Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease due to the peripheral resistance to aldosterone. Its clinical spectrum includes neonatal salt loss syndrome with hyponatremia and hypochloraemia, hyperkalemia, metabolic acidosis and increased plasmatic levels of aldosterone. Two genetically distinct forms of disease, renal and systemic, have been described, showing a wide clinical expressivity. Mutations in the genes encoding for the subunits of the epithelial sodium channels (ENaC) are responsible for generalized PHA1.SCNN1A  gene mutation, inherited from both heterozygous parents, was identified by next generation sequencing (NGS) in the homozygous child . A more detailed family history disclosed a possible related twenty-year-old girl, belonging to the same Sicilian small town, with referred neonatal salt loss syndrome associated to hyperkalemia, and subsequent normal growth and neurodevelopment. This second patient had a PHA1 clinical diagnosis when she was about 1 year old. The genetic investigation was, then, extended to her and to her family, revealing the same mutation in the homozygous girl and in the heterozygous parents.We hereby report on two Italian patients with generalized PHA1, coming from the same small town in the center of Sicily. The first patient is a male child, born from the first pregnancy of healthy consanguineous Sicilian parents. A novel SCNN1A gene mutation either in both patients or in other heterozygous family members, enabling also primary prevention of disease. Our report may broaden the knowledge of the genetic and molecular bases of PHA1, improving its clinical characterization and providing useful indications for the treatment of patients. Clinical approach must be personalized, also in relation to long-term survival and potential multiorgan complications.The neonatologist should consider PHA1 diagnosis in newborns showing hyponatremia, hyperkalemia and metabolic acidosis, after the exclusion of a salting-loss form of adrenogenital syndrome. The increased plasmatic levels of aldosterone and aldosterone/renin ratio, associated to a poor response to steroid administration, confirmed the diagnosis in the first present patient. An accurate family history may be decisive to identify the clinical picture. A multidisciplinary approach and close follow-up evaluations are requested, in view of optimal management, adequate growth and development of patients. Next generation sequencing (NGS) techniques allowed the identification of the  SCNN1A, sodium channel epithelial subunit alpha) is mapped in 12p13, the beta (SCNN1B) in 16p12.2-p12.1, and the gamma (SCNN1G) in 16p12 [Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease due to the peripheral resistance to aldosterone . Clinicain 16p12 . Congeniin 16p12 , 6.We hereby report on two Italian patients with generalized PHA1, coming from the same small town in the center of Sicily. Our report highlights how neonatologists should consider its diagnosis, after the exclusion of a salting-loss form of adrenogenital syndrome. We describe its rapid diagnostic suspicion, complex initial management, and the relevant role of next generation sequencing (NGS) techniques in defining the genetic profile of patients, as well as of carriers within families, also in view of primary prevention of disease.3−− 10.8 mmol/l, BE − 19.5 mmol/l) with normal anion gap were found. Urine analysis showed pH 5, while blood creatinine and urea nitrogen, as well as brain, heart and abdomen ultrasound examinations were normal, without revealing signs of hyperplasia of the adrenal glands and/or abnormalities of the kidneys and the urinary tract. Intravenous (IV) rehydration therapy with sodium chloride and sodium bicarbonate was started, for correction of hyponatremia and metabolic acidosis, respectively. Meanwhile, a treatment with fludrocortisone acetate (0.1 mg/day) and hydrocortisone (maximum dose 10 mg/m2/day) was also begun. Due to persistence of severe hyperkalemia, IV infusion of insulin and rectal, and subsequently oral, sodium polystyrene sulfate administration were performed. Breastfeeding was stopped, and the baby was fed with a special low-potassium formula. Hormonal parameters showed normal renin levels , significantly increased aldosterone ones , aldosterone/renin ratio , 17-OH progesterone , and low of ACTH  and cortisol  , coming from a small town in the center of Sicily. Anthropometric measures at birth were: weight 4170 g(98th centile), length 55 cm (> 99th centile), head circumference 35.8 cm (86th centile). Apgar score was 10/10. He was exclusively breastfed, and postnatal clinical course was uneventful. Jaundice due to increased indirect bilirubin levels appeared on the third day of life, and requested phototherapy for 5 days. On the eighth day of life, progressive feeding difficulties, poor sucking, hyporeactivity and lethargy, tachycardia and profuse sweating were suddenly observed. He was transferred to the neonatal intensive care unit (NICU), where a severe salt loss syndrome with hyponatremia and hypochloremia , hyperkalaemia (9.6 mEq/l), and metabolic acidosis , length 72 cm (98th centile), and head circumference 42.5 cm (25th centile)  [Within a detailed medical history of the family, a possible related twenty-year-old girl belonging to the same Sicilian small town, with referred neonatal salt loss syndrome and hyperkalemia, failure to thrive in the first year of life and subsequent normal growth and neurodevelopment, was found. This second patient had a PHA1 clinical diagnosis when she was about 1 year old. Referring to a remote and unfocused coefficient of consanguinity, the genetic investigation was, then, extended to this further proband and to her family, allowing the identification of the same mutation in the homozygous girl and in the heterozygous parents. She currently is under treatment with low-potassium diet, sodium chloride and ion exchange resin, and has a normal life.miliaria rubra, for the high sweat sodium concentration, are a clinical sign of disease, and they were observed in our first patient [SCNN1A gene mutation identified in the proband, not previously reported in the literature, is located in a splicing site [PHA1 occurs in two forms, renal and systemic, genetically distinct and with wide clinical expressivity. Patients with generalized PHA1 are at risk of fatal salt loss episodes with severe hyperkalemia, from the neonatal age to adulthood . Some of patient . Ocular  patient . The ana patient , 14, whiing site . In vitr+ Cl− cotransporter (NCC), alternative to ENaC, in the distal nephron of affected subjects [The progressive clinical improvement of our first patient, associated to gradual normalization of hormonal profile, depended on sodium chloride supplementation, steroid therapy, which gradually inhibits cortisol levels by stimulating the residual activity of mineralocorticoid receptors , and ionsubjects . In PHA1subjects . Our infSCNN1A gene [Few known mutations are associated with systemic PHA1. Most of them, including the one here described, involve the N1A gene . These aN1A gene , 19, 20.N1A gene . HoweverN1A gene , 21. IndN1A gene –24.In newborns with hyponatremia, hyperkalaemia and metabolic acidosis, a PHA1 diagnosis should be considered, after the exclusion of a salting-loss form of adrenogenital syndrome. In addition, if normal kidney function tests and anion gap, urinary pH 5 and lack of response to furosemide are present, a type IV renal tubular acidosis is outlined, and the diagnostic suspicion may be further strengthened. In affected subjects increased plasmatic levels of aldosterone and aldosterone/renin ratio, associated with poor response to steroid administration are specific markers of disease. An accurate family history is often decisive for the diagnosis. The treatment relies on high dose sodium chloride, fludrocortisone and glucocorticoids, insulin, and ion exchange resins. It may be useful to reduce potassium intake with the use of special formulas. Clinicians must gradually balance the doses of drugs, based on electrolyte and hormone levels. Respiratory infections must be prevented by close follow-up evaluations, as well as careful and complete vaccination schedule, including anti-pneumococcal and anti-hemophilus b doses. Thus, a multidisciplinary team is needed for optimal management and adequate growth and development of these patients .SCNN1A gene mutations in present patients (homozygous), as well as in their parents and in the aunt of one of them (heterozygous). Indeed, detection of genetic variants in affected subjects and healthy carriers may be relevant for suggesting new elements of the genotype-phenotype correlations, and for a precise reproductive counselling, also in view of primary and/or secondary prevention of disease [NGS techniques allowed the identification of  disease , 26, 27.


Pneumocystis pneumonia (PCP). Immunocompetent hosts can eliminate the fungus without symptoms, while immunodeficient hosts develop PCP with an unsuitable excessive inflammatory response leading to lung damage. From studies based on rodent models or clinical studies, this review aimed to better understand the pathophysiology of Pneumocystis infection by analysing the role of immune cells, mostly lymphocytes, according to the immune status of the infected host. Hence, this review first describes the immune physiological response in infected immunocompetent hosts that are able to eliminate the fungus. The objective of the second part is to identify the immune elements required for the control of the fungus, focusing on specific immune deficiencies. Finally, the third part concentrates on the effect of the different immune elements in immunocompromised subjects during PCP, to better understand which cells are detrimental, and which, on the contrary, are beneficial once the disease has started. This work highlights that the immune response associated with a favourable outcome of the infection may differ according to the immune status of the host. In the case of immunocompetency, a close communication between B cells and TCD4 within tertiary lymphocyte structures appears critical to activate M2 macrophages without much inflammation. Conversely, in the case of immunodeficiency, a pro-inflammatory response including Th1 CD4, cytotoxic CD8, NK cells, and IFNγ release seems beneficial for M1 macrophage activation, despite the impact of inflammation on lung tissue.The host immune response is critical in  Pneumocystis jirovecii pneumonia (PCP), otherwise known as pneumocystosis, remains nowadays one of the most frequent fungal infections in immunocompromised subjects with a worldwide prevalence of over 500,000 cases per year [per year . The numper year .Pneumocystis, knowledge about the fungus has progressed considerably. This micro-organism, initially identified as a protozoan parasite and designated as Pneumocystis carinii, has been classified as a fungus over the years and been renamed Pneumocystis jirovecii for the species specifically infecting humans [Pneumocystis infection pathophysiology must rely either on clinical studies or experiments on animal models.Over a hundred years after the first description of g humans . NeverthPneumocystis infection with the specific species P. murina for mice and P. carinii for rats. They have proved to be good models for Pneumocystis infection for their numerous similarities with the human infections. Humans and rodents notably share the dichotomous asymptomatic infection or interstitial pneumonia in immunocompetent or immunosuppressed hosts, respectively, and the type of immunosuppression at risk of PCP [Rodents are the main experimental models for k of PCP .Pneumocystis without clinical signs or the need for antimicrobial treatment. However, in the case of immunosuppression, the infected host is no longer able to eliminate the fungus, which then multiplies in the alveoli. In this case, an excessive and ineffective inflammatory reaction develops in the lungs, which leads to lung damage and a progressive dyspnoea, characteristic of PCP. Hence, the immune system can be either protective, if fully functional, or can be detrimental and greatly responsible for pulmonary degradation, in the case of immunosuppression [Both clinical studies and rodent models underline a critical role of the host immune response in the disease. Indeed, a host with a functional immune system is able to control and eliminate pression ,6.Pneumocystis infection remains unclear and controversial [Pneumocystis and on the lung damage classically observed in PCP.Numerous studies have evaluated the impact of host immune response during PCP, with a frequent focus on lymphocytes, which are the determinant immune cells in this fungal infection. Lymphocytes encompass a variety of cells with different functions, among which the main ones are CD4 T cells (TCD4), CD8 T cells (TCD8), and B lymphocytes (BL). Currently, the role and importance of the different lymphocyte populations during oversial ,7,8,9. CPneumocystis infections in physiological conditions with studies on infected immunocompetent rodents. Then, the objective of the second part will be to identify the immune elements required for controlling fungus growth and avoiding PCP. It focuses on specific immune deficiencies in human diseases or in rodent experiments and their consequences on Pneumocystis infection. Finally, we will study the effect of different immune elements in immunocompromised subjects during PCP, to better understand which cells are detrimental, and which, on the contrary, are beneficial in the context of PCP.To that end, we will first describe the immune response in the case of P. jirovecii. The first contact with the fungus usually occurs during childhood as evidenced by the high seroprevalence in infants [Pneumocystis infection is mainly asymptomatic in immunocompetent subjects, it is rather difficult to study the immune physiological response in humans.In the course of their lives, humans will frequently and repeatedly be in contact with  infants ,11. PCR  infants . Infecte infants ,13. SincPneumocystis-specific immunity, unlike most of the Pneumocystis infections in humans.Our knowledge is therefore largely based on murine models. An infected immunocompetent mouse can also eliminate the fungus without noticeable symptoms, within an average of five weeks  ,15,16. NPneumocystis sp through the respiratory tract, the Antigen Presenting Cells (APC) available in the lungs can recognise several wall or membrane compounds of the fungus, such as Major Surface Glycoprotein (MSG) or beta-D glucan (BDG). MSG and BDG act as Pathogen-Associated Molecular Patterns (PAMPs) that are detected by Pattern Recognition Receptors (PRR). PRR, such as the Toll-Like Receptors (including TLR2 and TLR4) and C-type lectin receptors (particularly Dectin-1 and Mincle) are expressed on the surface of APC, myeloid cells, and alveolar epithelial cells [Pneumocystis and induce an increase of macrophage inflammatory responses [Pneumocystis, Damage-Associated Molecular Patterns (DAMPs) produced by the host cells in reaction to a stress or injury also participate to the development of the inflammation by interacting, like PAMPs, with PRR [When rodents are infected with al cells ,19,20,21al cells ,23,24. Sesponses . Stimulaesponses . Besideswith PRR . Indeed,with PRR ,28,29. Twith PRR .Under the action of the APC and chemokines, leukocytes infiltrate the lung tissue. There is a significant increase in the cellularity of the lung tissue and the BAL, with a peak one to two weeks after infection, mainly due to an increase of lymphocytes but also to an influx of macrophages and PMN at the site of infection. Homeostasis returns progressively with the decrease of the fungal load .T lymphocyte characterisation in the lungs of infected immunocompetent mice showed a rapid increase of both TCD4 and TCD8 from the first week of infection, and a decrease around the third week post-infection ,15. In tPneumocystis sp. infection [Concerning the T helper cells, both Th1 and Th2 responses are stimulated with an increase in the cytokines IFNγ, TNFα and IL-4 ,16,30. Infection . The antnfection .Pneumocystis sp. predominantly present in the lungs (ascus or trophic forms) could influence the type of response, probably due to a difference in APC activation. The cystic stage would induce a predominant pro-inflammatory Th17 response with an influx of TCD8, while trophic forms would conversely cause a decrease in the interaction between dendritic cells and TCD4 [Pneumocystis murina infection in immunocompetent mice [Influenza, or any other context identified as a “danger” by the organism. As a germinal centre, they mimic the structure observed in lymph nodes i.e. an important conglomerate of BL surrounded by T lymphocytes and macrophages. It appears transiently two weeks after the infection and disappears with the elimination of Pneumocystis from the lungs [Some studies have observed that the stage of and TCD4 .Recentlyent mice . iBALT ahe lungs .P. jirovecii infections are usually asymptomatic. A study performed on non-immuno-depressed patients with chronic bronchial disease colonized by P. jirovecii, showed an increase in the blood number of total lymphocytes and TCD4, confirming the results observed in the lungs and lymph nodes of infected immunocompetent mice [Studies in immunocompetent humans are limited because ent mice .Pneumocystis [Pneumocystis in mice [Pneumocystis. Nevertheless, macrophages that harbour a predominant M2 profile of alternative activation, i.e. through cytokine activation of the Th2 response (IL-4 and IL-13 among others), are preferentially involved in immunocompetent mice. This M2 profile is consistent with the predominant Th2 profile reported in infected immunocompetent mice [Macrophages have a major role in the elimination of mocystis ,38. Theymocystis ,37. An i in mice ,40 but n in mice . Both M1ent mice ,43.Pneumocystis [P. murina. They can, however, potentialize macrophage clearing capacity when co-cultured with non-polarised macrophages [Immune cell-mediated cytotoxicity may also play a role in Pneumocystis elimination. NK cells and eosinophils have demonstrated a fungicidal capacity in vitro against mocystis ,45. In crophages ,47 and erophages .Pneumocystis sp. infection induces an influx and stimulation of various types of immune cells in the lungs, each with their own functions. To help decipher the cells that are truly essential for host defence against Pneumocystis, we reviewed the different immune deficiencies, particularly those associated with lymphocyte lineage, and their impact on the risk of PCP development.A deficiency in the cellular immune response is the main factor of susceptibility to the disease ,49. PneuPneumocystis; all the more so if they are targeted deficiencies. Indeed, whether it is an induced depletion in mice or an inherited or acquired (iatrogenic or infectious) immune deficiency in humans, the development (or not) of PCP under various immunodeficiency conditions are important clues to establish the physiological mechanisms indispensable for the defence against P. jirovecii infection, as demonstrated by the high risk of developing PCP in HIV-infected patients with a TCD4 blood count lower than 200 cells/mm3 [Pneumocystis for 2–6 months or until TCD4 recovers to >200 cells/mL [TCD4 lymphocytes are essential cells for clearance of ells/mm3 ,51. Mousells/mm3 ,52. Receells/mm3 ,54. Thisells/mm3 . TCD4 < cells/mL .Pneumocystis sp. infection is well established, the actual subpopulations of TCD4 that are required to clear the fungus are not identified. Indeed, TCD4 population contains various heterogeneous subgroups with specific functions that can even be antagonistic. The different T helper cells  and regulatory T cells (Treg) are the real conductors of the adaptive immune response through their production of cytokines, enhancing a pro-inflammatory cellular response, a humoral response, or an anti-inflammatory response.Although the major role of TCD4 during Pneumocystis infection, classical T helper cells  appear to have little involvement in the control of the infection in physiological conditions. Indeed, in immunocompetent mice, the inhibition of Th1, Th2, or Th17 cytokines  does not induce a PCP [Pneumocystis control by blocking the transducer and activator of transcription proteins of Th1 (STAT4-/- mice), Th2 (STAT6-/- mice), and both Th1 and Th2 (double KO) and confirmed that they have little impact [Pneumocystis burden with an increased Th17 response [Pneumocystis elimination.In ce a PCP ,42,57,58response , suggestPneumocystis infection. Yet, they might have an important role to play as mice deficient in IL-21 (which is highly secreted by Tfh) and patients with a mutation of IL-21 receptor are susceptible to developing PCP [Follicular helper TCD4 (Tfh), which are mainly involved in B cell selection and survival , have alping PCP .Pneumocystis infection is not clearer. In humans, subjects with IPEX syndrome  present a deficiency in the Treg-specific transcription factor FoxP3 and sometimes develop PCP [The contribution of Tregs to elop PCP . Howeverelop PCP ,63,64. Oelop PCP .PD-1 and CTLA4 are immune checkpoints that provide inhibitory signal to immune cells, among which activated lymphocytes . Checkpo3 does not appear to be sufficient to predict the risk of PCP in HIV-negative patients [Pneumocystis. Thus, the critical role of TCD4 could be linked to the communication with other cells, notably BL, as suggested by the observations of iBALT in immunocompetent host infected with Pneumocystis, containing closely intertwined B and T cells [In the context of PCP, the focus has long been on TCD4 depletion, mostly because of their remarkable correlation with PCP risk in HIV-infected patients. Nevertheless, the TCD4 threshold of 200 cells/mmpatients . No effe T cells .Pneumocystis.The role of BL in PCP has little been studied compared to TCD4 lymphocytes. However, there are many arguments in favour of an essential role of these cells in the physiological response against P. murina and develop PCP [Indeed, µMT-/- or Jh-/- mice deficient in mature BL are unable to eliminate elop PCP ,69,70. Ielop PCP ,72,73. Ielop PCP ,75. Howeelop PCP .BL deficiency implies a dysregulation of several immune mechanisms, including antibody production, but also impaired co-activation of TCD4 by BL. Both for the isotypic switch of antibodies  or for the activation of TCD4 cells, contact between BL and TCD4 cells is necessary, via the major histocompatibility complex (MHC) class II and the CD40L/CD40 system ,78.Pneumocystis-specific antibodies seem to play a partial and dispensable role in the defence against the fungus. Depletion of FcyR (Receptor for the Fc region of IgG) in the mouse model increases the fungal load but Pneumocystis is ultimately eliminated [iminated . Depletiiminated . In addiiminated ,81,82,83Pneumocystis sp. infection. In humans, the hereditary CD40L immune deficiency (present on TCD4) belonging to “X-linked hyper IgM syndromes” is at high risk of PCP [Pneumocystis infection.However, the interactions between BL and TCD4 appear to be critical for the control of k of PCP ,85. Simik of PCP ,86. Morek of PCP . This coPneumocystis infection, Th1 and Th17 responses increase significantly in parallel with the development of PCP in mice. The transfer of healthy BL in these mice reduces the overexpression of Th1 cytokines while decreasing the fungal load. This would be linked to the production of anti-inflammatory IL-10 by the BL, which inhibits Th1 and Th17 responses [In mice deficient for B cells functions (depleted of B-cell Activating Factor Receptor—BAFF-R-/-), Rong et al. showed that during esponses .To summarise, B cells have a critical role during PCP infection and their main implication may more concern the antigen presentation and TCD4 activation, rather than antibody production.Unlike TCD4 and BL, isolated TCD8 deficiency is not a risk factor for PCP. Infected mice depleted of their TCD8 by injection of specific anti-CD8 antibodies can eliminate the fungus with no delay in clearance and no observable lung histological lesions [NK lymphocytes have been little considered in the context of PCP until now. However, Kelly et al. observed that mice depleted only in NK lymphocytes (yc-/-) showed a high fungal load at 4 weeks post-infection comparable to that observed in the case of TCD4 depletion .P. murina in the absence of NK lymphocytes, which reveals a role of these cells in the activation of the adaptive immune reaction against the fungus [In addition, Warschkau et al. found an in vitro decrease in the production of IFNγ by splenic cells in contact with e fungus .In contrast to combined immune deficiencies (CID) or severe combined immune deficiencies (SCID) affecting more or less the entire lymphocyte lineage, subjects with neutrophilic immunodeficiency syndromes (NIDs), such as chronic septic granulomatosis, are not (or only slightly) at risk of developing a PCP . Using aPneumocystis, current data does not show whether these cells are simply able to accelerate clearance of the fungus or whether their specific depletion may cause PCP [An eosinophilia in blood or BAL is sometimes observed in PCP among HIV-infected patients or transplanted patients ,93,94. Sause PCP .P. carinii infected rats is associated with a marked increase in the fungal load, demonstrating their importance in the clearance of the fungus [The reduction of macrophages in e fungus . M1 polae fungus ,96. Simie fungus . Moreovee fungus . Anti-TNe fungus ,99,100, e fungus .Pneumocystis infection. Moreover, the communication between these two cells seems to be a critical step for an efficient adaptive immune response. The exchanges between these two types of lymphocytes can occur among the iBALT structure, as reported by Eddens et al. [To conclude, TCD4 lymphocytes (without known specific polarisation) and BL are essential cells to control s et al. . TCD8 cePCP occurs in a context of immunosuppression, i.e. in conditions where at least one of the elements essential to the control of the fungus is impaired qualitatively or quantitatively. An important challenge now would be to identify in humans or in animal models, the secondary immune responses that take place in this context of altered immunity and their beneficial effect on the clearance of the infection and/or their toxicity to lung tissue.Pneumocystis infection, this third part focuses on immune cells roles in condition of PCP. Thus, these mice are immunosuppressed by lymphocytes depletion, anti-CD4 antibodies, or corticosteroids. Then, the evaluation of the outcome of infected mice after the modification of one or more immune elements helps to better understand the secondary immune mechanisms that take place during PCP.In contrast to the second part of this review, where specific targeted depletions were evaluated during This third part of the review also includes clinical studies focusing on good or bad prognosis factors associated with this disease.Pneumocystis is not cleared and an impairment of lung function and lung volume capacity occurs gradually [P. murina infection in TCD4-depleted mice results in a much greater leukocyte infiltration than in immunocompetent mice, with a predominant influx of TCD8 and PMN [During PCP in an immunocompromised patient, radually ,106. P.  and PMN .Pneumocystis infection; however, they also are responsible for lung damage during PCP. Indeed, in SCID mice , the lung damage is lower than in mice depleted only in TCD4 [Lymphocytes are essential for controlling a  in TCD4 ,108. Mor in TCD4 ,108.A reconstitution without T-lymphocytes (anti-CD3 antibody) of infected SCID mice shows a marked improvement in lung inflammation with a decrease in mortality .Pneumocystis while at the same time being responsible for lung tissue damage during PCP.However, fully reconstituted infected SCID mice have a significant decrease of fungal load in comparison to T cell-depleted mice . TherefoPneumocystis elimination when others are toxic, indicating the need for further studies on TCD4 and TCD8 subpopulations.One can postulate that some T lymphocyte populations are actually beneficial for Pneumocystis sp: isolated reconstitution of activated TCD4 (from Pneumocystis-infected mice) in SCID mice is sufficient to eliminate the fungus [TCD4 lymphocytes have a decisive role in the control of e fungus ,110. Thee fungus . Howevere fungus ,110. In e fungus .Pneumocystis but are associated with lung inflammation.Moreover, the isolated presence of inactivated TCD4 without BL and TCD8 was also shown to be detrimental in a mouse model without clearance of the fungus . If prevSeveral studies focused on the TCD4 subpopulations, and particularly the Th1/Th2 balance, in order to understand which profile is associated with a better outcome of PCP.Pneumocystis from the infected lungs during PCP. Indeed, the addition of IL-12 (which stimulates Th1 differentiation) in infected mice depleted in TCD4 accelerated the fungus clearance and decreased mouse mortality. IL-12 addition was associated with an increased number of macrophages, TCD8 and PMN in the lungs, and higher IFNγ production [Pneumocystis elimination in TCD4-depleted mice [First, several studies revealed the value of a Th1 profile subpopulation in clearing oduction . Similarted mice ,114. Theted mice . In humated mice .On the other hand, the pro-inflammatory Th1 response is also pointed out as a response prolonging an inadequate inflammatory state during PCP that is toxic to the lungs . It is aPneumocystis, it can also be associated with damage to lung tissue and function. The deleterious aspect of this response can potentially be explained by an overproduction of mucus that can alter alveolar epithelial cells and may reduce gas exchange [For the Th2 response, although it is the predominant TCD4 profile in immunocompetent individuals infected with exchange ,117,118.exchange ,117.Thus, both unbalanced Th1 and Th2 responses could be responsible for pathological disorders. As shown by Swain et al., depletion of BL and TCD4 in infected mice results in a strong inflammatory Th1 response linked to oedema formation and albumin release in the alveoli. In contrast, BL and TCD8 depletion induces a major Th2 response which causes a significant increase in lactic dehydrogenase (LDH), which reflects an alteration of the pulmonary tissue .Pneumocystis clearance, when the anti-inflammatory IL-10 was administered intratracheally to TCD4-depleted mice [Regarding the Treg profile, the addition of CD25+ CD4+ T cells in infected SCID mice is beneficial on inflammation but not on the fungal load, which increases with this treatment ,119. Theted mice . Howeverted mice .Pneumocystis clearance during PCP but they must be activated and preferentially in the Th1 profile. This pro-inflammatory response is associated with the production of IFNγ, the influx of cytotoxic TCD8, macrophages and PMN in the lungs. The benefits of Th1 profile appear to be independent of the actual presence of TCD4 because the addition of IL-12 or IFNy in the absence of TCD4 is sufficient to improve PCP outcome [Pneumocystis itself [Pneumocystis could rely on the production of cytokines, such as IFNγ, which would in turn stimulate other immune elements.In conclusion, TCD4 have a major role in  outcome ,113,114.s itself . Thus, TPneumocystis load [In PCP, there is a great increase in the number of TCD8 in the lungs, especially if TCD4 depletion is significant . Their etis load .Pneumocystis in the absence of TCD4. They would constitute a secondary route of defence against the fungus. In two clinical studies on HIV-negative patients infected with P. jirovecii (mostly with auto-immune diseases), an overall TCD8 decrease in peripheral blood has also been reported as a poor PCP prognostic factor [Conversely, increasing the number of TCD8 by administration of IL-7 in TCD4-depleted mice further reduces the fungal load . TCD8 lyc factor ,124.However, TCD8 have been shown to have a deleterious activity in the lungs of infected immunocompromised mice depleted in TCD4. Indeed, in the case of double depletion of TCD4 and TCD8, the inflammation and clinical condition of the mice were less severe than in the case of isolated TCD4 depletion . FurtherPulmonary infiltration of TCD8 in infected mice depleted in TCD4 is, at least in part, linked to the production of type I IFN and TNFα. Depletion of receptors for these cytokines, decreases the number of TCD8 in the lungs and improves pulmonary lesions, without effect on the fungal load .From these different results, TCD8 seem to sometimes have a discordant effect on the clearance of the fungus. This might be explained by a heterogeneity of TCD8 cells population. Indeed, Mc Allister et al. showed an impact of TCD8 polarisation on PCP outcome in mice. Even if they observed a deleterious effect after total TCD8 transfer in infected SCID mice, these mice were protected from infection when they transferred Tc1-polarised (cytotoxic) TCD8 . Tc1 CD8Pneumocystis. Yet cytotoxic TCD8 showed no fungicidal activity against Pneumocystis in vitro. Nevertheless, they increased the activity of macrophages, probably with the production of IFNγ [Therefore, TCD8 seem to constitute a secondary immune response during PCP, in the case of TCD4 absence. Like TCD4 Th1 profile, Tc1 cytotoxic would be protective in the case of PCP, in relation to an increased clearance of  of IFNγ ,47.P. jirovecii infection also induces the production of specific antibodies (anti-MSG or anti-KEX1 for example) that are measurable over time [Next to cytotoxic TCD8 response, ver time ,128,129.P. murina-specific antibodies have been carried out on immunocompromised mice with PCP. They showed a significant reduction in fungal load and a reduction in lung inflammation in treated mice [Contrary to studies in immunocompetent rodents, little is known about the role of BL in the case of PCP (in immunosuppression condition). However, therapy trials based on the administration of ted mice ,130. HowP. murina despite the absence of TCD4 and BL, related to an increased phagocytic capacity of alveolar macrophages [Rapaka et al. reported the value of antibodies in eliminating the fungus by opsonisation in immunocompromised mice. Indeed, the introduction of a fusion protein Dectin-1/IgG1 Fc fragment (antibody that binds to BDG) led to an improvement in the elimination of rophages .Pneumocystis recombinant protein KEX1 on immunocompromised non-human primates induces robust and durable humoral responses [Pneumocystis infection in the primates [Assays of immunization with esponses ,133. In primates .Thus, antibodies produced by BL have shown interesting effects in PCP, both on fungal load and inflammation. However, this effect was observed transiently and was not sufficient to heal the mice from PCP. The humoral response seems to have at least an interest by increasing fungicidal phagocytic capacities of macrophages.Concerning macrophages, the increase in the number of lung macrophages and their fungicidal activity by addition of GM-CSF in the lungs of infected mice depleted in TCD4 induced a marked improvement in fungal clearance and decreased inflammation. Conversely, GM-CSF depletion in infected immunocompromised mice increased the fungal load and lung inflammation .Pneumocystis. The macrophage orientation towards M2 response has been shown to be beneficial in infected immunocompromised mice [In contrast to infected immunocompetent rats harbouring predominantly M2-polarised macrophages, rats immunocompromised by corticosteroid therapy have predominantly M1-polarised macrophages . Both M1sed mice ,136, evesed mice ,43.Apart from their phagocytic activity, macrophages also produce chemoattracting cytokines. In particular, they are the main producers of TNFα. When TNFα receptors (TNFR I and II) are depleted in infected TCD4-depleted mice, the production of chemokines such as MCP-1 or Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) is decreased as well as the lung infiltration of TCD8 and PMN. This reduces inflammation such as lung lesions and clinical impairment associated with the disease . Hence, Pneumocystis sp. [As for PMN, a high infiltration into the lungs of PCP patients, whether HIV-infected or not, has been shown to be a poor prognostic factor for the disease ,139,140.stis sp. . Hence, Pneumocystis [Stimulation of eosinophils by addition of IL-5 before infection in mice lacking TCD4 or completely deficient in lymphocytes leads to a better clearance of the fungus at 14 days post-infection, without any increase in the Th2 response. In the case of eosinophils depletion, the addition of IL-5 shows no more beneficial effect, confirming the implication of eosinophils . Their emocystis .Pneumocystis in vitro [Finally, NK cells have been little studied in PCP context. Yet, they might be of interest in PCP. Their high number in the blood was reported as a good prognosis marker in HIV-negative patients . NK cellin vitro . NK cellin vitro , such asPneumocystis but are also associated with enhanced lung inflammation. Conversely, the addition of Treg reduces lung inflammation but does not improve the clearance of the fungus. Inflammation appears to be an important process to clear Pneumocystis but can lead to pulmonary tissue degradation.According to all these data  and formation of germinal centres in the presence of IL-6, TGFβ, and ICOS. TCD4 and BL would activate macrophages and direct them towards M2 polarisation, perhaps through IL-10 production by BL. Activated M2 macrophages would thus eliminate the fungus by phagocytosis, with or without opsonisation.One hypothesis could be that Pneumocystis sp. infection in both humans and mice [Pneumocystis could be limited, considering the low risk of PCP in patients with Bruton’s disease or hereditary Stat3 deficiency [Pneumocystis infection in immunocompetent hosts [This hypothetical model is consistent with existing literature on PCP risk factors associated with lymphocyte populations (in mice and humans) and described in this review. Indeed, the immune response based on iBALT formation requires the communication between BL and TCD4, which has been characterised as crucial for controlling and mice ,71,84,86and mice , consistand mice ,42,105. and mice ,31. Finaand mice . In contficiency ,83. In tPneumocystis. However, IFNγ can activate macrophages in the M1 polarisation, which are able to phagocyte and eliminate Pneumocystis. Th1, Tc1, and NK cells also mediate inflammation observed in the lungs, associated to a pulmonary influx of PMN. Thus, although lung lesions observed in PCP are related to inflammation, this process seems to be of great importance in fungus clearance. This model of immune response in the immunocompromised host is consistent with some human and mouse studies that also find a favourable Th1/Tc1 response [Pneumocystis has also been demonstrated in mice [In immunocompromised hosts, pro-inflammatory cells  are associated with a better fungal clearance. The role of these cells in PCP seems highly related to their cytokine production, notably IFNγ, as the addition of this cytokine in the absence of TCD4 and TCD8 is sufficient to trigger the decrease of fungus load. IFNγ, for its part, don’t seem to have any direct lethal effect on response ,113,116. in mice .Pneumocystis. Indeed, the role and impact of each immune cell on the outcome of Pneumocystis infection seem to depend on the immune status of the host: a given response may be favourable or unfavourable depending on the existence or not of an immunosuppression. It is also likely that the type of immunosuppression may induce different secondary immune responses. Understanding these different types of response will be necessary for a more individualised and optimal management of PCP patients according to the underlying immunosuppressive factors.In conclusion, understanding the pathophysiology and the immune mechanisms of PCP requires a clear differentiation of the role of each actor of this response depending on the immune status of the host infected by


Lymphangiomas are rare and correspond to 0.7% to 4.0% of mediastinal tumors, and isolated mediastinal location occurs in 1% of cases. They are benign tumors that originate from a congenital malformation of the lymphatic vessels and are diagnosed more frequently in children less than 2 years of age. Chylous ascites is a clinical manifestation of thoracic duct lymphangioma and is composed of lymph accumulation caused by dilation of this lymphatic channel. It appears milky in the peritoneal cavity, containing triglyceride levels higher than 200 mg/dl. We report the case of a young patient with chylous ascites and lymphangioma of the thoracic duct, who was conservatively treated with octreotide and a low-fat diet with medium-chain triglycerides. They are congenital malformations, and more than 80% of patients are below 5 years age.We report the case of a woman with thoracic duct lymphangioma, which was investigated through chylous ascites that the patient initially presented, with a brief review of the clinical aspects, diagnosis, and treatment of this pathology. The aim of the study is to report the case of a young patient with chylous ascites due to thoracic duct lymphangioma who was treated conservatively with octreotide and a low-fat diet with medium-chain triglycerides (TCM). In addition to increasing the index of suspicion of this pathology so that the patients can be treated as earliest as possible.oC) which started four days after admission. She denied drug allergies, smoking, alcoholism, abdominal trauma, and a family history of cancer. She had been using oral contraceptives for three years and had excised a benign nodule in the right breast. Physical examination revealed a diffuse pain on deep palpation and abdominal distension. Laboratory tests revealed normal hematocrit (14.6%); 7750 leukocytes with 5% band forms; C-reactive protein: 23.8 mg/dL; and beta HCG less than 0.1 mIU/ml. Serologies for HIV, HTLV, viral hepatitis, syphilis, Chlamydia trachomatis, Chagas disease, and toxoplasmosis were negative, and cytomegalovirus IgG positive and IgM negative. Urine and blood cultures were negative; negative tests for rheumatic activity; triglycerides (≤ 150): 186 mg/dL, HDL (> 60): 36 mg/dL, LDL: (≤ 129) 129.8 mg/dL, and total cholesterol: (≤ 200) 203 mg/dL. CA 15.3, CA 19.9, CA 72.4, and CEA were normal, but CA 125 (≥ 35): 330.6 U/mL. Contrast-enhanced abdominal computed tomography (CT) showed a well-delimited cystic image without enhancement, located in the topography of the thoracic duct in the thoracoabdominal transition and in contact with the aorta, associated with massive ascites and mild bilateral pleural effusion  and constant, worsening on inspiration and at dorsal decubitus, improving with analgesics and associated with a high fever (39 effusion .Magnetic resonance imaging (MRI) of the chest with contrast revealed a cystic image at the thoracoabdominal transition, in the topography of the thoracic duct on the right of the aorta, measuring 4.0 cm x 3.8 cm x 2.2 cm, with homogeneous content and lobulated contours in the upper portion . The invStaphylococcus lugdunensis; and the search for fungi, mycobacteria, and oncotic cytology were negative. With a diagnosis of thoracic lymphangioma, the patient had a zero diet for 48 hours, an oral diet with medium-chain triglycerides for seven days, and was medicated with octreotide. There was a decrease in daily ascitic output in abdominal and chest fluid by CT control images , polymorphonuclear (31%), and eosinophils (4%); total proteins: 1.46 g/dL; glucose: 116.8 mg/dL; DHL: 93.7 U/L; triglycerides: 511.5 mg/dL; the culture showed l images , and cliThere were no changes in the pulmonary parenchyma that had normal attenuation coefficients, and there was no lymph node enlargement in the mediastinum or in the lower cervical chain. With no flow, the abdominal cavity drain was removed, and the patient was discharged from the hospital in good general condition, with guidance for specialized monitoring in the outpatient service of Thoracic Surgery.-Lymphangiomas are benign tumors originating from congenital malformations of lymphatic vessels and presenting as dilatations of variable volumes forming cysts that are lined with vascular endothelium, which impair the normal flow of lymph.,-,,,,,,,,Chylous ascites is also rare, with the incidence of hospital admissions estimated at approximately 1: 20,000 up to 1: 180,000, according to international studies.S. lugdunensis infections are infrequent, and the knowledge about clinical characteristics is lacking. Research is necessary to better clear the diversity and virulence of this species and its role in urinary and respiratory tract infections, peritonitis, and bacteremia.The peritoneal fluid analysis showed classical chylous ascites without infective features; nevertheless, some bacterial role in the thoracic duct change was not discarded. The present report is in agreement with the literature because it is a young patient without suspicion of a congenital entity, and asymptomatic before the current hospitalization. The initial manifestation that led to the consultation was a consequence of chylous ascites, and resulted in the diagnosis of thoracic duct lymphangioma as the etiology of chyloperitoneum. The characteristics of this lymphangioma as shown in imaging exams, are of a single mediastinal cyst, a rare variety among mediastinal tumors. As the treatment instituted was conservative, there was no anatomopathological study. With the possibility of the progressive growth of lymphangiomas over the years, the patient will remain under regular outpatient monitoring for periodic imaging control.Chylous ascites associated with thoracic duct lymphangioma is a therapeutic challenge even currently, as this is a very rare condition that is related to elevated morbidity. The authors believe that case studies can increase the index of suspicion in the first manifestation of ascites allowing that diagnosis and control be established early.


Cladosporium, Penicillium, Aspergillus, and yeast often appeared in the air or on wall surfaces during sampling. The indoor/outdoor concentration ratios (I/O ratios) demonstrated that the airborne concentrations of commonly found fungal genera outdoors were higher than those indoors (median I/O ratio = 0.65–0.91), excluding those of Penicillium and yeast. Additionally, the surface density  of individual fungi showed no significant correlation with the airborne concentration, excluding that of Geotrichum. However, if a higher surface ratio (>0.00031) of mold spots appeared in the total area of an indoor environment, then the concentrations of Aspergillus and Geotrichum in the air increased significantly. Our results demonstrated that the airborne concentration of indoor fungi is significantly correlated with the outdoor concentration. A higher density of surface fungi does not necessarily contribute to a high fungal concentration in the air. In contrast to fungal density, quantification of the surface fungal area is recommended to assess the risk of surface fungi propelling into the air.The rapid monitoring of total fungi, including air and surface fungal profiling, is an important issue. Here, we applied air and surface sampling, combined with digital image quantification of surface mold spots, to evaluate the contribution of surface fungi to airborne fungal concentrations.  Fungal spores are ubiquitous in indoor environments and often present as mixtures of fungal genera. Although fungal types and concentrations in nonproblematic indoor environments are considered to originate primarily from outdoor air ,2,3, molAspergillus spp., Penicillium spp., Cladosporium spp., Alternaria spp., and yeast [To identify the potential fungal reservoir, comparisons between indoor/outdoor fungal concentrations in the air have been used to document the presence of fungal contamination. In Taiwan, 2.9 to 31.4% of residential environments have indoor/outdoor concentration ratios (I/O ratios) higher than one for specific fungal species, including nd yeast . Thus, tnd yeast ,9.At present, although different sampling and analysis methods are available, there are still no widely acceptable standard protocols for measuring fungi in indoor environments. Sampling methods can be divided into air and surface sampling. Air sampling is commonly used to determine the fungal concentration and composition in the air by an active/passive sampling apparatus . Surface2) [Penicillium, Aspergillus, and Cladosporium, can be easily propelled into the air, whereas wet spores, such as those of Stachybotrys, Acremonium, and Trichoderma, are considered to produce mucilaginous masses that stick firmly to surfaces [In practice, almost all surface sampling addresses only the mold density on the surface per area  , and ver2) . Howeversurfaces . ConsequTo understand the contribution of wall fungi to the concentration of airborne fungi in residential environments, the aims of this investigation were (1) to evaluate the correlation between the environmental factors and airborne/surface fungi in the residential environment; (2) to determine the correlation among the fungal concentrations recovered from outdoor, indoor, and surface sampling; (3) to apply a simple digital image quantification method for calculating the total mold surface area and then use this indicator to evaluate the surface mold ratio, with a greater emphasis on airborne fungal concentrations.2 concentrations  and wind speed , were also measured.The average atmospheric temperature and relative humidity (RH) were 30.0 °C (from 25.1 to 36.2 °C) and 67.6% (from 45.3 to 88.8%) throughout the sampling periods, respectively. In addition, the average surface temperature was 31.4 °C (from 27.82 to 36.8 °C), and the mean surface RH was 68.2% (from 37.9% to 89.93%). Two other environmental parameters, the CO3) and outdoor air . However, the fungal concentrations in the indoor air were significantly lower than those in the outdoor air for Cladosporium (p = 0.038), Aspergillus (p = 0.003), and Fusarium (p = 0.024). Only the concentration of yeast was found to be significantly higher in indoor air than in outdoor air (p = 0.012). Due to the low detection rates of Geotrichum (12–15%) and Verticillium (3.88%), the concentrations of these two fungi are not presented in Cladosporium, yeast, Penicillium, Aspergillus, and Fusarium, regardless of indoor or outdoor air. Cladosporium ranked first, with more than 98% of house samples containing Cladosporium  , yeast (I/O = 1.56), and total fungi (I/O = 1.01), the I/O ratios of the fungal genera recovered from the air were less than 1  . In addis than 1 .n = 56). n = 47). 2. Except for Fusarium, fungal genera that appeared on wall surfaces also appeared in the air. Because the detection rate of certain fungi was low  to 10,253.3 CFU/cm2. In addition, approximately 49% of the household samples contained Cladosporium and yeast  and airborne fungal concentrations in the indoor environment was determined by Spearman’s rank correlation. Cladosporium, Fusarium, and Verticillium in indoor air were significantly correlated with temperature. In addition, the airborne concentrations of Penicillium were significantly related to high RH. However, Fusarium and Verticillium were negatively correlated with RH. Only Verticillium was correlated with wind speed. For CO2, all statistically significant correlations were negative correlations for the concentrations of airborne Cladosporium, Aspergillus, Fusarium, Verticillium, and total fungi.As shown in 2) and surface fungal concentrations in the indoor environment are shown in Fusarium was not observed on the surfaces of indoor environments, no correlation result is shown for Fusarium in Geotrichum and Verticillium were correlated with surface RH, and the total fungi were negatively correlated with wind speed. In addition, the surface densities of yeast and total fungi were significantly correlated with CO2 concentrations.The correlations among the environmental parameters  with their outdoor concentrations. In addition, the indoor concentrations of Cladosporium were correlated with almost all the fungi (except for yeast) detected in indoor air . Furthermore, the indoor yeast concentration was negatively correlated with almost all other indoor fungal concentrations (except for Cladosporium and Fusarium), although some correlations were not significant .The correlation between the concentration of indoor and outdoor fungal aerosols is shown in Geotrichum exhibited airborne concentrations that were significantly correlated with its surface density . However, when only surface fungi were compared, the densities of almost all fungal genera were significantly correlated with each other (except for yeast), with Spearman’s rank correlation coefficients ranging from 0.30 to 0.70 . For the surface yeast, the concentration was consistently non-significantly correlated with that of other fungal strains on the surface .Aspergillus and Geotrichum aerosols than the reference group (surface ratio = 0). In addition, a dose-dependent response was also observed for Aspergillus and Geotrichum, and only the highest level 3 surface area ratio contributed to a significantly higher indoor air concentration  than the reference group. The level 2 surface ratio increased airborne Verticillium concentrations by 39.8% compared to the reference group. However, this correlation was not significant for Verticillium (p = 0.108).In 2 concentration in the indoor air, which is the stipulated standard of Taiwan’s indoor air quality management act.Fungal sampling in this study was conducted in summer. A number of studies have shown that the concentration of airborne fungi in summer is higher than that in winter ,14,15. ACladosporium, Penicillium, and Aspergillus always appeared to be predominant in air sampling [Cladosporium, yeast, Penicillium, Fusarium, and Aspergillus, were also similar to a previous study’s findings of 100 to 102 CFU/m3 [Cladosporium usually dominated among all airborne fungi in indoor and outdoor air. Since Cladosporium is a well-known genus from the outdoor environment, a previous study demonstrated that the I/O ratio of Cladosporium might be less than 1 [Penicillium, which is associated with soil and plants. However, the I/O ratio of Penicillium was often greater than 1 [Penicillium contamination in an indoor environment when the I/O ratio is greater than 1, but contamination sources are rarely found.The most predominant airborne fungal genera in our study agreed with those in previous studies conducted in Taiwan ,13, excesampling . In addi2 CFU/m3 . Cladosps than 1   and Verticillium (median I/O = 0.78) not only exist in outdoor soil and plants but are also common plant pathogens [Penicillium is also associated with soil and plants [Cladosporium and Fusarium. The correlation between yeast and Fusarium may be related to the water activity (Aw). Other molds, such as Penicillium and Aspergillus, are considered to have lower Aw values (0.85 or less) and therefore should be drought tolerant [Fusarium and yeast grown at an Aw above 0.90 require higher amounts of available water [Taking into account the I/O ratios, higher fungal concentrations in outdoor air than in indoor air may reveal an exogenous source of indoor contamination. ar-round ,8,16,19. the air . Geotricathogens ,32. Althd plants , it can tolerant . In contle water ,34.Geotrichum. A previous study indicated that spores produced by specific molds cannot be easily aerosolized because of their sticky nature [Cladosporium, Penicillium, and Aspergillus may have more chances to be propelled into the air. However, the recommendation by the previous study was based on the number of visible mold spots to predict air concentrations, not the mold density on the surface per area. Theoretically, the higher the concentration of surface fungi that can produce dry spores, the higher the air fungal concentration. A significant correlation was only observed for Geotrichum, which is a fast growing fungus with a powdery texture, whose dry spores can easily disperse through the air [Geotrichum can limit Fusarium growth because of available resource competition [Fusarium on any surface in the current study. For other fungi, the low correlation between the surface and airborne fungal concentrations may be related to the fact that the naked eye can only observe mold spots of a certain size. In addition, some fungi growing at high Aw levels (such as yeast) tend to grow on relatively humid surfaces, and this kind of surface type and structure might have an impact on spore formation and therefore affect spore flux [In y nature . The dry the air ,36. Resepetition , and we ore flux ,37.Aspergillus or Geotrichum. Aspergillus and Geotrichum may generate dry spores from surface mold spots, and these spores tend to proliferate from air to other organic compounds, such as food, paper, and dust, among others. Compared to Geotrichum, Aspergillus has received more attention because of its serious health impact [Aspergillus species are commonly saprophytic fungi in nature, with small (2–3 μm) spores, and the concentration of Aspergillus spp. can significantly increase after a typhoon event [Cladosporium and Penicillium, which have dry spores, might also contribute to higher indoor concentrations. However, the result was the same as that shown in Cladosporium and Penicillium contributed by surface molds may be quite limited. This phenomenon may be related to the small sizes of some mold spots of Cladosporium or Penicillium, which are too small to observe. Moreover, all our sampling families had children, so parents may have frequently cleaned up obviously visible mold spots.As shown in h impact . Aspergion event . We origVerticillium species are often found in agricultural areas, indoor environments, waste composting facilities, and even during Asian dust periods in Taiwan [Verticillium, the surface area ratio at level 2 (0–0.00031) may be related to the airborne concentration. However, this correlation was insignificant. Although we mainly explored the correlation between surface mold area and the concentration of airborne fungi, mold-infested surfaces may release more than spores into the air; a previous study also found that fungi on the surface of kitchens and showers may release volatile organic compounds into the air [Alternaria spp. A previous study indicated that Alternaria is related to agricultural plants [Alternaria as our sampling sites were mainly in residential areas. Another possible explanation is that we applied the culture assay for fungal quantification. The disadvantage of the culture method is that it may underestimate the viable fungal concentration, so that samples of Alternaria may be unavailable. Currently, several DNA-based methods, such as polymerase chain reaction (PCR), are available, but these methods may not distinguish dead or viable fungal cells without support from other technologies. Consequently, the microscopic method applied in this study maybe a complementary assay to culture-based methods for further identification of fungi.Airborne n Taiwan ,40,41. F the air . Thus, tl plants ; thus, tAspergillus and Penicillium species have a white color; therefore, the surface areas determined by digital image quantification can be underestimated.Our study results recommend determining the surface area of visible mold spots, which may be important since a larger area of mold spot may be related to the airborne concentration of certain fungi. Quantifying areas by counting the number of pixels in a digital image has already been used in ecological and environmental studies ,45. CompThis study involved several major cities in northern, middle, southern, and eastern Taiwan, including Taipei, Hsinchu, Yunlin, Chiayi, Tainan, Kaohsiung, and Taitung, with a total of 52 living environments being studied from June to September. The participating households all responded by questionnaire that there were visible mold spots on the walls of their houses.3 was calculated based on the colony number, sampling time, and flow rate. For fungal species identification, all colony samples recovered from the MEA plates were subjected to lactophenol cotton blue staining and then identified under a microscope following the clinical laboratory handbook [Aspergillus, Penicillium, Cladosporium, Fusarium, Geotrichum, Verticillium, and yeast. To increase the chance of collecting a variety of molds in the air, we collected three samples at each location. After cultivation, we determined the total counts of individual mold colonies recovered from the three agar plates and calculated their airborne concentrations separately.At each sampling site, fungal aerosol samples were collected in triplicate using an Andersen 1-STG impactor . The pump and sampling apparatus were placed in the living room, bedroom, and outside entrance of each home. Among all the households included for indoor sampling, one had two master bedrooms and therefore had two bedroom samples. In another four households, because there was no obvious partition wall between the living room and the bedroom (studio apartment), only living room samples were collected. The sampling height was 1.2 m above the floor, within the breathing zone of individuals, at each location. The Andersen impactor was operated for 5 min with malt extract agar plates (MEAs) at a sampling flow rate of 28.3 L/min. All evaluated MEA plates collected from the field were placed in an incubator at 25 °C for 5 days. The fungal culture method recommended by the American Conference of Governmental Industrial Hygienists (ACGIH) was performed at room temperature (18 °C to 22 °C) ,9,16. Fihandbook . The rep2. To quantify the sampling area, we calculated the target surface pixels in digital images, a method that has been applied to measure the area of a single leaf and hospital surfaces in ecological and environmental studies [During the sampling period, visible molds on the surfaces of living room or bedroom walls were selected for swab sampling. Surface sampling was performed using a sterile water-moistened swab, and the surface was rubbed for 5 s. Subsequently, swabs were placed in a 2 mL tube containing 0.1% Tween 80 buffer and immediately transferred for culture. Aliquots (0.1 mL) of the liquid samples from the tubes were diluted 10- to 100-fold, plated on MEA plates, and then incubated for 5 days at 25 °C. Finally, the total colony counts of the target surfaces were calculated based on the dilution ratio, and the colony counts were adjusted by the surface areas to present the results in CFU/cm studies ,47. Briesurface and Areference paper are the areas of the target surface and reference paper, respectively, and Nsurface and Nreference paper are the pixel numbers of the target surface and reference paper counted in Photoshop within the same picture, respectively  were recorded using a KD AirBoxx real-time instrument  in all sampling locations. In addition to atmospheric RH, we also measured the surface temperature and moisture content of the target surface where visible molds appeared using an HP23-AW handheld instrument . Conversely, the wind speed was measured using an air velocity meter The temperature, atmospheric relative humidity (RH), and concentration of carbon dioxide (COβ − 1)) to obtain the percent changes. The limit of detection (LOD) was 2.36 CFU/m3 for airborne fungal samples and 1.33 CFU/cm2 for surface samples. The airborne or surface samples with individual fungal concentrations below the LOD were assigned a value of one-third of the LOD for the statistical analyses.Differences in airborne fungal concentrations between different sampling locations were determined by the Wilcoxon signed rank test. For the individual pairwise correlations between environmental parameters and airborne/surface fungal concentrations, Spearman’s rank correlation model was constructed. Finally, to further explore the correlation between the mold surface areas and their concentrations in the air, a multiple linear regression model was constructed. A natural logarithm transformation (ln-transformation) of airborne fungal concentration was performed before conducting the analyses. The corresponding regression coefficients were back-transformed (100 × (eGeotrichum. However, for some specific genera, including Aspergillus and Geotrichum, the surface area of visible mold spots was significantly related to their airborne concentration indoors. These results indicate the need to focus attention on visible mold spots on surfaces, which may contain fungal spores of certain allergens, such as Aspergillus, with the ability to further disperse from the surface into the air through environmental forces. In addition, this study also provides a convenient and rapid method for quantifying the area of surface mold spots. This method may be applied in future environmental monitoring or epidemiological studies.Rapid monitoring of fungi is important for evaluating the related health risks and maintaining good indoor air quality. This study demonstrated that the indoor air concentration of individual fungi is significantly correlated with the outdoor air concentration. Regarding the contribution of surface fungi to indoor air concentrations, the density of individual fungi on the surface had no significant correlation with indoor air concentrations, except for


Quinoa is an important crop for food security and food sovereignty in Ecuador. In this study, we evaluated the nutritional value, bioactive compounds, and antinutrient compounds of leaves and grains of the Ecuadorian quinoa variety Tunkahuan, and we identified significant differences between the nutrient content in the leaves and grains. The quinoa leaves presented a higher protein content than the grains, as well as inorganic nutrients such as calcium, phosphorus, iron, and zinc. Both the grains and leaves had an appreciable phenolic content. In addition, the quinoa grains presented a higher content of the antinutrient saponin than the leaves, while the leaves contained more nitrates and oxalates than the grains. Thus, quinoa leaves and grains exhibit excellent potential for application in the food and pharmaceutical industries. Quinoa is an important and environmental stress-tolerant pseudocereal with high nutritional value and a globally promoted cultivar for human consumption and nutrition . Quinoa Quinoa is a complete food with high-nutritional value due mainly to its high content of good quality protein and are also called “the mother grain” . The EcuMoreover, the grain contains adequate levels of micronutrients, such as vitamins and minerals . Some factors such as the differences in drying and storage conditions, harvest time and fertilization, soil structure, climate, and irrigation affect the macro and micronutrient contents . AntinutResearch on quinoa has primarily focused on the nutrient composition of seeds, protein quality, starch functionality, and incorporation into food products made with cereal flours . There aChenopodium quinoa Willd.), produced in Ecuador. Moreover, bioactive compounds present a better nutritional profile than grains. Some studies have evaluated the nutraceutical potential of quinoa leaves in the context of bioaccessibility and bioavailability of their phenolic compounds, with in vitro studies suggesting that they can help prevent cancer and other diseases related to oxidative stress ,15. The t-test) confirmed significant differences between the leaves and grains. The ash and protein contents of the leaves were higher than those of the grains. In addition, the lipid content (ether extract) in grains was higher than in quinoa leaves, maize, and wheat. The crude protein content in leaves was higher than quinoa grains. The crude fiber content in leaves and grains was higher than that in maize, wheat, and spinach leaves. The quinoa leaves contained more dietary fiber (soluble and insoluble) than the grains. t-test) confirmed the significant differences between leaves and grains and other plants. Essential amino acids (EAAs) such as phenylalanine, lysine, isoleucine, and threonine were found in higher proportions in the quinoa leaves than in the grains. In addition, the leaves presented higher lysine and isoleucine levels than grains of maize and wheat [The amino acid profiles of quinoa grains and leaves are presented in nd wheat . Other nThe macro and microminerals analyzed are listed in t-test) confirmed significant differences between leaves and grains in the content of antioxidant compounds, although the estimated antioxidant capacity was not significantly different. A high content of ascorbic acid was observed in the quinoa leaves, whereas it was not detected in the grains. Carotenoids, total phenols, flavonoids, and anthocyanins were also present in higher amounts in the quinoa leaves than in the grains. Among the antinutritional factors, saponin substances were present in higher concentrations in the grains than in the leaves, whereas nitrates and oxalates were more predominant in the leaves. The compounds with antioxidant properties analyzed in this study are shown in The proximate composition of quinoa leaves and grains has been demonstrated to be richer than that of other cereals and leaves. In another study, the crude protein content of quinoa leaves was reported to be 14.77 g/100 g (dry weight) . Recent Grain oil is of high nutritional quality, containing several essential fatty acids; the predominant oleic and linoleic acids confer several positive health effects ,25. BothThe major components of dietary fiber include cell wall polysaccharides . Dietary fiber is divided into soluble and insoluble fractions based on water solubility . In thisQuinoa is one of the few plant foods that provides essential amino acids. Thus, quinoa proteins are accepted as high quality protein because of their balanced composition of essential amino acids, with the exception of tryptophan ,5. The qThe macro and microminerals in quinoa leaves and grains were found at concentrations greater than those reported for maize, wheat, and spinach leaves. Several authors have reported that the mineral content of quinoa is adequate for a balanced diet . Thus, tBioactive compounds of quinoa were found mainly in the outer layers of the seeds, functioning as a chemical defense against insects and microorganisms ,34. StudSome studies have established the chemical structure of phenolic compounds that confers potent antioxidant properties exerted through different mechanisms. They have the ability to scavenge free radicals and chelate metal ions, inhibit oxidases, terminate radical chain reactions, and stabilize free radicals . Chenopodium species varied from 36.2 to 144.3 mg/100 g, and the predominant flavonoids were shown to be quercetin and kaempferol [In the case of flavonoids and anthocyanins, the leaves contained concentrations 98.47 times higher than those present in quinoa seeds. In another study, the flavonoid content of empferol . The rutempferol . The antempferol . As showempferol . Thus, oWith respect to antinutritional compounds, other researchers have reported saponin content of 1260 mg/100 g and 970 mg/100 g in quinoa seeds and leaves, respectively . The sapThe concentration of oxalates in the leaves was 66.46% higher than that in the quinoa grain. In another study, the oxalate concentration in amaranth and quinoa grains were 184 mg/100 g to 232 mg/100 g dry weight, respectively ,46.2O5 per hectare were applied for fertilization.Iniap Tunkahuan (Ecuador) quinoa leaves and grains were selected for this study. The plants were provided by the National Legumes Program and INIAP Andean Grains (Ecuador). They were grown in the Santa Catalina Experimental Station with the geographic location of altitude 3050 m.a.s.l, latitude UTM 9959382 m S, longitude 17 M0772618 m W. Ecuador does not have a defined season, but the quinoa was grown with an average rainfall of 600 mm, temperature variation (7–17 °C), and sandy loam soil (pH 6.5). In the case of the soil, the fertilization application was 80 kg of nitrogen and 40 kg of PThe quinoa leaves were harvested manually after cultivation for 80 days. The grains were harvested with a reaper for 170 days, threshed and classified in a Crippen Mfg. Inc  using sieves with pore sizes of 2.5 mm, 2.2 mm, and 2 mm. For this study, grains with an average diameter of 2.5 mm were used.The samples were taken to the laboratory, classified, placed in polypropylene bags, frozen at −20 °C for 24 h, and then freeze-dried using LABCONCO equipment . The seeds were cleaned of dust and other contaminants. The leaves and grains were milled  to a particle size of 250 µm, and the ground powder was packed in polypropylene bags and stored in a cool, dry place (4 °C) until further analysis. The protein, fat, crude fiber, and ash contents were determined according to the Association of Official Analytical Chemistry (AOAC) standardized methods . CarbohySDF and IDF were quantified by sequential enzymatic digestion of quinoa leaves and grains by heat-stable α-amylase, protease, and amyloglucosidase. The IDF was filtered, and the residue was washed with distilled water, ethanol, and acetone. The combined solution of filtrate and water washings were precipitated with ethanol for SDF determination. The precipitate was filtered and dried, and both residues (IDF and SDF) were corrected for protein, ash, and blank for calculation of fiber content (AOAC 991.43) .2 min−1; injection volume, 5 μL; run time, 45 min [The amino acid profile was determined using high-performance liquid chromatography. The processed leaf and grain samples were applied to an LC-10AS Shimadzu liquid chromatography system, which operated under the following conditions: oven temperature, 60 °C; excitation wavelength, 350 nm; emission wavelength, 450 nm; sample cooler temperature, 4 °C; eluent solution flow, 0.60 m, 45 min .The mineral content in quinoa leaves and grains was determined using wet oxidation and atomic absorption spectrophotometry on an AA-7000 spectrophotometer , except for phosphorus, which was analyzed by colorimetry . Total carotenoid content was determined using cold acetone and petroleum ether. Absorbance was measured at 450 nm. The carotenoid content was calculated using an extinction coefficient of 2500 . Ascorbic acid was determined by reflectometric analysis using a test strip that monitors the reduction of yellow molybdophosphoric acid to phosphomolybdenum blue by ascorbic acid. The processed quinoa samples were diluted in distilled water and stirred thoroughly. The test strip was then dipped in the solution, excess liquid was allowed to run off via the long edge of the strip on to absorbent paper, and then the strip was analyzed using a reflectometer. The results were expressed in mg/100 g dry weight . Polyphenolic compounds were quantified using Folin Ciocalteau 2N reagent; the absorbance was measured at 765 nm, and the results were expressed in mg gallic acid/100 g dry sample . Total aTrolox equivalent antioxidant capacity (TEAC) was determined by measuring reduction of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) in 80% methanol and comparing against a Trolox standard curve. TEAC was expressed as µmol Trolox equivalent/g dry weight . g. Then, sulfuric acid (72%) and distilled water were added to a portion of the extract and were incubated for 2 h at 100 °C. The strip with the sample was analyzed using a reflectometer. The results were expressed as mg/100 g (dry weight basis) [Nitrate content was determined using a test strip that measures the reduction of nitrate ions by a reducing agent. The processed quinoa leaf samples were added to distilled water and were homogenized and centrifuged for 10 min at 10,000× t basis) . v/v). Absorbance was measured at 528 nm and compared against a pure saponin standard. The saponin content was expressed as mg/100 g dry weight [Saponin content was determined by spectrophotometry. Samples were first homogenized with ethanol (25%); the extract was filtered, and then an aliquot of the extract was mixed with a color reagent  were then added to the ash. The solution was filtered, warmed to approximately 60 °C, and used for titration. An oxalic acid equivalent (45 Eq/g) was used for the calculation [Oxalic acid concentration was determined by titration with 0.001 N KMNOculation . t-test was used to identify significant differences between leaves and grains. The INFOSTAT software package was used for the statistical analysis [The samples were analyzed in triplicate, and the results are expressed as mean and standard deviation (SD). Student’s analysis . Quinoa is a pseudocereal with high nutritional value. The leaves and grains of quinoa could be considered functional foods as they contain adequate protein, dietary fiber, minerals, and important bioactive compounds. However, quinoa can also present antinutrient compounds, whose content can vary depending on the plant organ, and can be reduced using thermal processing. Moreover, the leaves have a higher mineral content and may help to overcome nutritional deficiencies. Antioxidant compounds such as ascorbic acid, carotenoids, and phenolic compounds are also highlighted. The leaves have antinutritional compounds; however, the levels found in the leaves are low and within what is allowed for human consumption. The results of the research show that the leaves can be used in culinary preparations, in the elaboration of functional foods, and in the extraction of bioactive compounds for the food or pharmaceutical industry.


A multivariable logistics regression revealed cues to action (p < 0.001), the perception of COVID-19 vaccination benefits (p = 0.002), and the perception of barriers (p < 0.001) that were associated with vaccine acceptance. Conclusions: The acceptance of the COVID-19 vaccine was low among international students. The correct and comprehensive beliefs of the target groups regarding the benefits and barriers of the vaccination must be raised. Various effective social strategies must be adopted to trigger the intention of COVID-19 vaccination. The study findings will inform the decisions of public health campaigners, aimed at reducing vaccine hesitation when the COVID-19 vaccine is widely available.Background: With the continuous large-scale development of the COVID-19 vaccine, the acceptance of vaccination and its influencing factors at the individual level have become crucial to stemming the pandemic. This study aims to explore the factors that influence the acceptance of the COVID-19 vaccine among international college students. Methods: The target population constituted international students pursuing various degrees in Jiangsu Province through an online cross-sectional study. A cluster random sampling was performed using a self-administered questionnaire. The Health Belief Model and Knowledge, Attitude/Beliefs, and Practice Theory served as the underlying theories to understanding the factors that influence vaccine acceptance. Results: We received 330 responses. About 36.4% intended to accept the vaccine. The acceptance varied across respondents’ place of residence, program of study, continent of origin, knowledge, susceptibility, severity, benefits, and cues to action ( Coronavirus disease 2019), caused by SARS-CoV-2, is an RNA virus that belongs to the family Coronaviruses. COVID-19 is a highly infectious disease with the following symptoms: fever, dry cough, fatigue, myalgia, and dyspnea 9, caused, but it Hence, vaccination, which is one of the avenues to prevent susceptible and infectious diseases, is required. For instance, influenza vaccination is one of the most convenient ways to control seasonal influenza, among other related diseases . VaccineHowever, a successful vaccination program is largely affected by the rate of acceptance, which is unsatisfactory across the globe. Kwok et al. (2020) reported that about 70% of the population they surveyed accepted to take the COVID-19 vaccine upon its readiness . About 2Various theories have been proposed to explain the behavior of people during disease outbreaks. Commonly used theoretical frameworks entail the Health Belief Model (HBM) and Knowledge, Attitude/Beliefs and Practice Theory (KABP). The HBM consists of constructs like perceived susceptibility, perceived severity, perceived benefits, perceived barriers, and cues to action . PerceivThe young population perceive themselves not to be susceptible to COVID-19, hence their inability to adhere to the COVID-19 safety protocols . HoweverWe undertook an online cross-sectional survey using a questionnaire to identify the factors influencing the acceptance of the COVID-19 vaccine among International students pursuing various degrees in Jiangsu Province of China. Cluster random sampling was used in the survey. Seven universities with international students were randomly selected in Jiangsu Province. Two classes were randomly selected from the selected universities. The online questionnaire was distributed by volunteers of the selected classes in each university via WeChat social group platforms. However, slow and limited access due to Internet services on mobile phones resulted in the failure of 25 students among the sample size not being able to respond to the questionnaire. Hence, 330 (93%) students were enrolled and successfully completed the survey. The survey was performed from 17 March to 26 March 2021. The questionnaire could only be accessed once from the same WeChat account. Participation was voluntary, with no incentive. This study was anonymous, as no information concerning personal identification  was collected. A duration of five minutes could be used to complete the survey. Participant’s completion of the questionnaire was regarded as consent for participation.Our questionnaire was constructed by reviewing similar works on vaccine acceptance ,18,24,25p < 0.05 (two-sided).The acceptance of the COVID-19 vaccine was our dependent variable, while our independent variables were the demographic variables, health beliefs, and knowledge. The health belief constructs and acceptance, which were measured on a five-point Likert scale of strongly agree or agree, was given a score of 1, while the rest were recorded as 0. However, two questions regarding acceptance: “I still have some concerns so I don’t want to get the COVID-19 vaccine right away” and “I’m still hesitant about the COVID-19 vaccine” were reverse-coded; hence, disagree or strongly disagree were recognized as 1. Next, all the questions were summed for each part, and a score of 3 or more was recognized to have that belief  or attitude (acceptance), while the opposite was given a score of ≤2. In determining the knowledge scores, responses were recorded as 0 for incorrect responses and 1 for correct responses. All the correct answers were summed up and grouped into two “knowledge Yes” and “knowledge No”. Respondents with a score >7 were given “knowledge Yes”, while those who scored ≤7 were given “Knowledge No”. Likewise, Crosstabs were used to know how the acceptance varied across the demographic variables, the constructs of the Health Belief Model, and knowledge. A multivariable logistic regression was conducted by using the demographic variables, health beliefs, and knowledge as the independent variables, while COVID-19 vaccine acceptance was regarded as a dependent variable. We analyzed our data using SPSS version 25.0 , with the statistical level set at Out of the 330 responses received, more than half of the respondents were males, within the 18–25 age group. They were primarily undergraduates who currently reside in China. Half of them were African, whilst 45.8% were Asian. The respondents revealed they were healthy (94.8%). The majority of the respondents’ average monthly expenditures ranged between 1000 and 3000 CNY (152.6 and 457.8 USD) .About 67% of our respondents had adequate knowledge about general vaccinations and the COVID-19 vaccine . HoweverOur respondent’s perceptions of susceptibility toward COVID-19 were low, with 38.8% reporting as susceptible . About 4Generally, about 50.9% of our respondents saw COVID-19 as severe , with 75The majority of respondents (64.5%) perceived the COVID-19 vaccine to be beneficial . They reMore than half of our respondents were hindered by barriers (58.5%) . They weApproximately 51.2% were influenced by cues , where 6The proportion of respondents accepting of the COVID-19 vaccine was 36.4% . About 3A total of 36.4% of the 330 respondents strongly agreed/agreed to be vaccinated. Columns four and five of p < 0.001) predicted vaccine acceptance the most, followed by benefits of the COVID-19 vaccine (p = 0.002), while barriers to receiving the COVID-19 vaccine hindered the vaccine acceptance (p < 0.001). The perception of susceptibility, severity, and being knowledgeable were not significant , where their respondents’ reasons for accepting the vaccine were partly down to reducing the chances of being infected, because being vaccinated would make them less worried about the disease, which eventually increased their level of confidence and perception about the COVID-19 vaccine . Again, Numerous studies have enumerated barriers as one of the major negative predictors of vaccine acceptance, as reported in the present study. Like most studies, side effects, authenticity, and safety were the most reported barriers highlighted by the participants in the current study ,31,32. TThe perceived susceptibility and severity of COVID-19 have been found by different studies as predictors of vaccine acceptance . HoweverA low level of knowledge has been one of the negative predictors of vaccinations, as revealed in existing studies . AlthougDespite the reasons explained above, there were several limitations in this study. First and foremost, this study used a self-reported questionnaire, which was subject to bias or mixed feelings among the respondents. Second, this was a cross-sectional study that provided a correlation explanation, but the causal relationship between vaccine acceptance, knowledge, and beliefs were not delved into. Third, our cut-off points used to classify the HBM constructs and knowledge were not referenced elsewhere, which may produce bias in the study results. Last, but not least, the scope of this study was limited to universities in Jiangsu Province; the results can be used as a reference but cannot be extrapolated to all international college students studying in China or other countries.This study showed that 36.4% of our respondents intended to accept the vaccine against the Coronavirus disease (2019). The HBM can be used as basis for campaigns. In the strategy of improving vaccine acceptance, in addition to popularizing the relevant knowledge and the threat from infection, key interventions should be provided to enhance the benefits perception of vaccination and to reduce the barrier perceptions. Appropriate measures to increase the cues should be considered, since cues to action in social environments are found to be very critical promoters.