficult to translate rodents’ sexual behavior parameters to human sexual behavior, this is highly likely and would be also not surprising (see ). IndeedThird, although many neurotransmitters and/or neuropeptides  may play a role in the determination of trait and behavioral impulsivity, excessive levels of sexual arousal and hypersexuality, hypersexuality and paraphilias  can be observed in Parkinson’s disease patients treated with dopaminomimetic drugs (from L-DOPA to direct dopamine receptor agonists) with the severity of these symptoms decreasing by reducing the dose of or by discontinuing the dopaminomimetic drugs used  [4 receptors are also involved, since (i) selective D4 receptor agonists induce penile erection when given systemically or into the PVN [4 receptor agonists also facilitate male rat copulatory behavior, an effect that is abolished by the prior treatment with selective D4 receptor antagonists, which exert only minor effects on the improvement of copulatory behavior induced by mixed D1/D2 dopamine receptor agonists [4 receptor agonists on erectile function and the copulatory behavior found in male rats may be the basis of a new pharmacological strategy for the therapy of erectile dysfunction in men. Accordingly, a few selective D4 dopamine receptor agonists , given at doses found able to induce penile erection and to facilitate sexual behavior in male rats [4 receptor agonists devoid of emetic properties are certainly worthy to be tested for their clinical use in men affected by erectile dysfunction, as already done for the mixed D1/D2 receptor agonist apomorphine. As recalled above, a formulation for the sublingual administration of this compound was made commercially available in the 1990s for the therapy of erectile dysfunction, but the success of this formulation was scarce  due to the emetic properties of this compound, which are incompatible with sexual intercourse [4 receptor agonists that facilitate erectile function but are devoid of emetic properties would result in a new strategy for the treatment of erectile dysfunction in men.In conclusion, the studies reviewed in this work confirm also that the facilitatory role of dopamine in erectile function and copulatory behavior is mediated mainly by dopamine receptors of the D2 family and show in particular that dopamine Dic area) . However the PVN ,155, andagonists ,151. Theale rats ,70, haveale rats . If bothercourse ,151. Thu


Nanocarriers are gaining significant importance in the modern era of drug delivery. Nanofiber technology is one of the prime paradigms in nanotechnology for various biomedical and theranostic applications. Nanofibers obtained after successful electrospinning subjected to surface functionalized for drug delivery, biomedical, tissue engineering, biosensing, cell imaging and wound dressing application. Surface functionalization entirely changes physicochemical and biological properties of nanofibers. In physicochemical properties, wettability, melting point, glass transition temperature, and initial decomposition temperature significantly change offer several advantageous for nanofibers. Similarly, biological properties include cell adhesion, biocompatibility, and proliferation, also changes by functionalization of nanofibers. Various natural and synthetic materials polymers, metals, carbon materials, functional groups, proteins, and peptides, are currently used for surface modification of nanofibers. Various research studies across the globe demonstrated the usefulness of surface functionalized nanofibers in tissue engineering, wound healing, skin cancers, melanoma, and disease diagnosis. The delivery of drug through surface functionalized nanofibers results in improved permeation and bioavailability of drug which is important for better targeting of disease and therapeutic efficacy. This review provides a comprehensive insight about various techniques of surface functionalization of nanofibers along with its biomedical applications, toxicity assessment and global patent scenario. Nanofibers have attracted considerable massive attention and emerged as novel nanomaterials due to potential physicochemical properties, surfactant free and, commercial potential ,2. TheirVarious methods for fabrication of nanofibers are summarized in Electrospun nanofiber is a multifaceted approach and received greater attention owing to the diverse prospective applications for drug delivery carriers, biomedical devices, and tissue engineering scaffolds ,8. ElectThe various techniques employed for the characterization of surface functionalization nanofibers are scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), Atomic force microscopy (AFM), X-ray diffraction (XRD), X-ray photoelectron microscopy (XPS), Energy-dispersive X-ray spectroscopy (EDAX), BET analysis and mercury porosimetry, etc. ,14.The extensively employed technique for the manufacture of nanofiber is electrospinning. It is considering as a simple, cost effectiveness and versatile method. These surface functionalized nanofibers have been particularly used in tissue engineering and wound healing because nanofibrous membranes resemble the natural extracellular matrix and that can foster the proliferation and migration of cells. Electrospun nanofibers can be used in wound healing due to its antibacterial activity, able to promote rapid hemostasis and having good biocompatibility to promote cell growth. There are various techniques employed for surface functionalization of nanofibers .Considering several applications of the electrospun nanofibers, there is a need to focus on safety issues. Additionally, safety of the materials used for human application must be examined according to ICH guidelines. In vitro evaluation in animal models and clinical testing in humans should be undertaken to assess the toxicity profile of nanofibers after surface functionalization. Therefore, sufficient toxicity profile is required for human use in accordance with the standard regulatory guidelines .The present review is focused to give detail information of electrospinning technique, methods of surface functionalization, reagents used for surface functionalization, and toxicity evaluation. Moreover, patent scenario and applications of surface functionalized nanofibers have also been discussed. This manuscript is the comprehensive literature providing valuable information on every aspect related to electrospinning and surface functionalization of nanofibers for advanced applications in the field of healthcare and biomedical sciences.Electrospinning is a traditional technique used for the fabrication of the submicron to nanoscale size nanofiber with different morphological structure such as non-woven, core-sheath, porous, aligned nanofiber. This different morphology is used for different applications . FabricaThis is a melt-based technique used for the development of polymeric nanofiber from the melted form of polymer and also, use for the elimination of non-volatile organic solvent from polymeric nanofiber fabricating process. Melt electrospinning set up quietly analogous to traditional electrospinning set up, they consist of a spinneret, collector, and high voltage supply. In this electrospinning technique, the molten stream of the polymer is forcibly expelled through the spinneret and the polymeric nanofiber mechanically drawn to the grounded collector with continual flow where solidification is attained by the help of rapid cooling to form nanofiber scaffold for biomedical application. However, the individual diameter of nanofiber developed by this technique generally is larger than compared to nanofiber obtained from the traditional technique because of high viscosity of molten polymer ,24. MeltIn recent years, co-axial electrospinning technique attracts to the researcher for formation of complex product of polymeric nanofiber for various biomedical applications. Advancement of this technique is beneficial for dual drug loading as well as sustained release application along with improving mechanical strength and functional properties without change in surface chemistry of polymeric nanofiber ,29. AddiTo overcome some limitations of the co-axial or blend electrospinning technique, emulsion electrospinning emerged as a novel alternative approach for the fabrication of core-shell nanofiber by preparing core-sheath emulsified solution . AccordiThis technique is also termed as Forcespinning™, centrifugal spinning, and rotor spinning. RJS has some advantages to other electrospinning methods, such as eco-friendly, inexpensive, aligned nanofiber mats as compared to the conventional high-speed rotary drum collector method. The quality as well as size of nanofiber is easily tunable by the help of processing condition. The disadvantage for RJS is it requires high temperature for fabrication process ,43. In tSeveral methods reported in the literature for surface functionalization of the nanofibers such as physical method include plasma treatment, physical vapor deposition, and ion beam implantation. Surface functionalization using chemical technologies involves surface grafting, cross-linking and chelation. These methods suffer from potential drawback and are not optimal. In the physical method, adsorption binding forces are weak, whereas chemical immobilization involves a harsh reaction condition, such as use of organic solvents, high temperature, and prolonged reaction time, which generate toxic allergens, cause degradation of material this method not suitable for biomedical application. The surface chemical composition of nanofibers is easily changed by plasma treatment method. After the plasma treatment, the change in the surface wettability and biological properties is reported in the literature. Plasma source selection is important for plasma treatment is based on its diverse functional group incorporated into nanofibers. Plasma source should be of sufficient strength to reach target surface, and covalent immobilization of bioactive molecules achieved. The surface hydrophilicity of nanofibers increases by air or argon plasma treatment . Plasma New technique in coating of nanofibers surface by physical vapor deposition uses nitrogen, oxygen, and acetylene, etc. These gases introduced into chamber during deposition for surface coating. It involves evaporation and metallic sputter coating of surface of nanofibers .The surface of polymeric nanofiber modified with ion beam implantation method. The modification with nitrogen ion treatment introduced amine or amide functionality on nanofibers surface. These functionalities on surface of nanofibers promote cell compatibility. This method has advantageous of controllability and reproducibility of results for surface coating. In the literature, PVA Poly  nanofibers treated with ion beam implantation to study the mechanical properties .The surface modification by chemical method includes surface grafting, cross-linking, and chelation, etc.2) by EDC cross coupling reaction on PLGA surface. First, carboxy group of Poly (acrylic acid) cross linked with amino group of PAMAM (G5·NH2) dendrimers, which further grafted to PLGA nanofibrous scaffold for gene delivery, as shown in PLGA electrospun nanofibers of uniform morphology pre-treated with positive charge diallyl dimethyl ammonium chloride and negatively charge Poly (acrylic acid) then immobilized of poly(amidoamine) (PAMAM) dendrimers electrospun nanofibers obtained by electrospinning were subjected to chemical modification by cross-linking with diamine, ethylene glycol, and thioamide. These nanofibers can be efficiently explored for biomedical application. The schematic presentation for chelation nanofiber as shown in 2 low plasma treatment was applied to replace routein roughening conventional process with sulphuric acid and potassium dichromate. In this work electroless deposition of copper on uniformly cover individual nanofibers surface for significantly change in surface morphology and conductivity observed and high surface area of Polyamide 6 nanofibers retained intactly. Similarly, nickel, gold, and copper used in the literature to give uniforms thickness of 160–400 nm ..73].Zinc oxide- and silver-based nanomaterials are also used for surface modification of nanofibers which resulted and significant efficiency. Hota et al., reported Surface functionalization of electrospun PAN nanofibers with ZnO-Ag heterostructure nanoparticles. In this research, surface functionalization of electrospun PAN nanofibers with ZnO-Ag heterostructure nanoparticles was catalyzed by various chemical pathways. The functionalized nanofiber showed antibacterial property. In the inhibition study it was shown that these surface modified nanofibers are excellent antimicrobial agents against E.coli and micrococcus leuteus ,75.Nanohydroxyapatite is a calcium-containing chemical compound. It is used to connect nanofibers to scientific labs for research into new potential treatments. Zhang et al., reported a bioinspired surface-functionalization approach to improve the osteogenesis of electrospun polycaprolactone (PCL) nanofibers. In this study, a versatile and effective approach was suggested to improve PCL nanofibers using bioactive nanohydroxyapetite using dopamine as an efficacious bioadhesive agent. In addition to woven, weft/interweave and PCL/PDHA copolymer nanofibers, PCL-PDHA blends significantly improved the bioactivity of nanofibers . The in Collagen is the most abundant structural protein in animals. Collagen is also used for surface modifications of electrospun nanofibers . Zhang eE. coli K12 was 800 microgram/mL for each CS and pDADMAC, as soon as immobilized, pDADMAC—functionalized nanofiber mats exhibited a higher inactivation of E. coli k12 (~97%) ..95].S. aureus, which is immobilized on to biocompatible fiber of cellulose, i.e., cellulose-chitosan. The lysostaphin functionalized cellulose fibers showed an activity against the S. aureus in an in vitro skin model which is obtained after processed as a bandage preparation. Moreover, it showed low toxicity towards keratinocytes, which will suggest its good compatibility and hence it can be a good antimicrobial material in wound healing. In this study, they prepared cellulose fibers by electrospinning solution of cellulose, cellulose-chitosan and cellulose poly (methylmethacrylate). These all are tested for the tensile strength testing, which showed reasonable mechanical strength (6 +/− 0.5 MPa) for oxidized cellulose fiber mats and tested for antimicrobial activity against S. aureus from which it is observed that lysostaphin-based cellulose fibers showed complete counteract of S. aureus population and hence it can be considered as an anti-infective, antistaphylococcal, biocompatible and stable bandages for wound healing application  using polymerization approach and fabricated electrospun nanofibers with gelatine. The authors synthesized GO by oxidizing pristine graphite powder and the incorporation of acetyl chloride into it, to offer an atom transfer radical polymerization (ATRP) microinitiator (GO-CL). With the help of gelatine, uniform, conductive and biocompatible nanofibers were prepared by electrospinning which were investigated for its various properties. The biocompatibility of this electrospun nanofibers were confirmed by assessing survival rate of human osteoblast MG-63 cells on nanofibers using MTT assay. Also, the electrospun nanofibers were analyzed for electrical conductivity which were found to be proper conductive for scaffolding. Authors reported simple, convenient and effective approach to preparation of GO-g-[P(HEM-g-CL)]/gelatine electrospun nanofibers with adequate biocompatibility, biodegradability and conductivity for different applications .An application of surface functionalization in planning some targeted strategies such as tumor targeting has been explored. Patil and his associates reported versatile surface functionalization technique for polymeric nanoparticles. The fact is when a dipole co-polymer, polymeric ethylene glycol (PLA-PEG) is introduced in the oil/water emulsion which is used in the polymeric nanoparticle formulation, PLA blocks the partitions into polymer containing organic phase while PEG blocks partitions into aqueous phase. Nanoparticles with PEG on the surface can be obtained after desolvation. The study facilitates IAASE approach, by simultaneous incorporation of biotin and folic acid which is known as tumor targeting ligands on drug loaded nanoparticles, which was confirmed by NMR, TEM, surface plasmon resonance and tumor cell uptake studies. Authors claimed that the new surface functionalization technique enables the development of targeted strategies employing multiple ligands on a single surface to target a particular tissue .Surface functionalization a simple technique can be used to functionalize the surface of nanofibers to enhance number of activities like antibacterial, antimicrobial, for developing targeted strategies, etc. Following is the case study related with applications of surface functionalization in increased anti-bacterial activity.Nirmala et al., prepared and characterized chitosan blended polyamide-6 nano fibers for human osteoblastic (HOB) cell culture applications, which was prepared by electrospinning process. In this study, they used chitosan which is a natural biopolymer which is non-toxic, possessing polycationic chelating and film forming properties, because of its active amino and hydroxy functional group.Chitosan is used because of its biodegradability, biocompatibility, non-toxicity, antimicrobial activity, and absorption potentiality. Also, an in vitro cytotoxicity study reveals that polyamide-6/chitosan blended nanofibers were non-toxic for the osteoblastic cell culture and their characterization using FT-IR spectroscopy indicates its increased stability due to amide group terminated polyamide-6 nanofibers and also it is observed that the bands were corresponding to amide II and amide III be decreased with increasing chitosan content ,104.The nanofiber technology become prevalent to overcome recent challenges in the biomedical field viz. burn and wound care, organ repair and treatment of various diseases. Even nanofibers can be engineered into advanced macro-scale structures. Moreover, activity and physicochemical properties of nanofibers could be enhanced via surface functionalization for biomedical applications, diagnosis, and targeted drug delivery.Wang and associates reported aminopropyltriethoxysilane (APTS)-mediated surface modification of nanohydroxyapatite employing various surface functional groups for potential biomedical applications. Authors tried to modulate nanohydroxyapatite surface with APTS and APTS amine groups which was further altered with acetic anhydride and succinic anhydride to produce neutral surface charge and nanohydroxyapatite particles with negative charged. These nanohydroxyapatite derivatives were further characterized and evaluated for cytotoxicity with the help of MTT  colorimetric assay of mouse fibroblasts (L929 cells). For these functionalized derivatives, in vitro hemolysis assay assessed for the determination of blood compatibility. The present research can be used for development of nanohydroxyapatite based composite materials for different biomedical application .Nanofibers have various application like immobilization of bioactive molecule. Electrospun nanofibers have been widely utilized as support materials for enzyme immobilization due to their various merits, including large specific surface area, high porosity, and interconnectivity which in turn helps in crosslinking, covalent coupling, encapsulation, etc.2 (PLGA-b-PEG-NH2) block copolymer was used to produce surface functionalized nanofiber. The synthesized nanofiber was prepared by primary amine on surface using lysoenzyme as a model enzyme. It immobilized lysozyme on its surface with improved activity due to its huge surface area [Kim et al., synthesized nanofiber by blend of the biodegradable poly (e-caprolactone) (PCL)and poly -poly(ethylene glycol)-NHace area .Electrospun nanofibers revealed as capable approach for implantable stage in cancer treatment, on-site delivery of therapeutic compounds and marginal adverse effect to normal tissue. Xu et al., developed BCNU—loaded implantable functionalized electrospun nanofiber for cancer treatment by using poly (ethylene-glycol)-poly (Lactic acid) (PEG-PLLA) diblock copolymer with controlled release of 1,3-bis(2-chloroethyl)-1 nitrourea(BCNU). ESEM  showed very smooth and uniformed BCNU-Loaded PEG-LLA fiber formation. The resulting nanofibers have antitumor activity with controlled release effect, and it is suitable for postoperative chemotherapy of cancer.Magnetic nanofibers are commonly used in various application like cancer treatment which responds to external magnetic field. As magnetic NPs can be precisely implanted in the tumor site which provides high loading and superior magnetic response. Susikala et al., reported implantable smart magnetic nanofiber for endoscopic hyperthermia treatment and tumor-triggered controlled drug release prepared using two component smart nanofiber matrix from mono disperse iron oxide nanoparticle (IONPs) and bortezmib (BTZ) as chemotherapeutic agent. The mussel-inspired surface functionalization using 2- ethylamine (dopamine) was done to conjugate the borate-containing BTZ anticancer drug through a catechol metal binding in pH-sensitive manner. It is applicable in both endoscopic hyperthermia and triggered controlled dung release. The functionalizes have an advantage due to low toxicity toward normal cell and highly effective in consideration of therapeutic efficacy .Cancer is already considered as the lethal disease in the world. It has been reported that liver cancer caused 466,100 new cases and 422,100 deaths in China alone in 2015. Chemotherapy is the most usual approach for treatment of liver cancer .2 nanofiber conjugated with folic acid. According to characterization results of SEM and TEM showed hollow structure of average outer diameter of 200 nm and wall thickness was 500 nm. The rate of drug release and dissolution profile of PTX was significantly improved. In vivo and in vitro experimentation showed the efficient inhibiting effect on the growth of liver cancer cells [Wu et al., fabricated the PTX -loaded mesoporous hollow SnOer cells .E. coli. The collection and inactivation of Escherichia coli K12 was examined using cellulose nanofiber mats that were surface-functionalized using three polyelectrolytes: poly (acrylic acid) (PAA), chitosan (CS), and polydiallyldimethylammonium chloride (pDADMAC). Hydrophilicity paired with neutral or positive charge improved the collection of E. coli K12, whereas hydrophilic cationic nanofiber mats exhibited the highest killing of E. coli K12 [Rieger et al., work on the concept of mat hydrophilicity and surface chemistry by polyelectrolyte functionalization nanofiber to control the collection and inactivation of coli K12 .Nanofiber plays the promising approach in the cell culture design. The morphological features of nanofibers simulate extracellular matrix that is in vivo microenvironment hence these are favorable substrates for regenerative medicine. Nanofibers used for the culture including human pluripotent stem cells known as oligopeptide-immobilized nanofibers. These are fabricated by employing synthetic or natural polymers, and ECM-modified nanofiber.Nirmala et al., developed chitosan blended polyamide-6 nanofibers by a via electrospinning process for human osteoblastic (HOB) cell culture applications using novel single solvent system. FT-IR and SEM showed the effective result, i.e., proper adhesion, viability and proliferation properties of osteoblast on the polyamide-6/chitosan blended nanofibers were analyzed by in vitro cell compatibility test .Li juanu et al., developed nanofiber functionalized magnetically responsive heparin—immobilized cellulose nanofiber composites were synthesized by wet—wet electrospining method by using—1-methyl-3-methyl imidazolin acelate ((EMIM)AC) into an aqueous coagulation bath Synthetic fiber developed in three types of nano composite fiber. These nano composite fibers were characterized by Thermogravimetric analysis, Liquid chromatography mass spectrometer fourier transform infra-red X-ray diffraction Spectroscopy. It showed anticogulant activity .Toxicity study is most important and, critical aspect for assessment of lethal dose and, noxious effect of surface functionalized nanofiber. In study report this can be determined by In vitro, In vivo assay. In vitro hemocompatibility of nanofiber can be examined by the hemolysis and cell viability assay to confirm the safety as well as biocompatibility of functionalized nanofiber material. MTT assay used for the assessment of metabolic activity of cells ,110. ActNanofibers are having broad spectrum biomedical, healthcare and theranostic applications. The nanotechnology is becoming prime choice of drug delivery across the globe due to its efficiency in drug delivery and disease targeting. The nanofibers are one of the prime carriers used across the world and contributing to the large extent to global pharmaceutical market. The surface functionalized nanofiber is the preferred choice of drug delivery in various infections, diseases, and disorders. The recent advancements in surface functionalization techniques are exploring the scope of multiple biomedical applications. In the future, the hybridization of two or more surface functionalization techniques for nanofibers may result in efficient drug delivery. The large scope of nanofiber technology in healthcare may result in improved global importance of nanofibers with large significant contribution to the economy of the pharma sector.


To the Editor:The authors reported no conflicts of interest.Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.The ,We thank Dr Timek for his comments regarding the elegant approach to investigating the implications of incorporating (reefing) the anterior mitral leaflet to the anterior annulus.We wish to clarify the issues that Dr Timek has raised with regard to left ventricular outflow tract obstruction and aortic valve incompetence. The pressure components of the hemodynamic parameters altered by the reefing intervention were measured with a solid-state pressure catheter in the left ventricle. We have demonstrated in a separate sheep model that left ventricular outflow tract obstruction causes an increase in intraventricular pressure rather than a decrease.Both reefing the anterior leaflet and insertion of the mitral prosthesis can cause aortic valve incompetence. We performed a detailed echocardiographic assessment at the time of initial reefing and releasing of the anterior leaflet in each sheep. This included an assessment of the left ventricular outflow tract. We did not repeat this assessment for subsequent interventions in the absence of suspicion based on altered hemodynamic parameters.We hope that our findings do add to the basic understanding of mitral valve physiology and acknowledge that there are limitations to the clinical extrapolation. We remain grateful to Dr Timek for his leadership in this field of research.


KMT2D and KDM6A. Here, we describe two cases of suspected KS that met clinical diagnostic criteria and had a high gestalt match on the artificial intelligence platform Face2Gene. Although initial KS testing was negative, genome‐wide DNA methylation (DNAm) was instrumental in guiding genome sequencing workflow to establish definitive molecular diagnoses. In one case, a positive DNAm signature for KMT2D led to the identification of a cryptic variant in KDM6A by genome sequencing; for the other case, a DNAm signature different from KS led to the detection of another diagnosis in the KS differential, CDK13‐related disorder. This approach illustrates the clinical utility of DNAm signatures in the diagnostic workflow for the genome analyst or clinical geneticist—especially for disorders with overlapping clinical phenotypes.Kabuki syndrome (KS) is a neurodevelopmental disorder characterized by hypotonia, intellectual disability, skeletal anomalies, and postnatal growth restriction. The characteristic facial appearance is not pathognomonic for KS as several other conditions demonstrate overlapping features. For 20‐30% of children with a clinical diagnosis of KS, no causal variant is identified by conventional genetic testing of the two associated genes,  We and others have previously described a gene‐specific KMT2D and KDM6A). DNAm analysis demonstrated a positive KS signature in one patient congruent with the subsequent result on genome sequencing that identified a single exon duplication in the KDM6A gene, consistent with a diagnosis of KS. In the other patient, a DNAm signature different from KMT2D and controls, suggested the genomic alteration was not in either KMT2D or KDM6A. Genome sequencing identified a pathogenic variant in CDK13, which causes a syndrome with cardinal distinctive features overlapping KS called CDK13‐related disorder.It is important to determine the exact genetic etiology in patients who meet the clinical diagnostic criteria for KS because the information informs recurrence risk, prenatal testing options, and potential gene‐targeted therapies , the two patients, along with individuals with pathogenic KMT2D (n = 9) and KDM6A (n = 1) variants at The Center for Applied Genomics, SickKids Research Institute. Whole‐blood genomic DNA from each subject was sodium bisulfite converted using the EpiTect Bisulfite Kit , according to the manufacturer's protocol. Modified genomic DNA was then processed and analyzed on the Infinium HumanMethylationEPIC BeadChip (Illumina 850K) according to the manufacturer's protocol. The raw IDAT files were converted into beta‐values, which represent DNAm levels as a percentage (between 0 and 1), using the minfi Bioconductor package in R as previously reported  model as previously described  , thus classifying samples as “KS” (high scores) or “not‐KS” (low scores). This SVM model was built as a tool for the classification of variants in KMT2D and KDM6A as previously described . Separate consent was obtained to use a 2‐dimensional facial photograph in the Face2Gene platform and to perform DNAm analysis (REB #1000066122).33.1A 9‐year‐old boy of British and Bulgarian ancestry, born to non‐consanguineous parents was first seen in the neonatal period for a genetics consult when he presented with congenital hyperinsulinism. His subsequent medical issues included global developmental delay, postnatal growth retardation, hypotonia in infancy, cortical visual impairment, seizures, and severe gastrointestinal reflux disease requiring G‐tube feeds. Magnetic resonsance imaging (MRI) of the brain showed remote bilateral matrix hemorrhages, but no structural malformations. He had renal cysts detected on ultrasound and a normal echocardiogram. His clinical examination at 10 months of age was in keeping with KS and in addition to his characteristic facial features Figure , he had KDM6A and KMT2D gene sequencing and multiplex ligation‐dependent probe amplification (MLPA) analysis and exome sequencing were all non‐diagnostic. DNAm analysis showed that this patient clearly classified as positive with the KMT2D signature , microcephaly, hypotonia, severe feeding issues in infancy, postnatal growth retardation, and esotropia. A brain MRI showed no structural abnormalities. She had a normal renal ultrasound and echocardiogram. On clinical examination, she was noted to have dysmorphic features consistent with KS, including high arched eyebrows and eversion of the lower eyelids Figure  and fetaKDM6A and KMT2D genes) via a commercial platform, which did not identify any pathogenic variants. Since we were still highly suspicious of a diagnosis of KS, we performed DNAm analysis. Unexpectedly, she had a unique DNAm profile that did not cluster with either typically developing controls or with individuals with KS and pathogenic KMT2D or KDM6A variants ‐ an intermediate SVM score (SVM score = 0.4) —also known as congenital heart defects, dysmorphic facial features, and intellectual developmental disorder—has previously been noted to present with a KS‐like phenotype  Figure . We perf4KMT2D or KDM6A. A functional test such as genome‐wide DNAm can be a useful adjunct to defining an accurate diagnosis. Here, we present two patients that illustrate how this approach can either reliably confirm that further assessment of a candidate gene is indicated or that an alternative diagnosis is more likely, in which case further testing using exome or genome sequencing should focus on potential pathogenic variants in other genes, in particular those that can be associated with overlapping phenotypes, for example, CDK13‐related disorder. For our patient with CDK13‐related disorder, although the variant would have been detected on exome sequencing, we opted for genome sequencing as this was available to us through a research study and provided the more comprehensive testing option. Genome sequencing has the added benefit that analytical detection of CNVs is at least equivalent to chromosomal microarray analysis, which is not the case for exome sequencing (Marshall et al., CDK13 variant impacts genome‐wide DNAm, the mechanisms of disease likely include features of epigenetic dysregulation. These data also suggest the existence of a gene‐specific CDK13 signature which is under investigation. Although CDK13‐related disorders have a similar facial gestalt to Kabuki, the distinctness of the genomic targets identified by the methylation pattern in Patient 2, indicate that the former has a separate pathogenic mechanism. This suggests that CDK13‐related disorder is likely to be a distinct clinical syndrome as opposed to “Kabuki syndrome 3.” As we learn more about the basic biology of conditions with overlapping phenotypes, the inclusion of both genotype and phenotype data into nomenclature, as recently proposed by Biesecker et al. (Despite the development of International Consensus criteria for the diagnosis of KS, it is useful to have alternative avenues to identify a molecular diagnosis if testing does not confirm a pathogenic variant in r et al.  will allKMT2D. At this time, there is not a DNAm signature for CDK13, although we anticipate in the future this signature will be defined. However, not all genes will be associated with a DNAm signature. If DNAm is not diagnostic, then reflexing to genomic sequencing would be the next logical step. A positive DNAm classification can establish a diagnosis, but additional testing would be required to identify the pathogenic sequence variant. Since DNAm analysis does not identify the causal variant, it cannot be a stand‐alone first‐line test. DNAm testing also has important limitations, which must be considered when the testing is utilized in a clinical context but might not be widely understood. The DNAm signatures developed are only validated for peripheral blood samples and may not apply to other tissue types. Sample size used for generating the signature, age at the time of blood sampling and other external factors can lead to reduced reproducibility in reliably using a specific signature for diagnosis (Chater‐Diehl et al., We suggest that in cases of suspected KS with negative targeted panel testing, utilization of genome‐wide DNAm and consideration of facial phenotyping tools could help provide direction for further genetic testing to improve the efficiency of the diagnostic workflow. DNAm can be used to streamline further genetic testing—in some cases suggesting more detailed analysis of a specific gene and in other cases expanding to genome‐wide sequencing, which is more costly and not universally available in the clinical setting. The utility of DNAm profiling in classifying VUS , arising from genomic sequencing, has already been demonstrated for a large number of rare neurodevelopmental disorders caused by pathogenic variants in genes that affect epigenetic regulation (Choufani et al., The authors have no conflict of interests to declare.Ashish Marwaha is the primary author of the manuscript, gathered clinical report data and helped direct data analysis. Gregory Costain performed the whole genome sequencing analysis and reviewed the manuscript. Cheryl Cytrynbaum and Roberto Mendoza‐Londono contributed to the clinical data reported and reviewed the manuscript. Lauren Chad reviewed the manuscript. Zain Awamleh, Eric Chater‐Diehl, and Sanaa Choufani performed methylation sequencing and analysis and reviewed the manuscript. Rosanna Weksberg secured ethics approval and funding for the data collection and analysis, she also co‐wrote the manuscript, directed data analysis/presentation and is corresponding author.


Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype–phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds ; (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436  Furthermore, NGS high-throughput platforms, which in recent years have seen radical improvements in quality, running times and cost, have revolutionised the diagnosis of health-related issues in animals and humans. This includes infectious disease diagnosis, where in-silico (or computer based) genetic data analysis is aiding and, in some cases, substituting more complex and costly laboratory techniques4. The COVID-19 pandemic is a testimony of the usefulness of this technology for both research and surveillance6. Similarly, by using NGS technologies such as sequencing the whole genome of bacterial isolates, transmission chains of pathogens and a global overview of their population structure is being identified, helping inform surveillance and to trace outbreaks7. For antimicrobial resistance (AMR) detection in bacteria, which is another global threat leading to decreasing therapeutic options and increasing treatment failures, antimicrobial susceptibility testing which produces a phenotype, is progressively being substituted by detection of the underlying genetic mechanisms using whole genome sequencing (WGS) of bacterial isolates. Nevertheless, performing correlations between pheno- and geno-types remains essential, as phenotypes are still accepted as the gold standard due to genotypes being based only on already known AMR genes so new variants may be missed. WGS analysis also facilitates the identification of bacteria such as Escherichia coli, their lineage, and plasmids, in addition to genetic features such as resistance to critically important antimicrobials for therapeutics, and chromosomal mutations, deletions and insertions that may be associated with AMR phenotypes13. Characterisation of bacterial plasmids, which often transfer AMR genes due to their mobility, is particularly important and has become more achievable by combining short and long read WGS so complete AMR plasmid genomes can be determined16.Next Generation Sequencing (NGS), a DNA sequencing technology, has become an established technique with hundreds of publications each year detailing the use and advancement of this technology, often replacing other gene-based typing tools such as PCRs, and microarrays17 reported at least 47 bioinformatics tools were freely available, and no doubt this has increased further. However, to understand the epidemiology of AMR in an One-Health context, it is vital to harmonise in-silico AMR detection methods, as has been established for bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA), where ideal requirements for molecular typing techniques have been clearly defined19. There have been similar discussions for in-silico AMR detection20, and although the recommendations have not been properly evaluated, a small multi-centre study with nine institutes that performed predictions of AMR genotypes from 10 samples harbouring carbapenem-resistant organisms, showed that differences in the database selected and gene coverage thresholds were some of the factors contributing to variation in AMR results21. Such evaluations are required at a much larger scale because supranational organisations such as the World Health Organisation (WHO), European Centre for Disease Control (ECDC) and the European Food Safety Authority (EFSA) have recommended the use of genomics within international surveillance programmes that compare AMR trends across countries in Europe and worldwide, to help tackle the spread of multi- and extensive drug resistant bacteria which are the cause of great concern24.However, a major barrier to the application of bioinformatics software for AMR detection beyond individual research applications, to diagnosis and national and/or international surveillance, is the standardisation of both DNA-based laboratory techniques and in-silico analysis. The availability of a plethora of bioinformatics tools and pipelines, with continual rapid advancement in this area, has resulted in no standardised methodology or nomenclature making comparisons across compartments  or institutes difficult. In 2019, Hendriksen et al.25.In-silico AMR detection methods are based on a three-step process: i) sample preparation such as bacterial culture and DNA extraction; ii) whole genome sequencing; and iii) in-silico analysis of data produced from isolate WGS. This process offers attractive possibilities for diagnostic test automation, including parallelising tests for multiple characteristics and even retrospective exploration for novel AMR genotypes without having to repeat steps i) or ii). AMR detection pipelines are based on existing knowledge of AMR genotype–phenotype associationsTo detect the genotype or underlying genetic mechanism for resistance by screening the WGS data obtained from isolated bacteria, the bacterial DNA is compared against a reference set of DNA sequences, also known as a database  containing the genotypes (i.e. AMR genes or point mutations) responsible for known AMR phenotypes.26 using the assembled genome contigs as a query against the database. While the first approach may be faster (< 10 min for a single core computer) and straight forward, it involves dealing with large raw data files . The second method may require a longer running time (~ 25 min) as the raw data files must be de novo assembled prior to comparison against the database. Many laboratories perform the assembly step as a routine for other purposes, so the extra running time might not be a burden. Once assembly for the bacterial genome has been stored, re-running the pipeline to screen for a novel AMR gene should be a matter of a few seconds.Bacterial DNA can be compared against the database using two different techniques: either by mapping the WGS short-reads onto the reference DNA sequences in the database, or by basic local alignment search27, nine partners compared the software performance of five pipelines for AMR detection based on WGS of E. coli isolates, and this paper describes that work. Each software detected the presence or absence of genes and point mutations associated with the sensitivity phenotype to 14 antimicrobials established for all isolates, which were taken as the “gold-standard”. This study restricted its scope to comparing the performance of pipelines in terms of their sensitivity and specificity to detect AMR, under the default settings defined by the pipelines’ authors. No attempt to evaluate the software installation process was considered in this study since most of the users agreed that IT support is provided institutionally. Isolate DNA extraction and sequencing protocols used by participating institutes were also not evaluated in this study.As part of the One-Health European Joint Programme Project ARDIG E. coli collected by nine different collaborating institutes working in the veterinary and human health sectors in Europe, were included in the study. Table A total of 436 E. coli did not show an even distribution of resistance for all 14 antimicrobials, although for most antimicrobials the numbers of resistant isolates in comparison to sensitive ones was more than 10% of the total, so enabled accurate sensitivity estimates to be made  in Europe. The unbalanced ratio between resistant and susceptible isolates for these antimicrobials will result in loss of precision for the sensitivity estimates. Nevertheless, sensitivity estimates for colistin was still calculated but for tigecycline, the sensitivity was not calculated due to the very low numbers of resistant isolates.Our panel of de Table . However28; APHA SeqFinder/Abricate14; WBVR BLAST (in-house pipeline); ResFinder/PointFinder30; ARIBA31. Some differences existed between the pipelines in their data input method or the algorithm used for detecting gene presence, which were part of the criteria for selecting these pipelines  pipeline settings and under the same interpretation conditions. Using these conditions, we concluded that no pipeline clearly stands out from the rest, in terms of performance and ease of output interpretation although some user preferences were noted from our questionnaire. Further, we observed that the performance of the pipelines depended in some instances on the antimicrobial for which the resistance determinant was being detected and therefore the ability to set individual thresholds for each database entry is an important feature but not widely available.Standardisation of any methodology is essential to enable comparison, as well as reproducibility across different sectors and countries, but can often be undervalued or overlooked. Monitoring systems that are harmonised already exists in areas such as AMR, which has been invaluable to determine AMR trends overtime across Europe although it only uses phenotypic testing resultsE. coli using underlying genetics7 is increasingly being used in place of serotyping34 to identify pathogens due to its ability to provide more detailed/accurate subtyping of populations, we believe a harmonised WGS method will do the same for bacterial characterisation, including for AMR. Therefore, our recommendations for harmonisation are as follows. Firstly, it may not be relevant which pipeline is used as long as it verifies a certain level of performance that can be agreed by the relevant scientific experts, depending on the application and the establishment of common inclusion/exclusion criteria of targeted matches. We propose that a control set of isolates are used to test and evaluate any pipeline with an appropriate representative sample and pre-set validation thresholds. For example, the collection of isolates used in this study may be appropriate for testing in-silico AMR pipelines, although any well validated set may be included, provided there is a balanced ratio between resistance and susceptibility of isolates to antimicrobials included in the test panel, e.g., to the EFSA panel of antimicrobials. However, the isolate panel will need to be regularly updated to incorporate isolates with new/novel AMR genes and the pipelines re-evaluated. However, for AMR pipelines, different bacterial species such as MRSA or Brachyspira, which may have different AMR mechanisms, a control set of isolates representative of AMR in those species will need to be included. The pipeline database will require to be updated to include species specific AMR genes/mutations and thresholds for these resistance determinants evaluated. For ease of evaluation and interpretation of pipeline results we recommend different species be tested separately using the same principles as performed in this study.We believe that the results of our study can be applied to inform future initiatives for harmonisation of results from WGS pipelines, whether for AMR or any other area of diagnostics and surveillance. Just as multi-locus sequence typing of Also, a certain level of pre-agreed performance in terms of minimum sensitivity and specificity thresholds, when comparisons are made between phenotypes and genotypes, should be used as a validation test for any AMR detection software. From the results of this study a sensitivity and specificity value of ~ 0.9, would be reasonable to use, although, for detecting resistance to HP-CIAs e.g. colistin or carbapenem, a more relaxed threshold may be used to maximise resistance detection, including of new gene variants. Secondly, we recommend unifying the databases used by different pipeline software for positive identification; unless genes present in databases, including their nomenclature, are harmonised, there will be differences in the output even from the same isolate test set. In the AMR context, AMR gene identifiers or sequences, including any chromosomal point mutations leading to reduced susceptibility, and the translation rules from genotype to phenotype, should be consistent and transparent. This will also help the naïve user in interpretation of genotypic data, in addition to promoting harmonisation. And thirdly, to allow greater access and usability of this technology for routine surveillance, the final output information should be standardised into user-friendly documents. This will enable individuals with minimal background in genetics to benefit from these softwares.As there are countless bioinformatics tools available, and many of them pursue similar aims but use different approaches with numerous fine tune adjustments, continual comparison of their performance is a difficult task. Our recommendation to achieve harmonisation does not require focusing on the best performing software, but on setting a common evaluation process based on universal minimal performance thresholds e.g. sensitivity and specificity measures applied to a representative testing sample set. In other words, we have made recommendations which will help towards creation of an appropriate structure for global standardisation of the bioinformatics component to enable genomic surveillance and diagnostics to become routine and standardised worldwide.E. coli isolates were provided by nine European institutes: the French Agency for Food, Environmental and Occupational Health and Safety, Lyon France (49 isolates), the Universidad Complutense de Madrid, Spain (50 isolates), the Institute Pasteur, France (50 isolates), the German Federal Institute for Risk Assessment, Germany (50 isolates), the Norwegian Veterinary Institute, Norway (50 isolates), the Wageningen Bioveterinary Research, The Netherlands (50 isolates), the University of Surrey, United Kingdom (50 isolates), the Animal and Plant Health Agency, United Kingdom (37 isolates) and Public Health England, United Kingdom (50 isolates).A total of 436 16, are available in the NCBI nucleotide archive under project number PRJNA805266.The raw WGS reads of isolates, generated from Illumina sequencing described elsewhere35. The antimicrobials were: Ampicillin, Azithromycin, Cefotaxime, Ceftazidime, Chloramphenicol, Ciprofloxacin, Colistin, Gentamicin, Meropenem, Nalidixic Acid, Sulfamethoxazole, Tetracycline, Tigecycline and Trimethoprim. The susceptibility of wild type E. coli to the panel were categorised as sensitive (S) or resistant ( R) by: Sensitive, when the isolate was inhibited at an antimicrobial concentration equal or lower than the established ECOFF value for the MIC, as described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)36 ; and Resistant, when the isolate was not inhibited at a specific antimicrobial concentration higher than the established ECOFF values36. The full S and R profiles for each isolate to the panel of antimicrobials, interpreted using ECOFFs, are provided in Table The sensitivity of isolates to the 14 antimicrobials used for AMR monitoring by the European Food and Safety Authority, was assessed using a standard MIC protocolFor some institutions MIC values were not available for part or all isolates for an antimicrobial, this has been marked with an asterisk in Table Description of the five AMR detection software used in this study are provided below:GeneFinder. Public Health England (PHE), UK28. URL: https://github.com/phe-bioinformatics/gene_finder. Version: 2.7. Operator: PHE. Language: python 2.7.5. Input format: FASTQ. Algorithm: mapping (bowtie 2.1.0). Reference database: provides three in house references sets in FASTA format for E. coli, Salmonella and Campylobacter. Users can incorporate their own reference set. Reference database used in this study: in house  and CARD . The database is provided with the tool. Detection: presence or absence of sequences and mutations. It also reports insertions, deletions, mixed positions and large indels. Possibility to set the similarity thresholds (between sample DNA and a reference DNA) individually for each gene. Quality metrics: coverage, similarity, depth and coverage distribution.APHA SeqFinder/ABRicate. Animal and Plant Health Agency (APHA), UK14. URL: https://github.com/APHA-AMR-VIR/APHASeqFinderVersion: 3.0. Operator: APHA. Language: python 3. Input format: FASTQ. Algorithm: mapping . APHA SeqFinder Reference database: provides three in house reference sets in FASTA format for AMR genes, mutations, plasmids, virulence factors and heavy metal resistances. APHA SeqFinder Reference database used in this study: in house . Detection: presence or absence of sequences and mutations. Quality metrics: coverage, similarity, depth and normalised depth by MLST genes. ABRicate19 is used in conjunction with SeqFinder as an additional filter. URL: https://github.com/tseemann/abricateVersion: 0.7. Language: perl. Input format: FASTA assembled contigs. (SPAdes 3.13.1). Algorithm: BLAST 2.7.0 or higher. ABRicate Reference database: same reference database as used for APHA SeqFinder (see above); it also provides additional databases which were not used in this study. Detection: presence or absence of genes. Quality metrics: coverage and similarity.BLAST, Wageningen Bioveterinary Research (WBVR), The Netherlands. Pipeline not published at the time of this study. Operator: WBVR. Input format: FASTA assembled contigs. Algorithm: raw reads are error corrected with Tadpole from the BBduk suite v38.71. Quality trimming to Q20 with BBduk. Genomes are assembled using SPAdes 3.13.1. Assemblies are compared to the reference database using BLAST version 2.9.0. (with filters: 98% sequence identity and 97% gene coverage). Reference database: ResFinder database (updated 10.02.2020). Reference database used in this study: ResFinder database (updated 10.02.2020). Detection: presence or absence of sequences and mutations. Quality metrics: sequence identity and gene coverage provided by BLAST.ResFinder v.3.2 + PointFinder v.3.1.030 Technical University of Denmark.URL: https://bitbucket.org/genomicepidemiology/resfinder/src/master/. Operator: The Norwegian Veterinary Institute (NVI). Language: python 3. Input format: FASTQ or FASTA assembled contigs. Algorithm: BLAST is used to analyse assemblies (FASTA files). Mapper KMA is used to analyse read data (FASTQ files). Reference database: ResFinder database (updated 10.02.2020) and PointFinder_database. Reference database used in this study: ResFinder database (updated 10.02.2020) . Detection: presence or absence of sequences and mutations. Quality metrics:ARIBA v2.1231, Sanger Institute, UK. URL: https: //github.com/sanger-pathogens/ariba. Operator: Universidad Complutense de Madrid (UCM). Language: python 3. Input format: FASTQ. Algorithm: mapping (Bowtie 2.1.0). Reference database: does not provide its own reference database but has an integrated method to download and standardise one from different sources such as CARD, ResFinder, ARG-ANNOT, MEGARes, NCBI, PlasmidFinder, VFDB, SRST2 and VirulenceFinder. Users can incorporate their own reference set. Reference database used in this study: ResFinder database (updated 10.02.2020)). Detection: presence or absence of AMR sequences only .It also reports genetic fragmentations, interruptions, and duplications. Quality metrics: gene coverage, sequence identity.All 436 isolates were run through each pipeline by five independent operators (one per pipeline). Result tables from the five pipeline runs were sent to each of the nine institutes who extracted the results corresponding to their isolates. For the following pipelines the antimicrobial class associated with each resistance gene was provided in the output to enable matching with the phenotype by operators: ResFinder; APHA SeqFinder; GeneFinder; WBVR Blast; for ARIBA prior knowledge from operators was required. The AMR genotype information  for each isolate was collated with the gold standard phenotype on a standardised form for each antimicrobial (Supplementary Table A bespoke R script was used to calculate the sensitivity and specificity and their 95% confidence intervals for each pipeline, for each antimicrobial, by using the information provided in Supplementary Table A questionnaire to evaluate user friendliness and quality control metrics of the pipelines output documents was sent to the 9 people, one at each institute, that were responsible for extracting the information for each of the 5 pipelines for their corresponding isolates. Three of the responses were from APHA, PHE and WBVR, who were also running their own pipelines, APHA Seqfinder, GeneFinder and BLAST; but both tasks were not carried out by the same person. The other six responses were from institutes with no link to any of the software used in the study, and an additional evaluation was performed on this subset.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Kaposi's sarcoma is associated with immunosuppression and human herpesvirus 8 infection, while rarely described in myeloid malignancies. Here, we illustrate a rare case of chronic myeloid leukemia treated with imatinib, a tyrosine kinase inhibitor, who developed a human herpesvirus 8‐related Kaposi's sarcoma. Human herpesvirus 8‐related Kaposi's sarcoma development is a rare complication in chronic myeloid leukemia patient under imatinib mesylate treatment likely, associated with its immunosuppressive activity. After one year of therapy, PET/CT scan re‐evaluation showed a significant reduction of FDG lymph nodal uptake, and in January 2016, clinical conditions worsened with anemia, increased HHV8 replication activity , and augmented FDG uptake by PET/CT scan while still in major molecular response. In May 2016, the patient started liposomal anthracycline at a cumulative maximal dose of 1260 mg/m2 for a total dose of 2142 mg and reduced imatinib mesylate to 200 mg daily. After two months of therapy, PET/CT scan showed a 50% reduction in FDG uptake and size of lesions, and after nine cycles of liposomal anthracycline, HHV8 replication activity was also decreased  until complete negativization in June 2021. At the time of writing, the patient is alive and is at the 49th cycle of liposomal anthracycline at 35 mg/m2 cumulative dose  without skin lesions. He is also monitored for anthracycline‐related cardiotoxicity with a 58% left ventricular ejection fraction by ultrasound assessment. Flow cytometry immunophenotyping from peripheral blood samples displayed normal frequency of CD19+ B lymphocytes (2%), while CD8+ T cells were slightly increased compared with CD4+ T cells (54% vs. 30%). The patient is still receiving imatinib mesylate at 200 mg daily, maintaining a deep molecular response with an overall survival of 13 years.A 70‐year‐old Caucasian male with a medical history of surgery for hemorrhagic gastroduodenal ulcer received a diagnosis of CML with the presence of BCR‐ABL fusion protein in 2009 outside our institution. He first received imatinib mesylate at 400 mg daily until 2010 when treatment was discontinued because of acute renal failure. In 2011, imatinib was reintroduced at 300 mg daily achieving a major molecular response, and the patient was admitted for observation at the Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi d’Aragona,” Salerno, Italy. In January 2013, red macules appeared to the right lower limb, and skin biopsy was performed showing an inflammatory purpura and fine‐needle aspiration of a right inguinal lymph node displayed reactive hyperplasia. Imatinib was stopped and a Positron Emission Tomography–Computed Tomography (PET/CT) scan was carried out displaying a high F‐18 fluorodeoxyglucose (FDG) radiotracer uptake in right inguinal lymph nodes. Because of disease relapse, imatinib was reintroduced at 300 mg daily in September 2013, achieving a major molecular response in January 2014; however, right iliac, obturator, and inguinal lymph nodes showed persistence of high FDG uptake by PET/CT scan, and lymph node biopsy displayed an infiltrate of KS cells positive for HHV8, CD34, and vimentin, while negative for HIV . The previous reported case, a 61‐year‐old Caucasian male, showed a KS arising after one year of treatment with 400 mg daily of imatinib mesylate in CML with the presence of BCR‐ABL p210‐b3a2 fusion protein. Our patient developed KS after five years of imatinib at lower dosage (300 mg daily) compared with the previously reported case who was treated with local skin lesion surgical removal resulting in continuous recurrence even after three years from diagnosis.+ T cell count, and a sustained response is observed in around 46% of cases, especially those subjects without visceral involvement.KS is a rare angioproliferative disorder frequently occurring in immunosuppressed subjects, such as HIV infected or transplanted patients,in vitro anti‐proliferative effects on CML progenitors by targeting cell cycle progression and pro‐apoptotic proteins and by promoting Natural Killer cell cytolytic activity against leukemic cells. Moreover, IFN‐α inhibits KS cell growth through downregulation of transcription and basic fibroblast growth factors, and by interfering with HHV8 viral life cycle.Before imatinib introduction, IFN‐α based regimens were the main treatment option in CML, and the combination of imatinib and IFN‐α induces a more rapid complete cytogenetic response at six months of therapy and a sustained treatment‐free response compared with imatinib alone, likely because of synergistic effects of these two drugs on immune responses and leukemic cell growth.+ T cell‐mediated responses against viruses likely by altering type I IFN‐mediated responses.Imatinib, the first‐in‐class tyrosine kinase inhibitor, mainly blocks BCR‐ABL1 activity and interferes with other tyrosine kinase downstream signaling, such as c‐kit and platelet‐derived growth factor receptor, that are also involved in KS progression. In HIV‐related KS and the reported KS arising from CML, lesion regression rate is low (around 40%) and recurrence is observed even under full dose imatinib treatment.In conclusion, HHV8‐related iatrogenic KS type is more frequently associated with immunosuppression after solid organ or hematopoietic stem cell transplantation and during chemotherapy; however, KS might rarely occur also under imatinib mesylate, a BCR‐ABL1 tyrosine kinase inhibitor, used for treatment of CML. Imatinib reduces CD8+ cytotoxic T cell and type I IFN‐mediated responses, thus decreasing antiviral and immunosurveillance functions of the immune system. We also encourage the use of standard chemotherapy for KS treatment in association with imatinib mesylate for CML control instead of IFN‐α, to minimize immunosuppressive synergistic effects of imatinib and IFN‐α and favor KS progression.V.G. and C.S. involved in conceptualization; M.D.A, L.P., V.G., and C.B. involved in clinical data; V.G. and M.D.A. involved in data curation and writing—original draft preparation; C.S. and P.Z. involved in writing—review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare no conflict of interest.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.