Lung cancer and pulmonary tuberculosis are two significant public health problems that continue to take millions of lives each year. They may have similar symptoms and, in some cases, are diagnosed simultaneously or may have a causal relationship. In tuberculosis disease, the chronic inflammation, different produced molecules, genomic changes, and fibrosis are believed to be important factors that may promote carcinogenesis. As a reverse reaction, the development of carcinogenesis and the treatment may induce the reactivation of latent tuberculosis infection. Moreover, the recently used checkpoint inhibitors are a debatable subject since they help treat lung cancer but may lead to the reactivation of pulmonary tuberculosis and checkpoint-induced pneumonitis. Pulmonary rehabilitation is an effective intervention in post-tuberculosis patients and lung cancer patients and should be recommended to improve outcomes in these pathologies. Lung cancer, the primary cause of cancer-related deaths worldwide, taking 1.8 million lives in 2020, is now recognized as a public health concern .Mycobacterium tuberculosis [M. tuberculosis is an intracellular organism that can spread efficiently through the respiratory system of the general population by causing life-threatening lung disease in immune hosts [M. tuberculosis, continues to be a leading cause of morbidity and mortality in the world [M. tuberculosis infected one-third of the population in 2021, producing disease in approximately 10% and killing 1.6 million people [Another significant global public health issue is tuberculosis (TB), caused by rculosis . M. tubene hosts . TB, a che world . M. tuben people ,6. The Cn people . Betweenn people . In 2021n people .M. tuberculosis is endocytosed and trapped in the phagosome of an alveolar macrophage [M. tuberculosis has developed unique mechanisms that either detoxify the toxic compounds before harm is done or repair the damage before it becomes fatal [M. tuberculosis to evade the phagosome and survive in the macrophage’s cytoplasm [Initial identification by macrophages is a key phase that mobilizes the immune response in both TB and cancer . In the crophage . T cellscrophage . To surves fatal . Additioytoplasm . This reytoplasm .Numerous studies have demonstrated how viruses cause genomic instability, which leads to cellular change . This isM. tuberculosis infection can lead to lung cancer in some cases [M. tuberculosis causes chronic inflammation, which in turn may lead to lung cancer [Many theories, including the suppression of the immune system, DNA damage, and the production of inflammatory factors, suggest that me cases . The essg cancer .M. tuberculosis modifies these signaling pathways to aid its survival [M. tuberculosis, is possibly the best researched of them [The two-stage carcinogenesis model can be used as a basis for the theory of involvement of chronic inflammatory processes in the tumorigenic process . The begsurvival . The sev of them . Althoug of them .M. tuberculosis in the lungs [M. tuberculosis manipulates the phagolysosome maturation process to avoid lysosomal sequestration and death so that it can survive and reproduce inside phagosomes [M. tuberculosis lives in the phagosome, where it multiplies; it breaches the phagosomal barrier and escapes into the macrophage’s cytoplasm, where it can multiply or spread through the necrosis of the infected cells, infecting other cells [M. tuberculosis is able to replicate in the cytosol are mainly unknown and require other studies [The resident alveolar macrophages are among the first cells to come into contact with the aerosolized he lungs . They enhe lungs . M. tubeagosomes . M. tubeer cells . However studies .M. tuberculosis infection [M. tuberculosis infection [M. tuberculosis is able to avoid being killed by innate immune cells and selectively colonize alveolar macrophages [M. tuberculosis after an aerosol-based infection [M. tuberculosis can acquire and maintain a slowed metabolic activity in the latent infection phase [There is evidence to show that active innate immune responses in local macrophages are linked to early clearance of nfection . In addinfection . Due to rophages . Most alnfection . To withon phase .M. tuberculosis, bacterial DNA may integrate to bronchial epithelial cells, causing neoplastic transformation [It has been suggested that TB-related chronic lung inflammation may induce clastogenic activity in the bronchial epithelium’s DNA . As an iormation . Lateralormation .M. tuberculosis can connect macrophages during the development of granulomas, a process similar to the mesenchymal–epithelial transition (MET) that takes place throughout malignancies [M. tuberculosis and other persistent infections activate transcription factors that trigger the MET, a crucial step in the development of cancer and metastatic disease [By causing E-cadherin expression to change into epithelioid morphology, gnancies . Additio disease .M. tuberculosis infection [Both TB and lung adenocarcinoma overexpressed MKI67, a nuclear protein that controls the regulation of several genes to promote tumorigenesis . Moreovenfection .M. tuberculosis growth in vitro, M. tuberculosis PtpA is a secreted, low-molecular-weight protein tyrosine phosphatase (PtpA) that is significant for M. tuberculosis pathogenicity in vivo [M. tuberculosis effector protein that targets MKI67 to control these activities [M. tuberculosis [Mycobacterium bovis BCG promotes the growth and migration of human lung adenoma A549 cells [While not necessary for  in vivo . In additivities . Moreoverculosis . In a mo49 cells .It is well-known that TB-related lung fibrosis and inflammation can cause genetic damage, which may raise the chance of developing lung cancer . AdditioA systematic review based on the risk of lung cancer after tuberculosis showed an increased risk when research was limited to people from “non-Westernized countries” . There wThe risk of having TB was shown to be correlated with cancer in a time-dependent manner, peaking 1 year before and after cancer diagnosis . In a stCommon signs of pulmonary TB and lung cancer include a cough, expectoration, fever, hemoptysis, weight loss, and shortness of breath . Due to High CA 19-9 levels as a tumor marker for gastrointestinal, pancreatic, and hepatobiliary cancers are suggestive of advanced illness and a bad prognosis . OverexpIn a study, 6683 individuals had been diagnosed with TB in total . Among t+ Th1, Th2, and Th17 cells, as well as CD8+ cytotoxic T lymphocytes [Protective immunity against pathogens such as viruses, bacteria, fungi, parasites, and malignancies is mediated by different subsets of effector T cells, such as CD4phocytes ,27. Thesphocytes .M. tuberculosis infection is immune response activation with type 1 T helper cells (Th1) and production of IFN-ɣ and TNF-α [Chronic infections like tuberculosis are comparable to cancer in that they both involve prolonged periods of high antigen exposure ,29. The nd TNF-α .M. tuberculosis infection [The expression of PD-1 on CD4 and CD8 T cells does rise after prolonged exposure to tuberculosis antigens, and inhibiting PD-1 or PD-L1 using an antibody boosts CD4 and CD8 T-cell activity . It is cnfection . T cellsnfection . T-cell nfection .The interaction between co-stimulatory and co-inhibitory receptors on T-cell surfaces (such as CD28 and CTLA-4), as well as CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells, is a crucial step in the T-cell-mediated immune response . Regulat+ T cells, and to enhance tumor-specific immune responses as a result [+ T cells, as it occurs in HIV infection [Checkpoint inhibitors are a rapidly expanding field of study in cancer immunotherapy, with potential applications to tuberculosis . Immune a result . The maia result . They rea result . Additioa result . Most ofa result . The abia result . One of nfection .+ T cells [M. tuberculosis antigen significantly activates PD-1 signaling pathways for both CD4 and CD8 T-cell immune response [M. tuberculosis infection promotes the tumor metastases in lung tissue, PD-1 signaling being required in this regulation [An essential factor of the interplay between hosts and pathogenic microorganisms is the inhibitory PD-L/PD-1 pathway . In pulm T cells . This maresponse . M. tubegulation .−/− animals more vulnerable to MTB-induced death [The PD-1/PD-L1 pathway’s function in MTB infections is debatable because of inconsistent results from mouse and human studies . Accordied death .M. tuberculosis H37Rv aerosol infection [An inhalation infection model was used to compare PD-1-deficient and control C57BL/6 mice to examine the function of PD-1 in aggressive TB . At diffnfection .M. tuberculosis H37Rv-infected animals with PD-1 deficiency had large localized necrotic regions in their lungs [−/− and wild-type mice developed extremely uncommon inflammatory nodules two weeks after infection [−/− mice developed necrotizing pneumonia with considerable lymphocytic infiltrates but few degenerating and fragmented neutrophils [In contrast to control mice, which exhibited fewer and less severe inflammatory lesions, ir lungs . Both PDnfection . Howevernfection . The granfection . In conttrophils . Extensitrophils . Withouttrophils . The widtrophils . The redtrophils . It has trophils . Immune trophils . In the trophils . Howevertrophils .M. tuberculosis infection, blocking PD-1/PD-L1 is insufficient to restore effector T-cell activity [M. tuberculosis infection are not exhausted T cells in the traditional sense, since they contribute to the maintenance of effector T cells that are specific for the antigen [Evidence suggests that in the event of a persistent activity . This isactivity . Further antigen . T-cell  antigen . As a re antigen . Some di antigen . As a re antigen .M. tuberculosis infection, and this effect is thought to be mostly mediated by the recovery of worn-out T cells [Another immunotherapy target is T-cell immunoglobulin and mucin domain-containing-3 (TIM3) . Contrar T cells . Further T cells . For bot T cells .M. tuberculosis are significantly amplified, demonstrating the crucial function of this co-inhibitory receptor in regulating inflammatory reactions to a highly immunogenic pathogen [M. tuberculosis infection is lethal, as opposed to the outcomes seen in chronic infection models, where blockage of PD-1 produced higher effector activities in T cells and a more robust response against the pathogen [Without PD-1, the inflammatory and necrotic responses to pathogen . When thpathogen .Immune checkpoints that regulate T-cell activation and regulatory cells of the innate and adaptive immune systems, such as Treg cells, myeloid-derived suppressor cells (MDSCs), and M2-type macrophages, all play a role in immune regulation . As a meM. tuberculosis spreads as lung cancer progresses [There is a connection between the two processes—lung cancer and TB . Lung caogresses .M. tuberculosis multidrug-resistant (MDR) strains, and the expansion of resistance to second-line anti-TB therapies reduces the range of available treatments [At this moment, the specific guidelines recommend that all patients, including those with HIV co-infection, who have not received treatment before and who do not exhibit signs of drug resistance, should be given an internationally recognized first-line treatment regimen utilizing medications with known bioavailability . Two moneatments ,40. Knoweatments ,40. Thereatments . These deatments . When aceatments . Investieatments .In a study including 84,984 patients with lung cancer diagnosed over a period of 8 years, 8015 were diagnosed with tuberculosis before or at the time of cancer diagnosis . For theMedical treatment options for lung cancer include chemotherapy, radiation, immunotherapy, and targeted therapy . These tPatients with lung cancer, especially those with non-small-cell lung cancer, have greatly benefited over the past 10 years from immunotherapies that manipulate the immune system to find and eliminate cancerous cells . In addiPulmonologists actively monitor and work with patients with lung cancer from the time of their first diagnosis and staging through treatment and restaging . In the Even while immune checkpoint inhibitors have proven to be a highly effective treatment for advanced NSCLC, they only help a very small proportion of patients . An immuImmune checkpoint inhibitors have the potential to cause severe pulmonary toxicity, which requires early diagnosis and treatment . The pneThere are still many knowledge gaps, even though several standards have made it simpler to classify pneumonitis severity and standardize treatment approaches . PotentiTherefore, there is a pressing need to improve communication between oncologists and pulmonologists in the multidisciplinary care of lung cancer patients, particularly for those receiving immunotherapy with immune checkpoint inhibitors who run the risk of developing immune-related pneumonitis .The therapy of patients with chronic respiratory disorders, such as COPD, bronchiectasis, and interstitial lung disease, among others, has been acknowledged to include pulmonary rehabilitation as a key component . Recent An increasing amount of data also indicates the advantages of PR for patients with chronic lung impairment brought on by previously treated tuberculosis .Patients report weight loss, asthenia, and exercise dyspnea during the active phase of TB because of the severe chronic inflammatory state that causes the release of many cytokines, TNF-α, and interleukins . As a reQuitting smoking at or around lung cancer diagnosis is also part of the PR programs . It was Pulmonary tuberculosis and lung cancer are at the top of the leading causes of morbidity and mortality worldwide, a situation worsened by the SARS-CoV-2 pandemic. Genomic modification, chronic inflammation, and fibrosis produced by tuberculosis may promote carcinogenic effects. Treatment used in lung cancer, for example, checkpoint inhibitors, may reactivate latent tuberculosis or aggravate its evolution. Special attention must be given to the treatment of coexisting pulmonary tuberculosis and lung cancer. Moreover, in both diseases, pulmonary rehabilitation plays a significant role, which in multiple situations is overlooked.


Anopheles spp. composition and abundance, and Plasmodium spp. infection burden are critical components of integrated vector management. The present study aimed to investigate the effect of landscape heterogeneity on entomological and parasitological indices of malaria in western Kenya.Identification and characterization of larval habitats, documentation of Anopheles mosquitoes were identified and characterized. Adult mosquitoes were sampled using pyrethrum spray catches (PSC). Finger prick blood samples were taken from residents and examined for malaria parasites by real-time PCR (RT-PCR).A cross-sectional entomological and parasitological survey was conducted along an altitudinal transect in three eco-epidemiological zones: lakeshore along the lakeside, hillside, and highland plateau during the wet and dry seasons in 2020 in Kisumu County, Kenya. Larval habitats for Plasmodium falciparum infection was associated with residency in the lakeshore zone, school-age children, rainy season, and no ITNs . Similarly, lakeshore zone and the rainy season significantly increased Anopheles spp. abundance. However, house structures such as wall type and whether the eave spaces were closed or open, as well as the use of ITNs, did not affect Anopheles spp. densities in the homes . Anopheles funestus (41.8%) and An. arabiensis (29.1%) were the most abundant vectors in all zones. Sporozoite prevalence was 5.6% and 3.2% in the two species respectively. The lakeshore zone had the highest sporozoite prevalence  and inoculation rates (135.2 infective bites/person/year). High larval densities were significantly associated with lakeshore zone and hillside zones, animal hoof prints and tire truck larval habitats, wetland and pasture land, and the wet season. The larval habitat types differed significantly across the landscape zones and seasonality .Increased risk of Plasmodium infections in humans emphasizes the importance of tailoring specific adaptive environmental management interventions to specific landscape attributes to have a significant impact on transmission reduction.The empirical evidence on the impact of landscape heterogeneity and seasonality on vector densities, parasite transmission, and The online version contains supplementary material available at 10.1186/s13071-022-05447-9. Plasmodium infection in western Kenya . In total, 2505 Anopheles larvae were morphologically identified belonging to four species, which included An. gambiae (s.l.), , An. funestus , An. coustani , and An. pharoensis . Furthermore, drainage ditch had the highest abundance (28.9%) of Anopheles larvae (Table An. gambiae (s.l.), An. funestus, and An. coustani, and An. pharoensis were found in the drainage ditch (29.1%), manmade pond (28.6%), and natural pond (30.4%), respectively. Anopheles pharoensis was only found in the drainage ditch (Table An. gambiae (s.l.) (n = 1500) proportion differed significantly across the larval habitat types , with manmade ponds (31.3%) having the highest composition. The proportion of A. funestus (n = 30) was highest in manmade ponds (36.7%); An. coustani (n = 15) was mainly found in manmade ponds (46.7%). Only the drainage ditch had An. pharoensis , and water algae were not significant predictors of larval density   times higher in the lakeshore zone and 1.81  times higher in the hillside zone. Anopheles larval density was 4.59  times higher during the wet season than the dry season . The mean Anopheles larval density in the hillside zone was 1.11 larvae per dip, with a high density of Anopheles larvae found in animal hoof print larval habitats (8.33 larvae per dip). The mean Anopheles larval density in the highland plateau zone was 0.64, with a high density collected from animal hoof prints (9.00 larvae per dip)  (n = 124), An. funestus (n = 89), An. coustani (n = 7), and An. pharaoensis (n = 1). The mosquito species composition differed significantly between topographical zones . Anopheles funestus (n = 89) was the most common primary vector in all the zones followed by An. arabiensis (n = 62), then An. gambiae (s.s.) (n = 2). Based on ITS2 (ITS2A/ITS2B primer set) PCR and subsequent sequencing results, 60 specimens were identified to be either An. sp. 1  or An. sp. 9  as described by Zhong et al. [Anopheles funestus and An. arabiensis were the most abundant species in the lakeshore zone, hillside zone, and highland plateau zone . Anopheles funestus had the highest sporozoite rate of 5.6% (n = 5/89), followed by An. arabiensis . Anopheles gambiae (s.s.) had only two samples with one infection at 50%. Across the landscape zone, the sporozoite rates were higher in the lakeshore zone , followed by the highland plateau zone  and the hillside zone    times higher in the lakeshore zone while there was no significant difference for the hillside zone (P = 0.917). The adult Anopheles were 2.17  times higher during the wet season than the dry season  Table . CompareP. falciparum infection prevalence . The prevalence of P. falciparum infection was 15.1% (130/862). Increased risk of P. falciparum infection was associated with residency in the lakeshore zone, school-age children, rainy season, and no ITNs  then hillside  zone compared to the highland plateau. The odds of P. falciparum infection was higher among the school-going children  compared to individuals ≥ 15 years. Not using an ITN was linked to a three-fold increase in the risk of P. falciparum infection . People who lived in brick or block houses had lower odds of P. falciparum infection  than those who lived in mud and wood houses. Residents were 1.47 times more likely to contract malaria during the rainy season  than during the dry season  was a more efficient vector than An. arabiensis (24.1 bites/person/year). The EIRs are influenced by topography [An increase in mosquito larval habitats leads to an increase in vector density, which eventually leads to an increase in malaria transmission, as has been observed elsewhere , 42, 44.pography , and theAnopheles larval density, with cultivated land negatively influencing larval density. The low density of larvae along cultivated land could be attributed to agricultural insecticides, which may interfere with the Anopheles species composition. During the rainy season, the reported productivity of larval habitats correlates with increased vector density and species richness [Anopheles mosquito larvae may increase during and immediately after the rainy season [Land use type surrounding the larval habitat was significantly associated with richness , 9, 39. y season .P. falciparum infection in the current study. It has been reported that school-aged children act as reservoirs for infectious gametocytes, spreading infection throughout the community [School-aged children were more likely to contract ommunity –50. The ommunity , 51, 52.Anopheles larvae identification.According to the current study, larval habitats are confined to the valley bottom, with high intensity of infection in lowland areas and few larval habitats in the highland plateau. This has resulted in a heterogeneous distribution of the vector and parasite burden, which has been documented , 39, 53.P. falciparum infection was linked to residency in the lakeshore zone, school-going age, living in mud houses, rainy season, and not using ITNs. Adult Anopheles abundance, on the other hand, was linked to landscape zones and seasonality. Furthermore, high larval densities were predicted by landscape of the lakeshore and hillside zones, animal hoof prints, and tire truck larval habitats, wetland and pasture land, as well as the wet season. The highest abundances of Anopheles larvae were found in drainage ditches and manmade ponds. Anopheles funestus and An. arabiensis were the most abundant adult vectors across the study zone landscape. Understanding the larval habitats of malaria vectors and reducing their availability is critical for malaria control and elimination through the implementation of target specific environmental management interventions, which can significantly reduce malaria burden.The risk of Additional file 1: Table S1.Anopheles larvae density in various larval habitat types across topography. Table S2.Anopheles species composition and sporozoite rates, human blood meal index, and entomological inoculation rates (EIR).


Staphylococcus aureus (S. aureus) causes a broad range of infections and is associated with significant morbidity and mortality. S. aureus produces a diverse range of cellular and extracellular factors responsible for its invasiveness and ability to resist immune attack. In recent years, increasing resistance to last-line anti-staphylococcal antibiotics daptomycin and vancomycin has been observed. Resistant strains of S. aureus are highly efficient in invading a variety of professional and nonprofessional phagocytes and are able to survive inside host cells. Eliciting immune protection against antibiotic-resistant S. aureus infection is a global challenge, requiring both innate and adaptive immune effector mechanisms. Dendritic cells (DC), which sit at the interface between innate and adaptive immune responses, are central to the induction of immune protection against S. aureus. However, it has been observed that S. aureus has the capacity to develop further antibiotic resistance and acquire increased resistance to immunological recognition by the innate immune system. In this article, we review the strategies utilised by S. aureus to circumvent antibiotic and innate immune responses, especially the interaction between S. aureus and DC, focusing on how this relationship is perturbed with the development of antibiotic resistance. S. aureus is mainly found living on the skin and mucus membranes of the anterior nares of most healthy human populations (20–80%) [S. aureus maintains a symbiotic relationship with the host but becomes virulent in deeper tissue sites accessed through trauma or parenteral route of infection [S. aureus are also associated with respiratory tract infections that range from asymptomatic colonisation to necrotizing pneumonia [20–80%) ,2. Under0–80% ,2.nfection . Staphylnfection . It is rnfection . Pathogeneumonia . This neneumonia . Diseaseneumonia .S. aureus infection is of high socioeconomic burden in both developed and developing nations [S. aureus infections. Infections caused by daptomycin-resistant S. aureus are particularly more prolonged and difficult to treat [S. aureus-host innate immune interactions may help to discover the fine details of pathogenesis of infections involving these bacterial strains and to develop effective therapeutic options. nations . The eme nations ,11 furthto treat ,11,12. AThe innate immune response is the first line of defence against invading pathogens. The innate immune system is comprised of diverse cells, proteins, and receptor systems that recognise pathogens and initiate a sequence of events that result in the production and secretion of inflammatory mediators . The subS. aureus infections [S. aureus are outlined in this review  α1–α3 inhibited HMGB1-induced NF-κB activation by binding with TLR4 in HEK-Blue hTLR4 and THP-1 cells [TLRs are the best-characterised pattern recognition receptors . They hafections , highligs review . TLR4, w-1 cells . WhetherS. aureus infection as it is highly expressed on resident macrophages and recruited neutrophils and monocytes [−/− mice have shown that TLR2 is required for survival, abundant cytokine secretion, and effective clearance of S. aureus from the brain [S. aureus phagocytosis [TLR2 can recognise a wide range of PAMPs derived from bacteria, fungi, parasites, and viruses  and geneonocytes . Studieshe brain . Even thocytosis ,44,45. Aocytosis .S. aureus [S. aureus. LTA is thought to be recognised by TLR2 in cooperation with CD36 [TLR2/TLR6 heterodimers recognise diacylated lipopeptides and peptidoglycans from . aureus . Lipoteiith CD36 . Indeed,ith CD36 . Of inteith CD36 .S. aureus infection models [S. aureus infection [S. aureus was demonstrated in murine bone-marrow-derived DC [S. aureus-released microvesicles by RAW 264.7 cells [S. aureus nucleic acids does appear to play a role in innate sensing of S. aureus but the reliance on a single endosomal TLR is less clear, with likely differences between different cell types.Endosomal TLR7, 8 (human), 9, and 13 (murine only) sense the presence of single-stranded RNA (ssRNA), DNA, and ribosomal RNA (rRNA), respectively. Studies have shown that single deficiencies in TLR7, 9, or 13 have little or no effect in host defences in n models . Howevernfection . A stronrived DC . In humarived DC ,25,26. R.7 cells . OverallC-type lectin receptors (CLRs) are a heterogenous group of transmembrane proteins, many of which are expressed on myeloid cells . CLRs poN-acetylglucosamine (GlcNAc) of teichoic acid of the S. aureus cell wall (WTA), inducing activation and innate cytokine responses by the DC subsets [S. aureus express structurally different WTA, which is instead recognised by macrophage galactose-type lectin receptor , expressed by macrophages and DC, also inducing proinflammatory immune responses [The C-type lectin (CLEC) receptor langerin  is expressed on Langerhans cells of the skin and subsets of conventional DC in mouse and human . Human C subsets . Certainesponses .S. aureus through binding to carbohydrate linkages of WTA [S. aureus bacteraemia [S. aureus infection in infants, which becomes redundant upon induction of antibodies against WTA [S. aureus infection, indicating a role for MBL in regulating cytokine release [S. aureus [Mannose binding lectin (MBL) is a serum glycoprotein of humans and animals that is involved in opsonisation of complement on the surface of s of WTA ,54. In hteraemia . The MBLinst WTA . Mice la release . MBL com. aureus .S. aureus, potentially dependent on host cell type and differences in lipid moieties present in different bacterial strains or cell wall changes induced upon acquisition of antibiotic resistance.Many other CLRs have been implicated in uptake and recognition of bacteria . It is lNLRs are located in the cytosol and respond to a range of both PAMPs and DAMPs . They arS. aureus infection and a significantly higher bacterial tissue burden. Their reduced viability is due in part to loss of NOD2 leading to defective neutrophil phagocytosis and reduced proinflammatory cytokines IL-1β and IL-6 and/or antimicrobial peptides, e.g., β-defensin production, against S. aureus infection [Mice deficient in the NLRC family member NOD2 have increased susceptibility to nfection ,33.S. aureus α, β-, and γ- hemolysins; Panton–Valentine leucocidin (PVL) [in (PVL) ; and leuin (PVL)  can actiin (PVL) ,62,63. Iin (PVL) .The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) family consists of three DExD/H box RNA helicases located in the cytosol: retinoic acid-inducible gene (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and laboratory of genetics and physiology-2 (LGP-2) ,66. BothS. aureus genomic DNA [S. aureus when compared with healthy controls—whether there is a link to protective immunity during septicaemia is unclear [RLRs are key sensors of virus infection that recognises intracellular RNA introduced to the cytosol ,77. Althomic DNA . In anot unclear .S. aureus [Cytosolic DNA sensing by the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway is implicated in host immune defence mechanisms. cGAS senses viral, bacterial, or host dsDNA aberrantly localised in the cytosol  and prom. aureus . S. aureus infection model, STING deficiency resulted in increased mortality and a higher bacterial burden in the lungs and bronchoalveolar lavage (BAL) fluid, alongside a severe destruction of lung architecture [S. aureus infection showed that S. aureus infection promoted IFN-β mRNA expression and TBK1/IRF3-dependent production of IFN-β in murine macrophages [S. aureus infection, as IFN-β is required to turn on downstream genes important for local inflammatory responses [S. aureus showed that approximately 95% of the induced genes within the first four hours were mostly regulated by TLR and STING signalling pathways [S. aureus [S. aureus.In a murine pulmonary itecture . Howeveritecture . Anotherrophages . Productesponses . RNA-seqpathways . This st. aureus . OverallS. aureus skin infections since they are able to phagocytose S. aureus and mediate caspase-1-dependent clearance through the activation of an inflammatory programmed cell death pathway, pyroptosis [KCs form the outermost barrier of the epidermis and are considered nonprofessional phagocytes, which are typically the first cells that encounter pathogens ,90. Theyroptosis .S. aureus sepsis, increased microbial load and mortality was noticed in mice lacking macrophages [S. aureus load and loss of normal alveolar structures. The antimicrobial peptide calprotectin present in monocytes and early macrophages successfully inhibits S. aureus growth in a mouse abscess model by reducing bioavailability of metal ions, starving the bacteria from essential nutrients [Macrophages are professional phagocytes and are critical for the clearance of invading pathogens from the site of infection. In macrophages, staphylococcal lipoproteins induce proinflammatory cytokines, NO, and reactive oxygen species (ROS) ,95,96,97rophages . Similarrophages ,101 and rophages , where lutrients ,104,105.S. aureus are phagocytosed by macrophages and trigger NLRP3 inflammasome activation [S. aureus infection induces a caspase-dependent pyroptotic cell death pathway in human macrophages by inhibiting mTORC1/STAT3 [S. aureus persists in intracellular vacuoles for 3–4 days before escaping into the cytosol [In humans, Extracellular Vesicles (EVs) released by tivation . AnotherC1/STAT3 . It is r cytosol . Treatme cytosol .S. aureus infections [S. aureus in humans, neutrophils can release NETs that trap the bacteria independent of ROS [S. aureus in vitro [Neutrophils are capable of eliminating bacteria by producing nonspecific antimicrobial molecules. In a wide variety of tissues and organs, defects in neutrophil number or function result in an increased susceptibility to fections ,111,112.fections ,114, indfections . For efffections ,117. In t of ROS ,119. Humin vitro . Howeverin vitro .DCs are the most potent antigen-presenting cell and are pivotal in the generation of adaptive immunity. Steinman and Cohn discovered the cell and applied the term “dendritic cells” based on their unique morphology . ImmaturDCs represent a heterogeneous population of cells and can be broadly divided into four categories based on surface marker expression and functional specialisation. The first two subsets, including the cross-presenting cDC1 and helper T-cell priming cDC2, comprise conventional DC (cDC) subsets ,131 and S. aureus by DC, much of it is either assumed from DC receptor expression and their known ligands [S. aureus interactions with other phagocytes [S. aureus, with few direct experiments investigating cDC subsets and their function either in vitro or ex vivo.While there is a body of literature focussing on the molecular mechanisms regulating innate recognition of  ligands  or findiagocytes ,135. Theagocytes ,137,138.S. aureus [S. aureus and upregulated expression of costimulatory markers MHC classes I and II, and increased production of proinflammatory cytokines and gamma interferon (IFN-γ) by CD4 and CD8 T cells [S. aureus by human pDC, demonstrating that activation is dependent on sensing of endosomal nucleic acids via TLR7 and TLR9 [S. aureus and produce tumour necrosis factor (TNF-α), IL-6, IFN-α, and upregulate CD86 expression [Study on BDCA1(+) myeloid DCs (mDCs) indicated that they can induce both innate and adaptive immune response against . aureus . In this T cells . Parcina T cells  examinedand TLR9 . Therefopression . CollectS. aureus143. Indeed, cDC-deficient mice (CD11c-DTR transgenic) exhibit increased bacterial loads, accelerated mortality, and more severe pathology than controls [S. aureus stimulation elicits cDC-dependent IL-12p70 [In murine models, it is clear that cDCs are important players in the recognition of controls . FurtherIL-12p70 , suggestS. aureus phenol soluble modulins in altering the Th balance in a murine infection model [S. aureus lab strain USA300 expressing PSM, when compared with knockout strains [More recently, Richardson and colleagues  demonstron model . This st strains .S. aureus is known to induce DC toxicity, with decreased numbers of murine splenic cDC1 and cDC2 during infection [S. aureus leucocidins in monocyte-derived DCs are primarily induced through the action of pore forming leucocidin and leukocidin AB [S. aureus has also been shown to survive and replicate following internalisation by CD11c+ splenocytes in vitro [S. aureus immunopathogenesis, especially so with regard to distinct primary DC subsets.In terms of DC pathogenesis, nfection . Recentlcidin AB . Howeverin vitro , comprisin vitro . It is tS. aureus has the capacity to survive even in the presence of an antigen-specific adaptive immune response [S. aureus to modulate innate immune defences.response . It possresponse . The remS. aureus that interfere with a variety of innate immune defences [Superantigen-like proteins (SSLs) are one of the major immune evasion molecules produced by defences ,148. Thedefences ,152,153.defences . SSL3 haS. aureus lab strain MSSA476 [S. aureus strains was localised within SCC, together with genes encoding fusidic acid resistance and genes providing resistance to methicillin. This protein exerted a specific inhibitory effect against TLR2-mediated NF-κB activation, JNK phosphorylation, and production of proinflammatory cytokines [S. aureus (both Methicillin-sensitive and -resistant S. aureus) strains, and found to downregulate the NF-κB pathway through inhibition of not only TLR2, but also TLR4, TLR5, and TLR9 [tirS mutant in an intravenous mouse infection model revealed that presence of TirS increased the bacterial load in multiple organs [The TLRs and IL-1Rs superfamily have a conserved cytoplasmic region, known as the Toll/IL-1 receptor (TIR) domain ,155,156. MSSA476 . TirS inand TLR9 . A compaAnother staphylococcal virulence factor, adenosine synthase A (AdsA), catalyses the hydrolysis of adenosine monophosphate (AMP) to adenosine and increases the overall abundance of extracellular adenosine. Interaction of adenosine with its receptor triggers anti-inflammatory signalling cascades that cause the inhibition of platelet aggregation , cellulaS. aureus employs an array of mechanisms to interfere with normal host endosomal acidification, which is required for the clearance of the pathogen and the induction of an adaptive immune response. In immature bone-marrow-derived DCs, incomplete assembly of the S. aureus enzyme V-ATPase results in reduced acidity of host phagosomes [S. aureus increases endosomal pH by converting ammonia to urea [S. aureus cell death [2- into H2O2 and reduce the bacterial cell damage [S. aureus also encodes accessory gene regulator (agr)-dependent factor(s) that induces autophagosome formation but prevents autophagolysosomal fusion events [S. aureus within an autophagosome phosphorylates mitogen-activated protein kinase 14 (MAPK14) and autophagy-related 5 (ATG5) in murine fibroblasts, preventing autophagosome maturation and, therefore, bacterial degradation [S. aureus can further escape into the cytosol from autophagosomes, where it proliferates and induces caspase-independent host cell death [agosomes . Similar to urea . Intracell death ,169,170,ll death . In the l damage ,172. S. n events . It has radation ,175. S. ll death .Klebsiella pneumoniae [S. aureus has also been shown to diminish macrophage efferocytosis and instead induce programmed necroptosis of neutrophils that have phagocytosed and/or been infected with S. aureus [S. aureus to colonise the lung [As elucidated for the gram-negative bacterium eumoniae , S. aure. aureus . The sec. aureus . This imthe lung .S. aureus produces a wide range of cellular and extracellular factors that helps them escape intracellular degradation and antigen presentation [S. aureus to host lysozymes is mediated by O-acetyltransferase A (OatA), which adds an acetyl group to N-acetylmuramic acid in the peptidoglycan of the cell wall [S. aureus produces PSM, which can form membrane pores in the phagosome facilitating escape from destruction and host antigen processing pathways [ess-encoded virulence factor EsxA prevents host apoptosis, therefore impeding clearance, while EsxB reduces cytokine production by primary human DCs [S. aureus can persist in human infection and serve as a reservoir for recurrent infection [entation ,180. Resell wall . The aceell wall . S. aurepathways ,184,185.pathways ,187. Theuman DCs . Host ceuman DCs ,190. Sevnfection ,194,195.S. aureus in the late 1940s resulted in the development of a semisynthetic penicillin (Methicillin) [S. aureus (MRSA) was reported and then spread worldwide. MRSA is now endemic in health care facilities in virtually all industrialised countries [S. aureus has the capacity to acquire antibiotic resistance [mec is central for antibiotic resistance, there is no direct evidence that SCCmec plays a clear role in MRSA virulence [The emergence of Penicillin-resistant icillin) . In the ountries . Communiountries ,198. Unlsistance ,201,202 sistance ,204. Ressistance . mecA gesistance ,204,205.irulence .S. aureus are generally thought to be associated with an overall change in the net charge of the bacterial membrane that results in the repulsion of the DAP antibiotic molecule from the cell surface [mprF is most consistently associated with the development of DAP-R in S. aureus both in vivo and in vitro [mprF is responsible for the cross-resistance of MRSA to DAP and Vancomycin (VCM) [S. aureus are those involved in phospholipid metabolism [S. aureus (and other Gram-positive organisms) harbor two (or more) cls genes (cls1 and cls2) and changes in this enzyme play a role in DAP-R by altering the pool of PG and Cardiolipin (CL) in the bacterial CM [The acquisition of MRSA resistance to the last-line antibiotics vancomycin and daptomycin has been recorded over the last 15 years ,208,209. surface ,211. Thein vitro ,213,214.in (VCM) ,216,217.tabolism . Importaerial CM ,219. Anoerial CM . GenomicS. aureus have been shown to be significantly less virulent than susceptible parent strains following daptomycin therapy and clinical failure [Interestingly, DAP-R clinical isolates of  failure . Cameron failure  demonstr failure .With the mutations conferring antibiotic resistance being well-characterised, growing evidence is emerging regarding a duality in function, whereby antibiotic resistance mutations further modulate immune effector mechanisms .S. aureus [Perhaps most well-characterised is that of the multipeptide resistance factor (MprF), which is an evolutionarily conserved protein expressed by various bacterial species including . aureus ,231,232.. aureus , serving. aureus ,234,235.. aureus . As prevcls2, and, partly, mprF, correspond to a significant reduction in the secretion of proinflammatory cytokines including TNFα, interleukin-6 (IL-6), and macrophage inflammatory protein-1β (MIP-1β) as well as expression of CD80 by cDCs in response to daptomycin-resistant MRSA [S. aureus also demonstrated that exposure to daptomycin alone or in combination with vancomycin or oxacillin  led to a downregulation of TLR1, TLR2, TLR6, and TLR9 expression and dampened macrophage inflammatory response with diminished TNFα secretion and reduced synthesis of Nitric oxide (NO) [S. aureus to evade neutrophil chemotaxis and represented a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack [Indeed, it has been demonstrated that the acquisition of specific point mutations in ant MRSA . In vitride (NO) ,230. In e attack . Mishra e attack .S. aureus has the remarkable capacity to also accumulate antibiotic resistance mutations that simultaneously mask ligands of critical innate sensors. Increased biofilm formation capacity in hospitalised patients by MRSA compared with Methicillin Sensitive S. aureus (MSSA) strains is considered an important virulence factor influencing its persistence in both the environment and the host [Aside from directly interfering with innate effectors, the host . In addithe host .S. aureus confer resistance to macrolide, lincosamide, and streptogramin group antibiotics, whilst simultaneously preventing detection through TLR13 [S. aureus 23S rRNA remain to be confirmed. The notion that bacteria can change throughout the course of infection to evade innate immune detection is indeed novel, and these findings set an important precedent for further investigation.Indeed, it has been shown that specific mutations in the 23S rRNA of gh TLR13 . TLR13 iS. aureus possesses a large repertoire of virulence factors including antibiotic resistance genes that can lead to suboptimal innate immune responses, opening a window to establish and sustain infection. The precise mechanisms defining how antibiotic resistance mutations affect the bacterial isolate’s ability to evade innate immune activation is of clear importance. Understanding how resistant S. aureus modulates innate cells, including DCs, to escape recognition is critical to restore immunogenic potential and to allow recognition and clearance.The innate immune response is the key to the prevention of infection and generation of adaptive immunity. Initial sensing of bacteria through PRR initiates the inflammatory cascade to release cytokines, antimicrobial peptides, and recruit antigen presenting DCs to further activate adaptive immune responses. However,


Apis mellifera is controlled by the complementary sex-determiner (csd) gene: females (queens and workers) are heterozygous at this locus and males (drones) are hemizygous. When homozygous diploid drones develop, they are eaten by worker bees. High csd allelic diversity in honeybee populations is a priority for colony survival. The focus of this study is to investigate csd variability in the genomic sequence of the hypervariable region (HVR) of the csd gene in honeybee subspecies sampled in Italy. During the summer of 2017 and 2018, worker bees belonging to 125 colonies were sampled. The honeybees belonged to seven different A. mellifera subspecies: A. m. ligustica, A. m. sicula, A. m cecropia, A. m. carnica, A. m. mellifera, Buckfast and hybrid Carnica. Illumina genomic resequencing of all samples was performed and used for the characterization of global variability among colonies. In this work, a pipeline using existing resequencing data to explore the csd gene allelic variants present in the subspecies collection, based on de novo assembly of sequences falling within the HVR region, is described. On the whole, 138 allelic sequences were successfully reconstructed. Among these, 88 different alleles were identified, 68 of which match with csd alleles present in the NCBI GenBank database.Sexual regulation in  The comoneybees . Honeybes drones . When diker bees . The csdm of csd ,6.csd gene is composed of nine exons divided into three clusters that encode an SR-type protein with a putative protein-binding function [csd alleles in Apis mellifera populations ranged from 10 to 13 [The function ,7. The tfunction ,8,9,10. 10 to 13 ,12. Subscsd alleles from hundreds of embryos collected from two colonies in each location [csd locus by sampling six purple eye drone pupae from 42 hives, obtaining both allele sequences from 35 of the 42 queens, and only one allele sequence from the remaining seven. Based on these 77 amino acid sequences, they identified 16 different alleles, six of which were new alleles that had not been sequenced previously [A. m. carnica, A. m. ligustica and A. m. caucasica) and 8 samples of honey collected in China, Hungary, Japan and Georgia. Among these samples, they found a total of 25 csd alleles [csd alleles [In detail, Cho et al. identified 18 distinct alleles by 27 workers sampled from a single hive in Michigan . In Slovlocation . In New eviously . In 2019 alleles . In 2021Apis mellifera subspecies they sampled  [csd sequences sampled worldwide, demonstrated that the total number of csd alleles in A. mellifera ranges from 53 locally to 87 worldwide [However, some studies propose that the number of circulating alleles in the worldwide honeybee population is significantly underestimated. Wang et al. found a total of 79 haplotypes by analyzing 50 workers for each of the six different gustica) . Lechnerorldwide . A recent comprehensive study conducted on 652 sequences found in GenBank confirmed that the number of circulating alleles is very high. In this set, which represents a vast geographic area and eight subspecies, 225 alleles were found, grouping sequences by identity and numbering them consecutively from the most common to the least (Amelcsd-HVR1-Amelcsd-HVR225) . The high number of csd alleles reflects the high honeybee genetic diversity due to adaptation to different global environmental conditions . Howevercsd alleles have been explored, usually based on Sanger sequencing. Currently, csd alleles of a diploid female honeybee are frequently determined through the analysis of csd sequences of six to eight male descendants by Sanger sequencing [Over the years, different approaches to determine quencing ,19,20,23csd alleles from honeybee queens and from honey samples. DNA was extracted from clipped wings of queen bees and worker bees, and locus targeted Illumina sequencing was performed, obtaining csd alleles directly from the diploid queens. In 2021, Bovo et al. [csd allelic variability from 12 different colonies. To further investigate the applicability of bioinformatic approaches for csd allele assessment and variability investigation from NGS data, we propose an alternative method based on local de novo assembly of Illumina reads matching the HVR, integrated by manual inspection and curation. The aim of this work was to exploit the existing Illumina genomic resequencing data collection described in Minozzi et al. [csd HVR allele-specific sequences from diploid worker bees, thus describing the inter- and intra- genetic variability of seven Apis mellifera subspecies on 125 diploid worker bees sampled in different Italian regions.In 2020, Kolics et al.  used a ho et al.  used a si et al.  and retrA. m. ligustica (n = 61) and A. m. sicula (n = 6); three allochthonous A. m. mellifera (n = 4), A. m. carnica (n = 8) and A. m. cecropia (n = 1); and two hybrids: A. m. carnica by A. m. ligustica hybrids (n = 2), and Buckfast bees (n = 43). One hundred twenty-five worker colonies were sampled between summer 2017 and summer 2018  in twelvcsd gene allelic variants present in the subspecies collection.All details of DNA extraction to sequence processing and alignment can be found in Minozzi et al. . Based ohttps://www.ebi.ac.uk/Tools/st/emboss_transeq/, accessed on 28 April 2021), and the two in-frame translations matching the csd amino acid sequences were retrieved [Apis cerana (GenBank QKY64514.1) was added to the dataset to be used for tree rooting. PhyML 3.0 [Samtools was used to extract reads belonging to each sample, which mapped in the genomic interval corresponding to exon 7 of the csd gene, harboring the HVR (Hav3.1_NC_037640.1:11771976-11772119) . Sample-etrieved ,13,15. Ietrieved . The HVRhyML 3.0  was usedApis mellifera NCBI nr database and retrieve csd HVR known sequences through tBLASTn (amino acid query versus translated database). Only identical hits (100% coverage and 100% similarity) were retained.CAP3-derived putative HVR amino acid allele sequences were used to blast the csd alleles using genomic resequencing data was applied to sequences from 125 honeybee diploid worker bees. In detail, quality-trimmed reads were mapped to the Amel_Hav3.1 honeybee genome, and 5310 mapped reads falling within the HVR genome coordinates (Hav3.1_NC_037640.1:11771976-11772119) were extracted and split according to the sample of origin. Sample-specific CAP3 generated contigs were inspected, and only sequences from the 87 samples for which the assembly procedure resulted in two contigs and not more than one singleton were retained. The two contigs sequences retrieved for each sample, supposed to represent the sequences of the two alleles, were translated into proteins. One hundred seventy-four sequences gained from 87 diploid samples were then filtered to 154 sequences due to 20 polypeptide sequences being truncated. Thus, among these 154 sequences, only those belonging to the 69 samples showing both alleles successfully reconstructed were kept, leading to 138 sequences.The pipeline aiming at the reconstruction of csd alleles represented in our colonies collection that were successfully reconstructed, are reported along with their sequence length in csd alleles ; A. m. mellifera belongs to the western and northern Europe lineage (M); A. m. carnica, A. m. cecropia and A. m. ligustica belong to the eastern Europe group (C); Buckfast and hybrid Carnica are crosses of European subspecies belonging to C type (EC) [A. cerana was used to root the tree. The honeybee samples sub-grouping does not strictly reflect the evolutionary lineages.A phylogenetic tree was constructed based on the 138 llection . The sevype (EC) . A. ceraA. m. ligustica, A. m. carnica and Buckfast). A. m. ligustica.Allele frequencies of alleles were computed and reported in A. m. ligustica specimens, we identified 49 different alleles. Among these, alleles 1, 11 and 32 were the most frequent in A. m. ligustica.Among the 68 sequences belonging to 34 Among the 42 sequences belonging to 21 Buckfast individuals, we identified 33 different alleles. Allele 9 was the most frequent, with a frequency of 9.52%.A. m. sicula, seven different alleles were identified; allele 30 was the most frequent, with a frequency of 25%,Among the eight sequences belonging to four A. m. carnica, we identified nine different alleles with a frequency of 20%; allele 25 was the most frequent.Among the 10 sequences belonging to five A. m. mellifera, A. m. cecropia) had a limited number of reconstructed csd HVR sequences , each bearing a different allele.The remaining three subspecies  belonging to two different geographical types [The phylogenetic tree  generateal types . This real types .A. m. ligustica, A. m. carnica and Buckfast. It is known that the three subspecies belong to the same evolutionary lineage C type  are shared between three different subspecies e C type  and oftea sample , identifon level .csd alleles in Apis mellifera populations with many alleles present in a single bee  and other alleles that show a high frequency [csd alleles by Bilodeau et al. in 2021 (named Allele Amel Csd-HVR57 in their report) [Furthermore, our results confirm the uneven distribution of requency . In detacsd gene of 125 diploid worker bees and investigated allelic variability in the complementary sex determiner gene (csd). We identified 88 different alleles, 68 of which match with csd alleles already present in the NCBI GenBank database, while 20 are newly described alleles. Given that the viability of the brood requires a high number of circulating alleles, high allelic variation should be a priority in both subspecies as well as in highly selected honeybee breeding lines. A representation of circulating alleles can be obtained by both traditional Sanger and NGS-based sequencing approaches. To this end, NGS-based csd assessment offers the availability of a method to rapidly generate csd alleles population information, starting from whole genome data created for selection. This information can enable beekeepers to monitor csd variability in selective breeding programs and to perform traditional selection at the same time. This solution should be actively promoted among bee-breeders.In conclusion, we used existing 150 bp Illumina whole genome resequencing data to explore the hypervariable region (HVR) of the


Microbes have dominated life on Earth for the past two billion years, despite facing a variety of obstacles. In the 20th century, antibiotics and immunizations brought about these changes. Since then, microorganisms have acquired resistance, and various infectious diseases have been able to avoid being treated with traditionally developed vaccines. Antibiotic resistance and pathogenicity have surpassed antibiotic discovery in terms of importance over the course of the past few decades. These shifts have resulted in tremendous economic and health repercussions across the board for all socioeconomic levels; thus, we require ground-breaking innovations to effectively manage microbial infections and to provide long-term solutions. The pharmaceutical and biotechnology sectors have been radically altered as a result of nanomedicine, and this trend is now spreading to the antibacterial research community. Here, we examine the role that nanomedicine plays in the prevention of microbial infections, including topics such as diagnosis, antimicrobial therapy, pharmaceutical administration, and immunizations, as well as the opportunities and challenges that lie ahead. Antibiotics and vaccines are among the greatest medical advances. Over the previous century, broad-spectrum medicines and vaccinations greatly lowered infectious disease morbidity and mortality ,2,3. InfThe pharmaceutical and biotechnology industries have been revolutionized by nanomedicine, or the application of nanotechnologies in medicine ,20,21,22It has been demonstrated that utilizing the host’s immune system to recognize and kill germs protects the host against microbial infection. Pathogen-associated molecular patterns help the innate immune system to identify pathogens that breach the host’s physical barriers . AntigenIsolated proteins, polysaccharides, and bare DNA are used to create next-generation bacterial vaccines, thanks to biotechnology . CompareNanoparticles deliver mucosal vaccinations well. Mucosal surfaces contain nearly 80% of immunocytes and are the first line of defense . A totalEffective non-inflammatory mucosal adjuvants include nanoemulsions, which are oil-in-water emulsions containing droplets on the nanoscale . PotentiBurkholderia pseudomallei   nanofibers containing Ag nanoparticles showed promising results [The advantages of the synthetic polymer analogs of CAPs include lower cost and improved enzymatic stability . Compara results .E. coli and S. aureus, compared to 20 g/mL for normal chitosan molecules. Chitosan nanoparticles kill fungi and Gram-positive bacteria more effectively than Gram-negative bacteria [Chitosan: Besides synthetic polymers, chitosan, which is a natural cationic polysaccharide polymer, exhibits antibacterial properties. Polycationic chitosan, and its derivatives, are antibacterial, due to their polycationic properties. The electrostatic contact increases the microbial wall permeability, and chelating essential trace metals inhibits enzymes . Due to bacteria . In addibacteria . These nbacteria .Figure Nanoparticles provide antibacterial agents, which is another important use. Nanoparticle-based medication delivery might overcome antibiotic systemic toxicity, drug uptake and efflux, biofilm development, and intracellular bacterial infection. Surface modification with targeting ligands or microenvironment responsiveness can focus the nanoparticles on the infection site, improving therapeutic efficacy and reducing antimicrobial medication adverse effects. The nanoparticle distribution of antimicrobial medications also improves the hydrophobic drug solubility, the systemic circulation time, the drug half-life, and the drug release, which may minimize systemic adverse effects and the administration frequency ,125,126.P. aeruginosa and E. coli, may also reduce the uptake of hydrophobic antibiotics, such as beta-lactams and macrolides [Antibiotic resistance prevents microbial cells from attaining harmful drug concentrations through reduced uptake and enhanced efflux . The outcrolides . An overcrolides . Two reccrolides ,130 havecrolides .Nanoparticle-based antibiotic delivery may help to fight biofilms and intracellular bacteria, which cause persistent infections that are hard to treat with traditional antimicrobials. Liposomes and lipid-/polymer-based nanoparticles shield antibiotics from enzymes and promote penetration, boosting their efficiency against biofilm-forming bacteria . LiposomE. coli was increased by a factor of 64 compared to vancomycin alone [Conjugating several antibiotic copies on nanomaterial surfaces can boost antibacterial effectiveness because some antibiotics interact with bacterial surface components. Au nanoparticles can attach antibiotic medicines to a solid surface and boost their antibacterial activity by interacting with the cell walls . The effin alone . Severalin alone .P. aeruginosa and E. coli [Nanoparticles also deliver nitric oxide (NO), which is a short-lived gaseous antibacterial agent. NO inhibits DNA replication, cell respiration, and reactive nitrogen intermediate production, which makes it antibacterial . These p E. coli . When NO E. coli . Besides E. coli . NO nano E. coli . An MRSA E. coli .S. aureus [A combination antibiotic treatment may prevent and treat drug resistance . Additiv. aureus .Antimicrobial drugs could be more effective if they were delivered to the location of infection via tailored nanoparticles rather than random ones. The treatment of slow-growing or dormant bacterial infections, which are notoriously difficult to treat and require regular high doses of antibiotics, may also benefit from this . Ligand-A low pH, enzyme overexpression, localized bacterial toxins, and ligand-targeted nanoparticle delivery are some of the other targeting strategies that have been used . AntibioThe enzymes and toxins that are produced by bacteria can be employed for site-specific applications. By incorporating themselves into the liposome membranes and releasing the encapsulated therapeutic drugs, novel liposomes that were generated by Zhang and colleagues can selectively deliver antibiotics to the areas of bacterial infection . In ordeAccording to the type and the place of infection, nanoparticles that are compatible with the biological environment should be used. In the following subsections, the studies that have evaluated different nanoparticles against infections in animal models are discussed.Bacteria easily settle in skin lesions including atopic dermatitis and chronic wounds, contributing to infection-induced inflammation and disease progression . Due to S. aureus and E. coli by 74% and 75%, respectively. Subcutaneous abscesses were cured by NIR exposure following nanocomposite injection.Garlic contains antimicrobial allicin . SharifiCarvacrol was incorporated into poly(-caprolactone) (PCL) nanocarriers and combined with hydrogel for topical distribution . MonoterS. aureus showed rapid antibiotic release. Mice received intracellular S. aureus subcutaneously. After two days, the animals received nanocomposite intravenously. After PBS and free-medication injections, the infected regions had 2.3 × 107 and 8.4 × 106 CFU/mL, respectively. The nanoparticles reduced the bacteria to 1.5 × 104 CFU/mL. The surfactants formed micelles. The antibacterial SMEs were cationic surfactants that formed nanoscale micelles [Yang et al.  created micelles . In the 2O3:Cr3+ is luminous [Pneumonia, TB, and cystic fibrosis are caused by respiratory tract bacteria. Nanoformulations were administered intravenously or intratracheally to animals with lung infections. Tigecycline was the model antibiotic that was encapsulated in ICAM1-conjugated β-Ga2O3:Cr3+ nanoparticles by Kang et al. . Inflammluminous . In ordeP. aeruginosa-induced lung infection. Inhaled tobramycin cannot permeate DNA-rich lung mucus [P. aeruginosa doubled the survival rate from 40% with free antibiotics to 80%. DNase-containing nanoparticles penetrated the cystic fibrosis sputum more effectively. Intratracheal PLGA nanoparticles carrying esculentin-1a cured lung infection in a study by Casciaro et al. [P. aeruginosa-infected mice. Free esculentin-1a had 17-fold less anti-P. aeruginosa action. Micelle nanocarriers were made by conjugating vancomycin with amphiphilic PEG-co-PCL copolymer via pH-cleavable hydrazone linkages [P. aeruginosa. The micelles reduced the lung bacterial load and the alveolar damage in P. aeruginosa-infected mice.Polymer-based nanocarriers alleviate ng mucus . Deacon ng mucus  created o et al. . PVA stalinkages . The nanP. aeruginosa infections received the nanosystem intravenously. Moxifloxacin could increase the survival rate from 20% to 40% following nanoparticulate encapsulation. Nanocomposite therapy eliminated the lung pathogen colonies. PEGylated phosphatidylcholine-rich nanovesicles were tested for infectious pneumonia treatment [A ROS-responsive 4-(hydroxymethyl) phenylboronic acid pinacol ester-modified α-cyclodextrin was coated with phospholipids in order to generate lipid-coated nanoparticles in order to deliver moxifloxacin to infected lung tissue and sustain drug release . In the reatment . CiproflS. aureus-recognizing cyclic 9-amino-acid peptide CARGGLKSC (CARG). CARG bound only to S. aureus in vitro. Intravenous CARG-conjugated nanoparticles had eight-fold more lung deposition than non-targeted nanoparticles. S. aureus that was instilled intratracheally into mouse lungs caused 67% mortality after 24 h. CARG-conjugated MSNs enhanced the survival rate to 100%. All MSN-treated mice survived for 20 days.Antimicrobial peptide NZX inhibits drug-resistant M. tuberculosis. Due to the macrophages’ high absorption of MSNs, Tenland et al.  tried toE. coli caused intestinal illness. Nanoparticles were taken orally daily for five days. The casein-coated nanomedicine reduced the bacteria colonization more than the free ciprofloxacin. Compared to the non-coated MSNs and the free peptides, the casein-coated nanoparticles lowered the intestinal TNF-α 1.5- and 2.2-fold.Oral antimicrobial nanoparticles treat gastrointestinal infections. Nanocarriers protect antibiotics against GI fluid breakdown. Bioadhesive nanoparticles prolong GI tract retention for oral bioavailability. Oral MSNs are suitable for GI medication enzymolysis protection. Zhao et al.  created H. pylori in the gastrointestinal tract led to a reduction in gastric ulcers and inflammation. Compared to omeprazole, amoxicillin, and metronidazole, this triple therapy was more effective against germs. In order to create biomimetic nanocarriers for targeting H. pylori, the gastric epithelial cell membrane was coated onto PLGA nanoparticles [H. pylori was attracted to the biomimetic nanocarriers 10 times more than to the uncoated nanoparticles. After the oral administration of the biomimetic nanoparticles and the free medicine, the bacterial burden in the stomachs of the infected mice was reduced from 1.6 105 CFU/g to 6.5 103 and 5.0 104, respectively.Montmorillonite is a smectic clay with mucoadhesive and EPS-attaching properties . H. pyloarticles . H. pyloAntibacterial nanoparticles have been used to treat systemic, bone, and vaginal infections. Systemic bacterial infections cause bacteremia and sepsis . Rai et S. aureus bone infiltration in mice. Boron was added to nanoparticles in order to prolong antibacterial action because its anticorrosive properties delayed Cu oxidation [S. aureus was inserted into the mice’s bones with a silk suture in order to cause osteomyelitis. The 1 mg/kg intravenous nanoparticles reduced the bacterial CFU 10-fold compared to the control. The S. aureus bone accumulation was also suppressed intramuscularly. Magnetic Fe3O4 nanoparticles and heat-disrupted biofilm were used to cure osteomyelitis [E. coli inoculation for five days caused vaginitis in mice. The nanoparticles reduced the CFU in vaginal washes. The histological epithelial exfoliation scores and the E. coli counts were consistent.Ag–Cu nanoparticles by Qadri et al.  eliminatxidation . The nanmyelitis . The S. myelitis . A PVA hmyelitis . VaginalThe good news is that nanosystem-based antibiotics, antitoxin compounds, and antimicrobial peptides have been transferred to the clinic after substantial research into revolutionary antimicrobial delivery systems to combat antibiotic resistance. Many are still undergoing clinical testing .In a Phase 1 trial in healthy volunteers, Lipoquin was used to inhale ciprofloxacin-loaded liposomes . In 21 aS. aureus’ α-toxin and P. aeruginosa’s type III toxins secretory moiety are in clinical trials [Antibacterial drugs may benefit from a nano-preparation that targets bacterial toxins. In 2016, the first human monoclonal antibody targeting Clostridium difficile toxin B was approved, which was bezlotoxumab . Monoclol trials . A broadl trials .Antimicrobial peptides have broad-spectrum antibacterial action and little resistance risk due to their fast death . AntimicNanotechnology is promising for microbial illness treatment. Due to its high adjustability and broad range of adaptation, antibiotics with nanomaterials are a more cost-effective option for macrophage persister cells and biofilm infections. Nano-antibiotic systems can target, penetrate, absorb, and change infectious microenvironments, and combine with other treatment techniques due to their nanomaterial design. Thus, nanomaterials have considerable potential to improve antibiotic efficacy. Clinical translation must first resolve various issues and testify carefully about in vivo toxicity and clinical effects. Nano-antibiotics for resistant bacterial infections will require long-term research and practice before their widespread use. Nanomaterials are still a promising antibiotic-resistance-fighting option. We think that nano-antibiotics can combat bacterial resistance and save more lives soon.


Protaetia brevitarsis seulensis widely distributed in Asian countries is traditionally used in oriental medicine. This study explored gene expression abundance with respect to wing development and metamorphosis in P. b. seulensis based on the large-scale RNA-seq data. The transcriptome assembly consists of 23,551 high-quality transcripts which are approximately 96.7% covered. We found 265 wing development genes, 19 metamorphosis genes, and 1,314 candidates. Of the 1,598 genes, 1,594 are included exclusively in cluster 4 with similar gene co-expression patterns. The network centrality analyses showed that wing development- and metamorphosis-related genes have a high degree of betweenness centrality and are expressed most highly in eggs, moderately in pupa and adults, and lowest in larva. This study provides some meaningful clues for elucidating the genetic modulation mechanism of wing development and metamorphosis in P. b. seulensis.A white-spotted flower chafer  Protaetia brevitarsis seulensis (Coleoptera: Scarabaeiformia: Scarabaeidae: Cetoniinae), commonly known as the white-spotted flower chafer, is widely distributed in Korea and southeast Asia, including China [ng China , 2. It bng China . Its larng China , 4. In Kng China . MoreoveP. b. seulensis habituated in China and South Korea has been reported in previous studies [P. b. seulensis genome was 751 Mb (including the scaffolds longer than 2 kilobases (kb)) with 327 scaffolds, and the N50 length of the assembly was 2.94 Mb. Further, 34,110 genes  were identified using Evidence Modeler. In South Korea, we sequenced P. b. seulensis genome using Illumina NextSeq500 and PacBio sequencing platforms, generating 277.7 gigabases (Gb), providing 190-fold coverage based on the 435-megabases (Mb) estimated genome size. The assembled P. b. seulensis genome was 692 Mb (including the scaffolds longer than 2 kilobases (kb)) with 302 scaffolds, and the N50 length of the assembly was 4.53Mb. A total of 19,371 genes were identified using a gene prediction algorithm.The draft genome of  studies , 7. In CGlyphodes pyloalis [Cryptolaemus montrouzieri [Bombyx mori [D. melanogaster and Blattella germanica reported general trends in modern insect evolution [P. b. seulensis.The transcriptome represents all genes expressed in a cell or a population of cells, providing a biological perspective on cellular processes. Novel high throughput sequencing technologies greatly facilitate the global analysis of the blueprint of development-related genes , 9. Tranpyloalis , Cryptolrouzieri , and Bombyx mori . The comvolution . TherefoD. melanogaster [P. b. seulensis, remains unclear and incomplete. By comparing the P. b. seulensis transcriptome according to the four different developmental stages , we determined the specific genes and related candidates involved in the wing development and metamorphosis.Identification of specific genes regulating wing development would provide insights for further study of the molecular mechanisms controlling metamorphosis development. Wing development and metamorphosis signaling pathways are highly evolutionarily conserved in different animals , 22. Hownogaster –26. KnowP. b. seulensis during its developmental period. Differentially expressed genes (DEGs) were identified. Their functions were analyzed based on the various types of databases. Protein-protein interaction network was constructed by applying the concepts of interlog to explore gene importance and their potential functions based on the network topology. Furthermore, these investigations were focused on developmental regulatory factors responsible for wing development and metamorphosis in P. b. seulensis focusing on the network system. These data can increase the understanding of the development of P. b. seulensis. By performing comparative analyses of the gene expression levels and different developmental stages of P. b. seulensis, we aimed to determine and discuss novel gene sets related to the wing development and metamorphosis in P. b. seulensis, possibly revealing insights at the molecular level.This study enhanced the understanding of wing development and metamorphosis in P. b. seulensis) managed by the insect breeding facility of the National Academy of Agricultural Sciences  was used in this study. Larva and adults were raised on fermented oak sawdust in a dark photoperiod continuous breeding room at 28℃±1℃, less than 70% relative humidity (RH). For samplings, three egg lumps, each consisting of 20 eggs, three third instar larva, three 15-day-old pupae, and three 10-day-old adults were randomly selected.The white-spotted flower chafer  was isolated for overall transcriptome sequencing. During the total RNA isolation, the larva were washed with PBS, sterilized with 70% ethanol, and anesthetized on ice. The whole body was fixed, the dissected skin was cut along the abdomen, and tissue was collected. From the prepared samples, we extracted mRNAs with TRIzol reagent . RNA quantification and quality check were carried out using ultraviolet absorption and gel electrophoresis. All the samples were washed with 70% ethanol to reduce microbial contamination from the surface. Fine powders in liquid nitrogen, excluding eggs, were quickly crushed after ethanol volatilization. During the extraction of eggs, three pestles were homogenized in a 1.5 ml tube containing TRIzol reagent. RNA quantification was carried out using ultraviolet absorption and gel electrophoresis was used to confirm its quality.The mRNA sequencing library was prepared according to the Illumina TruSeqRNA Sample Preparation Kit v2 . Total RNA of 1ug was used in poly mRNA selection using streptavidin-coated magnetic beads, followed by thermal decomposition of the selected mRNA. Subsequently, 200–500 bp fragmented mRNA was used as a template for cDNA synthesis using reverse transcriptase with random primers. The cDNA was converted into finally repaired double-stranded DNA and incorporated into 15 cycles of specific index adapters for multiplexing, purification, and amplification. The quality and functionality of the final amplification library were tested by Agilent 2100 BioAnalyzer  that uses highly sensitive DNA chips. The library was prepared following the Illumina Protocol. Random fragmentation of DNA or cDNA and binding of 5’ and 3’ adapters were also investigated. Alternatively, fragmentation and nodule responses could be integrated into one single stage, considerably improving the efficiency of library preparation. The adapter binding fragments were then PCR-amplified and gel purified. Further, the concentrations of each library were measured by real-time PCR. Agilent 2100 Bioanalyzer was used to profile the distribution of insertion sizes. To generate clusters, the library was loaded into a flow cell and fragments were captured on a surface-coupled oligo lawn that complements the library adapter. Subsequently, the fragments were amplified to different clone clusters by bridge amplification. After cluster generation, Illumina HiSeq was used to sequence the cluster library. Based on the manufacturer’s instructions  and pair-end sequencing, the TruSeq Rapid SBS kit or TruSeq SBS kit v4 was used for 100 cycles. The sequencing protocol used was HiSeq.TM2500 System User Guide #15011190 Revised VHCS 2.2.58.P. b. seulensis genome from China and gene sequences using the existing tuxedo protocol [HiSeq was used to generate a sequence of raw images. TM control software (HCS v.2.2.) and basic calls were made through integrated primary analysis software called RTA (v.1.18). The HiSeq-generated image TM2500 was converted into a nucleotide sequence by base call and stored in FASTQ format using the Illumina package bcl2fastq (v.1.8.4). Clean reads were generated by filtering dirty reads, including adapters, unknowns, or low-quality score bases, from raw ones. Clean leads were Remapped to the protocol . MismatcThe isolated RNA was sequenced using ISO-Seq sequencing methods familiar with long and short-read sequences. DNALink, a Korean service provider, carried out a complete experimental process. The Illumina pair-end sequence initially received technical artifacts ) and adapter filtering using the Trimmomatic-0.32 method . FinallyThe genes in the draft Kolbe were predicted using the in-house gene prediction pipeline, including the evidence-based gene modeler  were calculated as RPKM = (1000000*C)/(N*L/1000), where C = number of mappable reads in specific unigenes, N = total number of reads mapped to unigenes in a particular sample, and L = length of the unigene19 (see http://www.cs.cmu.edu/jernst/stem) was used to compare the trends exhibited in these four stages [Differential Gene Expression and Trend Analysis was conducted. Differential Unigene abundances were determined by performing independent alignments of short reads from four libraries against the set of ne19 see . During r stages . P-valueP. b. seulensis protein interaction network was constructed by mapping the interaction information (Flybase) of D. melanogaster into the P. b. seulensis species. The betweenness centrality of the node in the P. b. seulensis network with high centrality nodes signifies the important nodes in the signaling flow of the entire network.The https://github.com/apetkau/orthomclsoftware-custom), according to the standard protocol using a default inflation number of 1.5. We constructed a phylogenetic tree using IQ-Tree 1.5.1 (http://www.iqtree.org/release/v1.5.1/). A maximum likelihood phylogenetic tree was inferred from a concatenated amino acid data set containing 9,608 one-to-two orthologous genes shared by all 12 species by IQ-Tree 1.5.1 with the best fit model (LG+F+G4). In total, 269 wing development-related genes and 19 genes for metamorphosis were identified. We used conserved genes in five species within 12 coleoptera species. the half gap option of Gblock.To analyze gene family evolution in coleoptera, all-to-all BLASTP analysis was performed followed by Markov clustering to identify orthologous gene groups with OrthoMCL v.2.0.9 , larva (SAMN15647969), pupa (SAMN15647970), and adults (SAMN15647971) using the Illumina HiSeqTM 2000 sequencing platform. A total of -692.7 Mb reads , the differential expression along the stages was obtained, as listed in Using Short Time-Series Expression Miner software  for theD. melanogaster, the function of the D. melanogaster was transferred to assign genes related to wing development and metamorphosis using keyword mining  at the biological process level, 432 sequences (27.42%) at the cellular component levels, and 224 sequences (21.69%) at the molecular function level. P. b. seulensis unigene sequences mapped to the reference canonical pathways in the KEGG database were analyzed. Moreover, 1,183 unigene sequences were specifically assigned to the 236 KEGG pathways and 164 unigenes were involved in metabolic pathways. These pathways played dominant roles in the following pathways: Spliceosome, Ribosome, RNA transport, Protein processing in the endoplasmic reticulum, and others  and betweenness centrality. Betweenness centrality reflects the importance of genes in the network, with its score representing the minimum number of links connecting one protein to another in the network. Thus, a high betweenness centrality score implies that genes undergo a greater number of the shortest paths in the network. Therefore, the protein with the highest betweenness centrality score derives considerable importance from its position in the network. Additionally, the number of interacting partners (degree) is important for concluding network centrality.Physical and functional links of altered genes were investigated on the assumption that gene pairs that interact or share similar functions tend to interact within the cellular systems . The P. P. b. seulensis, the already known wing development and metamorphosis-related genes were mapped to the P. b. seulensis network. Subsequently, 162 Cluster 4 genes were mapped. As a result, 82 wing development and metamorphosis-related genes (30.7%) and 16 both Cluster 4 and wing development and metamorphosis-related genes were mapped in the functional association network  of P. b. seulensis is insufficient, annotations of the four stages of development indicate that the insect has a high proportion of functional genes [Although the full genome information of al genes –52. EdibMost interestingly, we identified Cluster 4 as a significant gene group because it contains several wing formation and metamorphosis-related genes. General expression patterns of genes belonging to Cluster 4 were significantly down-regulated transiting from eggs to larva, whereas their expression level was reused as they went to pupae, therefore showing a pattern of expression-level maintenance even in adults. This phenomenon was also clearly revealed in the Kr-h1 gene, known to play an important role as a wing formation and metamorphosis-related gene. Additionally, most genes involved in 20E signaling and JH signaling showed a similar expression pattern as those in Cluster 4. However, these genes are known for their major function in wing formation and metamorphosis and it showed an up-regulation of gene expression in all stages of development. These expression patterns are distinct from the expression pattern of the genes belonging to Cluster 4. They act as factors that regulate and command signal conductors at a higher level of wing development and metamorphosis regulation.P. b. seulensis. Future analysis of genes related to insect development and death will help in control. Additionally, the richness of single genes and expression data obtained from this study provides information on the identification of genes involved in the development of P. b. seulensis.Therefore, in this study, four developmental stages of transcriptome produced the genes and genome resources needed to investigate P. b. seulensis based on the large-scale RNA-seq data obtained from every different developmental stage, namely eggs, larva, pupa, and adults. When conducting three-time repetitive RNA-seq data productions, the results consistently exhibited high reproducibility. The resultant de novo transcriptome assembly consists of 23,551 high-quality transcripts, which are approximately 96.7% covered; out of 8,545 expressed transcripts, 5,183 correspond to the possible orthologs of Drosophila melanogaster. Finally, we found 265 wing development genes, 19 metamorphosis-related genes, and 1,314 potential candidates. Out of the 1,598 genes, 1,594, except for four wing development genes, are included exclusively in Cluster 4 among 11 major clusters recognized on the basis of gene co-expression patterns along developmental stages. The function interaction network and the network centrality analyses showed that the 1,598 genes highlighted with the 284 wing development- and metamorphosis-related genes appear with high degree of betweenness centrality, indicating their key roles in molecular signaling pathways. The Cluster 4 genes are expressed most highly in eggs, moderately in pupae and adults, and lowest in larva, implying that they are closely related to the four development stages, eggs, pupa, adults, and larva. This study provides meaningful clues in elucidating the genetic modulation mechanism of wing development and metamorphosis in P. b. seulensis.In this study, we explored gene expression abundance with respect to wing development and metamorphosis in S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis are—to various extents—contributors to the burden of gastrointestinal illness in high-income countries. Detection of these pathogens by microscopy examination is challenging because of the limited sensitivity and need for specific staining procedures. We developed and optimised a new multiplex real-time PCR assay for the simultaneous detection of Cryptosporidium spp., G. duodenalis and D. fragilis in clinical (stool) samples. The diagnostic performance of the assay was evaluated against a large panel of well-characterised DNA samples positive for Cryptosporidium spp. (n = 126), G. duodenalis (n = 132) and D. fragilis (n = 49). The specificity of the test was assessed against a DNA panel from other intestinal or phylogenetically related parasites (n = 105) and faecal DNA from individuals without clinical manifestations (n = 12). The assay exhibited a diagnostic sensitivity of 0.90–0.97 and a diagnostic specificity of 1. The limit of detection was estimated for Cryptosporidium (1 oocyst) and G. duodenalis (5 × 10−4 cysts). The method allowed the detection of four Cryptosporidium species  and five G. duodenalis assemblages (A–E) without cross-reacting with other parasites belonging to the phyla Amoebozoa, Apicomplexa, Euglenozoa, Microsporidia, Nematoda and Platyhelminthes. This newly developed multiplex real-time PCR assay represents a novel alternative for the rapid and accurate detection of Cryptosporidium, G. duodenalis and D. fragilis in clinical settings.The enteric protozoan parasites  Those most affected by diarrhoea are children and immunocompromised individuals living in developing countries with limited or no access to safe water and adequate sanitation. Even though diarrhoea-related mortality rates in resource-poor settings have decreased considerably, morbidity remains high [Diarrhoea is a major health problem worldwide. Only in 2017, almost 1.6 million people died from diarrheal diseases globally . This information would be extremely useful to help in ascertaining the true clinical significance of D. fragilis.Among parasitic pathogens, the protozoa globally . Cryptosnteritis ,4,5. Giaountries . Anotherisorders . Remarkaorganism . In contustralia . These dClassically, diagnosis of protozoan enteroparasite infections has been achieved by microscopical examination of faecal samples. Light microscopy of faecal concentrates stands as the preferred routine diagnostic method in many clinical settings. Although this technique is labour-intensive, lacks sensitivity and requires skilled technicians, its simplicity and low cost outweighs the above-mentioned limitations and makes microscopy suited for resource-limited laboratories, particularly those in endemic and high-prevalence areas . In contCryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis in human stool samples.In recent years, a wide diversity of in-house and commercial real-time PCR (qPCR) assays see  have beeCryptosporidium spp., G. duodenalis and D. fragilis.This was a retrospective observational study specifically conducted to develop, validate and standardise a novel multiplex qPCR method for the simultaneous detection of the diarrheagenic protozoan enteroparasites Cryptosporidium spp.  value, 32.6; range, 25.5–40.0) and Giardia duodenalis  were initially detected by a commercial multiplex qPCR platform . DNA samples positive for D. fragilis (n = 49) had initially been identified by an in-house singleplex qPCR  [n = 105) positive for enteric or phylogenetically related  parasitic species other than G. duodenalis and D. fragilis and those belonging to Cryptosporidium  were initially diagnosed by a variety of in-house single-round, semi-nested and nested PCR assays in place at the SNCM. DNA (n = 12) from stool samples of apparently healthy individuals was included for the same purpose. Whenever possible, species and genotypes/sub-genotypes were determined by PCR and Sanger sequencing methods at the time of initial diagnosis. The full dataset including all relevant information of the original DNA samples used in this study can be found in A total of 424 well-characterised genomic DNA samples extracted and purified from biological (mostly stool) specimens of clinical patients were used . These s.4–41.3) . DNA samCryptosporidium species most frequently found causing human cryptosporidiosis  [G. duodenalis genetic variants (assemblages A–E). For cross-reactivity evaluation, we purposely selected representative DNA samples of various taxonomic groups of pathogens, including amoebozoan (n = 38), apicomplexan (n = 41), euglenozoan (n = 13), microsporidian (n = 3), nematode (n = 8) and platyhelminthic (n = 2) parasites (The DNA reference panel included DNA samples of the four niculus) ,16 and oarasites .D. fragilis) were part of the DNA collection of the Parasitology Reference and Research Laboratory of the SNCM. Genomic DNA was extracted from 200 mg of faecal material using the QIAamp DNA Stool Mini Kit  according to the manufacturer’s instructions. Some DNA samples were from in vitro cultures or of animal origin, particularly those belonging to animal-adapted species/genotypes or rarely found circulating in humans . All DNA samples used in this study had been extracted during the period 2014–2019 and stored at –20 °C until testing.All DNA samples (except those positive for n humans . DNA samcowp1) gene sequence for C. hominis (GenBank accession No. DQ388389), the small subunit ribosomal RNA (ssu rRNA) gene sequence for G. duodenalis (GenBank accession No. M54878) and the internal transcriber spacer (ITS) gene sequence for D. fragilis (KY554844). Selected primer and probe sequences are detailed in C. hominis-specific (forward and reverse) primers and probe set comprised positions 237–261, 387–365 and 356–326 of DQ388389, respectively. The G. duodenalis-specific (forward and reverse) primers and probe set comprised positions 79–98, 141–126 and 105–124 of M54878, respectively. The D. fragilis-specific (forward and reverse) primers and probe set comprised positions 96–117, 193–169 and 169–144 of KY554844, respectively.PCR primers and detection probes were chosen using Primer Express software  based on the known oocyst wall protein 1 .Singleplex or multiplex qPCR reactions were carried out in volumes of 25 µL with PCR buffer  containing mutant Taq DNA polymerase, dNTPs and 5.5 mM of MgClCryptosporidium spp. (ROX fluorophore), G. duodenalis (FAM fluorophore) and D. fragilis (HEX fluorophore) typically generated fluorescence curves with qPCR CT values < 45.All samples forming part of the DNA reference panel were blindly analysed in duplicates to avoid bias. Samples positive for −7) of individual DNA samples positive for C. hominis, G. duodenalis and D. fragilis were used to estimate the relative limit of detection of the qPCR assay in both singleplex and multiplex formats. DNA isolated from 10,000 C. hominis oocysts and 500 G. duodenalis cysts  were used as starting concentrations for this purpose. No quantified starting material was available for D. fragilis, as cyst forms from this protozoon have not been unambiguously described in human clinical specimens [Ten-fold serial dilutions  are summarised in Cryptosporidium oocyst in both the singleplex and multiplex formats. However, the latter performed slightly poorer than the former in terms of efficiency . These results are consistent with the facts that: (i) a single Cryptosporidium oocyst contains four sporozoites, each with a haploid genome, resulting in an oocyst ploidy of 4N, and its cowp1 gene exists in a single copy, (ii) a fully differentiated Giardia cyst contains four nuclei, each with a ploidy of 4N, resulting in a cyst ploidy of 16N, and its ssu rRNA gene exists in multiple copies, and (iii) Dientamoeba is unknown with respect to ploidy and genome size, but the targeted gene (ITS) should be multi-copy, one for each ribosomal set of genes.The analytical sensitivity and efficiency of the qPCR assay for the detection and differentiation of G. duodenalis (limit of detection: 5 × 10−4 cysts) and D. fragilis (limit of detection: dilution 1 × 10−7). The multiplex format was more efficient than the equivalent singleplex formats in detecting G. duodenalis  and D. fragilis .The multiplex format of the assay was particularly sensitive for the detection of Cryptosporidium-positive samples included in the DNA reference panel, the multiplex assay was able to detect 119 . Samples belonged to C. hominis , C. parvum (gp60 genotype families IIa and IId), C. meleagridis (gp60 genotype family IIIg) and C. ubiquitum (unknown gp60 genotype family) , G. duodenalis (n = 10) and D. fragilis + G. duodenalis (n = 1), as previously determined during routine initial diagnosis. All these samples were also scored positive for G. duodenalis and D. fragilis by the multiplex qPCR assay and, therefore, not considered as false-positive results (The diagnostic performance of the multiplex qPCR assay is summarised in ily) see . The sev results .G. duodenalis-positive samples included in the DNA reference panel, the multiplex assay was able to detect 129 . Samples belonged to G. duodenalis assemblages A, B, C, D and E  and D. fragilis + Cryptosporidium spp. (n = 1), all of them previously identified during routine initial diagnosis . No attempts were made to determine the genotype of the D. fragilis isolates because of the limited intra-genetic diversity previously observed in this protozoon [D. fragilis-positive samples were co-infected either with G. duodenalis (n = 17) or Cryptosporidium (n = 1). All these concomitant infections were identified at initial diagnosis and therefore were not considered as false-positive results  with known partial ssu rRNA sequences from T. vaginalis (GenBank accession number JX943584), T. gallinae (GenBank accession number EU215373) and T. tenax (GenBank accession number D49495). In all cases, mismatches were found in 36–41% of the positions, making the amplification of Trichomonas spp. with our D. fragilis primer set highly unlikely.Out of the 49 rotozoon ,21. A to results . To discCryptosporidium spp., G. duodenalis and D. fragilis yielded positive results when tested in the multiplex assay, nor did the 12 DNA samples from stool samples of individuals without clinical manifestations produce any signal  was observed between the results obtained by multiplex qPCR assay and those previously generated by the reference qPCR methods at initial diagnosis .Cryptosporidium spp., G duodenalis and D. fragilis) in western countries. To do so, a large, carefully selected panel of well-characterised DNA samples was used. This is the strategy adopted in our laboratory for evaluating and validating PCR-based diagnostic tests [Here, we developed, optimised and evaluated a new multiplex qPCR assay for the simultaneous detection of the three most common and clinically relevant protozoan enteroparasites  reported using commercially available multiplex qPCR assays, including the Allplex GI-Parasite  [cowp1) compared to the multiple-copy nature of ssu rRNA and ITS. In contrast, cowp1 has increased specificity over ssu rRNA, reducing the rate of false-positive amplifications. Still, lower values of 0.86–0.92 have been reported with the RIDAGENE Parasitic Stool Panel II  [Cryptosporidium species  causing the bulk of human cryptosporidiosis cases globally.Our multiplex qPCR assay achieved a diagnostic sensitivity of 0.94 for the detection of h Korea) ,34, the h Korea) , the G-Dh Korea)  assays. Germany) ,34 and tGermany)  assays. G. duodenalis, our multiplex qPCR test achieved a diagnostic sensitivity of 0.97, a figure in the lower range of those (0.97–1) reported by the best commercially available assays. These include the Allplex (Seegene) [Regarding Seegene) ,34, the Seegene)  or the NSeegene) ,30 methoSeegene) ,33,34,35D. fragilis is a very common finding in human stool samples and is second only to Blastocystis sp. in terms of positive rates [D. fragilis is particularly difficult to identity using conventional microscopy examination [D. fragilis, have been developed and commercialised in the last decade  were not available for testing. Other potentially cross-reacting species that were not part of the reference panel included Cyclospora cayetanensis, Entamoeba coli, Endolimax nana and Encephalitozoon intestinalis, among others. Similarly, larger numbers of G. duodenalis assemblages C, D and E should be tested to confirm the diagnostic sensitivity achieved for isolates belonging to assemblages A and B. These limitations remain to be addressed in future works.Regardless of the pathogen considered, our multiplex qPCR assay produced diagnostic specificities of 1. This was somehow expected as the panel used for reference purposes included well-characterised DNA samples that were previously diagnosed for the potential presence of concomitant infections, minimizing the risk of obtaining false-positive results. Despite this effort, we are aware that some relevant pathogenic and commensal protozoan species were missing in our panel. For instance, DNA samples from host-adapted Cryptosporidium spp., G. duodenalis and D. fragilis, the most clinically relevant diarrhoea-causing protozoan parasites in high-income countries. The test can also be used either for screening or confirmatory purposes in endemic areas of resource-poor settings. Of note, detection of all three pathogens  by conventional microscopy examination is challenging because of the intrinsic difficulty of correctly identifying some of their morphological features or the need for specific staining methods  [The novel multiplex qPCR test described here exhibited excellent linearity and high analytical sensitivity and specificity values, providing a suitable alternative diagnostic tool for the molecular detection and differentiation of um spp.) ,40.