Mycobacteriumbovis and infrequently by other pathogenic mycobacteria. The problem of bovine tuberculosis is complicated when the infection is associated with multidrug and extensively drug resistant M. bovis. Many techniques are used for early diagnosis of bovine tuberculosis, either being antemortem or postmortem, each with its diagnostic merits as well as limitations. Antemortem techniques depend either on cellular or on humoral immune responses, while postmortem diagnosis depends on adequate visual inspection, palpation, and subsequent diagnostic procedures such as bacterial isolation, characteristic histopathology, and PCR to reach the final diagnosis. Recently, sequencing and bioinformatics tools have gained increasing importance for the diagnosis of bovine tuberculosis, including, but not limited to typing, detection of mutations, phylogenetic analysis, molecular epidemiology, and interactions occurring within the causative mycobacteria. Consequently, the current review includes consideration of bovine tuberculosis as a disease, conventional and recent diagnostic methods, and the emergence of MDR-Mycobacterium species.Bovine tuberculosis is a serious infectious disease affecting a wide range of domesticated and wild animals, representing a worldwide economic and public health burden. The disease is caused by  Mycobacterium tuberculosis (Mtb), the causative agent of human TB [M. bovis as a different species from Mtb [Tuberculosis affects a wide range of mammals and has been identified for thousands of years. When compared with other serious diseases, it is considered the biggest killer in the last 200 years . In 1882from Mtb . Bovine from Mtb . The surfrom Mtb . In addifrom Mtb .M. bovis and Mtb are the major causes of TB. They are highly pathogenic and may infect many animal species as well as humans [M. bovis being responsible for the vast majority of TB cases in cattle, it is not the exclusive cause of bTB [Mtb and M.caprae [Mycobacterium caprae is an essential factor in tuberculosis in ruminants [Among pathogenic mycobacteria, s humans . Despitee of bTB . Other m.caprae . Mycobacuminants .Mycobacteriumtuberculosis was frequently isolated from diseased cattle in several countries such as Ethiopia, Nigeria, Egypt, South Africa, and India [M. bovis is indistinguishable from that caused by Mtb and differentiation between them is very difficult to achieve either by clinical samples or cultivation [Mtb was reported to be isolated from bTB in buffaloes  [(Bison bonasus) or African elephants [nd India ,13,14,15tivation . Similarlephants ,17. Alsolephants .Mycobacterium bovis belongs to the Mycobacterium tuberculosis complex (MBTC) group that also comprises Mtb, M. caprae, M. microti, M. africanum, M. canettii, M. pinnipedii, M. bovis BCG, M. leprae, and the newly discovered M. mungi. These pathogens share an identical 16SrRNA sequence and up to 99.9% similarity at the nucleotide [cleotide .Mycobacteria are non-motile, non-capsulated, non-spore forming, with straight or slightly curved rods, are aerobic, oxidative, and of varying lengths (0.2–0.6 × 1.0–10.0 μm). Although they are cytochemically Gram-positive, the mycobacteria do not take up the dyes of the Gram-stain because the cell walls are rich in lipids, particularly mycolic acid. The mycobacteria are closely related to the genera Nocardia and Rhodococcus, which have a similar cell wall type .(a)Acid-fastness phenomenon attributed to high lipids content involving the mycolic acid hence, the name of acid-fast bacilli.(b)Glycolipid complexes have some significance in the granuloma formation.(c)Cords formation when grown on liquid media due to aggregation of complex lipids.(d)The hydrophobic character of the lipid layer makes it impermeable to chemical agents and difficult to be stained by ordinary procedures. In addition, it causes poor penetration of nutrients, hence the slow growth of the organism and long incubation period of the disease.(e)Resistance of mycobacteria to disinfectant agents, host’s immune system, and anti-tuberculous drugs ,22,23.Pathogenicity of mycobacteria is a multifactorial process that depends on the participation of several virulent factors of complex lipids in the cell wall, principally, in addition to protein and protein complexes. Mycobacteria possess a very thick cell wall containing a complex hydrophobic lipid; the cell wall core is composed of three attached molecules: peptidoglycan, arabinogalactan, and mycolic acid known as mycolylarabinogalactan–peptidoglycan complex. This complex is underneath a sheet like surface layer of glycolipids that made of superficial lipids as mycosides, cord factor, wax D, sulfolipid and sulfatides. This complex cell wall gives the pathogen some unique characteristics such as:Mycobacterium bovis are inactivated by prolonged exposure to heat, direct sunlight, and dry conditions. They are killed by temperatures of 65 °C and above for at least 30 min, and UV light. In contrast, they are resistant to freezing for prolonged periods. Under ordinary temperatures, M. bovis can persist in slurry and soil for at least 6 months [The tubercle bacilli survival in the environment is extremely variable and ranges from a few days to 2 years depending on temperature, sunlight, and relative humidity. In general, tubercle bacilli survive best in cool, dark, and moist environments (buildings and transport vehicles) shaded from direct sunlight . Mycobac6 months . The hig6 months .M. bovis, buffalo act as an important maintenance and reservoir host for the pathogen and maintain the disease transmission between different species. In addition, several wildlife species are considered reservoirs of TB, such as possum, wild boar, badger, and deer. Moreover, recent studies demonstrated the ability of members of MBTC, particularly M. bovis, to infect other livestock, such as goats, sheep, pigs, horses, camelids, and pets. Furthermore, alpaca has become a popular pet breed in many countries. There is a growing problem with tuberculosis in this species [Tuberculosis affects wildlife, livestock, and humans; hence, the extensive interfaces between these groups in developing countries is deemed a significant risk of transmission of the disease. Also, the movement of cattle from TB endemic areas was reported to be a great risk for TB breakdowns. Despite cattle being the main host of  species . So, the species .M. bovis can survive in protozoa (Acanthamoeba castellanii), potentially facilitating transmission by extending the survival of the bacteria in the soil) [Broughan et al.  studied he soil) . (D) WilDejene et al. tested the correlation between different risk factors and the prevalence of bTB in cattle and reported that the older the age and the lower the body condition the higher the chance of a positive bTB test result, but sex, lactation status, and reproductive status were not correlated with bTB status. At herd level, the pastoral production systems with transhumant herds had a higher bTB prevalence than sedentary herds . M. bovis infection is generally by direct contact with tuberculous animals as the organism may be excreted in exhaled droplets, saliva, milk, urine, feaces , semen, vaginal and uterine discharges, and discharges from open peripheral lymph nodes (LNs) [Transmission of es (LNs) ,31. The es (LNs) .M. bovis from human to cattle [M. bovis cannot transmit the infection to cattle. Contrarily, Mtb was frequently isolated from different animal species, justifying the possible transmission of the pathogen from human to animals [Mtb has been suggested to be transmitted to cattle through different routes, including ingestion of feed contaminated with infected sputum and/or urine from infected farmers [Considering human to cattle transmission, Romha et al. refuted the hypothesis of transmission of o cattle . The aut animals . The pos animals . Also, t animals . Moreove farmers .Following the entry of mycobacteria through mucous membranes or into alveolar spaces, they are phagocytized by macrophages then through the lining of bronchioles, enter the circulation, and are carried to LNs, parenchyma of lungs, or other sites. The virulent tubercle bacilli possess the ability to escape being killed by macrophages through mycobacterial lipids such as lipoarabinomannan and mycobacterial proteins of the antigen 85 complex. Eventually, mycobacteria survive and multiply within the phagosomes, and destroy the phagocytes . After tMycobacterium has a direct action in granuloma formation through the secretion of virulence factors such as ESAT-6 [The caseonecrotic granuloma is one of the hallmarks of TB, it is an attempt by the host to localize the disease process and to allow inflammatory and immune mechanisms to destroy bacilli and knowns as a tubercle. In addition, s ESAT-6 . After 1s ESAT-6 . The gras ESAT-6 . Tuberculosis spreads in the body by two stages, the primary complex and post primary dissemination. Tubercle of the primary complex in cattle is frequently detected in the lymph nodes of the head and thorax, involving the lung parenchyma only in 10–30%of cases . The priAs described above, the pathogenesis of bTB is complex and based on host immune responses to mycobacterial infection; infected animals can recover from bTB, progression of disease or persistence without progression . Mycobacterium spp. infection and mediated by T lymphocytes which release lymphokines that attract, immobilize, and activate additional blood-borne mononuclear cells at the sites where virulent mycobacteria, or their products exist. CD3+ and CD4+ T cells are the predominant lymphocyte subtype in granulomas of all stages. In addition, lower numbers of CD8+ T cells and γδ T cells can be found in Stages I–III. Many studies focused on the protective role attributed to CD4, CD8, γδ T cells and CD2; detection of changes in these cells considered as diagnostic markers for Mycobacterium spp. infection [The CMI response is the major host response to nfection . In addinfection .Diagnosis of bTB depends on traditional cellular immunity-based methods via tuberculin skin test (TST) and/or interferon gamma release assay (IGRA). In addition, antibody-based methods have recently emerged for diagnosis. Other methods, such as X-ray are also used to detect tuberculous lesions of tuberculosis and tuberculosis-similar diseases . FurtherTuberculin skin test is the recommended standard procedure for ante-mortem diagnosis of bTB, which evaluates a delayed-type hypersensitivity reaction in sensitized cattle following an intradermal injection of purified protein derivate (PPD) tuberculin from mycobacteria, by measuring changes in skin thickness after 72 h from injection. In Egypt, as well as many other countries, PPD is injected in the mid-cervical region , whilst in other countries as North America it is applied in the caudal fold of the tail  . The PPDM. bovis is used, despite its wide usage, high availability, and low costs, fails to differentiate between bTB infected cattle and others sensitized with M. avium complex or environmental mycobacteria. The second type is the comparative intradermal tuberculin test (CITT), in which both bovine and avian PPDs are injected simultaneously to increase the specificity of TST [There are two commonly used types of the intradermal tuberculin test. The single intradermal (SID), in which PPD of y of TST . In Egypy of TST .OIE (2009)  define tByrne et al. found a strong positive relationship between the reaction size of injected PPDs and the severity of PM lesions and suggested that increased reaction size is indicative of progressive lesions. The authors also found that dairy breeds exhibited a greater reaction size compared to other non-dairy breeds .Nocardia farcinicus causing non-specific responses. Additionally, they occur in early stages of the disease, where TB granulomas are too small or infrequent to be seen during PM examination, or in infected animals during the latency in which infection with M. bovis, but without disease. (4) False negative reactions and those given during the late stage of infection, particularly in severe and generalized cases where the animals are non-responsive for TST and known as the State of Anergy. Also, the false negative reactions are given in early cases until 3–6 weeks post-infection which is known as the pre-allergic period. Further false negatives include animals desensitized by PPD administration during the preceding 8 to 60 days, old cattle, early post-partum cows , and low potency tuberculin, subcutaneous injection , or bacterial contamination of the tuberculin [Despite its wide use worldwide, TST has several drawbacks: (1) Complexity. (2) The interpretation is subjective and differs between operators. (3) False positive reactions when the animals were TST positive but there is no PM lesions nor positive culture, known as Non-Visible Lesions (NVL). They are given by cross reactivity to other non-virulent mycobacteria, avian TB, human TB, Johne’s disease (JD), or sensitized by other allergens as berculin ,31,47.M.avium subspecies paratuberculosis (MAP), the causative agent of Johne’s Disease, such cross-reactivity lead to misdiagnosis as MAP shares structural proteins and virulence factors with M. bovis [Fasciola hepatica and Fasciola gigantica, may affect the accuracy of bTB but the direction of the effect differs among studies [M. bovis infection [There are various factors affecting the sensitivity and specificity of TST, that range from 55–99%, such as potency, purity, dosage, and biological activity of the PPD, as well as the inoculation site, misreading of the results and the genetic background of the animal ,47. In aM. bovis . In addiM. bovis . Further studies . It is snfection . Howell nfection . The stunfection .M. bovis AN5 and some of these components are present in nonpathogenic environmental mycobacteria [Bovine PPD is a mixture of protein, carbohydrates, and lipids obtained from bacteria . Therefobacteria .Two approaches have been used globally at the herd level to overcome the limited sensitivity of TST in case of high prevalence settings. The first is the use of in vitro ancillary tests to maximize the detected number of infected animals and the other is the depopulation of the herds whose reactors are detected. Nevertheless, high costs and economic and social implications make whole herd depopulation difficult and inapplicable, particularly in endemic setting . Bison bonasus) is extremely sensitive to mycobacterial infection and its ongoing restitution requires the improvement of ante-mortem diagnostic methods such as TST and IGRA [European Bison , and of high costs . IGRA alM. bovis infection in cattle [Novel biomarkers to distinguish between the healthy and infected animals are still urgently needed, particularly when TST and IGRA fail to detect the infection . Hence, n cattle . M. bovis in large amounts during the late stages of the disease [Both TST and IGRA detect the early bTB infection as they depend on the measurements of pathogen specific CMI responses . At adva disease ,75. Various factors affect the sensitivity and specificity of serological tests for diagnosis of bTB such as the stage of the disease, immune status of the animal, type of antigen used, and previous exposure to bovine tuberculin . The senM. bovis infection of 83.7% of animals that were ELISA positive 15–17 days after a negative TST, by histopathology and PCR [M. bovis within weeks after TST as a rapid and inexpensive way to improve bTB control. On the other hand, Casal et al. took the serum samples before the injection of PPD and evaluated the sensitivity of three different ELISAs in addition to TST and two IGRA tests for the diagnosis of bTB in cattle and concluded that in vitro diagnostic techniques maximized the detection of bTB infected animals [TST is known to boost the antibody response; thus, the use of ELISA without skin tests would further reduce their sensitivity . Testing and PCR . In the  animals . The aut animals . The autSeveral studies highlighted the great significance of MPB83 protein as a diagnostic antigen in serological tests and recommended its use because of the highest and earlier response that was triggered against it . Waters M. bovis strains from the three countries in an attempt to explain these geographical disparities. However, the study concluded that sequence variation in these genes does not explain the variation in sensitivity.IDDEX™ ELISA is a commercial kit recognized by OIE for detection of bTB infection in blood and milk serum samples depending on detection of antibodies against MPB70 and MPB83 antigens. It is used in several studies: Al-Fattli in Iraq used it as a screening test and reported a seroprevalence of 20.16% and 15.12% in blood and milk of lactating cows, respectively . HoweverM. bovis mpb70 antigen. Elsohaby et al. used it in conjugation with TB-Feron test (a type of IGRA test) as ancillary tests and both were able to reduce a significant number of false positive TST slaughtered cows [Other serological tests are used in several studies, such as rapid lateral-flow test for detection of antibodies against red cows .All the previous tests, TST, IGRA, and ELISA, have wide various limitations related to their sensitivity and specificity, thus, parallel use of more than one test and focusing on finding new cocktail mycobacterial antigens offer substantial advantages for maximizing the detection of infected animals ,51,76. Isolation and identification of the mycobacteria is still the international gold standard test in diagnosis of bTB . It can Mtb and M. avium that grow well on glycerol containing media and known as eugenic, M. bovis has a sparse thin growth on them and is called dysgenic, but it grows well on pyruvate-containing media without glycerol [M. bovis in the presence of glycerol is due to lack of pyruvate kinase enzyme [M. bovis on Lowenstein–Jensen slants [The most frequently media used for mycobacterial growth are Lowenstein–Jensen buffered egg potato medium, Middle brook 7H10, Middle brook 7H11, and Dubos Oleic-Albumin agar. Unlike glycerol . This dye enzyme . The gron slants . Zihel–Nn slants . M. bovis [M. bovis from bovine tissues is the Petroff method in which 4% NaOH solution is used as a decontaminant (OIE 2014).A range of pre-culture treatments, such as decontamination, homogenization, and concentration are conducted before inoculation on the media to facilitate the recovery of M. bovis . The mosMohamed et al., compared between BACTEC MGIT 960TM as a fully automated liquid-medium system and the conventional culture using Lowenstein–Jensen media for isolation of mycobacteria and found that automated system was more sensitive, faster, and revealed a higher recovery rate of mycobacteria than Lowenstein–Jensen media [2SO4) [2SO4 and inoculation of Middlebrook (7H11-A) was the best strategy for the primary isolation of M. bovis.Issa et al. in Brazil, compared three decontaminants used in mycobacterial decontamination: 2% sodium hydroxide (NaOH), 0.75% hexadecyl pyridinium chloride (HPC), and 5% sulphuric acid (H2SO4) . In addiFor achieving the best results, Soares Filho et al. recommended two sampling protocols for both PCR and isolation . For PCRDespite mycobacterial culture usually being regarded as the golden standard, it is time consuming and prolonged for several months, risky, laborious, with a high level of tissue sample contamination and decreased number of viable mycobacteria due to the decontamination methods ,97,98. IM. bovis immune response.Canal et al. described the characteristic histopathological pictures of tuberculous lesions that were classified microscopically into four stages  . Stage IM. bovis nor member of MBTC were detected either by molecular methods or cultivation over 3 months, but NTM and Actinomycetales were identified in the lesions. Despite high sensitivity, histopathology lacks specificity; McKinley et al. described a histopathological profile with encapsulation of granuloma, presence of Langhan’s cells, sometimes in association with epithelioid cells, lymphocytes, or neutrophils in tuberculous-like lesions . HoweveresxA and esxB genes, respectively [atpE and lpqT [IS1081 [IS6110. Numerous studies targeted the insertion sequence region IS6110 as it is found in all members of MBTC [PCR techniques are widely used for the diagnosis of bTB and have several advantages; they are quick, applied within a few hours which means rapid diagnosis and efficient control, overcome the lack of specificity of other traditional tests such as histopathology, and are able to identify the mycobacteria either from culture or clinical specimens ,103. Morectively , atpE anand lpqT , RvD1-Rvand lpqT , and ins [IS1081  and IS61 of MBTC ,106.IS6110 gene as it was able to detect 5 pg/μL of M. bovis specific DNA or even smaller quantities in tissue samples [Thacker et al. reported a high specificity of TaqMan Real-Time PCR targeting  samples . Despite samples . On the  samples . Several other reports detected lower sensitivity and specificity of PCR. The sensitivity of the molecular studies varies from 50% to more than 80% depending on the study and the employed methods . The comThe varying results of PCR technique performance are mainly because of technical differences in the setting up of assays, particularly during DNA extraction from lesions ,109, andThere is a significant variation of sensitivity of PCR assays related to the DNA extraction method used and there is not a definitive view for the best method of DNA extraction from bovine tissues . Hence, M. avium complexes [M. bovis from tissues and provides higher positive values than culturing [Despite their advantages, PCR techniques have several limitations; limited to the PM diagnosis , not speomplexes . In addiomplexes ,109. Helomplexes . In addiulturing . HoweverM. bovis in both blood and milk in dairy cattle, where the sensitivity of PCR conducted on blood and milk samples was 53–95% and 1–60% while its specificity was 95–100% and 43–99% for each sample, respectively [M. bovis in blood stream is needed to detect the proper sampling time because most of TST and IGRA reactor animals failed to be detected by PCR in blood samples [M. bovis, bacteremia has been assumed to be rare in cattle [Besides tissue samples, many PCR assays had been performed on blood and milk samples such as the Mataragka et al. study, which illustrated that PCR can be used as an early and sensitive indicator method to detect infection in pooled milk samples collected from the aged animals of a dairy farm, which could support TST monitoring and improve bTB control . In addiectively . The autn cattle .In the light of the aforementioned, bTB lacks a definitive gold standard diagnostic test  since noM. bovis isolates from Nile Delta and reported a predominance of isolates which were closely related to clonal complex (European 2). In addition, the authors reported two isolates belonged to M. bovis BCG group that are rarely isolated from animals [Whole Genome Sequencing (WGS) provides new insights into dynamics of disease transmission, host-pathogen interactions, and comparative analysis that elucidates key differences between the animal and human mycobacteria ,120. AbdM. bovis in buffalo tissues [® Molecular Transport Medium, in combination with Xpert® MTB/RIF Ultra assay (an automated cartridge-based semi-quantitative nested RT-PCR assay that detects DNA of MBTC and rifampicin (RIF) resistance in clinical specimens) as a safe and rapid PM screening test for M. bovis in buffaloes. In humans, Xpert MTB/RIF has recently become a significant breakthrough in TB diagnosis [Clarke et al. investigated an in-field sampling technique for rapid, safe detection of  tissues . The autiagnosis . This teM. bovis. This LAMP method was able to detect M. bovis within 40 min following incubation and results could be read with the naked eye following development of a color change [M. bovis infection in cattle and humans in resource-limited, endemic areas.Kapalamula et al. developed a loop-mediated isothermal amplification (LAMP) method for specific identification of r change . The autAdditional tools for diagnosing TB, such as lymph node biopsy and tracheobronchial aspirates, have been successfully implemented in wildlife ruminants. Didkowska et al. used tracheobronchial aspirates and ultrasound-guided biopsies for diagnosis of TB in European bison . In addiBioinformatics is the science of analyzing and managing biological data using computational tools and algorithms for expanding the use of biological, medical, behavioral, or health data. It uses genetic databases and biological samples and provides a time-saving and cost-effective tool to obtain an important data on gene and protein levels not easily obtainable by other techniques . ResearcMtb isolates were scattered among 4 lineages and 25 sub lineages [Mtb H37Rv strain [M. bovis and Mtb which were obtained from various hosts including humans and cattle [M. bovis isolates from different sources; dairy and beef cattle, as well as humans [Mtb isolates from cattle and their attendants (People near cattle) in South India [Mtb from cattle and their handlers and detected three isolate pairs that were highly related, of which two pairs were from handlers and one was from cattle, suggesting that Mtb transmission occurred between handlers, either directly or through an intermediate host. Also, it was suggested that Mtb was transmitted between two cattle who shared a highly related strain. Assal in Canada used bioinformatics tools for the prediction of extracellular proteins from M. bovis genome sequences and identified 96 protein candidates. In addition, the proteomics analysis identified additional 92 protein candidates secreted by M. bovis [In silico analysis tools have been highlighted for analyzing the sequenced genome of mycobacteria, studying the interaction between the gene mutations and anti-tuberculous drug resistance, and for prediction of the drug resistance particularly in humans ,129. A rlineages . In addiv strain . The stuv strain . The stuv strain . More rev strain . The autd cattle . The anas humans . Palaniyth India . The stuM. bovis .M. bovis isolates. Although several studies on the bioinformatics analysis of human TB and its related mycobacterial isolates are existing [Mycobacterium sequenced strains [In Egypt, studies that used bioinformatics tools regarding bTB are scarce; Abdelaal et al. in Nile Delta of Egypt used in silico analysis to detect lineages and spoligotypes of existing . Howeverexisting . Recentl strains . Future M. bovis and Mtb in slaughtered cattle, suggesting anthropozoonosis (transmission between humans and cattle) as a public health concern [The infection in humans, is primarily acquired following consumption of contaminated unpasteurized dairy products or, to a lesser extent, undercooked meat, causing extrapulmonary TB cases ,141. Fur concern .Mycobacterium bovis is also underestimated in humans because the commonly used diagnostic techniques do not allow identification of the pathogen at species level [M. bovis is naturally resistant to pyrazinamide (PZA), which is one of the key drugs used in the treatment of TB [M. orygis). M. bovis is considered the proxy for zoonotic TB, it is responsible for up to 37.7% of all human TB cases in Africa [M. canetti, M. caprae, M. microti, M. pinnipedii, M. mungi, and M. orygis [M. bovis transmission that govern bTB epidemiology and transmission to humans. These factors include demographic factors , feeding habits, people living in close contact with their animals, socio-economic status, illiteracy (lack of knowledge of zoonotic TB), the wildlife–livestock–human interface is particularly relevant in Sub-Saharan Africa [Zoonotic TB poses a significant hazard; in 2016, there were 147,000 new cases and 12,500 deaths in people worldwide . The trues level ,146. Zoont of TB . Zoonoti. orygis . Accordi. orygis . The hig. orygis . The exi. orygis . There an Africa .https://www.cdc.gov/tb/, accessed on 10 May 2022). Extensively drug-resistant tuberculosis (XDR-TB) is a rare type of MDR-TB in which at least four of the most powerful and core anti-TB drugs cannot act against bacterial activity. These drugs include INH and RIF, levofloxacin/moxifloxacin, and at least one second-line injectable drug such as capreomycin, amikacin, or kanamycin. Misuse or mismanagement are the major causes of the development of resistance to anti-TB drugs , accessed on 10 May 2022). According to a WHO report (2019), in 2018 alone, there were approximately half a million  new cases of RR-TB, of which 78% had MDR-TB. Moreover, among cases of MDR-TB in 2018, 6.2% were estimated to have XDR-TB [The development and emergence of multidrug resistance in animals has gained worldwide attention owing to the possibility of pathogen transmission to humans ,153,154.e XDR-TB .M. bovis is known to be naturally resistant to PZA owing to the presence of C→G point mutation in the pncA gene, at nucleotide 169, which confers natural resistance to PZA. Different reports documented an emerging trend in the incidence of INH-resistant M. bovis in Ireland, as well as an outbreak caused by multidrug-resistant M. bovis strains—resistance, at least to RIF and INH—is also documented in other literature with a dramatic impact [M. bovis clinical isolates including three MDR strains [katG and rpoB genes that conferring resistance to first line antibiotics in the MDR M. bovis strains. Furthermore, they confirmed the absence of genetic relationship between human M. bovis strains or between human and bovine strains. Recently, Abdelsadek et al. in Egypt tested 135 MBTC isolates obtained from both infected cattle and veterinarians or workers in contact with these animals [Mtb strain that was MDR to RIF, INH, and streptomycin, this was the first report of multidrug resistant (MDR)-Mtb originating from buffaloes [M. bovis strains that were resistant to ethambutol and ethionamide. Anne et al. emphasized the importance of more research on MDR strains of Mtb isolates from bovines particularly in endemic areas due to the high possibility of reverse zoonosis [The problem of MDR-TB was recently exaggerated by its emergence in animals. Tuberculosis is most commonly treated with pyrazinamide (PZA), INH, RIF, and ethambutol, followed by streptomycin and kanamycin . Previouc impact . Further strains . The aut animals . The stuuffaloes . In addizoonosis . Hence, zoonosis .Bovine TB is rarely treated in domestic livestock except in rare cases of valuable animals as zoological exhibits. Anti-tuberculous first-line chemotherapies that have the greatest activity include INH, RIF, PZA, ethambutol (EMB), and streptomycin (SM). Second line drugs include capreomycin (CAP), ethionamide (ETH), cycloserine, and thioacetazone . PrimaryThe control system for identification of bTB comprises TST, abattoir surveillance and PM confirmation. There is a dynamic link between these three arms and they could not be considered independent because the deficiencies in TST will manifest in an increased number of lesions at abattoirs. Likewise, missed lesioned animals at slaughterhouses will delay the detection of undisclosed transmission within herds .M. avium and do not follow the mandatory CITT procedure, in which both bovine and avian PPDs tuberculin are tested for.Complete eradication of bTB has not been achieved in any country due to the presence of major problems facing the eradication: (1) Breakdowns: disclosure of bTB affected animals either through detection of TST-positive animals after a successive number of free tests within herds or through confirmation of the infection within abattoirs by bacteriological culture. Breakdowns are caused by anergic carrier or a break in the security of the herd or infection from animals newly introduced to free herds. (2) Non-Visible Reactors (NVL) that create administrative and public relations difficulties. (3) Presence of wildlife reservoirs. (4) Large herds: as in North America and South America where cattle are run under very extensive conditions on large ranches or stations. Also, in intensive dairies where the complete depopulation of herds cannot be performed ,102. PosVaccination against bTB is rarely used for controlling the disease in areas with official control programs because BCG can induce a cellular immune response producing interference with the testing strategies using PPD as antigen (TST and IGRA). Lack of the available diagnostic tests to differentiate between infected and vaccinated animals is the main impediment . In addiChandran et al. demonstrated a novel BCG vaccine to protect animals against bTB. The authors also demonstrated a novel skin test for differentiating infected from vaccinated animals (DIVA) that will detect bTB . NeverthBovine tuberculosis stills representing a big challenge in developing countries, especially in Africa due to interactions between people, livestock transhumance, and wildlife. In addition, deficiencies in preventive and/or control measures, poor sanitation, veterinary and slaughterhouse services, and lack of political measures are the main causes of persistence of the disease in Africa . Regarding Egypt, the first record of bTB was by Piot Bey in 1917 . In 1920There are several challenges facing bTB control in Egypt; lack of financial support is a big challenge that prevents covering the entire cattle population, which estimated to be 4.9 million cows and 4.1 million buffalos, during the national control program testing by SID only a quarter of this population is tested. In addition, the single comparative cervical tuberculin test is not practiced by the general organization of veterinary services due to lack of financial support. Most abattoirs in Egypt do not have diagnostic facilities for rapid confirmation of grossly detected bTB lesions. Import of live animals from some endemic countries is recognized as a potential source of bTB transmission into Egypt. Moreover, the majority of cattle populations are raised by smallholders individually or with other animals such as sheep, goats, and probably camels, horses, dogs, and cats at rural areas in both Middle Delta and Middle Egypt regions, where they share the same pasture and the same shelter ,168,169.The number of bTB infected animals is growing in infected dairy herds in Egypt as recorded recently by Elsohaby et al. who reported a high prevalence ranging from 6% to 66% in blood and 35%–61% in milk samples using PCR, IGRA, and mycobacterial culture . The autThere is a markedly wide geographic variation in the prevalence of bTB in Egypt. Despite the underlying causes of this variation being poorly understood , it may Mycobacterium species. Our review is intended to renew interest in considering the emergence of infection caused by drug-resistant M. bovis as an important public health threat that menaces the success of TB control programs. Despite annual efforts, bTB continues to have a huge impact on human and animal health, particularly in developing countries. The absence of a gold standard single test to detect all cases of bTB, the absence of a realistic vaccine against the disease, and the zoonotic impact are the main challenges that should be considered in future research. This review has discussed bTB as a disease, conventional and recent diagnostic methods, and the emergence of MDR-


Molecular representation learning is a crucial task to accelerate drug discovery and materials design. Graph neural networks (GNNs) have emerged as a promising approach to tackle this task. However, most of them do not fully consider the intramolecular interactions, i.e. bond stretching, angle bending, torsion, and nonbonded interactions, which are critical for determining molecular property. Recently, a growing number of 3D-aware GNNs have been proposed to cope with the issue, while these models usually need large datasets and accurate spatial information. In this work, we aim to design a GNN which is less dependent on the quantity and quality of datasets. To this end, we propose a force field-inspired neural network (FFiNet), which can include all the interactions by incorporating the functional form of the potential energy of molecules. Experiments show that FFiNet achieves state-of-the-art performance on various molecular property datasets including both small molecules and large protein–ligand complexes, even on those datasets which are relatively small and without accurate spatial information. Moreover, the visualization for FFiNet indicates that it automatically learns the relationship between property and structure, which can promote an in-depth understanding of molecular structure.The online version contains supplementary material available at 10.1186/s13321-023-00691-2. Molecular representation learning is a fundamental task to learning the molecular structure–property relationship. Accurate and effective molecular representations have a pivotal role in drug discovery  and mateIn recent years, many effective GNNs in molecular representation learning have been developed. They make several adaptions in the inputs and network structures to make GNNs perform well on molecule datasets. Basically, these GNNs can be divided into two categories, i.e. 3D-unaware GNNs and 3D-aware GNNs. As literally, 3D-unaware GNNs do not utilize the 3D geometry of molecules, and they typically take predefined molecular graphs as inputs. One of the most famous 3D-unaware models is called DMPNN , which iIn this paper, we construct a force field-inspired neural network (FFiNet) that can utilize all the interactions in molecules. Force field, which is a simple approximation to calculate the potential energy in molecules, divides all the interactions in molecules into four parts, i.e., bonded interactions, angle bending interactions, torsion interactions, and nonbonded interactions. Intuitively, these interactions can denote the importance of source atoms toward target atoms, which can be associated with graph attention mechanisms. Following this idea, we construct a novel attention-based message passing scheme that calculates the importance scores of source atoms within three hops according to their interactions with target atoms. The proposed model shows state-of-the-art performance on extensive molecular property benchmark tasks, even on those small datasets without accurate information. Moreover, we also applied our model to represent large protein–ligand complexes and predict their property (i.e. binding affinity). The result shows that FFiNet can outperform all the baselines on the PDBBind dataset, which further indicates the competence of FFiNet for molecular representation learning. Furthermore, the visualization of the learned node features and attention weights agrees well with the intuition of the relationship between chemical molecular structure and property.The force field divides the total potential energy into four terms :1\documeThe overall architecture can be seen in Fig. r denotes the node index in the k-hop index, As shown in Table nsformer  did, andFrom the intuition that different hop atoms have different forces with the destination atom, we use axial attention to determine the contributions of these three outputs. We adopt dot production of k-hop outputs and input embeddings to get the axial attention score of different hops. Then the node embeddings are updated by a weighted sum of these three outputs. After the node updating steps are iterated for N steps, a readout function is applied to aggregate the output embeddings of all nodes into a graph-level representation. More precisely, we apply weighted sum and max-pooling to the node representations and concatenate the results as the graph representation. Finally, we use an MLP to give a final molecular property prediction. Besides, the model uses some performance-improving mechanics, such as residual connect , dropoutESOL, FreeSolv, Lipophilicity: Regression tasks for predicting log water solubility (ESOL), hydration free energy of small molecules in water (FreeSolv), and octanol/water distribution coefficient (Lipophilicity). The datasets have 1128, 642, and 4200 molecules, respectively. These datasets represent the molecules in SMILES format, which does not include the spatial information of molecules.BACE, BBBP, HIV: Binary classification tasks for predicting inhibition of human β-secretase 1 (BACE), the ability to penetrate the blood–brain (BBBP) inhibition of HIV replication (HIV). The datasets have 1513, 2039, and 41,127 molecules, respectively. These datasets represent the molecules in SMILES format.QM9: Quantum mechanical properties regression tasks with 12 targets. The dataset has 133,885 molecules and all the molecules in the dataset have accurate spatial information.ClinTox, Tox21, SIDER: Multi-class classification tasks for predicting toxicity  and sider effects of drugs (SIDER). The datasets have 1478, 7831, and 1427 molecules, respectively. The datasets have 2, 12, and 27 tasks, respectively. These datasets represent the molecules in SMILES format.We experiment on ten molecular property benchmarks from MoleculeNet , includiMany models can be used to give predictions for these benchmarks. Basically, these models can be divided into descriptors-based methods and graph-based methods. In this work, we compare FFiNet with multiple methods, including (1) SVM and RF, which are traditional machine learning (ML) tools for classification and regression; 2) GIN , GCN 1010, which GIN , GCTables Apart from molecular property prediction of small molecules, the prediction of protein–ligand binding affinity is also of vital importance in computational drug discovery . The firHerein, we use a well-known protein–ligand binding affinity benchmark (PDBBind)  to evaluAs shown in Fig. Since our model makes several changes compared with the previous GNNs, we do some ablation experiments to verify the necessity of each added module Table . To studThe FFiNet has achieved state-of-the-art performance on a variety of molecular prediction tasks. Therefore, it is worth investigating the interpretability of the model. Model interpretation can help us find how the model learns the molecule representations and what happened during the model learning process. Although neural networks are often called “black boxes”, we can explore the interpretability of the model by visualizing the features and attention weights. Taking the lipophilicity dataset as an example, we choose some molecules to visualize atom similarity, atom contributions, and attention weights Fig. . Since tFiguring out which parts of a molecule play a more important role in a certain property is helpful for the chemist to design molecules with the desired property and learn the nature of the property. Since our model uses weighted sum pooling and max pooling as readout functions, the weight that each atom obtained can represent the importance of atoms for a certain property to some degree. Taking the lipophilicity property as an example, we plot a heat map over molecules showing the atom contributions to the property Fig. b. As shoMolecular property is of vital importance in various fields, e.g., pharmaceutical and chemical engineering. There have been many attempts at the accurate prediction of molecular properties. Early attempts use molecule descriptors as input features and feed them into RF, SVM, or neural networks (NN). However, the generation of descriptors is time-consuming, and the descriptors-based methods cannot obtain satisfactory results due to the unchanged molecule representation. Recently, more and more research has been on applying machine learning to graphs, and many graph-based network architectures have been constructed, e.g., GCN, GAT, GIN. However, these models are designed mainly for citation graphs and knowledge graphs, which are not mainly designed for molecular representation learning. To this end, a series of well-designed GNNs are proposed for molecular properties, e.g., DMPNN, DimeNet. However, these models also do not uncover all the information about a molecule. Some of them lose spatial information, and some of them lose bonded or nonbonded information.In this work, we construct a theory-guided network based on the force field. Inspired by the force field, we devise an attention network including all the interactions in a molecule, i.e., bonded and nonbonded interactions. The bonded interactions include bond stretching, angle bending, and torsion, corresponding to spatial information of distance, angle, and dihedral. We utilize the spatial information with guidance from the functional form in the force field to include all these intramolecular interactions. Since FFiNet is less sensitive to the accuracy of spatial information, it enables quick screening for those molecules without accurate spatial information. In contrast to other tested models, FFiNet performs well across a wide range of molecular property prediction tasks. Even in the task with large molecule graphs, the model can also perform well. Moreover, the visualization of the hidden state, atom contributions, and attention weights provides access to model interpretation. The results indicate that our theory-based model has the interpretability to relate property with molecular structure.Our FFiNet can easily extend to other complex tasks, such as protein–protein interactions prediction or DNA-binding proteins prediction. These tasks need a model that has a sizeable receptive field and can model intramolecular interactions since their molecular size is very large and the nonbonded interactions often play a leading role in the properties. One potential limitation of the model is that in our message passing phase, a node involves two attention mechanisms, which need more parameters. Therefore, our following work is to find a generic global attention mechanism which can distinguish different hop nodes and nodes in the same hop with spatial information. All in all, the design philosophy of our model has great potential for driving the development of molecular representation learning.In addition, FFiNet has great potential to cover other more specific tasks by changing the applied force field. By extracting the embedding basis from other functional forms of force fields, it is simple to utilize other force fields for better representations of a certain class of molecules. For example, when the task is about macromolecules (such as the property prediction task of proteins), the functional form of CHARMM  and OPLSSince the datasets except PDBBind and QM9 do not provide the 3D structure of molecules, we generate atom positions with the fast ETKDG method . AlthougAdditional file 1. Additional model details, hyperparameters setting, and model interpretation.


Atypical meningiomas are a heterogeneous group of tumors, different to the benign WHO grade 1 meningiomas, and with sub-group varying levels of aggression and recurrence, making prognostication, and thereby management, often complicated for the clinician and the patient. In this edition of Frontiers, the Editors have selected key publications which may facilitate decision making for surveillance, treatment options, and even prognostication modeling using AI.Xiao et al., in which the authors utilized radiological features to pre-operatively predict brain invasion. As Neurosurgeons and clinicians, we routinely review all the radiological features the authors have used as objective measures in their radiological cohort—peritumoral edema as seen on FLAIR and post-contrast architecture of the tumor on MR. The use of an open source github Python package, in addition to the pathological confirmation of brain invasion and additional blood indices, leads to the extrapolation of this multi-faceted study of how a conglomeration of clinical factors may in the future lead to AI-hybrid decision-making for the optimal care of our patients.As we march through the twenty-first century with significant technological progress, specifically with reference to applications of artificial intelligence, the overlap with our partners in Molecular Biology, Radiology, Medical Physics, and Computational Biology becomes even more remarkable. An ode to this is the study by Millesi et al.'s study is of keen interest here, showing the potential strong prognostication role of DNA methylation in the often unpredictable course of WHO grade 2 meningiomas. I bring the reader's attention to the powerful heatmap in Figure 4 showing the correlation of DNA methylation patterns in response to clinical patterns and outcomes. While this is a small cohort of patients, the methylation was associated with a poor prognostic course and surprisingly, showed no association with meningioma location size, Simpson grade, or predicted meningioma methylation class.Hong et al. follows the therapeutic journey of a patient with recurrent and refractory skull base meningioma, and how the molecular markers, specifically of GNAS mutation, led to the multi-disciplinary team widening their treatment options to include tyrosine kinase inhibitor sunitinib.Continuing on the molecular biology theme, the case report by Gao et al. looking at the relationship of a vast array of hematological markers related to the progression and potential prognostication of WHO grade 2 meningiomas. For researchers interested in establishing or refining robust clinical prognostication models, I refer you to the authors' detailed work in Figures 2A,B.The fourth publication in this Frontiers series is the impressive work undertaken by We hope our colleagues and readers find this Research Topic to be of as much educational interest and to awaken as much academic fervor as it did for myself and my colleagues during the review of these manuscripts.MA, AM, and EP contributed to conception and design of this journal, reviewed the submitted manuscripts, and designated the Reviewers. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Molecular vibrations constitute one of the smallest mechanicaloscillators available for micro-/nanoengineering. The energy and strengthof molecular oscillations depend delicately on the attached specificfunctional groups as well as on the chemical and physical environments.By exploiting the inelastic interaction of molecules with opticalphotons, Raman scattering can access the information contained inmolecular vibrations. However, the low efficiency of the Raman processtypically allows only for characterizing large numbers of molecules.To circumvent this limitation, plasmonic resonances supported by metallicnanostructures and nanocavities can be used because they localizeand enhance light at optical frequencies, enabling surface-enhancedRaman scattering (SERS), where the Raman signal is increased by manyorders of magnitude. This enhancement enables few- or even single-moleculecharacterization. The coupling between a single molecular vibrationand a plasmonic mode constitutes an example of an optomechanical interaction,analogous to that existing between cavity photons and mechanical vibrations.Optomechanical systems have been intensely studied because of theirfundamental interest as well as their application in practical implementationsof quantum technology and sensing. In this context, SERS brings cavityoptomechanics down to the molecular scale and gives access to largervibrational frequencies associated with molecular motion, offeringnew possibilities for novel optomechanical nanodevices.Themolecular optomechanics description of SERS is recent, andits implications have only started to be explored. In this Account,we describe the current understanding and progress of this new descriptionof SERS, focusing on our own contributions to the field. We firstshow that the quantum description of molecular optomechanics is fullyconsistent with standard classical and semiclassical models oftenused to describe SERS. Furthermore, we note that the molecular optomechanicsframework naturally accounts for a rich variety of nonlinear effectsin the SERS signal with increasing laser intensity.Furthermore,the molecular optomechanics framework provides a toolparticularly suited to addressing novel effects of fundamental andpractical interest in SERS, such as the emergence of collective phenomenainvolving many molecules or the modification of the effective lossesand energy of the molecular vibrations due to the plasmon–vibrationinteraction. As compared to standard optomechanics, the plasmonicresonance often differs from a single Lorentzian mode and thus requiresa more detailed description of its optical response. This quantumdescription of SERS also allows us to address the statistics of theRaman photons emitted, enabling the interpretation of two-color correlationsof the emerging photons, with potential use in the generation of nonclassicalstates of light. Current SERS experimental implementations in organicmolecules and two-dimensional layers suggest the interest in furtherexploring intense pulsed illumination, situations of strong coupling,resonant-SERS, and atomic-scale field confinement. SchmidtM. K.; EstebanR.; González-TudelaA.; GiedkeG.; AizpuruaJ.QuantumMechanical Descriptionof Raman Scattering from Molecules in Plasmonic Cavities. ACS Nano2016, 10, 6291–629810.1021/acsnano.6b0248427203727.1Introduction ofa cavity-optomechanical framework to address SERS of molecules inplasmonic nanoantennas.BenzF.; SchmidtM. K.; DreismannA.; ChikkaraddyR.; ZhangY.; DemetriadouA.; CarnegieC.; OhadiH.; de NijsB.; EstebanR.; AizpuruaJ.; BaumbergJ. J.Single-moleculeoptomechanics in “picocavities. Science2016, 354, 726–72910.1126/science.aah524327846600.2Experimental proofof single-molecule optomechanical interactions by exploiting the extremefield localization of a plasmonic picocavity formed by single-atomprotrusions.ZhangY.; EstebanR.; BotoR. A.; UrbietaM.; ArrietaX.; ShanC.; LiS.; BaumbergJ. J.; AizpuruaJ.Addressingmolecular optomechanical effects in nanocavity-enhanced Raman scatteringbeyond the single plasmonic mode. Nanoscale2021, 13, 1938–195410.1039/D0NR06649D33442716.3Consideration ofthe full plasmonic response to correctly address the nanoscale optomechanicalinteraction which induces broadening and a spectral shift of the SERSsignal.SchmidtM. K.; EstebanR.; GiedkeG.; AizpuruaJ.; González-TudelaA.Frequency-resolvedphoton correlationsin cavity optomechanics. Quantum Science andTechnology2021, 6, 03400510.1088/2058-9565/abe569.4Detailed theoretical analysis of the color correlationsexhibited by scattered Raman photons.1las ≈ 1.5–2.5 eV, with ℏ being the reduced Planckconstant) interchange energy with molecular vibrations occurring atmuch lower energy ℏωvib ≈ 25–200meV (≈ 200–1600 cm–1). The resultinginelastic emission of Stokes and anti-Stokes photons at slightly smallerℏ(ωlas – ωvib) orlarger ℏ(ωlas + ωvib) energies,respectively, leads to a characteristic fingerprint of peaks in theemission spectra. This fingerprint is mostly determined by the chemicalbonds within the functional groups but is also sensitive to the temperature,the molecular surroundings, and the isotopic composition of the molecules,among other influences.Molecular vibrations containrich information about the physicaland chemical properties of molecules and their interaction with theenvironment. These vibrations can be addressed optically using Ramanscattering, a powerful characterization technique in which photonsof visible or near-infrared light ,sonators 1a. This 11In this Account, we describe the recently developedquantum optomechanicaldescription of (nonresonant) SERS that goes beyond this simple pictureand explicitly considers the inelastic interaction between cavityplasmons and molecular vibrations at the origin of SERS 1a. This 213 far below the ∼(λ/2)3 diffraction limitof free photons, leading to a huge increase in the SERS signal, astypically explained through the following classical picture.14 The local electric field exciting the molecule E(ωlas) is enhanced by a factor K(ωlas) = E(ωlas)/E0, with E0 being the amplitude of the incident illumination |2). This enhanced field induces a Raman dipole whose (frequency-shifted)emission rate is also strongly enhanced by the large local densityof states (LDOS) of the plasmonic resonance.15 When the optical reciprocity theorem is applied, this increase inthe emission rate can be related to the square of the field enhancementat the Raman emission frequency, |K(ωlas ± ωvib)|2. |4, which is usefulin estimating the order of magnitude of enhancement at the expenseof accuracy: the energy of typical vibrations ℏωvib can be comparable to the line width of plasmonic resonances,so K(ωvib) and K(ωlas ± ωvib) can be ratherdifferent. We note that the SERS signal scales with the fourth powerof the field enhancement, i.e., the square of theintensity enhancement, but in this classical framework,it remains linear with the intensity of the excitation laser.The total enhancement factorin 18 A related configurationthat has the advantage of being precise and relatively straightforwardto integrate with molecular self-assembled monolayers (SAMs) or with2D materials is the nanoparticle-on-mirror (NPoM) configuration, wherea metallic nanoparticle is deposited on a metallic substrate withthe SAM acting as a separator between them.19 Another important configuration which naturally produces a controllablegap is the junction between a tip and a substrate sandwiching moleculesin-between, as in scanning probe microscopy such as tip-enhanced Ramanspectroscopy (TERS).20The simpleenhancement estimate in 33.1S(ωlas – ωvib), Stokes line)or increased (S(ωlas+ωvib), anti-Stokes line) energy is associated with an inelasticprocess that creates or annihilates molecular vibrations, respectively. S(ωlas – ωvib)and S(ωlas + ωvib) arise from the (incoherent) population of vibrations, nb, at ωvib, and the incident laser intensity, Ilas, asnb = nbth =  for small and moderate laser intensity, Ilas), and g0 is the single-photonoptomechanical coupling rate. This g0 isthe key magnitude that determines the optomechanical interaction inSERS , Γ– (blue curve), andthe total optomechanical damping rate Γopt (graycurve) in the presence of a single plasmonic mode at frequency ωres. These three rates are proportional to the intensity ofthe incident laser Ilas (opt is negative when the illumination frequencyis blue-detuned from the mode (ωlas>ωres) and positive when red-detuned.According to ser Ilas 5 and 6.+ and Γ– (vib to γvib + Γopt. The sign of Γopt becomes particularlyimportant for large intensites where |Γopt| ≈γvib. A narrowing of the line width (under blue-detunedillumination) with increasing intensity is illustrated in The vibrational populationalso depends on the incident light intensityand frequency through Γ+ and Γ– 7, as obs3.2g0, which together with the losses and the frequencyand intensity of the incident light determines the nonlinear regimesof SERS. The coupling g0 can be obtainedby quantizing the plasmonic field and the molecular vibrations, followingthe standard procedure. The electric field operator Ê of a single plasmonic mode is mostly determined by the effectivevolume Veff describing the localizationof the electromagnetic field,25â† and â being the plasmoncreation and annihilationoperators, ε0 being the vacuum permittivity, εd being the (linear and nondispersive) relative permittivityof the surrounding medium, and u(r) beinga normalization function giving the spatial distribution and polarizationof the plasmonic field. This field induces a nonresonant polarizationof the electronic molecular transitions, given by the molecular polarizability k, and b̂† and b̂ as the creation and annihilation operators associatedwith the generalized coordinate Q k. Q k parametrizes the vibration (assumed to beperfectly harmonic) and depends on the properties of the molecule,including its mass.6 In the following text,we consider just a single vibrational mode and write g0 is the (single-photon) optomechanical coupling strength,given byp̂r isan induced dipole moment operator.13 Adetailed discussion of this framework can be found in ref  depend on the optomechanical coupling, d in ref .24 such as a Fabry–Pérot optical cavity where one ofthe mirrors supports mechanical vibrational modes. The connectionbetween these two previously unrelated disciplines is further emphasizedby an alternative interpretation of the interaction Hamiltonian ĤI.11 By adding ĤI to the Hamiltonian Ĥres = ℏωresâ†â describing the cavity mode at energy ℏωres, we obtain ĤI + Ĥres = ℏ(ωres - g0(b̂ + b̂†))â†â. Thus, the excitation of the vibrationalmodes induces a shift, g0(⟨b̂⟩ + ⟨b̂†⟩), of the cavity mode frequency . For example, in a Fabry–Pérotconfiguration, the mechanical oscillation of a macroscopic mirrorchanges the length of the optical cavity, modifying the resonant energyof the optical mode24 .27 As an example, g0/(2π) and of the vibrational frequency ωvib/(2π) in typical SERS situations, as compared to other cavityoptomechanical systems, greatly extend the optomechanical landscape.Although their interaction Hamiltonians are analogous, SERS andtraditional optomechanical systems explore a different set of energies,losses, and coupling rates. Plasmonic modes exhibit very large losses, but the vibrational frequencies ω4Ilas. Optomechanical pumpingand decay can lead to three different regimes of SERS as Ilas increases:+ and Γ– are small and the vibrational population nb is given by the thermal pumping nb = nbth. The Stokes S(ωlas – ωvib) and anti-Stokes S(ωlas + ωvib) scatteringrates are then proportional to the laser intensity : In most experiments,the laser intensity is sufficiently weak so that Γ+) rather thanby thermal processes so that the vibrational population nb in nb ∝ Ilas . Importantly, the emissionof an anti-Stokes photon requires the destruction of a vibration,so the anti-Stokes scattering is proportional to both Ilas and nb (1 (nb ≳ 1). These results are fully consistent with semiclassicalmodels of vibrational pumping28 and have also beendiscussed in studies of Stokes–antiStokes correlations.29 An analysis of the quadratic anti-Stokes signaldependence allows an estimation of the optomechanical coupling strength g0, as has been demonstrated for the NPoM constructs2 [g0 reached tens ofmeV, much larger than for any conventional cavity optomechanical system.Attempts to probe into the nonlinear regime of the Stokes signal usepulsed illumination,31 which better mitigates the damageto molecules while allowing large vibrational populations before bondbreaking.Intermediate illumination intensity: For larger laser intensity, the system enters the vibrationalpumping regime, where vibrations are predominantly created by theStokes processes induced by pumping , and the scalingof the anti-Stokes signal with laser intensity turns out to be linearagain.32Strong illumination (populationsaturation and parametricinstability): For very large intensities , whereas red-detuned illumination leadsto vibrational saturation and broadeningWe discuss in the We analyze in thissection the consequences of optomechanical couplingin the evolution of the SERS signal as a function of incident illuminationintensity, 58 but very often target samples are constituted by molecular assemblies.In NPoM configurations, for instance, hundreds to thousands of self-assembledmolecules can be placed in each NPoM gap.19 In typical theoretical calculations, these molecules are consideredseparately, adding the Raman scattering from each molecule as if theydid not interact. This simple approach is valid under weak illuminationconditions , but an extension of the optomechanicalmodel32 to consider multiple molecules reveals thatthe single-molecule description fails at large laser intensity Ilas. In such a situation, the molecule–moleculecorrelations established via their mutual Raman interaction throughthe plasmonic resonator become large enough to modify the emittedRaman signal, revealing the collective nature of the response33 of the full molecular assembly.In the last few decades, various SERS experimentshave shown theability to measure Raman scattering from single molecules,Ilas2 induced by vibrational pumping ,resembling superradiance.34 This superradiant-likescaling can be observed for weaker incident intensity if the temperatureis reduced, making experimental observation easier. A more pronouncedscaling with N is also attainable at large Ilas (at a suitable laser frequency), i.e., whenthe system approaches the parametric instability  that are fully included in the continuum-fielddescription can invalidate the single-mode approximation, particularlybecause these high-energy modes strongly influence the dyadic Green’sfunction that describes the self-interaction of the molecule  is compared with that obtained for the fullplasmonic response (red curve), showing substantial differences. Contraryto the single-cavity-mode approximation  has been created by the emission of a Stokesphoton so that the possibility of these two photons arriving at thedetector at the same time is typically much larger than the productof the individual probabilities.Typical SERS experiments obtain spectralinformation from the intensityof the scattered radiation. However, the emitted light also containsinformation on the photon statistics, which can be accessed, for instance,by measuring the second-order correlation between photons arrivingat two detectors in a Hanbury Brown-Twiss configuration. Furthermore,interest in spectrally filtering the light before detection 6a has re4 leads to two-dimensional correlations maps  mechanismsunearthed in SERS signalsare challenging to interpret because the complexity of organic molecule–metalinteractions and the rich dynamics governing plasmonic and moleculardecay can affect the emitted signal in many different ways. Nonlinearscaling of SERS signals and broadenings of Raman lines with laserintensity can have other origins besides optomechanical interactions,such as structural molecular changes during irradiation, heating,decay from excitonic states, or the decay of plasmons into hot carriers.57 is particularly promising becauseit allows for stronger (peak) laser intensities without harming samplesand for directly accessing the dynamics of vibrations by controllingthe delay between pulses. Pulsed illumination can also be useful tostudy the influence of intramolecular vibrational redistribution (IVR)58 (the interchange of energy between different vibrations) and environmenteffects on molecular vibrational states and thus on Raman signals.Such studies use picosecond pulses or two-dimensional femtosecondspectroscopies.60 Moreover, time-resolved measurementsof molecular dynamics can help to identify if the width of the Ramanlines is due to either pure dephasing or to other decay processes.In this context, the use of ultrafast pulsedillumination63 is still poorly understood,and the optomechanical framework introduced here can address thissituation. Furthermore, plasmonic structures can create regions ofstrong fields (hot spots) smaller than ∼1 nm3 viaatomically sharp features that form picocavities.64 These atomic-scale hot spots enable us to address individual molecularvibrations,2 including those that are inaccessibleto standard Raman spectroscopy, and even map them with submolecularresolution.66 A combined classical and quantum treatment can explain many of theresults observed in these picocavities,67 but a fulldescription of the optomechanical interaction in such a situationrequires further elaboration. Moreover, the study of nonresonant SERS,as described in this Account, can be completed with the inclusionof electronically resonant SERS processes, driving a new variety ofnonlinear effects.70Additionally, the optomechanical framework can be further generalizedin the future to other situations of interest in SERS. For example,the emission of Raman photons from systems where vibrations are stronglycoupled with infrared cavity modes71 usinga similar principle as in surface-enhanced sum frequency generation(SE-SFG57). These devices can exhibit afast response and a low level of noise as compared to current technologies.The first experimental demonstrations of IR detection have recentlybeen achieved.73 In conclusion, molecular optomechanicsbrings the field of optomechanics into the realm of the nanoscalewith organic molecules and solid-state phonons, introducing a newplethora of physical and chemical effects in SERS and thus openinga new technological avenue for molecular nanotechnologies.Advances in molecular optomechanics can alsobe useful in the designof new devices. It has recently been proposed that the sensitivityof the Raman signal to the molecular vibrational state can be exploitedto fabricate terahertz detectors


Antimicrobial resistance (AMR) is a quickly advancing threat for human health worldwide and almost 5 million deaths are already attributable to this phenomenon every year. Since antibiotics are failing to treat AMR-bacteria, new tools are needed, and human monoclonal antibodies (mAbs) can fill this role. In almost 50 years since the introduction of the first technology that led to mAb discovery, enormous leaps forward have been made to identify and develop extremely potent human mAbs. While their usefulness has been extensively proved against viral pathogens, human mAbs have yet to find their space in treating and preventing infections from AMR-bacteria and fully conquer the field of infectious diseases. The novel and most innovative technologies herein reviewed can support this goal and add powerful tools in the arsenal of weapons against AMR. The phenomenon called antimicrobial resistance (AMR), emerged as one of the leading public health threats of the 21st century. The review on antimicrobial resistance, commissioned in 2014 by the UK Government, estimated that AMR could kill 10 million people per year by 2050  that targeted the RSV fusion (F) protein : Multimerized antibodies that include several antigen binding sites are a potent tool of considerable interest in the field of therapeutic mAbs. In a recent study, Divine and colleagues established a new method, applicable to IgG antibodies, to increase antibodies valency through antibodies protein nanoparticles  Fc receptor, an intracellular detection system for opsonized pathogens that have evaded lysosomal degradation  while promoting their pairwise hetero-oligomerization after binding co-expressed antigens on the surface of the targeted cells (Oostindie et al., The technological and methodological advancements of the last 50 years brought the field of mAb discovery and engineering at their peaks. Despite that, only 26 mAbs against bacterial pathogens have so far reached clinical phases for efficacy evaluation and only five were approved by the FDA. Therefore, a further step forward needs to be made. The new technologies herein described can contribute to this effort and finally allow monoclonal antibodies to find their place in the fight against AMR.All authors contributed to the conceptualization, writing, and final revision of this manuscript.


Conclusion: The total coronary revascularization rate after CCTA was 39.2% and not different among low, intermediate, and pre-test probability groups. These findings support the role of CCTA as an excellent gatekeeper in patients with suspected obstructive CAD even beyond pre-test probability calculation models. Purpose: We assessed the application of appropriate use criteria of coronary computed tomography angiography (CCTA) in comparison to invasive coronary angiography results and revascularization rates in patients with coronary artery disease (CAD). Methods: 1305 patients referred to invasive coronary angiography (ICA) after CCTA were evaluated retrospectively. The primary indication for CCTA was assessed according to the consensus for intermediate-risk (15–85% pre-test probability) into appropriate (A), inappropriate (I), and uncertain while referring to published guidelines. Patients’ risk factors, angina, and heart failure symptoms (Canadian Cardiovascular Society classification (CCSC), New York Heart Association (NYHA); clinical data; and ICA results were gathered. Results: Of 1305 patients referred to CCTA prior to ICA, 496 (38.0%) were appropriate, 766 (56.9%) inappropriate, and 43 (3.3%) uncertain. Of 766 patients with inappropriate CCTA referrals, 370 (48.3%) were classified as “inappropriately low” (<15% pre-test probability) and 396 (51.7%) as “inappropriately high” (>85%) in regard to the recommended CCTA utilization. Sub-analysis of the adherence to the appropriate use criteria did not differ between the source of the referring physicians . Obstructive CAD with subsequent revascularization rates  did not differ significantly between the appropriate (38.3%), inappropriate (41.0%), or uncertain (23.3%) groups ( The primary and accurate detection of coronary artery disease has always been a challenging task for clinicians but remains paramount in the practice of cardiology . CardiovApart from primary and secondary prevention of CAD, an early and correct diagnosis is crucial to reducing mortality and healthcare costs. Pre-test probability models for CAD were published earlier and, due to over-and underestimated risk rates, revised and republished ,4,5,6,7.Coronary computed tomography angiography has emerged over the past decade as a non-invasive tool for the diagnosis of coronary artery disease. Due to substantial technological advancement, refinement in clinical imaging protocols, the fast-growing feasibility of CT, and low patient radiation exposures, CCTA rivals conventional catheter angiography in the ability to illustrate coronary artery anatomy . MoreoveA total of 9578 patients were retrospectively screened over 4 years; 7881 were excluded due to known CAD, lack of previous CCTA, and ACS. Of the remaining 1697 patients, 392 were excluded due to incomplete documentation and lack of follow-up. Thus, data from 1305 patients referred to ICA after pathologic CCTA were included in our final analysis. We defined a pathologic CCTA as a stenosis of >50% in one or more epicardial vessels. The indication of CCTA was made by board-certified cardiologists or specialists in internal medicine from primary, secondary, or tertiary referral centers. The study was approved by our local institutional review board; patients’ informed consent forms were waived. A flowchart illustrating patient selection is shown in Inclusion criteria: age > 18 years, CCTA prior to invasive angiography, complete documentation. Exclusion criteria: known CAD, previous invasive coronary angiography (ICA), percutaneous coronary intervention (PCI), coronary artery bypass graft (CABG), acute myocardial infarction, unstable angina, renal insufficiency (GFR < 30 mL/m²/min), hyperthyroidism, iodine allergy, pregnancy. CCTA was performed using different scanner types with a minimum of 64 slices. The assessment was made by one experienced observer  or a resident in cardiac imaging training with subsequent discussion by an experienced level 3 observer. Obstructive CAD was defined as a stenosis > 50% in one or more epicardial vessels. All previous imaging exams and reports were acquired, and the indication for CCTA was reviewed and retrospectively classified as appropriate (A), uncertain (U), or inappropriate (I) following consensus papers ,15.Cardiovascular risk factors  were gathered for each patient. Symptoms (according to the Canadian Cardiovascular Society (CCS) scale and New York Heart Association (NYHA) score) at the time of ICA referral were categorized, and the patients’ respective pre-test probability was calculated according to the modified Diamond and Forrester plot. Clinical follow-up was performed using patient chart reviews and included ICA results and revascularization rate.t-test or one-way ANOVA. Categorical variables are given as absolute values and percentages and were compared with Chi-squared and Fisher’s exact test as appropriate. Multivariate linear regression analysis was performed to adapt for significant differences in known CAD risk factors. A statistically significant difference was defined as a p value < 0.05. To perform statistical analyses, SSPSTM software  was used. Quantitative variables are expressed as mean ± SD. In case of normal distribution, further comparisons were made using The final analysis included 1305 patients  referred to ICA due to pathological CCTA. Of those, the CCTA indications of 496 (38.0%) were appropriate, 43 (3.3%) were uncertain, and 766 (56.9%) were inappropriate (n = 370) of the referrals defined as inappropriate were asymptomatic or presented with atypical chest pain classified as CCS 0. Considering symptoms, age, and sex that defined the respective pre-test probability for epicardial coronary artery disease, we found that 48.3%  presenting with more typical angina, dyspnea, or mixed symptoms (combination of CCS or/and NYHA classifications), adjusted for age and sex and with the addition of existing risk factors, had a higher pre-test probability and should have been referred to functional testing or directly to invasive diagnostic angiography. p < 0.001). Overall cardiovascular risk profiles were intermediate–high in all appropriateness groups; there was a significant difference between inappropriate and appropriate referrals in female patients .The overall mean age was 63.9 ± 9.6, with a significantly lower mean (52.6 ± 5.8) in uncertain referrals compared to appropriate (62.2 ± 9.3) and inappropriate : of 766 inappropriate referrals, 57 (7.4%) were admitted by a tertiary center, 63 (8.2%) by a secondary, and 646 (84.3%) by a primary care physician. On the other hand, most appropriate referrals also came from primary referring sources. The absolute numbers of patients attributed to the three categories of appropriateness and the respective referral sources are displayed in The CAD pre-test probability rates were calculated following adapted ESC guidelines  definingp = 0.071) , without differences between the groups (= 0.071) .p = 0.068). The rates of percutaneous coronary interventions were similar; the rate of CABG was higher in the inappropriate group.Interestingly, coronary revascularization rates did not differ significantly between the appropriate and the inappropriate groups . The previously cited study from Bahrain found a higher number of uncertain CCTA indications (20.1%) but applied a different rating system . The majNevertheless, this highlights the high uncertainty in clinical decision making in an insufficiently attributed patient cohort that requires the physician’s judgment to determine the pathway for an individual clinical scenario .In accordance with the guidelines, asymptomatic patients not requiring CCTA could be further risk-stratified with a simpler approach using coronary calcium scanning added to various standard risk scores ,20,21,22The rate of patients with significant CAD in ICA after inappropriate use of CCTA was high. Revascularization in these patients was performed at the discretion of the operator or the heart team’s decision. This may be explained by the definition of appropriate and inappropriate CCTA indications. The consideration of the high number of inappropriate CT referrals only included patients with no, atypical, or typical symptoms, which may explain the unexpected ICA-triggered numbers of revascularization procedures. We see this underlined by a more precise clinical evaluation of chest pain symptoms with newly defined angina classification and the introduction of dyspnea as an additional feature in the newer chronic coronary syndrome ESC guidelines .Of our patients, 105 had left main or/and severe three-vessel disease requiring subsequent CABG. Previous studies in asymptomatic patients, mainly diabetes subsets, revealed high rates of obstructive CAD in CCTA ,25. ThisIn addition, our patient cohort, preselected due to the retrospective approach, shows high cardiovascular risk profiles. Therefore, the proportion of patients referred to CCTA due to a too-low initial probability of CAD (classified as inappropriate) and undergoing subsequent revascularization was as high as 36%. The distribution of these risk factors was similar in the appropriate and inappropriate groups, causing a preselection bias.Another issue is that we depend on a large diversity of referring sources and cannot judge how accurate individuals’ knowledge of existing appropriate CT guidelines is. Moreover, the timing of easily available CT investigation before invasive angiography was certainly co-influenced by longer schedule delays of other imaging or functional ischemia testing modalities. This study was performed retrospectively with an inherent bias of only including patients after ICA and correlating their results with previous CCTA findings. In a second step, the appropriateness of the CCTA referrals was investigated. We therefore have a predefined patient population but wanted to draw a real-world clinical scenario. A second limitation is that the study design is single-centered, but the large number of patients and the variety of CCTA referring physicians were an elementary step of the investigation. A large number of CCTA patients with nonpathologic reports not referred to ICA was excluded and could have served as comparing group; nevertheless, this group would only be comparable in terms of the need for revascularization. This population, of course, was guided to primary prophylactic measures and/or medical treatment depending on the CCTA reports.In our single-center evaluation, CCTA use diverged from the current appropriate use criteria. We observed a large number of patients with obstructive CAD and subsequent revascularization, regardless of the initial appropriateness recommendation. CCTA, therefore, may have an incremental prognostic and clinical value in patients beyond the intermediate pre-test probability classification and should be considered a valuable diagnostic tool. It should be pointed out, however, that a more precise exploration and definition of clinical chest pain symptoms may avoid inappropriate CCTA referrals, especially in higher pre-test probability populations.


Klinefelter syndrome (KS) is the second-most prevalent chromosomal disorder in men, though late diagnosis is very common and 50–75% of men remain undiagnosed. Evidence suggests that men with KS have impaired quality of life (QoL) but research on how the diagnosis of KS is associated with different QoL domains and what factors influence patients’ QoL is limited. This study aimed to provide a systematic review of the published evidence on factors that influence QoL in men with KS.Systematic review and meta-analysis with narrative synthesis.Medline, Cochrane, Embase, Psychinfo, CINAHL, BASE and relevant publication reference lists were searched in January 2021. Eligible studies included randomised control trials, cohort studies, cross-sectional studies and epidemiology studies on KS and its effect on QoL and all domains of World Health Organisation (WHO) Quality of Life 100 (WHOQOL-100). Clinical studies with no date restriction published in English were included.P < 0.00001).Thematic analysis was completed on 13 studies, with a meta-analysis of intelligence quotient completed on 7 studies. Twelve out of the 13 studies suggested that KS negatively affected the QoL outcomes and KS was associated with impairments in physical, psychological, level independence and social relationship domains of WHOQOL-100. Meta-analysis suggested that men with KS have significantly lower full-scale Intelligence Quotient vs controls (This is the first evidence synthesis of QoL in men with KS. Current evidence suggests that combined physical and psychological impairments affect men with KS who also experience impairments in relationships and independence in society. Further research is needed to identify factors that influence the QoL in men with KS. Klinefelter Syndrome (KS) first described by Harry Klinefelter in 1942 . It affeThe extent of mosaicism in KS causes an array of cognitive, psychosocial, and physical symptoms which can affect men at varied degrees of severity. These include hypogonadism, gynecomastia, tall stature, small phallus, reduced level of intelligence, depression, autism traits, schizotypal traits and social anxiety which lead to impaired quality of life (QoL) , 7, 8, 9Evidence suggests that patients with KS have more impaired QoL compared to healthy controls; however, research on how the diagnosis of KS affects a patient’s QoL is limited , 12, 13.There is limited knowledge of the various symptoms, outcomes and patient experiences, which may result in health and social inequalities for patients with KS. A greater understanding of the associations between KS and the domains of QoL can better support clinical decision-making and meet the condition-specific needs of patients with KS.The objective of this study was to conduct a systematic review with meta-analysis to provide new insights and further understanding of QoL in patients with KS and to answer the following research question:What is the association between Klinefelter syndrome and the WHOQOL-100 domains/facets of QoL?Due to the many factors influencing QoL in patients with KS, the World Health Organisation (WHO) Quality of Life 100 (WHOQOL-100) was adopted as the overarching framework to underpin this systematic review. The WHOQOL-100 is a validated psychometric scale which can be used to measure QoL as an overall construct and across its six QoL domains: ‘overall QoL’, ‘physical health’, ‘psychological health’, ‘level of independence’, ‘social relations’ and ‘environment’ . The subThe systematic review followed the Preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines for quantitative systematic reviews , and theAll empirical studies involving male children and/or adults diagnosed with KS and measuring a quantifiable factor of QoL that could be defined within the WHOQOL-100 were reviewed for eligibility. No publication date restrictions were imposed (see The search was completed on 21 January 2020 using the following databases: MEDLINE (1946 to present), APA Psychinfo (1956 to present), Embase (1974 to present), CINAHL (1963 to present), Cochrane (2005 to present) and grey search via Bielefeld Academic Search Engine (BASE). A secondary search, using the same strategy, was run covering the period between 21 January 2020 and 20 April 2021 to ensure no recent studies were missed; no new studies were included.Each database was searched individually, the search keywords included ‘Klinefelter Syndrome’ and MeSH terms ‘48, XXYY Syndrome’, ‘49 XXXXY Syndrome’, ‘XXXY Males’, ‘XXY Syndrome’, ‘XXY Trisomy’ and ‘XXYY Syndrome’ all combined with ‘OR’. Keywords for QoL factors, combined with ‘OR’ included: ‘physical health’, ‘psychological health’, ‘level of independence’, ‘social relations’, ‘environment’, ‘spirituality’, ‘faith’ and ‘personal beliefs’; both groups were then combined with ‘AND’. A full search from CINAHL and Medline is included in the https://rayyan.ai/) using pre-specified inclusion/exclusion criteria (The eligibility assessment was performed in a blind independent review by two authors (BM and SL); all disagreements were resolved by consensus and did not require a third reviewer. The blind review for abstracts and full text articles was done in Rayyan QCRI systematic review manager (criteria . The PRIInformation was extracted from each study on the following: (i) number of participants; (ii) study settings, study country and study design; (iii) outcome measures related to the WHOQOL-100 framework (domains/facets) of QoL such as ‘physical health’, ‘social relations’, ‘psychological’, ‘environment’, ‘level of independence’ or ‘religion/personal beliefs/spirituality’; (iv) comparison groups where possible .The Joanna Briggs quality appraisal tool for cross-sectional studies/cohort studies  was usedThe SWiM reporting items protocol  was adops.d. from KS and control groups where reported. Accepted categories for Cohen’s d effect sizes as small (0.2), medium (0.5) and large (0.8) were applied  between patients with KS and controls.Data were too heterogeneous for meta-analysis due to the vastly different outcome measures included; therefore, to aid comparability, Cohen’s d was calculated by extracting the mean difference and  applied . Where s. Studies had a mixture of patient-reported, parent-reported, or physician-answered questionnaires. A total of 665 records were identified from the initial search of which 13 studies, 12 cross-sectional and 1 cohort, met the inclusion criteria and 7 studies were suitable for additional meta-analysis . The totThe quality of the included studies has an impact on the confidence of findings within the review. First, when assessed many studies did not discuss or include strategies to deal with confounders. Secondly, normative data and population averages were used for controls in three studies which lowers the comparative domain score for those studies , 22, 23,Sampling methods included non-probability using snowballing , self-idP ≤ 0.05) between patients with KS and controls for the outcome measures: Personal Well-being Index (PWI)  , WHOQOL-ex (PWI) , Short Fex (PWI)  and for ex (PWI)  subitems , psychological , social relations , environment )  in all domains of WHOQOL-100 between KS patients and healthy controls  in all Q-LES-Q sub items. Q-LES-Q subscales had significant differences favouring controls in scales , leisure time activities (P = 0.05), social relations (P = 0.003), general activities (P = 0.045)).Rapp  0.635)) . Similarcontrols . Fabrazzet al. found that patients with KS had significantly worse physical health compared to controls (P < 0.001) for the following parameters: hypogonadism, gynecomastia, undescended testis, osteoporosis, tremor, varicose veins, pulmonary embolism or leg thrombosis, heart valve disease, dental problem, gingiva, chronic headache, fatigue and anxiety  compared to three other groups of patients with disorders of sexual development (DSD): females with congenital adrenal hyperplasia, females with XY- DSD, males with XY-DSD  was identified between controls and patients with KS in each of these outcomes: autism spectrum quotient (AQ), gender identity/dysphoria, neocriticism, extraversion, conscientiousness, attention switching, imagination, communication, global severity index (GSI), mini mental state examination (MMSE), positive symptom distress index (PSDI), social skills including social behaviour and negative assertion (P < 0.001) between patients with KS and reference population was also reported by Herlihy et al. regarding the psychological measures of body-self relations, self-esteem, sexual identity and psychological distress  as measured by Autism spectrum quotient; both studies and a total of (n = 77) participants were included  in seven studies, six cross-sectional and one cohort longitudinal . This inet al.  of patients with KS scored mild, moderate or severe on all domains of the Social Responsiveness Scale (SRS) except for the social awareness domain  when measuring social responsiveness using SRS in patients with KS when compared to controls  did not complete high school, 55% completed high school and 10% studied further than high school (P < 0.05), concentration difficulties (P < 0.005), speech difficulties (P < 0.05), lack of self-confidence (P < 0.05), particularly dependent on parents (P < 0.025) (Acquiring new information and skills can be linked to school attainment and completing education. Herlilhy h school . Results< 0.025) .et al. found that patients with KS (n  = 132) were significantly less likely than controls to complete high school (P  < 0.001) and at least 1 year of higher education (P  < 0.01) (P < 0.001) for patients with KS when compared to healthy controls  and found that 13.6% completed post-graduate education, 23.9% college, 22.6% part of college education, 13.6% technical school, 22.2% high school and 4.1% completed elementary or junior school  earned less than AUS$30,000, 27% earned between AUS$30,000 and 69.999 and 30% earned more than AUS$70,000  had significantly lower (P < 0.001) household income compared to healthy controls (Skakkebaek  < 0.01) . School controls . TurriffS$70,000 . Similarcontrols .This is the first systematic review to provide an in-depth analysis of the associations between KS and QoL. The WHOQOL-100 provided a framework which allowed sufficient synthesis for many parameters and domains of QoL, which highlighted a disparity in the QoL between patients with KS and controls. Furthermore, the meta-analysis from the included studies indicates a lower full-scale IQ is associated with KS diagnosis.n = 829) across 12 studies included in this systematic review reported that KS had negatively affected the QoL outcome measures. When calculated between patients with KS and controls, a significant effect size (Cohen’s d) was present in most outcomes measured (91.8% of 49). The effect size within this review further quantifies the difference between KS and controls in outcome measures associated with QoL providing hence evidence of the negative impact of the KS diagnosis on patients’ QoL.Almost all patients , psychosis, personality disorders and developmental disorders , 47.et al. noted the importance of IQ as a predictor of future success  achievement and worse behaviours than healthy controls at all levels of education (School attainment and behaviour were measured by five studies which found that boys with KS had significantly lower (ducation , 33, 57.ducation . AlthougThis systematic review found that the diagnosis of KS has a significant negative impact on the patients’ erectile function and sexual satisfaction, which is most likely secondary to testosterone deficiency and psychological disorders associated with KS . FurtherIn conclusion, our systematic review with narrative synthesis and meta-analysis, guided by the WHOQOL-100 as an overarching framework, provides evidence that patients with KS have impaired QoL compared to healthy males. Although evidence for overall QoL outcomes was limited, subgroup analysis helped to provide a greater understanding of the WHOQOL-100 subgroups, and the extent to which each of these are affected by patients with KS. Further research is needed to understand the impact the diagnosis of KS has on patients’ QoL. A significant finding from this systematic review was the lack of a condition-specific PROM for patients with KS. Development and validation of a KS-specific PROM that would encompass all domains of QoL for this patient group and would provide a quantifiable and validated measure for QoL is therefore essential.Sofia Llahana: Occasional scientific consultant and conference fees for Bayer, Novartis, Pfizer and Ipsen. Unrestricted educational grants from Sandoz and Bayer. Senior Editor for Endocrinology, Diabetes and Metabolism Case Reports. Channa Jayasenna: Investigator- led grant by Logixx Pharma Ltd. The other authors have nothing to disclose.No funding has been received for this systematic review. S L is funded by an NIHR Clinical Lectureship. C N J is funded by the Imperial NIHR BRC & NIHR Post-Doctoral Fellowship.B M and S L conceived the presented idea. B M developed the conceptual framework, theory, methods and searching under supervision of S L, B M and S L completed the blind review of studies. B M completed the collection of data and analysis from the included studies. All authors B M, S L, S G, and C J contributed to the discussion of results and provided critical feedback to all aspects of the manuscript which helped shape the final manuscript. B M, C J and S L completed the abstract. B M and S L took the lead on writing the main body of text.