Pseudomonas aeruginosa is a leading cause of chronic respiratory infections in people with cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD), and acute infections in immunocompromised individuals. The adaptability of this opportunistic pathogen has hampered the development of antimicrobial therapies, and consequently, it remains a major threat to public health. Due to its antimicrobial resistance, vaccines represent an alternative strategy to tackle the pathogen, yet despite over 50 years of research on anti-Pseudomonas vaccines, no vaccine has been licensed. Nevertheless, there have been many advances in this field, including a better understanding of the host immune response and the biology of P. aeruginosa. Multiple antigens and adjuvants have been investigated with varying results. Although the most effective protective response remains to be established, it is clear that a polarised Th2 response is sub-optimal, and a mixed Th1/Th2 or Th1/Th17 response appears beneficial. This comprehensive review collates the current understanding of the complexities of P. aeruginosa-host interactions and its implication in vaccine design, with a view to understanding the current state of Pseudomonal vaccine development and the direction of future efforts. It highlights the importance of the incorporation of appropriate adjuvants to the protective antigen to yield optimal protection. Pseudomonas aeruginosa is a motile and aerobic Gram-negative bacillus, with great diversity and adaptability in a wide range of environments, including non-clinical  and clinical settings  [Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter [P. aeruginosa caused 32,600 infections among hospitalised patients and 2700 estimated deaths in the United States [P. aeruginosa infections; however, the pathogen quickly develops tolerance to these agents due to its intrinsic resistome [P. aeruginosa was included by the World Health Organisation (WHO) among the “critical” group of pathogens for which new antibiotics are urgently needed [orisers) . As an orobacter ,3. In 20d States ,5. A broy needed .P. aeruginosa is responsible for opportunistic infections in immunocompromised individuals and patients with malignant diseases or HIV infection [P. aeruginosa are a significant co-morbidity in patients with cystic fibrosis (CF), bronchiectasis, chronic obstructive pulmonary disease (COPD) or ventilator-associated pneumonia (VAP) [P. aeruginosa [P. aeruginosa is a major threat due to its propensity to adapt and acquire resistance to antibiotics. Consequently, vaccines have the potential to be more effective interventions for the prevention and treatment of Pseudomonas infections. Optimal vaccine design must consider complex host–pathogen interactions to identify effective antigens and delivery systems. Hence, this review describes the host immune response against P. aeruginosa; the factors that allow the adaptation of the pathogen to the host; and the numerous vaccine candidates and adjuvants that have been evaluated over half a century of Pseudomonas vaccine development.nfection . Moreoveia (VAP) ,10. Accoruginosa . P. aeruP. aeruginosa possesses a wide arsenal of virulence factors that contribute to its pathogenicity  to release a wide variety of toxins and hydrolytic enzymes that attack the host to both the intracellular and extracellular milieu [P. aeruginosa, inhibiting host protein synthesis due to its ADP-ribosylating activity, ultimately leading to irreversible cell death [P. aeruginosa further contributes to the disruption of the respiratory epithelium by degrading lung surfactant and disrupting tight junction integrity [P. aeruginosa overcome oxidative stress in the host are also expressed, including catalases , alkyl hydroperoxide reductases and superoxide dismutases [P. aeruginosa survival in this environment, where this essential nutrient is scarce [r milieu ,23. The r milieu . Exotoxill death . Pyocyanll death . In addill death . The prontegrity ,29,30. Msmutases . Products scarce ,33.P. aeruginosa with an extraordinary plasticity that facilitates successful colonisation of a broad range of niches [P. aeruginosa to persist in the respiratory tract of susceptible patients, thereby establishing chronic infections even for decades, especially in people with CF [P. aeruginosa is exposed to a diverse range of stressors in the CF lung, including osmotic, oxidative and nitrosative stresses, sublethal concentrations of antibiotics, and the presence of other microorganisms [P. aeruginosa populations. Thus, a multitude of genomic and phenotypic adaptations that promote bacterial survival by attenuating virulence and avoiding immune recognition have been reported [O-antigen and structural modifications of lipid A), (vi) selection against T3SS and loss of cytotoxicity, (vii) reduction in communication systems (QS), (viii) change in iron uptake strategy from siderophores towards haemoglobin utilisation, (ix) acquisition of antibiotic resistance and (x) loss of virulence [The regulation of all these virulence factors is cell density-dependent via the release of autoinducers of four quorum sensing (QS) systems . They are interconnected in a hierarchical manner, creating a highly adaptable network that responds to external stressors and provides f niches . This ad with CF . CF is a with CF . P. aerurganisms . Hence, reported ,38. Thesirulence ,40,41,42P. aeruginosa infection. Emond et al. observed that variants in the DCTN4 gene were associated with the age of first P. aeruginosa airway infection, time to chronic P. aeruginosa infection and mucoid P. aeruginosa in individuals with CF [P. aeruginosa in both mice and humans, since it transports a bacterial attachment-promoting metabolite (l-arginine) out of the host ASL [P. aeruginosa pulmonary colonisation in 210 CF patients, suggesting that T2R38, which encodes a taste receptor, is a novel modifier gene in CF [P. aeruginosa pathogenesis and the adaptation of the bacterium to the host environment.In addition to the environmental impact of CFTR mutations there is also variability between CF patients, despite identical CFTR genotypes, indicating that other genetic factors contribute to the severity of lung disease. This heterogeneity can be explained in part by the presence of modifier genes . Interes with CF . Furtherhost ASL . More rene in CF . These aP. aeruginosa expresses powerful agonists of Toll-like receptors (TLR), TLR2, TLR4, TLR5 and TLR9, which recognise lipopeptides, LPS, flagellin, and non-methylated bacterial CpG DNA, respectively  by the release of OMVs, promoting TLR-dependent responses in epithelial cells through the delivery of proteins and LPS. The OMVs activate NF-κB signalling and mitogen-activated protein kinase (MAPK) in epithelial cells [P. aeruginosa infected cells [al cells ,69. Impoed cells .P. aeruginosa pathogen-associated molecular patterns (PAMPs) elicits a potent inflammatory response, which is critical for the recruitment of neutrophils and macrophages, facilitating the bacterial clearance  and the release of bactericidal proteins from their acidophilic granules [The recognition of learance . Howeverd humans ,75,76,77tivation ,74,78,79lsewhere . The neugranules . Hence, P. aeruginosa in the lung [P. aeruginosa, but also for the phagocytosis of dying neutrophils, thus limiting neutrophil-induced tissue damage [P. aeruginosa is found in the intracellular environment of various types of mammalian cells, including macrophages. Recently, Garai et al. demonstrated that P. aeruginosa can initially reside in phagosomal vacuoles and then be detected in the cytoplasm of macrophages, indicating a phagosomal escape. Intracellular bacteria may eventually induce macrophage lysis, in an ExoS-dependent manner. Therefore, T3SS and ExoS, whose expression is modulated by MgtC and OprF, are key actors in the intramacrophage lifestyle of P. aeruginosa [Macrophages are the first cells to encounter the lung . They arthe lung . Alveolae damage . There iruginosa .P. aeruginosa [P. aeruginosa acts as a binding acceptor molecule for C3b to initiate the formation of the membrane attack complex (MAC). Mishra et al. demonstrated that C3b binding was significantly reduced in an OprF-deficient P. aeruginosa strain [P. aeruginosa infections; however, the relative importance of these pathways and how they are integrated in vivo remain unclear.The complement system is also necessary for the survival of mice after pulmonary infection with ruginosa . The Opra strain . The innP. aeruginosa infection is not eradicated during the acute phase, it evolves into a chronic infection characterised by a mucoid biofilm [P. aeruginosa infection is characterised by a Th2-skewed response with the upregulation of IL-5 and IL-13; higher B-cell sensitivity to IL-4; low levels of IFN-γ and elevated levels of IL-10, which further downregulate IFN-γ, and decrease co-stimulatory molecules in macrophages. This response hinders antigen presentation and the successful immune response of the host against P. aeruginosa infections [The resolution of the acute inflammatory response requires attenuation of pro-inflammatory pathways. Regulatory T cells (Tregs) inhibit the secretion of pro-inflammatory cytokines and secrete anti-inflammatory cytokines, while dendritic cells initiate adaptive responses. If the  biofilm . Persist biofilm . The adafections . The relfections ,87,88. Cfections .P. aeruginosa had greater levels of IL-3, IL-4, and secreted immunoglobulin A (Th2 markers) and lower IFN-γ secretion, compared with intermittently colonised patients or those without P. aeruginosa [P. aeruginosa, reflected in the reduction in bacterial counts within the lungs of mice or superior survival rates of Pseudomonas infected mice relative to the control groups  formation is also a hallmark in rognosis .P. aeruginosa infection, the airway of paediatric CF patients is associated with an elevated level of B lymphocyte differentiation factor (BAFF), indicating that BAFF expression is not specific to P. aeruginosa infection and may be a characteristic of the CF airway. Furthermore, despite the presence of this potent B-cell activator, chronic colonisation is common, suggesting that this response is ineffective [P. aeruginosa infections remain unclear, and better knowledge of the underlying mechanisms, such as maturation of avidity/affinity, class change, memory formation, and cytokine synthesis, is needed to understand this phenomenon [Regardless of ffective . The reaenomenon .+ B cell activation and the production of immunoglobulins during acute pulmonary P. aeruginosa infection [P. aeruginosa infection is not associated with clinical improvement [P. aeruginosa IgG may lead to the formation of circulating immune complexes, which are deposited in the lower airways tissue, triggering tissue damage and long-term deterioration of lung function [Pan et al. demonstrated that IL17-γδ T cells are involved in CD19nfection . Thus, Infection , supportrovement . Indeed,function .P. aeruginosa respiratory infections, as it is the predominant antibody isotype in the mucosal immune system, lining in the respiratory tract [P. aeruginosa in nasal secretions and saliva correlates with the infection status of CF patients  [Immunoglobulin A (IgA) is also of great importance in the humoral response against ry tract . The conuginosa) ,105.P. aeruginosa is still far from being understood. However, high antibody production is a characteristic of P. aeruginosa infections, which has not only proven to be ineffective in the clearance of the pathogen but also to be detrimental when the infection becomes chronic. A better understanding of the humoral response, such as IgG avidity for antigens or the role of IgA in mucosal immunity, is needed to find better methods of diagnosis and treatment of acute and chronic infections generated by P. aeruginosa, thus reducing the morbidity and mortality of susceptible individuals such as CF patients.The role of B cells in the defence against P. aeruginosa infection has been difficult in the absence of mouse models capable of developing spontaneous lung disease. CF mouse models do not develop distinctive features such as mucus plugging, chronic bacterial infections, or persistent inflammation [P. aeruginosa [P. aeruginosa infection to three months. The authors used CFTR-deficient mice, which they chronically colonised using P. aeruginosa embedded in agar beads by intratracheal instillation. The persistence of P. aeruginosa had a greater effect on inflammation and lung damage than the Cftr mutation itself, deepening our understanding of the pathogenesis and progression of CF airway disease and P. aeruginosa chronic infections [P. aeruginosa in agarose or agar beads physically retains the bacteria in the airways and creates an environment that mimics the biofilm of bacteria and microaerobiosis present in the CF lung. Such retention prevents physical elimination and leads to the persistent stimulation of host defences typical of CF [P. aeruginosa infections, there is no single animal model that fully reproduces the complexity of CF in humans and therefore provides the real picture of the development of P. aeruginosa infection in the patient [The study of the host immune response to chronic ammation ,106, whiammation . The disammation . Some moruginosa . Cigana fections . Embeddial of CF . Althoug patient .P. aeruginosa for over half a century, and several antigens and strategies have been examined. However, despite extensive efforts, there are no approved P. aeruginosa vaccines to date. The vast majority of vaccines have been evaluated in preclinical studies (O-polysaccharide-exotoxin A conjugate (Aerugen®)  . There a complex .®, Parke-Davis, Detroit, MI, USA) [P. aeruginosa nor clinical benefit was achieved [O-antigen, elicited protective antibodies on its own and was non-toxic, non-pyrogenic and immunogenic in pre-clinical studies [P. aeruginosa serotypes are responsible for life-threatening infections, limiting the utility of monovalent vaccines. Consequently, a heptavalent version with different OPS serotypes was prepared; however, antagonistic reactions resulted in limited levels of opsonic antibodies being detected [Lipopolysaccharide is possibly the most widely characterised and investigated vaccine candidate due to its surface accessibility and high immunogenicity. The first LPS vaccine created was a heptavalent formulation based on LPS extracts from seven different strains  , which wMI, USA) ,223,224.achieved ,199. Simachieved ,201,225.achieved ,226, but studies ,228. In  studies . Neverthdetected .P. aeruginosa immunotype 5 conjugated to ETA was safe upon intraperitoneal administration to mice or guinea pigs, and was not subject to toxic reversion when exposed to physiologic temperatures [® , containing the most frequently encountered serotypes in the clinical setting, underwent phase II evaluation with 59 healthy volunteers [P. aeruginosa chronic infection  was associated with better preservation of lung function [A conjugate vaccine formed by the OPS from eratures . When teeratures . A heptaeratures . Based ulunteers . The sinlunteers . In a folunteers ,232. Aftlunteers ,233,234;lunteers ; (iii) rfunction ,115,204;function .Conjugates of OPS to different carrier proteins, such as bovine serum albumin (BSA) , tetanusThe immunogenicity of alginate, also called mucoid exopolysaccharide (MEP), was not realised until the discovery of naturally elicited anti-MEP antibodies in CF patients in the 1980s ,240,241,O-acetylated form of alginate was covalently linked to ETA [O-acetyl groups contribute to the binding epitope of MEP-specific opsonic antibodies [Neisseria meningitidis serogroup B [Both molecular size and dose of alginate are key parameters for eliciting opsonic killing antibodies after immunisation with this exopolysaccharide . A triald to ETA . It provd to ETA . It was tibodies . Subsequtibodies . A polymtibodies . Later, tibodies . Similartibodies , a synthtibodies , diphthetibodies , OMVs ofogroup B  or PLGA ogroup B .P. aeruginosa infection and it showed high protection in pre-clinical studies with a mouse burn model [P. aeruginosa infections. In 1991, a phase I clinical trial on 220 healthy volunteers vaccinated intramuscularly with six different monovalent flagellar preparations showed no adverse side effects, and induced high and long-lasting antibody titres in both serum and secretory immune system (SIS) in all individuals [P. aeruginosa strains exhibiting flagella subtypes included in the vaccine were significantly less frequently isolated from vaccinated volunteers than from placebo controls [P. aeruginosa acute infection was determined to be 34% of participants, while 51% of patients with a chronic infection were protected. Ultimately, the primary outcome of 66% protection was not achieved [The flagellum is a key virulence factor for the successful establishment of rn model ,123. Conividuals . In 1995ividuals . Finallyividuals . P. aerucontrols . Howeverachieved .P. aeruginosa pneumonia model, achieving 83.3% survival after 125 h [P. aeruginosa challenge, generating robust immune responses. For instance, a recombinant type-a FliC induced both cellular (IL-4 and IFN-γ) and humoral (IgG and IgM) immune responses in mice [Since then, few advances have been made with flagella alone as a vaccine antigen. Intact polymeric flagella, which also contains FliD cap protein and other basal body compounds, was superior to monomeric FliC at inducing protective antibodies in a mouse er 125 h . Neverther 125 h . Two stuer 125 h ,253. Recer 125 h , type-b er 125 h , or a coer 125 h , showed P. aeruginosa may be a better approach, and various vaccines have been tested in animal models, such as (i) type-a and -b flagellins with entire polymannuronic acid alone or with whole alginate [Combinations of flagella with other immunogenic compounds of alginate ,130, -based approaches have been evaluated. Ideally, a pilin-based vaccine should target conserved regions, such as the N-terminal domain, to ensure broad protection, but the hydrophobicity and poor accessibility of this domain prevents it from being a good vaccine candidate . BecauseIntact PilA has also been investigated, either alone or in combination with other antigens. Mice intratracheally immunised with pilin protein showed a significant improvement in survival (80–100%) in response to challenge, and the bacterial load was reduced up to 5 logs after 18 h . HoweverP. aeruginosa strain 1244 naturally presents an O-antigen repeating unit covalently linked to each pilin monomer. When mice were immunised with a strain 1244 PilA-glycoprotein-based vaccine, it provided O-antigen-specific protection via the mucosal and systemic routes of immunity [The immunity . In a buimmunity . Moreoveimmunity . More reimmunity . These r380-706) and a fusion PilQ380-706-PilA protein, which produced specific opsonic IgG antibodies in rabbits that inhibited twitching motility, suggesting that they could be used as vaccine antigens [Synthesis of consensus peptide immunogens that represent the various pilin serotypes to achieve broad protection without the use of a vaccine cocktail has yielded promising results. Cross-reactive polyclonal sera were elicited, generating protective high antibody titres ,262,263;antigens ,265. Finantigens . HoweverP. aeruginosa strains (CFC-101) was tested in three clinical trials, showing toxicity issues without clinical benefit [6OprF190–342–OprI21–83) was produced, and resulted in a synergistically enhanced protection in an immunosuppressed mouse model [The most widely investigated outer membrane proteins as vaccine antigens are the major porin protein F (OprF) and the lipoprotein I (OprI). Various studies demonstrated that these OMPs are surface-exposed, highly conserved among all different serotype strains and immunogenic ,267,268, benefit ,211,212. benefit ,150,274, benefit ,270,271. benefit . Later, se model . Subsequse model .P. aeruginosa infections. Moreover, although the differences were not statistically significant, a higher infection rate was observed in the treatment group (11.2–14%) than in the placebo group (6.1%), suggesting that IC43 vaccine may affect P. aeruginosa virulence rather than clearance [P. aeruginosa infection prior to the development of effective IgG immune response [Thirty-two volunteers were vaccinated intramuscularly with the systemic formulation of the His-tagged hybrid protein, which was well-tolerated. Moreover, a significant rise in complement-promoting, opsonising antibody titres against both OprF and OprI was detectable up to six months after the third vaccination . Robust learance . A confilearance . Severalresponse . Recentlresponse .The mucosal formulation of the OprF-OprI hybrid protein vaccine was examined as an intranasal vaccine in eight healthy volunteers, and showed elevated IgG and IgA levels in the airways, which persisted for up to one year and had no side effects ,219. A sP. aeruginosa strains, produced antibodies that neutralised ETA cytotoxicity, and conferred protection in a burn wound mice model  [Other approaches involving OprF and OprI include mice immunisation with the N-terminal domain of OprF, which elicited specific and protective IgG1 and IgG2 antibodies . Co-admi10 days) . Both Op10 days) ,133,134.10 days) .P. aeruginosa challenge demonstrated in mice immunised with chimeric viruses expressing different epitopes. Influenza virus [P. aeruginosa cellular inclusions engineered to display OprF, OprI and AlgE antigens in the surface induced a protective Th1-cellular response associated with the production of antibodies that reacted with different strains and possessed opsonophagocytic killing activity [Viral vector systems presenting protective epitopes of OprF have provided encouraging results with strong IgG and/or IgA antibodies titres and protection against za virus , cowpea za virus ,280, tobza virus  and adenza virus ,284,285 activity .P. aeruginosa strains in a murine acute pneumonia model showed that porin protein OprH was the most highly expressed OMP, but immunisation of mice with OprH and curdlan adjuvant  and the carboxyl terminus. Indeed, immunisation with antigens containing these regions provided comparable survival rates with immunisation with whole PcrV (65%), with a significant increase in IgG1 and IgG2 titres [NH) combining two immunodominant domains of PcrV triggered a Th2-mediated immune response, and while no mice in the unimmunised group survived, 50–60% of immunised mice were protected [PcrV was the first protein of the T3SS translocation apparatus (PcrV) used as a vaccine antigen, which resulted in protective immunity and enhanced survival in challenged mice, both in lung infection and burn models ,289. Imm2 titres . Compari2 titres . Intrana2 titres . An optirotected . Howeverrotected . Overallrotected ,164,168.3 triggered a Th2-biased immune response and enhanced protection over immunisation with a single unit of the three antigens in an acute pneumonia mice model  [A recombinant PcrV with a L-homoserine lactone autoinducer showed over 75% survival when tested in a burn wound sepsis mice model . A trivaurvival) . Finallyurvival) .Other proteins of the T3SS translocation apparatus have also been evaluated. A reverse vaccinology approach identified that PopB possesses strong T-cell epitopes and non-protective B-cell epitopes . IntranaP. aeruginosa isolates [Widespread expression of this exotoxin by most clinical isolates ,291 and isolates ,293 ratiisolates ,299,300.isolates ,170,171.isolates . Mixed risolates ,156,202.P. aeruginosa chronic lung infection model [An elastase toxoid created by site-directed mutagenesis showed good levels of protection in mice , while aon model . When suon model . Althougon model , some ston model ,302,303.on model .Although surface-exposed and -secreted bacterial components comprise the majority of vaccine antigens evaluated, bacterial proteins that are not usually surface exposed have also shown protection. Despite its usual periplasmic and cytoplasmic location, KatA catalase showed a significantly greater clearance of both homologous and heterologous strains in mucosally immunised rats, which correlated with an increase in anti-KatA IgG and IgA titres in BAL . They lafpvA gene [Iron uptake system proteins, including FpvA, FoxA, and HasR were highly expressed during infection in a murine acute pneumonia model . UnfortupvA gene  may hampRecently, HitA, a ferric iron-binding periplasmic protein induced high levels of IgG1 and IgG2a specific antibodies in mice when administered subcutaneously in combination with a high dose of BCG plus IFA, followed by two booster doses of combined HitA and IFA .Vaccines involving components of the QS system seem to have limited potential. Immunisation of mice with the autoinducer of the Las QS system  conjugated to KLH , KatA 1 or PcrV P. aeruginosa. Although an orally administered live P. aeruginosa PAO1 strain failed to protect immunised rats against subsequent pulmonary infection [®) induced protective immune responses in the lung, enhanced bacterial clearance  and reduced mortality [2O2 to deactivate P. aeruginosa strains showed potential, resulting in reduced toxicity and retention of more complete epitopes compared to formaldehyde deactivation. Indeed, it stimulated opsonic protection (IgG1 and IgG2a) and induced IgG3 at later stages and IgM at earlier stages in mice [Construction of a vaccine using whole-cell killed or live-attenuated bacteria is a less developed strategy for nfection , mucosalortality ,182,183.ortality . In a phortality . Neverthortality . Recentl in mice .aroA deletion mutant of P. aeruginosa (PAO1ΔaroA), which is deficient in aromatic amino acids synthesis, was safe and enhanced titres of opsonic antibodies, primarily directed against the O-antigen [aroA) protected mice and rabbits against lethal pneumonia caused by LPS-heterologous strains [aroA was tested in a murine corneal infection model, outer membrane antigens, but not the LPS O-antigen, were identified as the immune effector, providing broader coverage against LPS diverse strains [P. aeruginosa strains, including PAO1ΔaroA, stimulated a multifactorial response, involving both humoral and cellular elements without provoking immunologic interference [murI) lacking an enzyme involved in D-glutamate biosynthesis, an essential component of peptidoglycan. Both humoral and cellular responses were triggered when delivered intranasally in a two-dose vaccination schedule, leading to murine survival of 86–88% against lethal pneumonia [Live-attenuated vaccines have also been developed. An -antigen , and fiv-antigen . By cont strains . When PA strains . Mucosalrference . Recentlneumonia .P. aeruginosa cannot replicate in the host and was designed to overexpress the T3SS. In a murine acute pulmonary infectious model, it was safe and elicited a strong humoral response with high antibody titres to OprF and PcrV, as well as activation of the Th1, Th2, and especially Th17 pathways [Since live-attenuated vaccines may retain residual virulence that could make them unacceptable for human use , Meynet pathways .P. aeruginosa pneumonia model [FliC that reduced its ability to interact with TLR5 while maintaining immunogenic properties overcame this issue. DNA immunisation with OprF also showed potential, eliciting OprF-specific IgG1 opsonic antibodies that reduced bacterial burden in the lungs of a mouse model of chronic pulmonary infection [oprL and oprF genes as monovalent vaccines, a fusion OprL-OprF vaccine or a divalent combination (OprL and OprF). They all induced a humoral response based on serum antibodies, IL-2 and IFN-γ, and protected chickens against P. aeruginosa challenge, with the divalent combination showing the greatest survival (80%) [P. aeruginosa, which elicited IFN-γ and IgG2a antibodies, but failed to protect mice against P. aeruginosa challenge  [toxA gene encoding a non-cytotoxic product, which stimulated production of neutralizing antibodies that protected mice against challenge with the wildtype ETA [P. aeruginosa challenge [P. aeruginosa pneumonia when administered via electroporation [DNA vaccines are increasingly being investigated for many vaccine applications as their ease of development and stability offer considerable advantages over protein sub-unit vaccines. DNA vaccines encoding type-a or b flagellins from different serotypes showed strong immunogenicity and efficiently protected mice against challenge with homologous but not heterologous strains in a murine ia model . Antibodnfection . Protectnfection ,194. Recal (80%) . A Herpeurvival) . The featype ETA ,311. Lathallenge . Finallyporation . DNA vacVaccine adjuvants enhance and/or shape antigen-specific immune responses. Currently, subunit vaccines are attractive due to their high safety profile; however, they show a reduced immunogenicity which necessitates the inclusion of adjuvants ,314,315.Adjuvants can be classified as delivery systems (DS) and immunostimulators (IMS), or a combination of both. DS work as carriers with which antigens are associated, creating local pro-inflammatory responses that recruit innate immune cells to the injection site. This type of adjuvant is well represented by liposomes or virosomes . ImmunosAlthough many molecules have adjuvant effects, only a handful have been licensed for use in humans . AluminiPseudomonas vaccine formulation or do not specify the contribution of this molecules to the protective effect of the immunisation. Hence, knowledge about the role of adjuvants in P. aeruginosa vaccines is limited, leaving one of the key questions unanswered: what immune response should the adjuvant trigger to provide effective protection against this pathogen? Although this has not been fully elucidated, recent studies have provided great insights  and aluminium phosphate (AlPO4), commonly referred to as “alum”, are the most widely used adjuvants in preclinical and clinical trials of P. aeruginosa vaccines [3 was used in the phase II study of the Pseudomonas IC43 (OprF/I) vaccine and it did not improve the immunogenicity of IC43 [P. aeruginosa pneumonia. A marked increase in anti-PcrV IgG1 titres but a decrease in IgG2a titres was observed, indicating a Th2-biased immune response and highlighting a key limitation of alum, it induces strong Th2-mediated immune responses, as a poor inducer of cellular immune responses [P. aeruginosa infections is characterised by a skewed Th2 response [P. aeruginosa infections [P. aeruginosa, and may even be detrimental.Aluminium hydroxide (Al(OH)vaccines ,154,217.vaccines ,320. Antvaccines ; furthervaccines . Al(OH)3esponses . As mentresponse , with hifections . Thus, aEscherichia coli LPS), provide hints about the harmful versus beneficial immune responses against P. aeruginosa [P. aeruginosa PilA (r-PilA), generated robust Th1 and Th2 type responses (IgG1 and IgG2a), compared to the use of rPilA + alum. The addition of NLX did not alter the IL-4 (Th2 marker) production, but it produced a significant increase in INF-γ and IL-17, suggesting that NLX shifts the Th2 response towards a more balanced Th2/Th1 response [P. aeruginosa  compared to immunisation with r-PilA + alum (approximately 40%) [Studies in mouse models comparing alum with other adjuvants, such as naloxone  or deoxygenated lipooligosaccharide  .P. aeruginosa, while mice given OMP + alum showed 20 to 50% survival. The improved protective effect of the CIA06 adjuvant may be related to increased opsonophagocytosis, along with elevated Th1 and Th17 cell responses [The use of dLOS combined with alum (CIA06 system) also improved the immunogenicity of an OMP vaccine. Mice immunised with OMP+ CIA06 showed 60 to 90% survival after 8 days of infection with esponses .P. aeruginosa vaccines is CpG ODN, a short, single-stranded synthetic DNA fragment containing an unmethylated CpG sequence (CpG motif) that is relatively common in the genomes of most bacteria and DNA viruses [P. aeruginosa pneumonia, with 73% of vaccinated mice surviving, compared with 30% of the mice vaccinated with PcrV-alum or CpG alone [P. aeruginosa vaccine consisting of O-polysaccharide serotype IATS-1 coupled to Toxin A [Another licensed adjuvant evaluated in  viruses . This ad viruses . IntranapG alone . Anti-PcpG alone . Previou Toxin A  and in a Toxin A .P. aeruginosa infections include AS04 and MF59® [28-294-OprI25-83-Hcp11-162 (POH), no improvement was observed with either relative with the use of alum in models of murine pneumonia  [P. aeruginosa vaccines.Other FDA-approved adjuvants for use in human vaccines that have been used in pre-clinical studies of nd MF59® ,325. How titres) , consequP. aeruginosa and that a shift to Th1 or Th17 responses may improve the efficacy of P. aeruginosa vaccines. This, together with the importance of mucosal immunity, which is strongly related to the IL-17 family of cytokines on different epithelial barrier surfaces [P. aeruginosa, mainly by increasing Th17 cell responses [Taken together, it is clear that Th2 cell responses are not optimal to achieve protection against surfaces , has ledesponses ,94.P. aeruginosa. Immunisation with PopB/PcrH+curdlan induced a systemic and mucosal Th17 response, but it did not increase the protective effect of immunisation . This protective efficacy was antibody-independent but IL-17-dependent [P. aeruginosa infections. Immunisation with PLGA-entrapped PopB/PcrH or curdlan-PopB/PcrH resulted in Th17 responses three to four times higher than in mice immunised with PLGA or PopB/PcrH alone. This was reflected in the protective effect of immunisation . Six days after infection, mice vaccinated with PLGA-PopB/PcrH or curdlan+PoB/PcrH had significantly better survival  than mice vaccinated with PopB/PcrH alone  or PLGA alone [P. aeruginosa. Schaefers et al. also reported a lack of antibodies with opsonophagocytic killing activity against P. aeruginosa suggesting a Th17- mediated protection [Curdlan is a component of the inner fungal cell wall, a covalently linked branched glucan moieties β- linked to other polysaccharides and proteins. It is a mast cell chemoattractant (MC) , mediateependent ,92, emphGA alone . These sotection .+ memory T cells in the lung compared with the control groups . Immunisation with OMPs+ dmLT protected the mice against lethal P. aeruginosa lung infection, and was associated with the early production of IFN-γ and IL-17 (Th1/Th17 responses). Mice immunised with OMPs +dmLT showed 53% survival in 10 days, while all other groups completely succumbed to infection within 1–3 days. Again, these data highlight the role of Th1/Th17 responses in protection against P. aeruginosa. The ability of dmLT to support antibody responses in addition to Th1/Th17 cellular immunity is significant and offers a clear advantage, particularly for those pathogens that require both humoral and cellular responses for protection [P. aeruginosa infections.The mucosal adjuvant LT R192G/L211A) or dmLT consists of an 84 kDa polymer protein that is distinguished from its parent molecule, thermolabile enterotoxin (LT), by the replacement of two residues in subunit A, a glycine with arginine (R192G) and alanine with leucine (L211A). It induces strong IL-17 secretion and antigen-specific Th17 responses ,328. Bakotection . Notwithotection ,92, poteG/L211A oP. aeruginosa infection has also been examined. Krause et al. evaluated the systemic and mucosal immunogenic properties of a non-human primate-based adenovirus vector, AdC7, expressing P. aeruginosa OprF (AdC7OprF) and compared it with a human serotype (Ad5OprF), using an agar-beads chronic infection model in mice [P. aeruginosa. The AdC7OprF vaccine induced protective immunity against P. aeruginosa despite the presence of lower total systemic anti-OprF IgG titres. Both Ad5OprF and AdC7OprF induced an OprF-specific IFN-γ response in lung CD3+ T cells, while IFN-γ levels in mouse-derived CD3+ T cells that had received Ad5Null or AdC7Null were low [P. aeruginosa [The contribution of adjuvants to the protection against chronic  in mice . Intramu in mice . Immuniswere low . The dirruginosa .Salmonella or Francisella tularensis as antigen delivery systems stand out among the attempts to improve the mucosal vaccine response to P. aeruginosa with mixed results. In general nasal and oral vaccination with attenuated Salmonella mutant strains induce a mixed response which is biased towards a Th1 response [Salmonella species for the nasal or oral delivery of Pseudomonas OprF or OprI, induced IgG and IgA antibodies. In contrast, in murine models, subcutaneous vaccination with an S. Typhimurium LH430 expressing OprF-OprI achieved the highest levels of protection and specific immunoglobulin titres than oral immunisation [S. enterica type III secretion effector protein, SseJ, and the P. aeruginosa PcrV elicited detectable levels of antigen-specific IgG in all mice, reduced bacterial loads in the spleens and lungs and the levels of proinflammatory cytokines (TNF-α and IL-6) and most of the vaccinated mice survived. In contrast, Salmonella expressing SseJ-OprF/I was not able to induce a specific IgG response and did not show significant protection against P. aeruginosa challenge [Salmonella for the delivery of polysaccharide antigens, such as PA103 serogroup O11 O-antigen [F. tularensis has also been used for the delivery of P. aeruginosa PilA, OprF, and FliC. Mice immunised with LVS expressing PaFliC, produced high levels of antibodies specific for P. aeruginosa, demonstrating the potential of this bacterial strain for the delivery of P. aeruginosa antigens [Finally, the use of live attenuated pathogens such as response . Preclinresponse ,331 and response  involvinnisation . Importahallenge . Other g-antigen ,337,338.antigens .Pseudomonas has not yet yielded a marketable product. However, many lessons can be drawn which may improve future efforts. Most of the P. aeruginosa vaccine candidates developed to date were based on conventional protection mechanisms, i.e., opsonophagocytic antibody-mediated killing and/or antibody-mediated toxin inhibition [P. aeruginosa respiratory infections occur via interactions between the pathogen and the mucosa of the respiratory tract, so achieving mucosal immunity should be a goal for an effective vaccineOver 50 years of research into the development of a vaccine against hibition . Howeverhibition . P. aeruP. aeruginosa infections, so vaccines that elicit such responses may not be optimal. Th17 cell responses stand out as being key to protection against P. aeruginosa respiratory infections since they can increase mucosal immunity, as demonstrated with Th17 response-inducing adjuvants [Pseudomonas infections, as suggested by the use of alum+NLX, and, interestingly, with the use of non-human adenovirus vectors in chronic infections [Salmonella or F. tularensis is an interesting strategy for achieving mucosal immunity. While these studies give us hints, it remains unclear what the most effective immune response to combat P. aeruginosa is. A deeper understanding of host-pathogen interactions will facilitate the development of antigen–adjuvant systems capable of eliciting both cellular and humoral responses which work synergistically to give robust protective immune responses against P. aeruginosa infections.On the whole, Th2 cell responses do not appear to be protective against djuvants ,137,323.fections . MoreoveThe effectiveness of any vaccine depends on the correct selection of the antigen and adjuvant. Protein antigens show the most potential as they allow the identification of most protective epitopes, also facilitating the manufacture and optimisation of synthetic peptides and chimeric proteins. They can be easily and safely produced with relatively inexpensive large-scale purification. However, protein antigen variability and loss of expression during chronic infection is a challenge. Consequently, OMPs, which are surface exposed and far more conserved than other virulence factors, may result in broader coverage and better protection. Periplasmic proteins have greater solubility and are easier to purify, and their essential functions suggest that they will be conserved across serotypes, hence offering a broader coverage; however, further investigation of their potential in human trials is required.Although a multitude of bacterial components showed potent immunogenic properties, none of them alone conferred the expected benefit in the clinical setting. Therefore, vaccines that present multiple antigenic components may be a more effective strategy, with the potential to induce a more complete and effective response. DNA vaccines, which have shown good survival rates in general, offer the possibility to simultaneously target different antigens. They can be easily prepared and harvested in vast quantities. However, it is a newly developed field, needing further evaluation in clinical studies. Eventually, the use of a multivalent vaccine that combines accessible and conserved antigens may ensure broad coverage and clinical efficacy.P. aeruginosa despite many years of effort is clear evidence of the great challenge involved and the need for further progress in understanding the complex Pseudomonas-host relationship at each stage of infection. The adaptability of P. aeruginosa and the large arsenal of virulence factors further complicate the realisation of a vaccine; however, the emergence of antibiotic multi-resistant strains and the susceptibility of vulnerable groups to Pseudomonas infections drives us in our quest to find effective antigen–adjuvant systems for the generation of a vaccine against this challenging pathogen.The lack of a licensed vaccine against


For high-grade soft tissue sarcomas (STS), combined modality treatment with surgery and radiation therapy is the standard of care. The addition of chemotherapy has been shown to decrease the risk of local recurrence and improve survival. Evaluating treatment response with surrogate modalities such as MRI, CT and PET imaging have substantial limitations. Pathologic necrosis of the surgical specimen is a direct indicator of the effect of treatment on tumor cells. Studies in STS and other malignancies have shown that increasing rates of treatment-induced tumor necrosis correlate with improvement of oncological outcomes and survival. However, the relationship between pathologic response and outcomes of specific neoadjuvant treatments for STS remains indeterminate. We hypothesized that sequential neoadjuvant chemotherapy and radiation yields higher rates of pathologic complete response (pCR) than neoadjuvant radiation or chemotherapy alone. Our results indicate that neoadjuvant chemotherapy and radiation yields superior pCR compared to other neoadjuvant regimens.p = 0.003 in univariate (UVA) and OR 24.7, 95% CI = 2.88–211.2, p = 0.003 in multivariate (MVA), respectively) and NA-XRT  was significantly associated with non- pCR when compared to NA-CRT. The analysis also demonstrated that grade 3 tumors, when using grade 2 as reference, also had significantly higher odds of achieving pCR . (4) Conclusion: NA-CRT yields superior pCR compared to other neoadjuvant regimens. This extends to higher grade tumors.(1) Background: Pathologic necrosis of soft tissue sarcomas (STS) has been used to determine treatment response, but its relationship to neoadjuvant treatments remains indeterminate. In this retrospective, single institution study, we hypothesized that neoadjuvant chemoradiation (NA-CRT) yields higher rates of pathologic complete response (pCR) than neoadjuvant radiation (NA-XRT) or chemotherapy (NA-CT) alone. (2) Methods: Patients with extremity STS between 2011–2020 who received neoadjuvant treatment were included. pCR was defined as percent necrosis of the surgical specimen greater than or equal to 90%. (3) Results: 79 patients were analyzed. 51.9% of the population were male with a mean age of 58.4 years. 49.4% identified as Non-Hispanic White. Twenty-six (32.9%) patients achieved pCR while 53 (67.1%) did not. NA-CT (OR 15.82, 95% CI = 2.58–96.9,  Soft tissue sarcomas (STS) are rare tumors derived from mesenchymal cells . STS invTreatment approaches for STS vary across sarcoma centers of excellence . For higSurrogate modalities to measure tumor treatment response such as MRI, CT, and PET imaging, remain with substantial limitations. Pathologic necrosis of the surgical specimen is a direct indicator of the effect of treatment on tumor cells. Studies in STS and other malignancies have shown that increasing rates of treatment-induced tumor necrosis correlate with prognostic oncological outcomes and survival ,12,13,14A retrospective review was conducted on 289 patients diagnosed with extremity STS between 2011 and 2020 at the University of Miami. This analysis was approved by the University of Miami Institutional Review Board 20190880 and due to the retrospective nature of this study, informed consent was waived. All patients were older than 18 and treated with NA-CT, NA-XRT, or NA-CRT followed by oncologic resection performed by the Orthopedic oncology team.Exclusion criteria included patients who did not receive neoadjuvant therapy followed by an oncologic resection. Patients with low-grade sarcoma, rhabdomyosarcoma, extraosseous Ewing, primitive neuroectodermal tumors, osteosarcoma, chondrosarcoma, Kaposi’s sarcoma, angiosarcoma of the scalp/face, or any sarcoma of the head and neck were excluded based on RTOG 0630  and RTOGAge was calculated from date of birth to date of tissue diagnosis. Survival status was taken from the medical record as was gender, race, and ethnicity. Patients identified their gender as male or female. Ethnicity was classified as Hispanic or non-Hispanic. For race, patients identified as White, Black, Asian, multiracial, or unknown. The date of diagnosis was the date of pathologic confirmation of STS. Tumor location was classified as originating in the upper or lower extremity. Tumor grade and histology were identified on the pre-operative pathology report. The following data were obtained from the surgical pathology report: percent necrosis, margin status, distance from closest margin, tumor size, treatment effect, and number of positive nodes. Status at last follow-up was categorized as alive without recurrence, alive with local regional recurrence, alive with distant recurrence, or deceased. All staging for analysis was based on the American Joint Committee on Cancer (AJCC) Staging System for STS of the trunk and extremities, 8th edition. Pathologic complete response (pCR) was defined as percent necrosis of the surgical specimen greater than or equal to 90% based on criteria from the ARST1321 trial . Recurrep < 0.05.Patient demographics and disease characteristics were summarized using descriptive statistics. For continuous variables, mean, standard deviation, median, minimum, and maximum values were reported. For categorical variables, frequencies were reported. Univariable and multivariable analyses (UVA and MVA) were performed to examine association between covariates and pCR using logistic regression. Stepwise variable selection was used for identifying independent variables in MVA. Overall survival analysis was performed using Cox proportional hazards regression for univariable analysis only due to small number of deaths. Kaplan Meier approach was used to evaluate survival curves among categorical groupings. Associations between distant recurrence and covariates were examined using Fine-Gray competing risks regression due to presence of competing risks . Pre- and post-treatment tumor size in centimeters (cm) was examined using logistic regression analysis. All statistical analyses were conducted using statistical software SAS version 9.4 and R version 3.6.3. Statistical significance was considered when Clinical characteristics of the entire cohort by pathologic response are depicted in p = 0.010) but not among the entire cohort (p = 0.161) or among patients who did not achieve pCR (0.997) (Treatment and post-therapy characteristics of the entire cohort by pathologic response are depicted in  (0.997) . Patholop = 0.003 in UVA and OR 24.7, 95% CI = 2.88–211.2, p = 0.003 in MVA, respectively) and NA-XRT  was significantly associated with non- pCR when compared to NA-CRT. Grade 3 disease was also significantly associated with achieving pCR in UVA when compared to Grade 2 disease . Logistic regression analysis was performed to determine the effect of treatment on pCR . Variablp = 0.1809) (p = 0.9353) as represented on Kaplan–Meier curve (Univariable Cox models for OS were performed. . No vari 0.1809) . The fiver curve . At two er curve .p = 0.003 in UVA and OR 24.7, 95% CI = 2.88–211.2, p = 0.003 in MVA, respectively) and NA-XRT  when compared to NA-CRT. The analysis also demonstrated that grade 3 tumors, when using grade 2 as reference, also had significantly higher odds of achieving pCR . These findings suggest that sequential neoadjuvant chemotherapy and radiation confer significantly greater degrees of pCRupon surgical resection as well as combined neoadjuvant therapy is effective for higher grade tumors compared to NA-CT or NA-XRT alone at achieving pCR. Within the different treatment groups, we determined that over half of those treated with NA-CRT (55.0%) achieved a level of pathologic necrosis of at least 90%, whereas in the groups with NA-CT and NA-XRT, only 5.0% and 15.8% achieved at least 90% necrosis, respectively. Our results demonstrate that the odds of achieving a pCR are significantly decreased with NA-CT  [Prior studies have suggested NA-CRT is superior to solely preoperative radiotherapy, with NA-CRT increasing the likelihood of R0 resection . A meta-< 0.001) . Smaller< 0.001) . Our stuThe prognostic value of necrosis within a resected tumor has been the center of much debate as it continues being assessed in association with survival metrics. A study examining 113 individuals with sarcoma treated to achieve a median necrosis of 90% across samples found no difference in groups who had >95% necrosis and <95% necrosis in five-year follow-up for local control, disease-specific survival, and overall survival . Other sLimitations of retrospective studies apply to our single center study. Chief among the limitations is the rarity of STS, accounting for the small sample size accrued over the nine-year time frame. Additionally, the lack of availability of comparable data across all 289 screened patients factored into the findings. Many patients went to different hospital systems for their surgeries and as a result did not have pathologic necrosis reported in their pathology and were excluded on that basis. Another limitation is the selection bias. Larger tumors (greater than 5 cm) are more likely to receive chemotherapy than smaller tumors due to higher risk of local and distant recurrence. In addition, patients with poor performance status are more likely to receive NA-XRT due to inability to tolerate chemotherapy. Additionally, challenges associated with follow-up led to exclusions due to incomplete records, limiting potential survival analyses. Our findings indicate that the use of sequential neoadjuvant chemotherapy and radiotherapy before surgical resection yields superior pCR. This finding extends to higher-grade tumors. Further prospective research into this approach will examine toxicity associated with greater therapeutic load, utilize the percentage decrease in tumor size in calculating percent necrosis, as well as apply correlative imaging through PET/CT or advanced MRI techniques to further quantify reductions in tumor burden and viability. Expanding the number of eligible patients for study could also add power to the study in determining whether pathologic complete response could serve as a surrogate for measures of survival outcomes. Sequential NA-CRT before surgical resection yields superior pCR when compared to NA-XRT and NA-CT. This finding extends to higher grade tumors. Although there was a trend toward pCR correlation with superior OS when compared to non-pCR, the association was not significant.


Chlamydia pneumoniae (C. pneumoniae), periodontal pathogens including Porphyromonas gingivalis , Helicobacter pylori (H. pylori) and human immunodeficiency virus (HIV). Overall, activation of TLR2 and 4 seems to have a profound impact on infection-related atherosclerosis. This article reviews the role of TLRs in the process of atherosclerosis after C. pneumoniae and other infections and the current status of treatment, with a view to providing a new direction and potential therapeutic targets for the study of ASVD.Atherosclerotic vascular disease (ASVD) is a chronic process, with a progressive course over many years, but it can cause acute clinical events, including acute coronary syndromes (ACS), myocardial infarction (MI) and stroke. In addition to a series of typical risk factors for atherosclerosis, like hyperlipidemia, hypertension, smoking and obesity, emerging evidence suggests that atherosclerosis is a chronic inflammatory disease, suggesting that chronic infection plays an important role in the development of atherosclerosis. Toll-like receptors (TLRs) are the most characteristic members of pattern recognition receptors (PRRs), which play an important role in innate immune mechanism. TLRs play different roles in different stages of infection of atherosclerosis-related pathogens such as  Atherosclerotic vascular disease (ASVD) is a chronic process, with a progressive course over many years, but it can cause acute clinical events, including acute coronary syndromes (ACS), myocardial infarction (MI) and stroke . HyperteChlamydia pneumoniae (C. pneumoniae) [Porphyromonas gingivalis  [Helicobacter pylori (H. pylori) [cytomegalovirus (CMV) [Epstein-Barr virus (EBV) [human immunodeficiency virus (HIV) [herpes simplex virus-1 (HSV-1) [hepatitis C virus (HCV) [Although the traditional risk factors of atherosclerosis, such as hyperlipidemia, hypertension, smoking, have been efficaciously cut down, the occurrence of atherosclerotic disease is still high. In addition, 30–50% of patients lack these typical risk factors, suggesting that other unknown factors are related to the pathogenesis . Atherosumoniae) , Porphyrgivalis) , Helicob pylori) , cytomegus (CMV) , Epsteinus (EBV) , human ius (HIV) , herpes  (HSV-1) , HSV-2 [ (HSV-1)  and hepaus (HCV) . A largeus (HCV) , 20. The−/−) mice and LDL receptor deficient (LDLR−/−) mice [−/− and LDLR−/− mice [−/− mice, TLR9 has been observed to spread inflammation [−/− mice fed with high-fat-diet (HFD) and a TLR9 agonist reduced the severity of the disease [TLRs are the most characteristic members of pattern recognition receptors (PRRs) and play vital roles in innate immunity mechanisms . Located/−) mice , 25. TLR−/− mice , 27. In −/− mice . In respammation . Interesammation . TLR7 acammation . In addi disease . The mecThe present review aims to summarize the latest progression and focus on the role of TLRs between microbial infection and atherosclerosis, hoping to provide a reference and treatment for strategy of atherosclerosis. We pay special attention to three research areas: (1) the biological characteristics and functions of TLRs are systematically summarized; (2) advances in research on the relationship between different pathogens and atherosclerosis, and the role or potential role of TLRs in inducing or accelerating atherosclerosis; (3) current status and potential therapeutic targets of antibiotic and vaccine therapy for infection and targeting TLRs in atherosclerotic diseases. Finally, we summarize the full text, trying to explore the future research direction and potential drug treatment targets of atherosclerosis.The research history of TLRs dates back to the last century and as early as 1991 the homology between the Drosophila toll receptors and the interleukin-1 (IL-1) receptors was identified by Gay and Keith . In 1994TLR ligands are extremely extensive, each type of them has a series of ligands with a certain degree of specificity. Most TLRs are homodimers, but some are heterodimers. TLR2 has been proved to be able to sense lipopeptides: it is heterodimerized with TLR1 to recognize triacyl lipopeptides from Gram-negative bacteria and mycoplasma, or heterodimerized with TLR6 to recognize diacyl lipopeptides from Gram-positive bacteria and mycoplasma. The structure of the two dimers was finally solved in 2007 , 44. TLRThe specific response initiated by an individual TLR depends on the recruitment of a single or specific binding adapter protein containing the TIR domain. These adaptors are MyD88, MyD88-adaptor-like , TIR-domain-containing adaptor protein inducing IFNβ , TRIF-related adaptor molecule  and sterile α- and armadillo-motif-containing protein (SARM). SARM has now been shown to interact with TRIF and thereby interfere with TRIF function . MyD88 iThe two pathways have different dynamics. TLR stimulation triggers the binding of MyD88, which interacts with interleukin-1 receptor-associated kinase 4 (IRAK4) which is the most upstream serine/threonine kinase of the complex . IRAK4 pC. pneumoniae, Periodontal pathogens, H. pylori, CMV, EB and the like. As major members of the host's innate immunity, TLRs play an indispensable role in the atherosclerosis caused by pathogen infection. However, there is a significant difference in the strength of the data that supports its association. Table Current epidemiological, human, and animal experiments indicate that chronic infectious diseases or biological pathogens that cause these infections are associated with progression of atherosclerotic CVD. These pathogens include Chlamydia pneumoniae is an obligate intracellular pathogen with unique developmental cycles and survival strategies in host cells and is an ordinary cause of community-acquired pneumonia [C. pneumoniae had a raised risk of MI and ASVD compared with controls [C. pneumoniae and atherosclerosis were reported by a series of studies [C. pneumoniae can induce atherosclerosis or accelerate the pathological progress of atherosclerosis. Studies have presented that C. pneumoniae infection promotes plaque progression in ApoE−/− mice, diet-induced hyperlipidemic mice (LDLR−/− and C57BL/6J mice), while C. pneumoniae has no effect on plaque development [C. pneumoniae have failed to observe the deterioration of atherosclerosis [C. pneumoniae infection depends on the arterial response to hyperlipidemia. A study directly inoculated C. pneumoniae into porcine coronary artery  and pulmonary artery, resulting in thickening of the temporal lobe of the largest coronary artery, but no thickening of the pulmonary artery [C. pneumoniae can reduce the availability of endothelial nitric oxide in ApoE−/− mice, leading to endothelium-dependent relaxation damage. These experiments indicate that besides hyperlipidemia, vascular damage was identified as a prerequisite for C. pneumoniae infection to promote atherosclerosis. In animals with advanced atherosclerosis, C. pneumoniae infection did not accelerate the size of lesions, but the production of MMP-2 and MMP-9 and the decrease of fibrous cap area were observed, indicating that the degree of C. pneumoniae promoting lesions decreased with the progress of atherosclerosis [C. pneumoniae promoting atherosclerosis is to induce chronic inflammatory response, leading to tissue injury, repair and fibrosis. Infected monocytes/macrophages can transfer C. pneumoniae infection to smooth muscle cells (SMCs) and ECs. C. pneumoniae up-regulates the expression of integrin beta 2 on the surface of macrophages, thus promoting macrophage adhesion to endothelium. ECs infected with C. pneumoniae can upregulate the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), VCAM-1, P-selectin and E-selectin, thereby increasing the adhesion and migration of leukocytes to endothelium [C. pneumoniae infects ECs resulting in up-regulation of PDGF-B transcription, while PDGF-B contributed to SMCs proliferation and intimal thickening [C. pneumoniae infection in normal rabbits increases intimal thickening compared with controls, and the presence of C. pneumoniae in aortic tissue is related with the level of PDGF-B [Chlamydia pneumoniae promotes the activation of macrophages, ECs and SMCs through the above mechanisms.neumonia , 61. Saicontrols . Subsequ studies . The evielopment . Howeverclerosis . Interesclerosis . This exy artery . In addiclerosis . The mecothelium . In addiickening . Coombesf PDGF-B . ChlamydC. pneumoniae infection. The main molecules involved are TLR2 and 4. Massari et al. reported that TLR4 recognizes LPS and cHSP60 on the surface of Chlamydia [C. pneumoniae have been presented to activate macrophages via the TLR4 and MAPK pathways [C. pneumoniae can also induce the production of vascular endothelial growth factors (VEGF) and MMP-9 in human ECs via TLR2/4-dependent pathway [C. pneumoniae ligands activates the MyD88-dependent pathway in immune cells and then upregulates the expression of CD68, nitric oxide (NO), human beta-defensin-2 (HBD-2), pro-inflammatory cytokines [C. pneumoniae can induce foam cell formation and LDL oxidation. Cao et al. reported that foam cell formation induced by C. pneumoniae is modulated by TLR2 but not TLR4 [C. pneumoniae can affect atherosclerotic plaque development in a TLR2/4/MyD88-dependent manner by promoting foam cell formation and enhancing activated DCs in plaques. LXR signaling can counteract C. pneumoniae-induced atherosclerosis by reducing cholesterol efflux and blocking TLR-dependent proinflammatory signaling [C. pneumoniae promotes foam cell formation through activation of TLR2 and TLR4 signaling, but not TLR3, and activates downstream MyD88/TRIF-dependent pathways. They also demonstrated that LXR agonists can in turn reduce C. pneumoniae-mediated IRF3 activation in macrophages [C. pneumoniae can negatively regulate ABCA1 expression by the TLR2-NF-κB and miR-33 pathway in THP-1 macrophage-derived foam cells. ABCA1 is the key to macrophage cholesterol efflux and has a protective influence on atherosclerosis. Decreased expression or localization of ABCA1 on the cell membrane may lead to cholesterol accumulation in macrophages and aggravate atherosclerosis [TLRs play an important role in activating macrophages and ECs, inhibiting cholesterol outflow and promoting foaming after hlamydia . Outer mpathways . Paolill pathway . The intytokines . In the not TLR4 . Howeverignaling . In the rophages . Futhermclerosis .P. gingivalis, the best-studied bacterial pathogen associated with severe PD, is a Gram-negative anaerobic bacterium in which the host’s immune responding to the organism destroys the dental support structure [P. gingivalis and periodontal disease are risk factors for diabetes, premature labor, and atherosclerotic disease and the presence of P. gingivalis has been detected in atherosclerotic plaques [P. gingivalis in aortic plaque by Fluorescent in-situ hybridization (FISH), and reported that chronic oral infection with P. gingivalis results in a specific immune response, significant increases in oral bone resorption, aortic inflammation, viable bacteria in oral epithelium and aorta, and plaque development [P. gingivalis was reported to be refrained by monoclonal antibodies to TLR2 and 4, CD14 and β2 integrin [P. gingivalis demands TLR2 to induce oral inflammatory bone loss in mice, and P. gingivalis infection promotes atherosclerosis in hyperlipidemia mice with increased expression of TLR2 and TLR4. Hayashi et al. proved that TLR4 has atherosclerosis protection in response to P. gingivalis infection [P. gingivalis [P. gingivalis has the ability to regulate C5a signaling in neutrophils, which leads to the degradation of MyD88 by C5aR-TLR2 cross-talk involving the ubiquitin ligase Smurf1, reducing the production of inflammatory markers and resulting in reduced antimicrobial killing. Alternative TLR2 signaling through Mal and PI3 kinase (PI3K) in the absence of MyD88 to cause inflammatory activation [P. gingivalis infection in the oral cavity can result in significant TLR2-CD36/SR-B2-mediated IL1β release and promote plaque progression [P. gingivalis was confirmed to induce TLR-4 mediated ICAM-1, VCAM-1 and expression of the ox-LDL receptor-1 (LOX-1) in mice [Periodontal disease (PD) is a polymicrobial dysbiotic inflammatory disease with substantial inflammatory destruction of the supporting tissues, including gingival tissue, periodontal ligament, and alveolar bone. tructure . A serie plaques , 80, 81.elopment . Proinflintegrin . Frank anfection . In the ngivalis . P. gingtivation . Brown egression . HSP 60  in mice .P. gingivalis, other significant periodontal pathogens also support the potential connection between PD and ASVD. Treponema denticola (T. denticola) is a predominantly subgingival oral spirochete closely associated with periodontal disease. Chukkapalli et al. detected T. denticola clusters in both gingival and aortic tissue of infected ApoE−/− mice by FISH and reported that T. denticola infection increased alveolar bone resorption and aortic plaque area in mice, and changed the gene expression related to atherosclerosis [T. denticola cells and its major outer sheath protein (MSP) induced innate immune responses through TLR2-MyD88, whereas its lipooligosaccharide (LOS or glycolipid) induced a macrophage response through TLR4-MyD88 [T. denticola periplasmic flagella activate the innate immune system through TLR2 [In addition to clerosis . Nussbauugh TLR2 .Tannerella forsythia (T. forsythia) is a Gram-negative anaerobic organism that inhabits the subgingival cavity and initiates connective tissue destruction and alveolar bone resorption in PD. Lee et al. reported T. forsythia and its major surface virulence factor, BspA could induce foam cell formation in THP-1 cells and accelerated the progression of atherosclerotic lesions in ApoE−/− mice [T. forsythia, which is likely to increase the risk of atherosclerosis [T. forsythia infection promoted alveolar bone resorption, increased serum inflammatory markers and lipid profile changes in ApoE−/− mice, but did not increase the growth of aortic plaque [T. forsythia and atherosclerosis and the role of TLRs in them.−/− mice . Ardila clerosis . Chukkapc plaque . Onishi c plaque . These cFusobacterium nucleatum (F. nucleatum) is another significant periodontal pathogen with oral infection. Heat-shock protein GroEL of F. nucleatum has been reported to induce the expression of monocyte chemokines and cell adhesion molecules, and to promote the progression of atherosclerosis in ApoE−/− mice [F. nucleatum enhanced the vascular inflammation, changed the lipid profile and the gene expression of the aorta in ApoE−/− mice, and significantly reduced the number of atherosclerotic plaques in the aorta, which suggested that this member of the oral microbiome has a potential protective function [−/−TLR4−/− mice infected with polymicrobial infection including P. gingivalis, T. denticola, T. forsythia, and F. nucleatum, atherosclerotic plaque progression was significantly reduced. However, bacterial genomic DNA was detected in multiple organs indicating an intravascular dissemination from gingival tissue to heart, aorta, kidney and lungs, suggesting that TLR2 and 4 were dispensable for systemic spread after polybacterial infections but TLR2 and 4 deficiency markedly reduces atherosclerosis induced by oral bacteria [−/− mice . Velsko function . Recentlbacteria . These rbacteria .Collinsella, while that of healthy people is rich in Roseburia and Eubacterium by shotgun sequencing [Bacteroides) and an increase in the OUT 657  and OUT 990 (Clostridium subcluster XIVa) in fecal samples of ASVD patients. The abundance of gut microorganisms such as Bacteroides, Clostridium, and Lactobacillales has been shown to predict ASVD, suggesting that atherosclerosis may be regulated by gut microbiota [−/− mice without alterations in circulating cholesterol levels, suggesting that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors may serve as a potential therapeutic approach for the treatment of atherosclerosis [In addition to the oral cavity, the gut is a likely source of microorganisms that could influence ASVD. A growing body of literature showed that the permeability of oral and intestinal epithelial barrier increased, which made a small number of pathogens or its products enter the system circulation, inducing chronic low-grade inflammation and promoting inflammatory diseases including atherosclerosis . Karlssoquencing . Emoto ecrobiota . Maitra crobiota . Sayin ecrobiota . Short ccrobiota . SCFA cacrobiota . TMAO, ocrobiota , 106. Itcrobiota . A choliclerosis . These rclerosis .akkermansia muciniphila reduced the expression of CCL2 and ICAM-1 in atherosclerotic lesions, thus reducing macrophage infiltration. This effect was abolished when mice were infused with LPS with subcutaneously implanted osmotic pumps [Bacteroides fragilis (B. fragilis) signals through TLR2 directly on Foxp3(+) regulatory T cells to promote immunologic tolerance [The commensal gut microbiota is a tonic activating factor of TLRs and other PRRs in the intestine . Maitra ic pumps . In addiolerance . Jackel olerance . It is nHelicobacter pylori is a Gram-negative spiral microeosinophilic bacteria, most of which has a disposition to colonize in the gastric mucosa and cause lifelong inflammation in human gastric mucosa. It is closely related to a series of gastric diseases such as chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer [H. pylori infection rate in patients with coronary atherosclerosis was significantly higher than that in healthy people [H. pylori and atherosclerotic diseases. In the past 30 years, a large number of studies have confirmed its relationship with atherosclerosis, but some studies have reached contradictory conclusions. For example, Vijayvergiya et al. reported higher levels of anti-H. pylori IgG in the serum of CAD patients than in the control group [H. pylori and MI, and found that H. pylori infection was significantly associated with the risk of MI [H. pylori infection may contribute to the formation of atherosclerosis [H. pylori DNA was not detected by detecting 50 exfoliated plaques in the neck of Japanese patients [H. pylori play different roles in promoting the development of atherosclerosis. Vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA) protein encoded by the cytotoxin-associated genes pathogenicity island (cagPAI) are the two major H. pylori virulence factors [H. pylori infection is more significantly associated with ASVD than CagA-negative H. pylori infection. Three animal studies have used H. pylori SS1 to inoculate gastric infections to explore whether H. pylori infection promotes the progression of atherosclerosis, but inconsistent results have been obtained [H. pylori PMSS1 strain, which can effectively transfect CagA into the host, was inoculated into mice with gastric infection, confirming that CagA derived from gastric epithelial cells infected by H. pylori promotes macrophage-derived foam cell formation and atherosclerosis [H. pylori can increase the adhesion of aortic ECs and promote atherosclerosis through NLRP3-IL1β signaling pathway. It further suggests that CagA, as a key virulence factor of H. pylori, is related to the occurrence and development of atherosclerosis, but the specific mechanism needs to be further studied.c cancer . In 1994y people . This reol group . Liu et sk of MI . Howeverclerosis . Hagiwarpatients . These c factors . Some meobtained , 123. Alclerosis . Our resH. pylori infection mediated by TLRs mainly focus on gastric diseases. LPS derived from H. pylori is regarded as a direct stimulator of innate immunity mediated by TLR4. LPS of H. pylori activates the NF-κB pathway by ligating with TLR4, and then activates the IL-8 pathway [H. pylori is not related to TLR4. In addition to TLR4, TLR2 has also been reported to be involved in the activation of signaling pathways after H. pylori infection. Triantafilou et al. demonstrated that cell activation induced by LPS from H. pylori and P. gingivalis is mediated by TLR2 and requires the formation of a heteromeric receptor complex containing TLR2, TLR1, CD36 and CD11b/CD18 [H. pylori. Other studies have shown that TLR2 and 4 cooperate to cope with H. pylori infection. For instance, LPS of H. pylori can enhance the expression of TLR2 through TLR4 signal [H. pylori also enhanced the expression of TLR4 through MAP/ERK 1/2 kinase pathway [H. pylori induces IL-10 and IL-12 via TLR4/MyD88 signaling, and induces less IL-6 and IL-1β in TLR2-deficient macrophages, indicating that H. pylori activates TLR2 and 4, resulting in the secretion of different cytokines by macrophages [H. pylori-flagellin A (FlaA). Generally speaking, TLR5 signal induces IL-8 secretion through p38-map kinase signal by recognizing bacterial flagellin. However, some studies have shown that FlaA plays a weak role in IL-8 production [H. pylori and produce IFN I [H. pylori could induce pre-inflammatory cytokines such as IL-6 and IL-12 [H. pylori and LPS, the reactivity of TLRs in gastric mucosa infected by H. pylori is uncertain. The role of TLRs in gastric mucosa infected by H. pylori provides a possible mechanism for H. pylori to promote the development of atherosclerosis. Whether TLRs mediate the occurrence and development of atherosclerosis in H. pylori infection through similar signaling pathways in gastric mucosa is uncertain, and further research is expected.Pathological studies of  pathway . However11b/CD18 . Uno and4 signal . During  pathway . Howeverrophages . In addioduction . TLR9 hace IFN I . Rad andnd IL-12 . BecauseThere is increasing evidence that HIV infection and subsequent inflammation accelerate atherosclerosis. Imaging studies using coronary CTA showed that the prevalence of coronary atherosclerosis in HIV males was 59.0%, compared with 34.4% in control group . Except TLRs seem to play an irreplaceable role in immune activation and dysfunction caused by HIV infection. It is reported that the ssRNA oligonucleotides rich in guanosine (G) and uridine (U) from HIV-1 can be specifically recognized by TLR7 and 8 . NeutropIn addition to the above four pathogens, other pathogens have been identified to be associated with ASVD. For instance, CMV protein was detected in the arterial cells of atherosclerotic patients . High leC. pneumoniae, P. gingivalis, H. pylori, CMV, HIV, HCV, etc. Scientists have conducted a series of antibiotic experiments against these microorganisms. In addition, vaccine experiments against these microorganisms and endogenous antigens such as LDL and apoB-100 are also underway.Given that innate and adaptive immunity is a major feature of atherosclerosis, it is important to consider specific strategies for immunization as a new method to the prevention and treatment of atherosclerosis. The most important challenge is to identify specific antigens associated with atherogenesis, and targeting these antigens may beneficially affect the progression of atherosclerosis. As mentioned above, many exogenous antigens have been identified in atherosclerotic plaques, including C. pneumoniae infection accelerated the thickness of the intimal wall and the degree of atherosclerosis in a rabbit model, and treatment with azithromycin lead to attenuation of atherosclerosis [C. pneumoniae infection increased the size of lesions in mice, but azithromycin did not reduce the size of aortic lesions [−/− model mice infected with P. gingivalis [P. gingivalis can significantly reduce the size of circulating inflammatory markers and atherosclerotic lesions [H. pylori infection in gastrointestinal tract [Muhlestein and his colleagues found that clerosis . However lesions . Madan engivalis . Amar et lesions . Ayada eal tract . In addial tract , 166. ThP. gingivalis accelerated atherosclerosis, and its immunity reduced the extent of atherosclerosis [P. gingivalis outer membrane protein, which confirmed that the size of atherosclerotic lesions in the aortic sinus of mice was significantly reduced, and the concentration of circulating inflammatory cytokines was decreased [−/− mice were immunized with S. pneumoniae, the specific antibodies to oxidized LDL increased and atherosclerosis decreased [S. pneumoniae. It has been proved that immunizing ApoE−/− mice with PC conjugate results in the increase of PC antibody and ox-LDL antibody and the decrease of atherosclerosis. These results suggest that immunization against exogenous antigens may be a new objective for the treatment of atherosclerosis.Given the large number of studies confirming the close association between infection and atherosclerosis, some researchers have investigated the effects of vaccination against these microorganisms on the progression of atherosclerosis. In animal models, exposure to clerosis . Koizumiecreased . Binder ecreased . Anti-phThe exciting results of animal experiments have driven clinical trials to explore the role of vaccination in reducing atherosclerotic events. Lamontagne et al. conducted a hospital-based case–control study and found no hypothetical protective effect of the pneumococcal vaccination  in patients vaccinated 1 year before MI. Conversely, if vaccinated two or more years before admission, the correlation was stronger  . HoweverIn addition to vaccination against exogenous antigens, many studies have been conducted to explore vaccination against endogenous antigens. The most common research antigens are natural or modified homologous LDL and lipoproteins containing ApoB100, a major component of LDL. Because LDL has the strongest pathogenic relationship with atherosclerosis, antigens from LDL and ApoB100 are the most potent targets for vaccine development. Immunization of animals with homologous natural or ox-LDL in vaccine formulations containing different adjuvants demonstrates atherosclerotic protection . However−/− mice by about 35% [−/− mice. However, RS-LPS reduced lesion formation in ApoE−/− diabetic mice, suggesting the potential environmental dependence of TLR4 blockade on atherosclerosis [The role of TLRs in atherosclerosis has led researchers to explore the therapeutic potential of targeting TLRs in atherosclerosis and related diseases. Over the past decades, further attempts have been made to identify specific agonists and antagonists of TLRs. The initial clinical application of TLR therapy mainly focused on tumors and infections, but now more and more molecules are targeted at immune diseases. Animal models of atherosclerosis suggest that inhibiting TLR-dependent signals or limiting arterial exposure to PAMPS may provide therapy for the treatment or prevention of atherosclerosis . Statinsbout 35% . Therefoclerosis . A new pclerosis . Opn301,clerosis . Most exC. pneumoniae, periodontal pathogens including P. gingivalis, H. pylori, CMV, EBV, HIV, HSV-1, HSV-2, and HCV. However, despite the pathophysiological correlation, clinical studies of anti-infection have not achieved satisfactory results. The reason may be due to the following three points. First, long-term antibiotic treatment can lead to drug resistance. Second, antibiotic treatment may be too late to produce significant clinical effects. Third, persistent low-grade chronic inflammation is ineffective for antibiotic treatment. Recently, a hypothesis has been proposed that infection plays a “match” role for atherosclerosis. After igniting this pathological process, the fight against this “spark” does not inhibit the progression of atherosclerosis. Therefore, the fact that anti-infective treatment is ineffective does not deny the potential link between infection and atherosclerotic disease. More research is needed to determine the exact role of microbial infections in atherosclerosis.Over the past few decades, our understanding of the classical risk factors for atherosclerotic progression, the complex interactions of inflammation and immune activation has grown rapidly. There is an emerging epidemiological link between infection and ASVD. Chronic infections and atherosclerosis have a variety of biological mechanisms, and there is sufficient experimental evidence to support the atherogenic properties of various bacterial and viral infections. The most convincing evidence is that infectious agents are detected in the atherosclerotic vascular wall, and pathogen-specific antibodies are associated with atherosclerosis. These pathogens include C. pneumoniae infection, inhibiting cholesterol outflow and promoting foam formation. P. gingivalis infection can accelerate atherosclerosis in hyperlipidemic mice and increase the expression of TLR2 and 4. LPS derived from H. pylori is regarded as a direct stimulator of innate immunity mediated by TLR4. The crucial role of TLRs in gastric mucosa infected by H. pylori provides a possible mechanism for H. pylori to promote the development of atherosclerosis. TLRs seem to also play an irreplaceable role in immune activation and dysfunction caused by HIV infection. Overall, the activation of TLR 2 and 4 seems to provide a new mechanism for infection-related atherosclerosis.As the most characteristic member of PRRs, TLRs are highly evolved conserved proteins. By binding with a series of specific endogenous and exogenous ligands, it plays an important role in innate immune mechanism. TLR signaling pathway can be broadly classified as a MyD88-dependent pathway that drives the production of inflammatory cytokines and a TRIF-dependent pathway responsible for the production of IFN I and inflammatory cytokines. The role of TLRs in the development of related atherosclerosis has just begun to be discovered. Both critical and resident vascular cells express multiple TLRs in the initiation and progression of atherosclerosis, suggesting that these receptors and their ligands are key players in atherosclerosis. Many experiments have proved that TLRs are involved in signal pathways that affect the progression of atherosclerosis after microbial infection. For example, TLR2 and 4 play an important role in activating macrophages and ECs after C. pneumoniae, periodontal pathogens, H. pylori and CMV. In our efforts to reduce atherosclerosis, determining the right target pathway has been our Achilles heel. Is it the microbe itself or the myriad of signaling pathways, including TLRs that are activated in chronic infections, which ultimately lead to atherosclerosis? More research is needed to demonstrate the hypothesis that pathogens contribute to atherosclerosis through TLR activation. Identification of pathogenesis of microbial induction may reveal drug targets for therapeutic intervention or prevention of atherosclerosis through vaccines or immunomodulatory therapy. Since the direct role of these pathogens in atherosclerosis remains uncertain, it is unclear whether vaccines against these microorganisms are useful strategies for treating human atherosclerosis. In this conjecture, the development of atherosclerotic vaccines for various signaling pathways associated with infection may be promising.In summary, this review highlights the role of TLRs in the pathogenesis of atherosclerosis associated with microbial infections, particularly


Mycoplasma pneumoniae. Medline/Pubmed, Embase, the Cochrane Library, and ISI Web of Science were searched through June 12, 2019 for relevant studies that used ICTs for the detection of M. pneumoniae infection with polymerase chain reaction (PCR) or microbial culturing as reference standards. Pooled diagnostic accuracy with 95% confidence interval (CI) was calculated using a bivariate random effects model. We also constructed summary receiver operating characteristic curves and calculated the area under the curve (AUC). Statistical heterogeneity was evaluated by χ2 test or Cochrane’s Q test. Thirteen studies including 2,235 samples were included in the meta-analysis. The pooled sensitivity and specificity for diagnosing M. pneumoniae infection were 0.70 (95% CI: 0.59–0.79) and 0.92 (95% CI: 0.87–0.95), respectively. The positive likelihood ratio (LR) was 8.94 (95% CI: 4.90–14.80), negative LR 0.33 (95% CI: 0.22–0.46), diagnostic odds ratio 29.20 (95% CI: 10.70–64.20), and AUC 0.904. In subgroup analysis, ICTs demonstrated similar pooled sensitivities and specificities in populations of children only and mixed populations (children + adults). Specimens obtained from oropharyngeal swabs exhibited a higher sensitivity and specificity than those of nasopharyngeal swab. Moreover, pooled estimates of sensitivity and accuracy for studies using PCR as a reference standard were higher than those using culture. The pooled sensitivity and specificity of Ribotest Mycoplasma®, the commercial kit most commonly used in the included studies, were 0.66 and 0.89, respectively. Overall, ICT is a rapid user-friendly method for diagnosing M. pneumoniae infection with moderate sensitivity, high specificity, and high accuracy. This suggests that ICT may be useful in the diagnostic workup of M. pneumoniae infection; however, additional studies are needed for evaluating the potential impact of ICT in clinical practice.The aim of this study was to evaluate the diagnostic performance of immunochromatographic tests (ICTs) for the detection of  Mycoplasma pneumoniae is an important cause of respiratory tract infection (RTI) in school-age children and young adults [M. pneumoniae is responsible for approximately 10–40% of community-acquired pneumonia (CAP) cases [M. pneumoniae infection is primarily known to present with a mild clinical course [M. pneumoniae-infected individuals [g adults –4. M. pnP) cases , 5, risiP) cases –8. M. pnl course ; howeverl course , 10. Extividuals .M. pneumoniae lacks a cell wall, and therefore, β-lactam antibiotics, which are active against most respiratory bacterial pathogens for RTI in children, are ineffective against M. pneumoniae [M. pneumoniae infection leads to the use of appropriate antibiotics. However, it is difficult to distinguish M. pneumoniae from other causative microorganisms of RTI early during the clinical course based on patient history, symptoms, physical examination, or a chest radiograph. Therefore, laboratory confirmation of the microorganism is crucial for planning the appropriate management [eumoniae . A prompnagement –16.M. pneumoniae diagnosis, M. pneumoniae are fastidious and cultivation may require weeks for growth. Therefore, culturing is not routinely performed in clinical practice [M. pneumoniae-specific antibody is indicative of a recent infection; however, false-negative test results often occur early in the course of illness [M. pneumoniae-specific IgG titer ≥ 4-fold during acute and convalescent phases of the clinical course also implies recent infection [M. pneumoniae IgM antibodies, has been developed and is commercially available [M. pneumoniae infection as M. pneumoniae IgM antibodies may persist for several months [M. pneumoniae detection; however, it requires complex and time-consuming sample pretreatment, skilled technical ability, and expensive equipment [While microbial culturing has been a gold standard for practice . Serolog illness , 18. An nfection ; howevernfection , 18. Immvailable , 21. Howl months . Polymerquipment , 24.M. pneumoniae have been developed, including loop-mediated isothermal amplification (LAMP) [Recently, several techniques for the rapid diagnosis of n (LAMP) , 26 and n (LAMP) –30. LAMPn (LAMP) , 32. Altn (LAMP) , it requn (LAMP) , 35. Then (LAMP) , 37.Streptococcus pneumoniae and Legionella pneumophila serogroup-1 [M. pneumoniae antigen  has also been developed and is commercially available [M. pneumoniae have not currently been systematically reviewed or integrated. Therefore, the aim of this systematic review and meta-analysis was to integrate and assess the evidence for the diagnostic accuracy of ICT for M. pneumoniae infection.The immunochromatographic test (ICT), often referred to as a lateral-flow assay, is a popular application of enzyme-immunoassays that utilize antigen and antibody properties as a sample passes along a membrane –41. ICT ogroup-1 , 46. ICTvailable , 47–50. This review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement (PRISMA) . The proMycoplasma pneumoniae, immunochromatography and lateral flow assay. The search strategy included “Mycoplasma pneumoniae AND immunochromatography OR lateral flow assay”  independently screened the titles and abstracts for potential relevance and conducted full-text reviews of the selected publications. Any disagreements were resolved by a third reviewer (IKM) following a discussion with all three reviewers. Author names, country of origin for the study, publication year, study design, study period, age distribution of the study population (children were defined as ≤ 18 years of age), participant gender, index test assay, index test target, index test company, reference standard, type of specimen, sample size, and data regarding true positive, false positive, true negative, and false negative were extracted. If studies consisted of multiple groups, each group was treated as a single study. If there was insufficient information to construct the 2 × 2 table, we attempted to contact the corresponding authors by e-mail.The validity of the included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool . QUADAS-Summary estimates of sensitivity and specificity along with 95% confidence intervals were calculated based on a bivariate random effects model . From th2 test analysis (p < 0.05 indicated significant heterogeneity). If heterogeneity between studies existed, a bivariate random effects model was adopted [Summary receiver operating characteristic (SROC) curves and the area under the curve (AUC) obtained from the fitted bivariate random effects model were used to summarize the overall test performance. SROC curves were plotted with the confidence region and prediction region. Heterogeneity of sensitivity and specificity was assessed by visual inspection of forest plots and by χ adopted –57. A fiWe planned subgroup analyses prior to starting the evaluations as heterogeneity among the studies was expected. The following variables were expected to be possible sources of heterogeneity: age of the population , type of index test and type of specimen, reference standard used, and blinding procedures. Potential publication bias was visually assessed using a funnel plot , 59. TheTotally, 66 articles were retrieved. After the removal of duplicate articles and exclusion of studies based on titles and abstracts, 13 articles remained for full text review. Four of the 13 studies were excluded as two studies did not provide sufficient data for generation of a 2 × 2 contingency table and the other two studies did not use PCR or microbial cultures as reference standards. Of the nine studies not excluded, several evaluated more than one reference method  and diff®  was the most frequently assessed with eight (57.6%) of the studies evaluating this ICT [The data sets were extracted from the 13 articles and consisted of 2,235 samples. Descriptive characteristics of the studies are summarized in this ICT , 47–50. this ICT , 61 and this ICT , 50. No Results of QUADAS-2 assessment for evaluating the quality of the studies are shown in 2 = 63.75; p < 0.0001) and specificity . Taking into account the statistical heterogeneity, a meta-analysis was performed using a bivariate random effects model. Funnel plot asymmetry (p = 0.0001 from Egger’s test) revealed the existence of publication bias among the included studies . Similarly, the overall specificity was estimated to be 0.92 . DOR, as shown in According to our covariate significance test using a bivariate random effects model, the index test assay was the only significant heterogeneity factor . HoweverICT showed similar pooled sensitivity and specificity in populations of children and mixed populations (children + adults). The specimens obtained from oropharyngeal swabs showed a higher sensitivity and specificity than those from nasopharyngeal swabs. In addition, the pooled estimates of sensitivity and accuracy for studies using PCR reference standards were higher than those using microbial culturing .® brand of ICT [® for M. pneumoniae infection were 0.66 , 0.89 , 6.00 , 0.39  and 15.70 , respectively , with modest sensitivity (0.70) for the diagnosis of M. pneumoniae infection. The SROC AUC was 0.904, which indicates that ICT was highly accurate in the diagnosis of M. pneumoniae infection. This means that if a test result was positive, it was unlikely to be a false-positive result [M. pneumoniae infection for a patient with respiratory symptoms and a positive ICT result and can then start proper antibiotic treatment to control the infection. Unfortunately, negative ICT results cannot be used to definitely rule out M. pneumoniae infection [The current systematic review and meta-analysis is the first to establish an overview of the diagnostic accuracy of ICT for e result . Therefonfection . TherefoM. pneumoniae infection using ICT has the potential to decrease disease burden by the early prevention of outbreaks in closed populations, such as schools, colleges, and nursing home [M. pneumoniae infection can cause significant morbidity, and even mortality, in patients of extreme age [M. pneumoniae infection in these patients may be specifically beneficial.Regardless, the easy-to-perform, rapid, accurate diagnosis of ing home , 65. ICTM. pneumoniae (MRMP) has recently increased worldwide, reaching prevalence rates up to 80–90%, especially in Asian countries [M. pneumoniae, alternative antibiotic treatment including tetracyclines or fluoroquinolones is warranted in severe cases [M. pneumoniae is resistant to macrolide or not, until additional genetic testing for MRMP strains become available, rapid M. pneumoniae diagnosis can provide a clinical basis for the use of alternative antibiotics when no clinical improvement is observed with the use of macrolides as the first line of antibiotics [Moreover, the prevalence of macrolide-resistant ountries –70. MRMPountries , 71. As re cases , 70–73. ibiotics .M. pneumoniae infection was similar between the groups containing both adults and children and the groups containing only children. This finding suggests that ICT may be used regardless of patient age. Nevertheless, clinical studies evaluating ICT focusing on adult populations and studies that compare the accuracy of ICT between children and adults are still warranted.As for the target age group, our study demonstrated that the diagnostic accuracy of ICT for ® kit. Pooled estimates of the sensitivities, specificities, and AUC for Ribotest Mycoplasma® was lower than that of other index tests. The other commercial ICT kits used included the FUJI DRI-CHEM IMMUNO AG Cartridge Myco  and the Quick Chaser® Auto Myco M. pneumoniae antigen detection kit , which uses silver amplification. These have been shown to exhibit high sensitivity and specificity , even surpassing Ribotest Mycoplasma®  in head-to-head comparisons [The only significant heterogeneity factor in our covariate significance analysis was the index test used. The majority of index tests used in the studies analyzed was the commercially available Ribotest Mycoplasmaparisons . Test reM. pneumoniae, whether OP swabs or NP swabs, remains controversial [M. pneumoniae than that of NP swab specimens when the samples were concomitantly obtained [M. pneumoniae density in the NP swab specimens, which were collected in a blinded fashion, and the larger OP swab tip, which was able to reach deeper into the airway resulting in a specimen higher load of M. pneumoniae. In addition, it has been reported that a higher copy number of M. pneumoniae is found in the alveoli than on the epithelium of the upper respiratory tract [The best sampling site for detecting oversial –77. In oobtained . The autry tract , 79.M. pneumoniae diagnosis. M. pneumoniae culturing has specific short-comings, including being less sensitive, difficult to perform, and requiring longer than PCR to obtain results [Microbial culturing and PCR are currently the most commonly used reference standards for  results , 80–84. M. pneumoniae strains. In addition, industrial sponsorship, inclusion/exclusion of comorbid conditions, duration of clinical symptoms prior to testing, time lapse before specimen processing, and blinded assessment of ICT were so rarely reported that we were not able to evaluate their effects.It is noteworthy that our current study had several limitations. First, there were no studies included that assessed the difference in ICT performance between macrolide-sensitive and macrolide-resistant M. pneumoniae infection, regardless of patient age. This suggests that ICT is a useful test during the diagnostic workup of RTIs. Major practical advantages of ICT are its user-friendly format and short time requirements . ICT could function as the point-of-care in clinical practice, instead of serology, PCR and microbial culturing, especially in resource-limited settings. If physicians are aware of the limitations of ICT, such as false negative results, they could make educated decisions in using ICT to implement appropriate antibiotics stewardship and infection control as well as to help make decision regarding the use of other diagnostic modalities. However, additional studies regarding the potential impact of ICT in clinical practice are necessary. These include the cost-effectiveness of routine ICT use and whether ICT may allow for decreases in additional diagnostic tests and result in reducing the excessive use of macrolides.ICT is a rapid and easy-to-use detection method with moderate sensitivity, high specificity, and high accuracy in diagnosing S1 Checklist(DOC)Click here for additional data file.S1 Search strategy(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Fig(EPS)Click here for additional data file.S2 FigSummary points of the sensitivity and specificity, summary receiver operating characteristic (SROC) curve, 95% confidence region, and 95% prediction region are shown. The area under the curve of the SROC for the immunochromatographic tests (ICTs) was 0.786.(EPS)Click here for additional data file.