To investigate the association between thyroid functions and the oral microbiome diversity.Data from the US National Health and Nutrition Examination Survey  were analyzed. Thyroid functions were defined using thyroid hormones and related biomarkers. Oral microbiome was measured using the observed number of amplicon sequence variants (ASVs) and the Bray-Curtis dissimilarity. Linear regression was used to estimate the average change (β) and 95% CI for the number of ASVs against thyroid functions, adjusted for sociodemographic variables, health conditions, urinary iodine status, and periodontitis. Non-metric multidimensional scaling (NMDS) was used to analyze the Bray-Curtis dissimilarity.P = .002), between free thyroxine groups (P = .015), and between thyroglobulin groups (P = .035).A total of 2943 participants were analyzed. The observed number of ASVs has a weighted mean of 128.9. Self-reported thyroid disease was associated with reduced number of ASVs , if only adjusted for sociodemographic variables and health conditions. In the fully adjusted model, compared to normal thyroid function, both subclinical and clinical hyperthyroidism were associated with reduced number of ASVs . Thyroid peroxidase antibody level higher than the reference range was associated with higher observed ASV . NMDS analysis suggested significant difference in oral microbiome composition between free triiodothyronine groups (Hyperthyroidism was associated with reduced oral microbiome diversity. Free triiodothyronine, free thyroxine, and thyroglobulin levels may alter the oral microbiome composition. Humans and microbes have coexisted and coevolved in a symbiotic relationship . How humPrior studies have documented the close relationship between the oral microbiome and health status. Diseases not limited to oral diseases, but including gastrointestinal, neurological, endocrine, immune, and cardiovascular diseases, can lead to changes in the oral microbiome . ClinicaThyroid function is crucial for maintaining normal metabolic function, including energy expenditure, lipid and glucose metabolism, and thermogenesis , 13. AbnIt is reasonable to hypothesize a reciprocal relationship between thyroid function and the oral microbiome: the oral microbiome participates in the regulation of metabolic processes that involve thyroid function, while altered metabolic processes due to thyroid dysfunction can lead to changes in the oral microbiome. However, information on the relationship between the oral microbiome and thyroid function is limited. Within this context, this study aims to investigate the association between thyroid function and the oral microbiome diversity using the newly released oral microbiome data from the US National Health and Nutrition Examination Survey (NHANES) .https://www.cdc.gov/nchs/nhanes/index.htm). Informed consent forms are obtained from all NHANES participants.The NHANES is an ongoing survey, conducted by the National Center for Health Statistics (NCHS) of the Centers for Disease Control and Prevention (CDC), to measure the health and nutrition status of the civilian noninstitutionalized US population ≥2 months of age. The NHANES study protocol is described in detail on the NCHS website , total and free triiodothyronine (TT3 and FT3), thyroglobulin (TG), thyroglobulin antibodies (TGAb), thyroid peroxidase antibodies (TPOAb), and thyroid stimulating hormone (TSH). These measurements were conducted at the Collaborative Laboratory Services  for NHANES 2009-2010 and 2011-2012, and at the University of Washington  for NHANES 2009-2010 using the same methods.According to the laboratory procedure manuals, the reference ranges for these thyroid measures were: 0.30-5.60 uIU/mL for TSH, 80-180 ng/dL for TT3, 6.10-12.20 ug/dL for TT4, 2.50-3.90 pg/mL for FT3, 0.60-1.60 ng/dL for FT4, 0-4.0 IU/mL for TGAb, 0-35.00 ng/mL for TG, and 0-9.00 IU/mL for TPOAb. We grouped the study population according to these reference ranges. For TSH, TT3, TT4, FT3, and FT4, we defined 3 groups: 1) below the reference range; 2) within the reference range; and 3) above the reference range. For TG, TPOAb, and TGAb, 2 groups were defined: 1) within the reference range; 2) higher than the reference range.Additionally, we defined subclinical and clinical hypothyroidism and hyperthyroidism according to TSH, FT3, and FT4 for each participant: 1) subclinical hypothyroidism, if TSH was above the reference range, and both FT3 and FT4 were within the reference ranges; 2) subclinical hyperthyroidism, if TSH was below the reference range, and both FT3 and FT4 were within the reference ranges; 3) clinical hypothyroidism, if TSH was above the reference range, and either FT3 or FT4 was lower than the reference range; 4) clinical hyperthyroidism, if TSH was below the reference range, and either FT3 or FT4 was higher than the reference ranges; and 5) normal, if TSH, FT3, and FT4 were all within the reference ranges.Therefore, the following variables were used to represent thyroid function in this study: self-reported thyroid diseases, subclinical hypothyroidism, subclinical hyperthyroidism, clinical hypothyroidism, clinical hyperthyroidism, TSH group, TT3 group, TT4 group, FT3 group, FT4 group, TGAb group, TG group, and TPOAb group.Oral microbiome testing was performed using oral rinse samples that were originally collected to study the prevalence of oral human papillomavirus in the US population . DetailsAlpha diversity is a measurement of the microbiome diversity within a single participant, typically representing community richness and community evenness. Four alpha-diversity metrics, including observed ASVs, Faith's Phylogenetic Diversity, the Simpson index, and the Shannon-Weiner Index, were available in NHANES data. The study focused on the observed number of ASVs, a simple measure of alpha diversity that represents the number of unique microbial taxa detected in a sample. This metric is commonly used to compare the richness of different samples or to investigate changes in diversity between exposure groups. The observed number of ASVs appeared to reach saturation by a rarefaction value of 10 000 , and thiBeta diversity is a measurement of the microbiome diversity between participants, represented by pairwise dissimilarity of participants. In this study, we considered Bray-Curtis dissimilarity . The BraPeriodontitis was considered in this study, as it is closely related to the oral microbiome diversity. We defined 3 periodontitis groups according to prior publications on oral health in NHANES : a) seveWe used linear regression to estimate the average change (β) and corresponding 95% CI for the 4 alpha-diversity metrics against the thyroid function variables. Because current knowledge regarding the association thyroid functions and the oral microbiome is still limited, we consider 3 different models to examine how different confounder sets influence our estimates. Covariates selected for adjustment in these models were considered to have direct effects on both thyroid function and oral health, or to be the proxy variables for the unmeasured confounders. Model 1 adjusted for basic demographic variables race , age , gender . No missing values were reported in these covariates. Model 2 additionally adjusted for basic socioeconomic and health-related variables, including education , ratio of family income to poverty threshold , body mass index , hypertension , type 2 diabetes , and self-reported general health conditions . These variables are widely considered as confounders in health research. Model 3 further additionally adjusted for variables specifically related to thyroid functions and oral health, including urinary iodine status  and periodontitis . Missing values in the covariates were treated as not missing completely at random for Taylor series variance estimation as presented in the NHANES tutorials.We used non-metric multidimensional scaling (NMDS), which is commonly regarded as the most robust unconstrained ordination method, to analyze and visualize the overall microbiome compositional difference between participants based on the beta-diversity Bray-Curtis dissimilarity . AnalysiWe conducted a sensitivity analyses to analyze the other 3 alpha-diversity metrics, Faith's Phylogenetic Diversity, the Simpson index, and the Shannon-Weiner Index, in Model 3 to test whether different metrics would alter the results.P values were 2-sided, and P < .05 was considered statistically significant.All statistical analyses were weighted by appropriate NHANES sample weights and performed using SAS 9.4. The NMDS and ANOSI were conducted using the “vegan” package in R version 4.2.2 . All P vIn the weighted population, less than 10% self-reported a history of thyroid diseases. According to the thyroid hormone measures, 87.3% of participants had normal thyroid function. Subclinical hypothyroidism had the highest prevalence, of 1.9%, while both clinical hypothyroidism and clinical hyperthyroidism had a prevalence of 0.4%.No significant association was observed with most thyroid hormone and biomarker groups, including TSH, TT3, TT4, FT3, FT4, TGAb, and TG; however, a significant positive association with microbiome diversity was observed for high TPOAb. TPOAb was not significantly associated with microbiome diversity in Model 1 , but this association became significant and stronger in Model 2 , and further strengthened in Model 3 .When using other alpha-diversity metrics other than the observed number of ASVs , subcliP = .001), even though the difference was small in the figure. Although significant associations were observed for subclinical and clinical hyperthyroidism with the observed number of ASVs, no significant difference in the microbiome composition was observed when comparing subclinical or clinical hyperthyroidism with normal thyroid function groups . A significant difference in the microbiome composition was observed across FT3, FT4, and TG groups . For FT3, the microbiome distribution of higher group was narrower and appeared to be a subset of the lower group. For FT4, the non-overlapped area between higher and lower groups was non-ignorable. For TG, although a significant difference was noted, the distribution was quite similar between the normal and higher groups. Borderline significant difference was also observed for TSH (P = .076), for which the lower group demonstrated a narrower distribution of microbiome composition compared to the higher group.Analysis of beta diversity shows the difference in the oral microbiome composition across different thyroid function groups . Self-reThis is a large-scale study to investigate the association between thyroid functions and oral microbiome diversity. We found that both subclinical and clinical hyperthyroidism was associated with reduced oral microbiome diversity, and high TPOAb was associated with higher microbiome diversity. In terms of the microbiome composition between participants, findings from this study suggested difference in the composition across FT3, FT4, and TG groups. Overall, the study provides evidence for an association between the oral microbiome and thyroid function, especially hyperthyroidism.Porphyromonas gingivalis), demonstrated the capabilities to alter the downstream gut microbiome [Microbial diversity is often closely related to the health status of the host. Generally, in the absence of oral diseases, healthier hosts have a higher oral microbiome diversity. In this study, we observed both positive and inverse associations with different thyroid functions. To our knowledge, only one prior study linked higher TSH with increased oral microbiome diversity . A similcrobiome , 33.Bifidobacterium and Lactobacillus in the gut microbiome were significantly lower in 14 patients with hyperthyroidism than in 7 healthy controls [In a pilot clinical research, researchers found that the levels of controls . Patientcontrols . Additiocontrols . These fHowever, the evidence for hypothyroidism and human microbiome is more mixed. One study suggests that the primary hypothyroidism is related to a significant reduction in the number of strains producing propionic and butyric acid in the gut of patients . On the One interesting observation is that the significant association was observed with clinical or subclinical hyperthyroidism, but not with individual triiodothyronine or thyroxine levels. Prior research demonstrated that clinical and subclinical hyperthyroidism are associated with adverse health outcomes including heart failure, osteoporosis, and dementia, particularly among populations aged 65 or above . In compMoreover, experiment studies suggest that human microbiome is widely involved in triiodothyronine and thyroxine metabolism , 45, 46.It should be noted that some significant associations in this study were found for the observed number of ASVs, but not with other alpha-diversity metrics. These additional metrics usually consider other aspects. Specifically, Faith's Phylogenetic Diversity takes into account the evolutionary relatedness of microbial taxa in a community . The SimThe strengths of our study should be considered in light of its limitations. First, NHANES is cross-sectional, thus we cannot examine the temporality between thyroid function and the oral microbiome diversity. It is unclear whether thyroid dysfunction leads to changes in the oral microbiome, or vice versa, or the relationship is reciprocal. However, as the first large-scale study to link the oral microbiome with thyroid function, findings from this study still provide insights for future directions. Second, the composition of the human oral microbiome can be influenced by various factors, including the intake of food and beverages, the availability of endogenous nutrients, the host immune system, medication treatments, and systemic diseases. However, despite these potential influences, the oral microbiome exhibits significant resilience, particularly when compared to the microbial community in the large intestine. Apart from excessive and frequent consumption of fermentable carbohydrates or supplementation with nitrates, the diet has minimal impact on the composition of the oral microbiome . Third, Overall, this study is a large-scale study to investigate the association between the oral microbiome and thyroid function and fill the knowledge gap in this area. In this study, hyperthyroidism was associated with reduced oral microbiome diversity, while high TPOAb levels were associated with increased oral microbiome diversity. Some microbiome compositional differences were observed between FT3 and FT4 groups. All these findings advance our understandings of the role of the oral microbiome in human health and its link with thyroid function, providing insights for future research.


The neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte ratio (PLR), and systemic immune-inflammatory index  have been described as potential blood-derived inflammatory biomarkers in several diseases. Rheumatoid arthritis is an inflammatory disease that has been related to an increased risk of cardiovascular (CV) disease. In the present work, we analyze how these hematological composite scores of inflammation are related to classic CV risk factors and subclinical atherosclerosis in patients with RA. In this cross-sectional study that included 430 patients with RA, the NLR, MLR, PLR, and SIRI scores were calculated. Multivariable analysis was performed to examine the relationships of these composite blood scores with subclinical carotid atherosclerosis and with traditional cardiovascular factors, producing a complete profile of lipid molecules and insulin resistance or indices of beta-cell function, and a Systematic Coronary Risk Assessment (SCORE2) calculation. C-reactive protein and disease activity were significantly and positively associated with the four blood composite scores. SCORE2 was significantly associated with higher values of SIRI, NLR, and MLR, but not PLR. These relationships were maintained when SCORE 2 was considered categorical; patients in the very high CV risk category had higher values in all hematological composite scores, except PLR. In the multivariable analysis, SIRI and NLR were independently associated with higher levels of beta cell dysfunction. In conclusion, SCORE2 and the values of the hematological composite scores were positively correlated in patients with RA. In addition, there were some relationships of these scores with traditional CV risk factors, with their association with beta cell dysfunction being the most consistent. White blood cells are the largest group of inflammatory cells, and their various subtypes, including neutrophils, lymphocytes, and monocytes, are essential inflammatory markers. The role of platelets as a marker of inflammation has also been recognized, since they have different functions in inflammatory responses. In this regard, white blood cells and platelets are present in the chronic inflammatory environment, with a role in the secretion of cytokines, proteases, angiogenic factors, and chemokines . For thiRheumatoid arthritis (RA) is an inflammatory, peripheral polyarthritis of unknown origin that predominantly affects multiple joints in a symmetric pattern. RA is considered an inflammatory state in which acute-phase reactants are elevated. Furthermore, the magnitude of the increase in these acute-phase reactants is directly associated with the extent of inflammation and synovitis. Certainly, the increased risk of cardiovascular (CV) morbidity and mortality in RA has been recognized, with accumulating evidence that RA is an independent CV risk factor . ExtensiIn the present work we set out to analyze, in a large series of patients with RA, how the combined blood indices are related to traditional CV risk factors, including a complete lipid profile and indices of insulin resistance and subclinical atherosclerosis.2; cancer; or any other chronic diseases such as hypothyroidism, heart or respiratory diseases, and nephrotic syndrome were excluded. Additionally, patients with evidence of active infection were also excluded. The study protocol received approval from the Institutional Review Committees at the Hospital Universitario de Canarias and the Hospital Universitario Doctor Negrín, both located in Spain. All participants provided informed written consent . The research adhered to relevant guidelines and regulations, and the principles outlined in the Declaration of Helsinki.This was a cross-sectional study involving 430 consecutive patients diagnosed with rheumatoid arthritis (RA). All participants were aged 18 years or older and met the 2010 classification criteria established by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) . RheumatIndividuals included in the study completed a questionnaire regarding cardiovascular risk factors and medication use and underwent a physical examination. Body mass index (BMI), abdominal circumference, and systolic and diastolic blood pressure were assessed under standardized conditions. Information on smoking status, diabetes, and hypertension was collected. In patients diagnosed with rheumatoid arthritis (RA), disease activity was assessed through various measures, including the Disease Activity Score (DAS28) in 28 joints , the CliCardiovascular risk score (SCORE2) was calculated according to the 2021 European Society of Cardiology guidelines on cardiovascular disease prevention in clinical practice . SCORE2 Carotid ultrasound examination was conducted to assess carotid intima-media thickness (cIMT) in the common carotid artery and detect focal plaques in the extracranial carotid tree among patients with rheumatoid arthritis (RA) . The Esap-values were below 0.20 in the univariable analysis of hematological scores. All analyses were conducted using Stata software, version 17/SE , with the two-sided significance level set at 5%. A p-value of less than 0.05 was considered statistically significant.Demographic and clinical characteristics of patients with RA are presented as means (standard deviation) or percentages for categorical variables. For continuous variables that did not follow a normal distribution, data are reported as medians and interquartile ranges (IQR). The association between disease-related data and blood composite scores (continuous dependent variable) was examined using multivariable linear regression analysis, with adjustments made for confounding variables. Multivariable beta coefficients in linear regression must be interpreted as the average amount by which the dependent variable increases when the independent variable increases by one and other independent variables are held constant. Confounders were selected from demographics and traditional CV risk factors if their 2 and an abdominal circumference of 97 ± 13 cm. Traditional cardiovascular (CV) risk factors were common among the patients, with 22% being current smokers, 13% having type 2 diabetes, 32% classified as obese (BMI ≥ 30 kg/m2), and 34% having hypertension. Additionally, 32% of the patients were taking statins. The mean carotid intima-media thickness (cIMT) was 696 ± 131 microns, and 42% of the patients had carotid plaques. The median SCORE2 value for the entire population was 3.7 (IQR 1.8–5.9)%. According to SCORE2, 62% of the subjects were categorized as having low or moderate CV risk, while 25% and 13% were classified as having high and very high CV risk, respectively. A total of 430 patients with rheumatoid arthritis (RA) were included in this study. In the studied cohort, the median duration of the disease was 8 years, with an interquartile range (IQR) of 4 to 15 years. At the time of the study, the mean level of C-reactive protein (CRP) was 2.7 mg/L, with an IQR of 1.3 to 6.1 mg/L, and the mean erythrocyte sedimentation rate (ESR) was 18 mm/1st h, with an IQR of 7 to 32 mm/1st h. A total of 72% of the patients tested positive for rheumatoid factor, while 65% tested positive for anti-citrullinated protein antibodies. Disease activity, as assessed by the Disease Activity Score based on 28 joints and ESR (DAS28-ESR), had a mean value of 3.1 ± 1.4. Prednisone was being used by 36% of the patients, and 87% were taking at least one conventional disease-modifying antirheumatic drug (DMARD), with methotrexate being the most frequently prescribed DMARD (73%). Additionally, 19% of the patients were undergoing anti-tumor necrosis factor therapy. For further details on treatment frequencies and historical disease-related data, please refer to The median duration of the disease in our cohort of was 8 (IQR 4–15) years. The mean values of CRP and ESR, at the time the study was performed, were 2.7 (IQR 1.3–6.1) mg/L and 18 (IQR 7–32) mm/1st h, respectively. Seventy-two percent of patients were positive for rheumatoid factor, and 65% for anti-citrullinated protein antibodies. Disease activity measured via DAS28-ESR was 3.1 ± 1.4. Thirty-six percent of the patients were on prednisone and 87% were taking at least one conventional disease-modifying antirheumatic drug (DMARD), with methotrexate being the most widely used (73%). Nineteen percent of the patients were receiving anti-tumor necrosis factor therapies. The frequency of use of other treatments and historical disease-related data are shown in The relationship of CV risk factors, including SCORE2 and disease-related data, to hematological markers (as dependent variables) is shown in About disease-related data, several significant associations were found between disease characteristics and hematological inflammation markers . In thisThe influence of peripheral blood scores (as independent variable) on lipid pattern molecules and insulin resistance indices is shown in The relationship of hematological composite scores with glucose homeostasis molecules and insulin resistance indices was only evaluated in non-diabetic patients and if glucose was less than 110 mg/dL (n = 338). In this subgroup, after multivariable analysis, SIRI and NLR were associated with significant and higher levels of beta cell function—HOMA2-B%. Furthermore, no associations were found between carotid IMT and plaque and blood inflammation scores .In the present work, we studied, for the first time, the relationships of several composite blood scores with the CV comorbidity that occurs in patients with RA. According to our results, these markers correlate with SCORE2, and higher values of them are associated with beta cell dysfunction in this population. We therefore believe that hematology score levels, which reflect inflammatory status, may be useful diagnostic surrogate biomarkers for CV disease in patients with RA.RA patients have been reported to have higher peripheral blood score values than controls. In a recent meta-analysis comprising thirteen studies on neutrophil-to-lymphocyte ratio (NLR) with a total of 1550 patients with RA and 1128 healthy controls, as well as eight studies on platelet-to-lymphocyte ratio (PLR) with 380 RA patients and 305 healthy controls, both NLR and PLR were found to be significantly elevated in RA patients compared to the control groups. This was also the case in a study of 1499 RA patients from five hospitals and 366 healthy volunteers serving as controls, in which NLR, MLR, PLR, and SIRI were significantly increased in RA patients . FurtherHowever, there is some controversy regarding the relationship between hematologic composite scores and disease activity in RA. In a study involving 547 adults diagnosed with RA, it was observed that patients with active disease displayed higher neutrophil-to-lymphocyte ratios (NLRs) and platelet-to-lymphocyte ratios (PLRs) compared to those in remission. However, the discriminatory ability of NLR and PLR in detecting disease activity was found to be lower than that of CRP and erythrocyte sedimentation rate ESR. Additionally, the combination of NLR and PLR did not significantly enhance the diagnostic value of CRP and ESR . Based oSCORE2, a state-of-the-art algorithm incorporating conventional cardiovascular risk factors, is widely utilized to estimate the 10-year risk of developing cardiovascular disease for the first time. In our work, SCORE2 showed a positive and significant association with higher values of SIRI, NLR, and MLR, but not PRL. This relationship was also significant when SCORE2 was considered categorical and hematology score levels were compared between the very high and low or moderate risk categories. In this regard, we are not aware of any study that has studied this relationship in the general population. However, the predictive ability of peripheral blood scores for CV events has recently been reviewed. After analyzing a total of 13 studies with 152,996 participants, the overall pooled results showed that SIRI was significantly associated with an increased risk of CV disease . This inIn relation to this, a retrospective study involving 48 patients with RA found that SIRI was independently linked to the development of ischemic stroke in individuals with RA . The facIn our study, we found no relationship between hematological scores and lipid profiles, but we did find a relationship between SIRI and NLR and greater beta cell dysfunction after multivariable adjustment. It is known that RA patients have insulin resistance and that this is partly explained by the inflammatory state of the disease . AdditioFinally, we did not observe a relationship between hematological scores and the presence of carotid plaque or cIMT. Therefore, the role of CV risk factors in the development of atherosclerotic disease in RA is clearly greater than that of hematological markers.Several studies have studied the relationship of blood composite scores with CV disease in the general population. In a report of 158 patients with chronic kidney disease, red blood cell distribution width correlated with CRP, central diastolic blood pressure, and albuminuria . AdditioThere are several limitations to our study. One of them is the absence of a control group. However, many studies have already explored the role of blood composite scores in CV disease, the prediction of CV events, and other CV comorbidities in the general population. Second, we could not evaluate the causal relationship between hematological score parameters and the outcomes of our study due to its cross-sectional design. Third, the exact biologic mechanisms underlying the associations described in our work need to be clarified by further studies. Fourth, we think that the combination of some of these scores could be helpful in measuring systemic inflammation. However, there are still no studies that have validated these combinations. Fifth, although we registered the use of antihypertensive therapy, we did not consider the types of diuretics used by the subjects.In conclusion, hematological composite scores are related to SCORE2 risk calculations and to several CV comorbidities in patients with RA.


Stroke represents the third cause of long-term disability in the world. About 80% of stroke patients have an impairment of bio-motor functions and over half fail to regain arm functionality, resulting in motor movement control disorder with serious loss in terms of social independence. Therefore, rehabilitation plays a key role in the reduction of patient disabilities, and 3D printing (3DP) has showed interesting improvements in related fields, thanks to the possibility to produce customized, eco-sustainable and cost-effective orthoses. This study investigated the clinical use of 3DP orthosis in rehabilitation compared to the traditional ones, focusing on the correlation between 3DP technology, therapy and outcomes. We screened 138 articles from PubMed, Scopus and Web of Science, selecting the 10 articles fulfilling the inclusion criteria, which were subsequently examined for the systematic review. The results showed that 3DP provides substantial advantages in terms of upper limb orthosis designed on the patient’s needs. Moreover, seven research activities used biodegradable/recyclable materials, underlining the great potential of validated 3DP solutions in a clinical rehabilitation setting. The aim of this study was to highlight how 3DP could overcome the limitations of standard medical devices in order to support clinicians, bioengineers and innovation managers during the implementation of Healthcare 4.0. Stroke is a cerebrovascular accident that caused 5.5 million deaths in 2016 and represents one of the most important causes of disabilities in the world . MoreoveThe sites of brain damage, as well as their extent, influence the stroke’s outcome . In detaThe clinical signs could also include dysphagia, impairment in speech, behavioral functions, sensory functions, visual functions and cognitive functions such as attentional deficits, memory, orientation and awareness ,5,6, redHowever, in chronic stroke patients, residual disability may include mobility disorder, poor balance, gait impairment and limited physical activity . ConsequNevertheless, over half of stroke patients with upper limb impairments fail to regain arm functionality, resulting in a serious loss in terms of independence and social activities. Therefore, the use of the upper limbs is essential to interact with the environment and with people, and 87% of stroke patients showed hand paralysis with impairment of the clamp and grab movements and a negative impact on the activities of daily living (ADLs) .In these patients, the use of upper limb orthotics or splints, devices externally applied that modifiy the structural and functional features of the muscle-skeletal system, is widely accepted .Orthoses can be classified as standard or custom-made. Standard orthoses are less expensive but might provide less comfort to a patient compared to a custom-made device, as they come in limited sizes and are challenging to adapt. Alternatively, a traditional custom-made orthosis, namely, high-temperature and low-temperature thermoplastic orthosis, has the potential to enhance comfort and be more adequate, although the manufacturing process is more challenging . The conWhen clinical requirements demand a simpler design, custom orthoses may be handcrafted from low-temperature thermoplastic sheets molded directly on the patient’s body. However, such orthoses come with several functional and aesthetic limitations. Patients often experience problems with proper skin ventilation and maintaining cleanliness and dryness of their splints .Furthermore, conventional orthoses are not easily adaptable to morphological changes , and modifications require highly skilled personnel and additional working hours ,19. DespMoreover, since they are handmade, the risk of complications and discomfort, particularly skin breakdown and poor fit, depends largely on the skill and experience of the technician .Orthoses also include exoskeletons, devices made to produce a task-oriented motor training, to assist the patient in the execution of a particular movement. The exoskeleton has a structure associated with an engine, and it works with an input; for example, the EMG surface signals from the patient’s muscles ,23. The In the last 20 years, new techniques have been employed in rehabilitation with the aim of personalizing clinical devices/therapies, starting from the patient’s morphology to ensure the greatest comfort during task-oriented motor training; among these, 3D printing (3DP) seems to play a key role. Three-dimensional printing or additive manufacturing consists of the construction of a three-dimensional object from a digital 3D model or a computer-made model (CAD). The production of a 3D object can be achieved through different technologies belonging to seven families: powder bed fusion, binder jetting, directed energy deposition, VAT polymerization, material jetting, material extrusion and sheet lamination.Three-dimensional printing (3DP) transforms 3D designs into life-changing products, including patient-friendly pharmaceutical products as well as bio-inspired medical devices, resulting in the next revolution for the pharmaceutical and medical device industries  .Healthcare utilizes all of the AM families (ASTM F42—Additive Manufacturing) and the related 3D printing technologies, such as VAT polymerization via stereolithography (SLA)  or digitVAT polymerization is the family commonly used to produce dental and orthopedic implants, with medical industry applications ,42,43,44To overcome the aforementioned limitation, it is possible to use material jetting technologies, which offer the potential to print multi-material and color inks deposited with a layer height of about 14µm, with the related high aesthetic impact.PBF, DED and BJ technologies selectively fuse ,40,49 orFinally, SL, by using foils, supports the creation of 3D colored objects useful for teaching activities or phantoms ,54.Furthermore, 3DP applications and properties are strictly related to 3D printing technologies and natural or artificial materials (biocompatible or not) ,56. ABS Finally, to produce living tissue and organ bioprinting requires the combination of biological/synthetic materials ,68,69, eThe mechanochemical behavior of 3D-printed biomedical materials plays a pivotal role in assuring adherence to the final application functionality. These materials are exposed to mechanical stresses or chemical reactions that may influence their properties , requiring the accurate study of mechanochemical systems ,73 and bTherefore, to give a base for large-scale production, it is essential to identify or develop new cost-effective biocompatible and sterilizable materials  that areThen, starting from the design of the final application, it is possible to work with many different materials ,88, depeIn complex surgeries, 3DP can provide customized models on which the surgery team can train and improve pre-operatory planning. Three-dimensional printing also has the potential to train new surgeons by overcoming the observational learning method in favor of trial and error , with a Moreover, 3DP is a valid approach in the production of bioscaffolds for regenerative medicine, meeting the growing demand of human tissues and organs, upregulating regenerative cellular behaviors and improving cell attachment, proliferation and differentiation ,94.However, one of the main field of application of 3DP is represented by orthoses, that show clinical effectiveness through biomechanical and functional parameters and interesting results in terms of pain reduction, comfort, quality of life and patient satisfaction, and they are useful both in hospitals and in domestic rehabilitation programs ,96,97,98In fact, the advantage of 3DP orthoses lies in the possibility of customizing orthoses, providing an individualized shape that can adapt to complex anatomical curves with optimal pressure points, a high ventilation surface, good aesthetics and a light weight . FinallyMoreover, the augmented design possibilities can meet the needs of subjective treatment, also in pediatric or complex cases , and impEnergy consumption in 3DP processes is influenced by several factors; for example, an increasing resolution implies extending the production time, resulting in higher energy consumption . In thisOn the other hand, 3DP orthoses present some weaknesses: although the time of production can be shorter in comparison to traditional devices , the cosIn conclusion, 3DP orthoses have the potential to replace traditional technology thanks to cost-effective  personalThis systematic review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis for Network Meta-analysis (PRISMA-NMA) guidelines and the Cochrane Handbook for Systematic Reviews of Interventions  and invoThe aim of this review is to investigate the potential of 3D printing technology in the rehabilitation of stroke survivors, focusing on the improvement of upper limb function. PROSPERO registration: CRD42017065491.We utilized the PICO  approach: (P) post-acute, subacute or chronic stroke patients; (I) 3D-printed orthosis; (C), comparison between rehabilitation with 3D-printed orthosis and conventional rehabilitation, comparison between rehabilitation with 3D-printed orthosis and conventional orthosis (non-3D-printed orthosis); (O) preventing spasticity complications, improvement of the quality of life, pain and edema. Additional relevant trials were identified by manually reviewing the reference lists of the selected publications.We included trials that met the following inclusion criteria: stroke survivors ; 3DP orthosis; upper limb rehabilitation; RCT ; case control study; feasibility study as study design; full-text availability; written in English. The exclusion criteria comprised studies concerning lower limb rehabilitation in which only healthy subjects were included and which focused on cognitive conditions  or those which did not focus on stroke .The articles were independently screened by title and abstract by 3 reviewers. The full texts of the articles that were unclear from the title or abstract were reviewed in accordance with the selection criteria. Three reviewers extracted the data from the selected studies on a Microsoft Excel sheet. In case of disagreement, consensus was achieved with a fourth reviewer. Data extracted from each paper included the author and year of publication, subject characteristics , descriptions of intervention (3D-printed orthosis) and outcome measures.We used a modified variant of the STROBE criteria to conduct the methodological evaluation, using the ten criteria detailed in 3.10. Two authors independently assessed the score, and disagreements were evaluated and resolved by consensus. A numerical scoring system  was used to rate the items, and the studies were classified as follow: high risk of bias, score < 6; low risk of bias, score > 6. Then, to carry out the clinical review, we examined previous guidelines, research queries, adequate evidence, study quality, synthesis of results and their correct interpretation .The literature search generated 218 articles: 32 items from Pubmed, 78 from Scopus and 108 from Web of Science. Eighty documents were excluded for being duplicates and 138 articles were screened. Finally, 16 articles fulfilled the inclusion criteria; we excluded 6 articles after the quality assessment [A total of 109 patients  were included in the research analyses. A summary of the selected articles is shown in Three trials were RCTs ,109,110.The rehabilitation methods varied greatly in terms of the length, frequency and volume of training.Huang et al. tested the effectiveness of the 3DP orthosis in a task-oriented approach. Patients were divided into two groups , both completing a 4-week protocol and 2-week follow-up consisting of 30 min of therapist training and 30 min of daily home training. Both groups received the same amount of training, and the intervention group wore the orthosis throughout the protocol, while the control group was trained without the device .Zheng et al. tested the 3DP orthosis and typical thermoplastic devices supported by the intervention and control groups, respectively. Both groups received the selected orthoses, for 4 to 8 h per day for 6 weeks, in order to perform the conventional rehabilitation therapy .Yang et al. tested the 3DP orthosis in a six-week regimen. Both groups underwent conventional rehabilitation for 30 min, three times per week. The experimental group wore the 3DP brace for 6 h a day at home, while the control group had no brace and was advised to perform a series of independent handgrip exercises at home .The remaining eight studies were all feasibility studies.Chen, Zhou and Ben Abdallah tested the device efficacy after a rehabilitation training period (of variable duration) characterized by a series of exercises carried out independently at home by patients ,111,113.The other authors ,115,116 Studies regarding robotic or exoskeleton hand orthoses set a range of motion limit in order to prevent excessive hyperextension stress in patients with anatomical limitations, such as spasticity and range of motion deficits due to muscle fibrosis or myotendinous retractions. In these cases, applying excessive hyperextension force can lead to injuries that can even worsen the state of spasticity ,114.Three studies reported no safety issues regarding increased spasticity and skin damage ,112,115.Two studies ,110 asseThe other trials did not evaluate side effects.The technologies principally used in the selected studies belong to the material extrusion family (ASTM 52900-15), which is recognized as being a more flexible, not expensive, ergonomic (reduced weight) and sustainable (reduced waste) fabrication approach. Accordingly, eight of the ten described studies exploited the related benefits, and seven of them used PLA or TPU materials to build dedicated electronic cases too. The remaining experiments adopted selective laser sinter technology to fuse TPU and PA medical grade powders without adding toxic fillers, while Chen et al. described medical devices obtained from photo/thermal active polymers via CLIP (continuous liquid interface production) and MJP (multi-jet printing) technologies, respectively.In detail, three authors developed via 3DP only the splint or the finger mechanisms ,111,113;Ben  and YangChen  reached Finally, to assure patient safety and ergonomics with materials directly in contact with the skin, Dudley  reportedHuang  developeLaser-based technologies were also used by Toth  to fabriThree-dimensional-printed devices achieve the same elastic modulus of not-printed ones via SLS; MJP-processed actuators reach values between 87.12–94.1%, while CLIP technology does not overcome 7.27% compared with the standard TPU finger-extension component.Upper limb function has been assessed using validated scales such as the manual function test (MFT) ; Fugl-MeThe rate of perceived exertion (RPE) scale was used to evaluate exertion during the BBT test . The modADL were examined by some authors through the use of a range of tests such as pencil holding, eraser holding, handle grip, vertical handle, opening door handle, tool holding, plastic cup holding  and the Satisfaction was assessed using the Likert scale ,115; OPUWearing time during rehabilitation programs was evaluated in ,95.p = 0.00012). Differences were also found in handle grip , tool holding (p = 0.0066), vertical handle gripping (p = 0.0011), plastic cup holding (p = 0.0303) and opening a door handle (p = 0.0303). Eraser holding was the only activity that did not improve (p = 0.1212).Toth et al.  reportedPark et al.  reportedp = 0.034) after the 4-week training program, but only the patients in the experimental group maintained their improvement during the 2-week follow-up period (p = 0.042). Palmar pinch force improved significantly during the program and follow-up  in contrast with no significant change in the control group. Lateral pinch force increased significantly  in both the experimental and control groups. Grasp force improved in both groups, showing better results in the 3DP (29.1% against 6.6%) orthosis group.Huang et al.  used FMADudley et al.  evaluateBen et al.  showed tp = 0.028) and lateral pinch force ; palmar pinch force improved but did not reach statistical significance (p = 0.068). The participants’ ARAT score increased from 20.33 to 31.33 (p = 0.026).Chen et al.  found a Huber et al.  found thp = 0.03) and at Pos6 (2.0 (0.6) p < 0.01 for wrist flexors) when compared to Pre0. No significant changes in MAS were found in the control group; a slow decreasing trend was observed. The FMA-UE score improved significantly at Pos3 (Pre0 27.8 (11.7), Pos3 30.5 (12.0), p = 0.02) and Pos6 (36.8 (11.2) p < 0.01) in the experimental group. In the control group, a slight improvement was found at Pos3 and a significant increase at Pos6 (Pre0 21.6 (11.5), Pos3 23.0 (12.7) Pos6 26.0 (14.7), p = 0.01).Yang et al.  assessedp = 0.02). Changes in extension (mean increase of 10.3° (3.8°) vs. control group 3.3° (2.9°); p < 0.001) and ulnar deviation angles (mean increase 5.3° (6.0°) vs. control 1.1° (2.6°); p = 0.028) were significantly higher in the experimental group, as with the FMA-UE (1.3 (1.0) score improvement vs. control 0.2 (0.3)) and swelling scores. No statistically significant between-group differences were found for flexion and radial deviation angles, changes in VAS scale and subjective sensation score.Zheng et al.  observedIn Zhou et al. , after 2Patients were satisfied with the use of the orthosis in ,114  and comfortable (OPUS 4.2 ± 0.5) in .p < 0.01), ease of use (p < 0.01) and wearing time (p < 0.01). Pain ratings showed no difference between the two groups, with participants still experiencing mild annoying pain.In Yang et al. , particiIn Huang et al., the authors analyzed the time of use in both groups, showing more training time in the 3D-printed device group  ; in ChenAverage scores of 4 on QUEST (“quite satisfied”) and 90 out of 100 on the SUS scale were obtained in [Huber et al. compared a 3DP orthosis with a commercial orthosis and found no significant difference in the usability categories between the two devices .Many studies reported a small patient sample as a limit to their research ,113,115.In Park et al., the shape of the orthosis proved to be a limitation in completing two tasks  in the CAHAI-9 protocol chosen to evaluate its performance. The authors also report that the tension cables can break during use . In HuanFour of the ten studies considered 40%) ,109,114 0% ,109,1Stroke is the one of the leading causes of death and long-term disability worldwide. Patients affected by hemispheric stroke complain of a reduction in their upper limb motion ability in approximately 80% of the cases in the first days after stroke; that persists in 30% to 66% of cases at 6 months and is associated with higher levels of anxiety and reduced perception of health-related life quality. The improvement of upper limb function plays a role in stroke rehabilitation . In thisThis review investigated the role of 3D-printed upper limb orthoses in the rehabilitation of stroke patients, focusing on the technological advances and solutions of personalized orthoses and active exoskeletons.The studies reported a greater improvement in patients treated with 3DP upper limb and hand orthoses in terms of grip force and motor functions ,110,118.Park et al.  reportedOur review confirms the results of a recent article by Schwartz et al. examining the clinical use of 3D-printed orthoses for upper extremity musculoskeletal conditions such as fractures, wrist pain, overuse conditions or trauma. The authors evaluated outcomes such as range of motion, side effects, measures of grip and pinch strength, pain, measures of patient satisfaction (standardized and non-standardized) and patient functionality.This improvement is achieved even in the neurological affection of the upper limb. In particular, all studies we included that used the 3D-printed orthosis showed a reduction in pain , an increase in patient satisfaction in terms of comfort, ease of use, weight of the orthosis, time and independence in activities of daily living  and improved range of motion .Furthermore, in patients with upper limb spasticity, the use of 3DP orthoses appears to have a positive effect on spasticity compared to the control groups ,110,118,The ability to customize orthoses is one of the main advantages of 3DP. In fact, it is possible to create a personalized shape for each patient that can adapt to complex anatomical curves with optimal pressure points, a high ventilation surface and good aesthetics ,119. HowRegarding manufacturing, the traditional method of standard orthoses personalization is relatively long. It is necessary to manually correct the shape and dimensions of the orthosis depending on the patient’s anatomy ,120,121.According to Keller et al. , a well-One of the main limitations of 3DP is the manufacturing of large sizes of orthoses determined by the limited capacity of most common printers ,19. HoweOn the other hand, the results show that the comfort and satisfaction of patients wearing 3DP orthoses is higher than patients who have used traditional orthoses ,114,116.The most commonly used materials in 3DP are plastic polymers; therefore, lighter devices can have problems with brittleness and warping. This is more evident in lower limb orthoses where devices must withstand continuous mechanical stress during walking . A commoA further cause of fragility in 3D-printed parts is the anisotropic print direction in the fused deposition modeling process. This limitation can be solved by choosing a layer stacking scheme or changing the printing direction to achieve a better load distribution. In addition, alternative 3D printing techniques such as selective laser sintering, material jetting and vat photopolymerization can be integrated to achieve isotropic strength in printed modules .In this context, to reduce the environmental impact of 3D printing orthotics, the disposal and recycling of devices and scrap material must be considered. After cleaning and shredding, polymers can be re-extruded into filament. However, this process is still far from being cost-effective, and successive heating cycles and residual impurities reduce the physical properties of the filament, which may not be desirable for the production of an orthosis ,132. HowMoreover, other polymers, e.g., ABS, polycarbonate, polypropylene, high- and low-density polyethylene, polystyrene and polyethylene terephthalate, are commonly recycled .The growing scientific interest in the production of 3D-printed orthoses is relatively recent, and the included studies have all been published in the last six years.A strength of this review is represented by the specific condition of the included patients, which gives more specific insight into the impact of 3D-printed orthoses on stroke survivors.The results show how 3DP devices improve upper limb functionality and strength, with important implications for ADL improvement and user satisfaction in stroke patients. In addition, the ability to customize the orthosis allows for greater comfort and prolonged use, exceeding the limitations of traditional orthoses. These results pave the way to the integration of 3D printing in rehabilitation.However, the current literature on this topic is represented by a small number of studies conducted on a small number of patients, and future research is needed to improve the accuracy and efficiency of additive manufacturing for capillary diffusion in the medical field. In this context, artificial intelligence could play a role in the customization process to improve orthosis design based on patients’ needs. Moreover, the development of new biocompatible materials, absorbable materials and printing techniques could allow a wider utilization in a clinical setting with respect to environmental pollution .Three-dimensional printing technologies enable the production of complex medical devices to meet customization costs and patient needs, reducing risks, improving patient outcomes and reducing lead times via delocalized productions.Moreover, considering the negative effect of stroke on upper limbs, 3DP orthoses have shown interesting results in terms of functional improvement, satisfaction and cost. Three-dimensional printing could, therefore, represent a cost-effective and valid solution in a clinical setting, with the possibility of designing upper limb orthoses tailored to the patient’s needs which consequently have to be progressively introduced inside medical SOPs (standard operating procedures) during rehabilitation.Unfortunately, standardization, regulatory compliance, quality control and material limitations, such as the utilization of AM families for sensors or wearable devices, are challenging to address.The materials used in AM must be biocompatible, sterilizable and safe for human use, assuring strength, flexibility and wear resistance for long periods (without losing performance levels). Moreover, medical device production costs , and the related accuracy, are still associated with the technologies used and the post-processing, consequently reducing the fast scalability of skilled labor able to produce medical devices.


Bone marrow adipocytes (BMAs) are the most plentiful cells in the bone marrow and function as an endocrine organ by producing fatty acids, cytokines, and adipokines. Consequently, BMAs can interact with tumor cells, influencing both tumor growth and the onset and progression of bone metastasis. This review aims to systematically evaluate the role of BMAs in the development and progression of bone metastasis.in vivo studies. The results were synthesized using descriptive methods.A comprehensive search was conducted on PubMed, Web of Science, and Scopus electronic databases, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement standards, to identify studies published from March 2013 to June 2023. Two independent reviewers assessed and screened the literature, extracted the data, and evaluated the quality of the studies. The body of evidence was evaluated and graded using the ROBINS-I tool for non-randomized studies of interventions and the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool for The search yielded a total of 463 studies, of which 17 studies were included in the final analysis, including 15 preclinical studies and two non-randomized clinical studies. Analysis of preclinical studies revealed that BMAs play a significant role in bone metastasis, particularly in prostate cancer followed by breast and malignant melanoma cancers. BMAs primarily influence cancer cells by inducing a glycolytic phenotype and releasing or upregulating soluble factors, chemokines, cytokines, adipokines, tumor-derived fatty acid-binding protein (FABP), and members of the nuclear receptor superfamily, such as chemokine (C-C motif) ligand 7 (CCL7), C-X-C Motif Chemokine Ligand (CXCL)1, CXCL2, interleukin (IL)-1β, IL-6, FABP4, and peroxisome proliferator-activated receptor γ (PPARγ). These factors also contribute to adipocyte lipolysis and regulate a pro-inflammatory phenotype in BMAs. However, the number of clinical studies is limited, and definitive conclusions cannot be drawn.The preclinical studies reviewed indicate that BMAs may play a crucial role in bone metastasis in prostate, breast, and malignant melanoma cancers. Nevertheless, further preclinical and clinical studies are needed to better understand the complex role and relationship between BMAs and cancer cells in the bone microenvironment. Targeting BMAs in combination with standard treatments holds promise as a potential therapeutic strategy for bone metastasis. Once boBased on this knowledge, recent research has focused on the potential relationship between BMAs and cancer bone metastasis. Studies have shown that BMAs can regulate the migration, invasiveness, and survival of tumor cells –21. Thes22.1in vitro and in vivo) and clinical studies (Study design). Studies from March 22, 2013, to July 12, 2023, were included in this review if they met the PICOS criteria. The following were the exclusion criteria: studies evaluating 1) BMA function and/or role in primary cancer, 2) the role of mesenchymal stem cells (MSCs), 3) the efficacy of adipose-derived stem cells (ADSCs) as drugs for medical intervention, 4) mathematical modeling tool construction, 5) BMA variation in physiological conditions, 6) omental adipose-derived cells, 7) tumor progression in obesity, and 8) articles with partial data. Moreover, reviews, letters, comments to editor, meta-analysis, case reports, protocols and recommendations, editorials, guidelines, and articles not written in English were excluded.The PICOS model  was used to project this review: 1) studies that evaluated BMA function and role in bone metastasis in cells, animals, and patients (Population), submitted or not 2) to a specific intervention (Interventions), 3) with or without a comparison group (Comparisons), 4) that described BMA function and role in bone metastasis (Outcomes), in 5) preclinical  statement . The sea2.3After the duplicate elimination by a public reference manager (Mendeley Desktop v.1.19.8), the potential pertinent articles were screened using the title and abstract by two reviewers (FS and DC). Studies that did not meet the inclusion criteria were eliminated, and any disagreement was resolved through debate until a consensus was reached or with the involvement of a third reviewer (GG). Finally, the remaining studies were comprised of the final stage of data extraction.2.4The data extraction and synthesis started with cataloging the details of the studies. To increase validity and avoid omitting potential findings for the synthesis, two authors (FS and DC) extracted and made a table  tool  individually analyzed the methodological quality of the included studies. In case of disagreement, they tried to reach a consensus; if this failed, a third reviewer (GG) made the definitive decision. The methodological quality of included clinical studies was evaluated by the ROBINS-I tool for the assessment of the risk of bias in non-randomized studies of interventions . The metLE) tool , which h33.1in vitro and in vivo, five only in vitro, and two only in vivo. The search strategy and study inclusion and exclusion criteria are specified in The initial search found 484 studies. Of those, 110 were detected using PubMed, 217 using Scopus, and 157 using Web of Science. After screening the title and abstract, 53 articles were uploaded to Mendeley Desktop version 1.17.9 to remove duplicates. The resulting 26 complete articles were reviewed to determine whether the publication met the inclusion criteria, and 17 were considered eligible for the review. From the reference lists of the selected articles, no extra publications were found. Of the articles eligible for the review, two were non-randomized (retrospective) clinical studies, while the remaining 15 were preclinical studies, of which eight were both Of the preclinical studies, eight were on prostate cancer bone metastasis, one on breast cancer bone metastasis, one on melanoma bone metastasis, two both on breast and melanoma cancer bone metastasis, one on multiple myeloma bone metastasis, one on lung cancer bone metastasis, and one on specimens of different primary tumors bone metastasis from humans. Concerning the clinical studies, one was on breast cancer bone metastasis, while the other evaluated bone metastasis from different types of primary cancer. 3.23.2.1in vitro and in vivo models of marrow adiposity. They showed that adipocyte-exposed cancer cells (PC3) exhibit increased expression of glycolytic enzymes, higher lactate production, and reduced mitochondrial oxidative phosphorylation, indicative of the Warburg phenotype. They also revealed that PC3 induces lipolysis of adipocytes as a potential maintenance mechanism. Additionally, adipocytes were found to drive the metabolic reprogramming of cancer cells through the oxygen-independent mechanism of hypoxia-inducible factor 1α (HIF-1α). Importantly, the metabolic signature observed in adipocyte-exposed cancer cells mimics the expression patterns observed in patients with metastatic bone disease. Another study demonstrated that soluble factors released by human primary BMAs can sustain the migration of prostate cancer cells in a CCR3-dependent manner, suggesting the potential involvement of the CCR3 pathway in prostate cancer cells homing to bone  the implication of the directed migration of CCR3 in vitro to the BMA production and 2) the enhancement of CCR3 that expresses cells in different human bone metastatic sites. These results suggested that the CCR3 pathway might be potentially implicated in prostate cancer cells homing to the bone. Wang et al. (in vitro and in vivo models. They demonstrated that a high-fat diet (HFD) in nude mice leads to dyslipidemia and specific alterations in the bone marrow, including increased adipocyte area and number, elevated level of free fatty acids (FFAs), and a decline in osteoblasts’ area and number. Furthermore, HFD stimulated COX2 expression and suppressed osteoprotegerin (OPG) expression in the bone marrow microenvironment. In a small cohort of patients, caprylic acid was identified as a specific FFA with higher levels in patients with prostate cancer bone metastases (n = 8) when compared to those without bone metastases (n = 8) or healthy controls (n = 16)  with caprylic acid resulted in increased adipocyte differentiation and PPARγ expression, along with a subsequent reduction in osteoblast number. Treatment of BMAs with caprylic acid in vitro promoted BMSC-derived adipocyte differentiation, COX2 expression, PGE2 production, and antagonized osteoblastic differentiation and OPG release  model permitted us to authenticate this signature. Conditional media secretome analyses demonstrated that fibronectin and collagen type 1 were critical bone-secreted factors able to regulate tumor protein kinase A (PKA)  . In vivo release . These f release . Additioy et al. , demonstteoclast . They futeoclast , using a A (PKA) .3.2.2in vivo model of breast and melanoma cancer bone metastases. They initially characterized the effects of BMAs using the MDA-MB-231 breast cancer cell line in male immunocompromised RNU rats and established a malignant melanoma B16F10 model in male C57BL/6 mice. To translate their findings to patients, they used typical diagnostic techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) combined with molecular analysis. The results demonstrated that breast and melanoma cancer bone metastases exhibited greater bone destruction in animals with high body weight, which is associated with a distinctive bone marrow environment characterized by enhanced bone marrow fat. In humans, BMAs were found to be co-localized with growing cancer cells. In addition, by analyzing the antagonization of PPARγ as a treatment for inhibiting bone tumor metastasis growth, they showed that targeting adipocytes through the inhibition of PPARγ, especially in overweight individuals, could reduce skeletal metastasis spreading and protect against cancer-associated bone loss. Similarly, Gregoric et al. , which is located adjacent to S100A8/A9 genes on chromosome 1q21.3.In a recent preclinical o et al.  investig3.2.5In a recent study by Sato et al. , the rol3.3Two retrospective clinical trials were found, one focused on breast cancer bone metastasis and the other on bone metastasis from various primary cancers including prostate, breast, lung, hepatoma, and rectal. In the first study, Goupille et al.  evaluate3.4in vivo studies (n = 10), the risks of bias, reported in in vivo studies did not declare the method of sequence generation (n = 10). All the studies displayed that concerning baseline characteristics, groups were similar (n = 10). In approximately 80% of the studies (n = 8), the allocation was adequately concealed, while in two studies, it was not. One study  were linked with bone metastasis development in premenopausal women.in vitro, in vivo, and clinical, systematically emphasizing the role of BMAs in the onset and progression of bone metastasis for the first time. Nevertheless, it is worth mentioning some limitations of this systematic review. The number of relevant articles included in the review is low, and some of the preclinical articles included had a high risk of bias in almost all the papers. Finally, inherent bias associated with the retrospective and descriptive nature of clinical study cannot be excluded.The strength of this review lies in its systematic approach and comprehensive evaluation of studies retrieved from three different databases: PubMed, Scopus, and Web of Science. Furthermore, the review included all types of studies, including Given the abundance of BMA in the bone marrow and their control over the formation and endocrine functions, targeting BMAs and their products could be a novel therapeutic strategy against metastatic bone disease. To date, therapeutic options for bone metastasis, such as bisphosphonates and monoclonal antibodies, although effective in reducing the prevalence of skeletal-related events in prostate and breast cancers, remain palliative. Based on the results of this review, it is evident that treatment strategies for bone metastases should target multiple components of the bone metastatic niche, including BMAs. Targeting BMAs in conjunction with standard treatments may provide a promising therapeutic alternative for bone metastasis in prostate and breast cancers. However, further preclinical and clinical studies are needed to better evaluate and understand the complex connections between BMAs and cancer cells in the bone microenvironment.The original contributions presented in the study are included in the article/Conception and design of the work: FS. Acquisition of data for the work: FS and DC. Analysis of data for the work: FS, DC, MS, VB, and GG. Interpretation of data for the work: FS and DC. Drafting the work: FS and DC. Revising the work: FS, DC, MS, VB, CE, and GG. Approval for publication of the content: FS, DC, MS, VB, CE, and GG.


Durvalumab is an immune checkpoint Inhibitor (ICIs) that is used in the treatment of malignant tumors, such as lung cancer and melanoma. ICIs are associated with immune-related adverse events including autoimmune encephalitis, although both paraneoplastic phenomena and ICI treatment may lead to autoimmunity.BR1 and GAD65 antibodies and both diabetes and autoimmune limbic encephalitis. Because he was followed prospectively as part of a treatment study, we had access to repeated serum samples and cognitive assessments over time prior to developing encephalitis and diabetes, in addition to later assessments. A high titer of GABABR1 antibodies appeared early, while GAD65 antibodies appeared later with a lower titer in parallel with the development of diabetes. As he subsequently developed clinical signs of encephalitis, verified by EEG and brain MRI, he also had CSF GABABR1 antibodies. Durvalumab was discontinued and steroid treatment with subsequent plasmapheresis were started, resulting in reduction of both CSF and serum antibody levels. Clinical signs of encephalitis gradually improved.We describe a 72-year old male patient with small-cell lung cancer, who during adjuvant treatment with Durvalumab developed GABAThis case illustrates the importance of being aware of possible serious autoimmune adverse reactions, including neurological syndromes such as encephalitis, when treating patients with high risk of para-neoplasia with ICIs. In addition, the case shows the development of autoantibodies over time. Because he participated in a clinical trial, serial cognitive tests and serum samples collected at several time points prior to the development of encephalitis were available for analysis.2The patient was a 72-year old man, who until recently had been working full time in a cognitively demanding job. In 2009, he had an anterior-wall myocardial infarction and emergency PCI. He had surgery for hernia in the spring of 2022 but was otherwise healthy. He had smoked for 35 years, until 2007.2.1In May 2022 (2 body surface area (BSA), durvalumab 1500 mg iv. on day 1, and etoposide 200 mg/m2 BSA po. on days 2–4 with or without thoracic radiotherapy and was randomized to therapy without concurrent radiation. At the start of each cycle, he had dexamethasone 8 mg for 3 days.He was included in an RCT of combination therapy with carboplatin (AUC 5 mg*min/ml), etoposide 100 mg/mst cycle, he developed petechia on his chest and thrombocytopenia. For the 2nd cycle, he therefore received a reduced dose of carboplatin/etoposide (60% of AUC 5).Shortly after the 1nd cycle, showed considerable reduction of lesions in the right lower lobe and lymph nodes in the mediastinum and right hilum, but new lesions in corpus of vertebra T8 and possibly a pathological fracture of the 8th rib. The liver metastases were reduced in size, from 55 to 25 mm largest diameter for the largest lesion. After the 4th cycle, the pulmonary lesions were smaller, with a remaining 4 mm nodule in the right lower lobe (th week and received the 5th cycle in September 2022 (week 16). In October 2022 he received prophylactic cranial irradiation (PCI) of 25 Gy in 10 fractions and continued with durvalumab monotherapy (1500 mg iv. every 4 weeks). In December 2022, he was in good general condition, ECOG 1, but had challenges with diabetes control. CT evaluation early January 2023 showed stable disease in the thorax/abdomen, and there were no metastases on brain CT.He completed four cycles of systemic therapy. CT thorax/abdomen after the 22.2One week later (week 17), at consultation for evaluation for PCI, he was diagnosed with diabetes mellitus with blood glucose 26.7 mmol/L, HbA1c 64 mmol/mol (reference 20–42), c-peptide 230 pmol/L (fasting reference 300-1480). A diagnosis of diabetic ketoacidosis was confirmed and insulin treatment was started.Serum IA2 and GAD65 antibodies were not detected using an ELISA-based test from RSR Limited, . In May 2022, before the start of immunotherapy, he had no confirmed diagnosis of diabetes.2.3In January 2023, during 4 days of hospitalization for compression fracture of vertebra T7 after a fall, he had spontaneous alterations of cognition. The examination revealed no neurological abnormalities, and a brain CT was normal.A few days later, he was admitted to the stroke unit of the hospital after a 10 min episode of dysphasia with spontaneous normalization. Brain CT, including angiography and perfusion images, showed no evidence of stroke. However, he had minor electroencephalogram (EEG) abnormalities with a left-sided frontotemporal dominance and reduced cognition, with 26/30 points on the mini mental status examination (MMSE). An epileptic seizure could not be excluded, but this was not treated. He was discharged to his home with an outpatient appointment for a new brain MRI and follow-up for treatment of his SCLC.In February 2023 (week 41), he was admitted to the Department of Pulmonary medicine because of increasing confusion. His wife described seizure-suspect episodes with strange swallowing noises, no eye contact, and tremor-like convulsions of the upper extremities, followed by incoherent speech. In addition, his general condition had worsened, with increasing gait disturbances and imbalance, and visual hallucinations.2.4At admission to the Department of Pulmonary medicine, he had no neurological abnormalities. During the next days, he became disoriented and periodically dysphasic. Brain MRI on day 4 after admission showed high T2/FLAIR signal and increased volume in the left temporal lobe and hippocampus with no gadolinium contrast enhancement  and glutamic acid decarboxylase 65 (GAD65)  Figure 3the Euroimmun autoimmune encephalitis mosaic 6 cell-based assay  or the Ravo PNS 14 line assay .After 10 days of treatment, he still had poor balance/gait, was not oriented for time/place and person and had cognitive difficulties. There was no clinical improvement since hospital admission. Plasma exchange (PLEX) was therefore started, with sessions every other day using a Spectra Optia apheresis machine , in total five sessions. Each time, 1.2–1.3 plasma volumes were removed with 4% albumin as substitution fluid and acid citrate dextrose solution as anticoagulant. He continued with oral prednisolone 80 mg per day with gradual tapering of the dose.BR1 and GAD65 and CSF positive for GABABR1, but negative for GAD65  on the MMSE and 19/30  on the Montreal cognitive assessment (MoCa) scale , were seen only in serum from week 16 to 39  at several time points before the diagnosis of encephalitis, collected as part of the RCT. The serum samples were screened for relevant antibodies. There was a dip in the longitudinal MoCa scores at the time of encephalitis development and a slight improvement with encephalitis treatment in parallel with the observed clinical improvement. Detectable antibody titers of GABA16 to 39 Table 1.3We have described a patient with SCLC, who developed an ICI treatment-associated limbic encephalitis and was treated with high dose steroids and subsequent PLEX.BR1 and GAD65 autoantibodies over time.Serial cognitive assessments with MoCa  showed aBR1 was present with a high titer in serum before starting treatment with durvalumab, suggesting paraneoplastic association with SCLC. When the patient developed encephalitis, the serum anti-GABABR1 titer was lower, but anti-GABABR1 was also detected in the CSF, and it is therefore likely that this antibody was associated with development of limbic encephalitis. In a similar case report of a patient with SCLC and encephalitis after durvalumab, both serum and CSF were positive for anti-GABABR antibodies , and with a lower titer than GABAith time . This isBR1 antibodies, but 4 months before onset of the symptoms of encephalitis. Therefore, we cannot exclude that the PCI may play a role in the pathogenesis here, e.g., by direct radiation-induced tissue damage, direct immuno-modulating effects, potentiation of side-effects from durvalumab.There is accumulating evidence that ionizing radiation of the brain may lead to cognitive deficits, also after low doses, possibly through mechanisms such as chronic neuroinflammation and altered immune reactions , 12. RadOur patient received high-dose steroids with little or no early clinical improvement and worsening of his diabetes. For irAEs that are not corticoid-responsive, PLEX or IVIG are alternative treatments as has also previously been suggested for anti-GAD-65 limbic encephalitis . In thisPLEX is recommended as add-on treatment to immunosuppressives in severe and life-threatening irAEs , and recThis case raises several questions, e.g., how to balance effective cancer treatment versus severe encephalitis symptoms and treatment including if durvalumab could be restarted when the SCLC progresses. Because of the severe symptoms of encephalitis, we were reluctant to reintroduce durvalumab, as reported for patients with encephalitis associated with other ICIs , althougTo our knowledge, this is the first case report of a patient with an n-irAE, that has been followed with serial measurements of autoantibodies from the debut of SCLC, through development and treatment of ICI-associated encephalitis. Our longitudinal data may contribute to a better understanding and awareness of the risk of using highly potent ICI treatment.In conclusion, this case demonstrates that treatment with ICIs must be followed with vigilant monitoring for possible irAEs that can have serious or fatal consequences for the patient if left untreated.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.Ethical approval for reporting individual cases or case series is not required by our institution or local regulations. The patient and his wife provided their written informed consent for the publication of anonymized patient information in this article, and they received the final manuscript for comments prior to submission. Written informed consent was obtained from the participant/patient(s) for the publication of this case report.TM: Data curation, Investigation, Writing – original draft, Writing – review & editing. KS: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – review & editing. AA: Data curation, Investigation, Validation, Writing – review & editing. AG: Conceptualization, Data curation, Investigation, Writing – review & editing. BG: Conceptualization, Data curation, Investigation, Writing – review & editing. KN: Conceptualization, Data curation, Investigation, Writing – review & editing. CV: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. CL: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – review & editing.


Pneumocystis jirovecii (PJ) is an opportunistic pathogenic fungus, and PJ pneumonia (PJP) is a commonly problem in HIV-positive patients. While PJP is not caused by HIV, it generally advances rapidly and can quickly lead to severe respiratory failure. To improve pediatricians’ understanding of the condition and aid early accurate diagnoses and therapy, we examined the clinical characteristics of five instances of non-HIV related PJP (NH-PJP) in children and the efficacy of metagenomic next-generation sequencing (mNGS) in its diagnosis.From January 2020 to June 2022, five children with NH-PJP were admitted to the PICU of the First Affiliated Hospital of Zhengzhou University. We retrospectively summarize the clinical presentation, previous histories, routine laboratory findings, treatment, outcome of regression, and results of mNGS in these five children.Five male children between the ages of 11 months and 14 years had an acute onset on NH-PJP, three of the children had chest tightness after activity, shortness of breath and paroxysmal dry cough, — and two had high fever and dry cough. All five of the children had several flocculent high-density pictures in both lungs at the beginning of the disease, and lung auscultation revealed coarse breath sounds in both lungs, one of which was accompanied by a modest quantity of dry rales. PJ nuclear sequences were found in one patient and four patients’ blood and alveolar lavage fluid. All five children were treated with Trimethoprim-sulfamethoxazole (TMP-SMX) in combination with Caspofungin and corresponding symptomatic treatment. Four patients were cured and one patient died.Children commonly encounter an initial exposure to NH-PJP, which manifests as a high fever, dry cough, chest discomfort, dyspnea that worsens over time, fast disease progression, and a high death rate. The clinical presentation of children with PJ infection should be taken into consideration along with the results for diagnose. mNGS has higher sensitivity and a shorter detection period compared to identification of PJP. With the use of chemotherapy or immunosuppressive medication in recent decades, the prevalence of non-HIV related PJ (NH-PJP) has been rising annually  immunocompromised conditions, including but not limited to hematologic malignancies, solid tumors, rheumatic diseases, long-term systemic use of corticosteroids , use of immunosuppressive agents , solid organ transplantation and hematopoietic stem cell transplantation;(2) typical clinical manifestations of PJP, including fever, cough, dyspnea, and progressive hypoxemia; (3) radiologic findings suggestive of PJP in bilateral lungs that newly emerged on CT scans; (4) BALF and/or blood samples were collected for mNGS. Furthermore, we collected patients’ baseline information, clinical features, laboratory and imaging examination results, diagnoses, treatments, and outcomes. Patients were excluded if they met any of the following criteria: (1) age≥18 years old; (2) HIV infection; (3) mNGS was not performed; (4) medical record was incomplete.2.22.2.1First 3-4mL of blood was drawn from patients, placed in EDTA tubes, and stored at room temperature for 3–5 minutes before plasma separation and centrifuged at 1,600 g for 10 min at 4°C within 8 hours of collection. Plasma samples were transferred to sterile tubes. DNA was extracted from 300 μL of plasma using the TIANamp Micro DNA Kit  following the manufacturer’s instructions. The extracted DNA specimens were used for the construction of DNA libraries.BALF was collected based on the standard clinical procedure. Briefly, 20 mL saline was injected into a segmental bronchus and drawn back after a while. Next, 3 mL of BALF was inactivated at 65°C for 30 minutes immediately after collection. Subsequently, a 1.5-mL microcentrifuge tube with 0.5-mL sample and 1 g 0.5-mm glass beads were attached to a horizontal platform on a vortex mixer and agitated vigorously at 2800–3200 prm for 30 min. Finally 0.3-mL sample was collected into a new 1.5-mL microcentrifuge tube and DNA was extracted using the TIANamp Micro DNA Kit  according to the manufacturer’s instructions.According to the protocol of the BGISEQ-50 sequencing platform, the DNA library was constructed through DNA fragmentation, end-repair, adapter-ligation, and PJR amplification. The constructed library was qualified by Agilent 2100  and Qubit 2.0. The qualified double-strand DNA library was transformed into a single-stranded circular DNA library through DNA-denaturation and circularization. DNA nanoballs (DNBs) were generated from single-stranded circular DNA using rolling circle amplification (RCA). The DNBs were qualified using Qubit 2.0. Qualified DNBs were loaded into the flow cell and sequenced  on the BGISEQ-50 platform.2.2.2ftp://ftp.ncbi.nlm.nih.gov/genomes/). RefSeq contains 4,061 whole-genome sequences of viral taxa, 2,473 bacterial genomes or scaffolds, 199 fungi, and 135 parasites associated with human diseases. The number of unique alignment reads was calculated and standardized to get the number of reads stringently mapped to pathogen species (SDSMRN) and the number of reads stringently mapped to pathogen genus (SDSMRNG).High-quality sequencing data were generated by removing low-quality and short (length < 35 bp) reads using in-house software, followed by computational subtraction of human host sequences mapped to the human reference genome (hg19) using Burrows–Wheeler Alignment. After the removal of low-complexity reads, the remaining data were classified by simultaneously aligning to four Microbial Genome Databases, consisting of viruses, bacteria, fungi, and parasites. The four Microbial Genome Databases were downloaded from NCBI  and regular maintenance therapy with prednisone (10 mg/d) during the treatment of the underlying disease.Case 1, male, 3 years old, with underlying disease of autoimmune encephalitis, was treated with rituximab  at presentation.Case 3, male, 8 years old, was in a post-transplant state and was taking tacrolimus regularly.Case 4, male, 14 years old, in post-renal transplant status, on regular Tacrolimus.Case 5, male, 6 years old, with underlying disease of special sarcoma, treated with VDC regimen  before the onset of the disease. five children had no abnormal personal, growth and developmental, or family history.3.2Cases 1, 4, and 5 started with chest tightness after activity, shortness of breath, and paroxysmal dry cough, and developed fever with a fever peak of 39°C or higher on the 2nd_4th day of illness. Cases 2 and 3 started with fever peaking at 39°C with dry cough. Five children developed respiratory distress within 3_4 days after the onset of the disease and were mechanically assisted with ventilation on day 3 in case 1, on day 10 in case 2 and on day 7 in cases 3, 4 and 5. In case 2, coarse breath sounds and a few dry rales could be heard in both lungs at the onset of the disease, and in the other four children, coarse breath sounds could be heard in both lungs as far as possible.3.3Peak LDH (233_2002) U/L, peak BDG (10.00_2470.6) pg/mL, and CD4 lymphocyte count (18.32_985)/μL in five children Table 1.3.4All five children showed multiple flocculent high-density images in both lungs on chest CT Figure 23.53.5.1The five children had gene sequence numbers of 340, 3, 1797, 27025, and 63156, respectively, with mNGS showing positive PJP. These children were co-infected with other pathogens, such as Streptococcus pneumoniae and cytomegalovirus Table 2.3.5.2Case 1 and case 4 results suggest positive, case 3 and case 5 results suggest negative, case 2 was not tested.PJ and encapsulation were seen in the BALF of case 1 .3.6Enterococcus faecalis and Streptococcus vestibularis. After 9 days of combined treatment with meropenem and teicoplanin and 3 days of caspofungin treatment, the child still had an intermittent fever and respiratory distress, and the oxygen saturation was maintained at about 90% by transdermal oxygen measurement under 5 L/min of face mask inhalation. Case 1, 3, 4 and 5 were cured and Case 2 was dead , a negative serum BDG is sufficient to exclude PJP; however, a positive serum BDG is not specific for the diagnosis of PJP, necessitating further tests to confirm the diagnosis .In addition, despite the great sensitivity of the BDG test, it can still give false positive results in cases of hemodialysis, gram-negative bacteremia, severe mucositis or intravenous immunoglobulin and certain antibiotics. Therefore, the BDG level alone cannot be used to diagnose PJP . Clinicain vitro culture, PCR) in the detection of MTB, bacteria, fungi, mycoplasma, and viruses (Polymerase chain reaction (PCR) for PJ was initially developed in the 1980s with primers against the pneumocystis mitochondrial large-subunit ribosomal gene . PCR has viruses .The advantages in mNGS over traditional detection methods include a shorter detection cycle, a wider detection range, and a higher positive rate. This makes mNGS particularly suitable for conditions such as pathogenic bacteria that cannot be clarified by traditional detection methods or for clinical aspects that require rapid clarification of pathogenic bacteria. In comparison with BDG and hexamine silver staining, mNGS showed a sensitivity of 100% and a specificity of 96.3% in the diagnosis of PJP, according to research by However, case 2 tested negative when the blood sample was tested on day 5 after the onset of the disease and only tested positive when the blood sample was tested again for mNGS on day 10 after disease onset. In four of the five children in our study, PJ was measured when BALF was tested within 3_5 days after the onset of the disease. This phenomenon suggests that BALF should be utilized as the test sample as much as possible in the clinical use of mNGS for the diagnosis of PJP. If only the blood specimen can be tested and the clinical manifestations and laboratory findings are highly suggestive of PJP, PJP cannot be excluded because of the first negative result, and mNGS should be performed at least once, but ideally more times. In addition, it is important to keep in mind that the microbial sequences reported by mNGS include potentially pathogenic organisms. As a result, the clinical setting must be properly considered when interpreting mNGS data. Although the list can help to qualify the range of pathogenic organisms, mNGS cannot yet accurately determine whether the flora is colonized or infected.PJ was detected in all five patients using mNGS with a sensitivity of 100%, whereas when PJ and encapsulation were discovered using pathological staining and microscopy on tissues from four patients, the sensitivity was only 50%. The mNGS experimental cycle is shorter than the pathology assay, with data available in 48 hours as opposed to the pathology assay cycle, which is typically 3 days, and the mNGS assay was more sensitive in this study. The pathology results are more influenced by the technician’s experience, while it is not necessary for the technician to be familiar with PJ for mNGS to simultaneously identify and compare all microbial genomes in a sample for analysis. The diagnosis PJ infections can be greatly aided using mNGS, which can also help doctors to quickly identify pathogenic organisms and administer prompt therapy to enhance the effectiveness of their interventions.Several limitations of the study should be mentioned. First, the major limitation of this study is the small number of patients, due to the rarity of the disease. Second, this is a single center retrospective study; thus, intrinsic bias was unavoidable. Third, diagnostic performances of mNGS and PCR were not compared in this study because PCR of PJ was not routinely performed in our hospital. This line of query should be explored in future work.In conclusion, to lessen the burden of NH-PJP patients, there is an urgent need for improved preventive and treatment measures. This is supported by the epidemiology, morbidity, and mortality of NH-PJP. Patients with suspected PJP should start anti-PJP specific medication immediately to prevent treatment delays brought on by diagnostic procedures such as bronchoalveolar lavage, which increases the need for mechanical assisted ventilation and disease mortality. The most crucial element in reducing NH-PJP related mortality is a straightforward, precise, and easily accessible diagnostic approach. The combination of BALF-mNGS should be used clinically whenever possible to improve the detection rate of NH-PJP, but the results should be interpreted with a comprehensive consideration of the actual clinical situation because mNGS is a new microbiological assay with some advantages over traditional assays in identifying Pneumocystis.https://www.ebi.ac.uk/ena/browser/view/PRJEB59277.The original contributions presented in the study are publicly available. This data can be found here: The studies involving human participants were reviewed and approved by The First Affiliated Hospital of Zhengzhou University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JN and JW collected the original clinical data and processed the statistical data. JN drafted and edited the manuscript. PJ and MZ participated in the design, and EW revised the manuscript. All authors contributed to the article and approved the submitted version.