Drain-associated intracerebral infections are life-threatening emergencies. Their treatment is challenging due to the limited penetration of antibiotics to the site of infection, resulting in potentially inadequate exposure. The emergence of multidrug-resistant pathogens might force the use of off-label intrathecal (IT) doses of antibiotics. We reviewed the literature on general aspects determining intrathecal dosing regimen, using pharmacometric knowledge. We summarised clinical experience with IT doses of antibiotics that are usually not used intrathecally, as well as the outcome of the cases and concentrations reached in the cerebrospinal fluid (CSF). Factors determining the IT regimen are the size of the ventricle system and the CSF drainage volume. With regard to pharmacometrics, pharmacokinetic/pharmacodynamic indices are likely similar to those in non-cerebral infections. The following number (N) of cases were described: benzylpenicillin (>50), ampicillin (1), ceftazidime (2), cephaloridine (56), ceftriaxone (1), cefotiam (1), meropenem (57), linezolid (1), tigecycline (15), rifampicin (3), levofloxacin (2), chloramphenicol (3) and daptomycin (8). Many side effects were reported for benzylpenicillin in the 1940–50s, but for the other antibiotics, when administered correctly, all side effects were minor and reversible. These data might help when choosing an IT dosing regimen in case there is no alternative option due to antimicrobial resistance. The incidence of community-acquired meningitis caused by multi-resistant micro-organisms is still low; however, these infections after neurosurgery might become more prevalent due to the overall rise in antibacterial resistance ,2. InfecBoth the blood–brain barrier and the blood–CSF barrier protect the central nervous system (CNS) from toxic compounds present in the systemic circulation. However, in the treatment of cerebral infections, both barriers complicate the achievement of adequate concentrations of antibiotics at the site of infection. Therefore, in some cases, extremely high intravenous doses have been administered, such as 24 g/24 h cefotaxime, 15 g/24 h meropenem or 8 g/24 h imipenem ,4,5. AnoCurrently, the number of antibiotics that are frequently administered, intrathecally, is limited. Only polymyxin B is registered by the FDA, and the EMA recommends maximum IVT doses of colistin of 125,000 IU ,7. HowevFor several antibiotics, IT use has been reviewed ,9,10. ThAs antibacterial efficacy is dependent on a certain antibiotic exposure that needs to be reached at the site of infection, the amount of antibiotics reaching the CSF as well as the resulting concentration–time profiles in the CSF are of importance. Several factors might be taken into consideration in choosing a dosing regimen for IT administration. Both the blood–brain barrier (BBB) and the blood–CSF barrier (BCSFB) are lipid layers surrounding the CNS aiming to protect the brain from toxic agents present in the systemic circulation. These barriers thereby limit the penetration of antibiotics to the site of infection when treating meningitis or ventriculitis. For example, the penetration of meropenem into the CSF in patients with (suspected) ventriculitis has a mean of only 9–18% ,16, and Meningeal inflammation during infection leads to the opening of tight junctions in the BBB. The level of inflammation, which is more pronounced in meningitis as compared to that in ventriculitis might, for some antibiotics, lead to an increased penetration into the CSF ,18. ThisStreptococcus pneumoniae and Escherichia coli might result in a relatively low permeability, while the penetration into the CSF in infections caused by Haemophilus spp. and Listeria monocytogenes is higher .,82.81,82Quinolones are known to have a potent seizure-inducing activity ,119. OveMycobacterium tuberculosis. Intraventricular doses of 1–2 mg per dose were administered for a prolonged duration without causing serious side effects. In one case, the levofloxacin dose was calculated assuming a CSF volume of 120 mL and a target CSF concentration of 8–10 mg/L [Two cases of intraventricular administration of levofloxacin have been described and summarised in –10 mg/L . During –10 mg/L . Both paChloramphenicol is an antibiotic that is little used in industrialised countries, but it is included in the WHO list of essential medicines to be used for several indications, such as meningitis . Three cIn all three cases that were described in detail, no side effects were reported . But, inDaptomycin is a cyclic lipopeptide that is used to treat infections due to Gram-positive micro-organisms. After systemic administration, daptomycin penetrates poorly into the CSF compartment, due to its low lipophilicity, significant protein binding and large molecular weight, thus resulting in insufficient concentrations to treat intracerebral infections ,121. In Intrathecal administration of the antibiotics described in this review is off-label and is limited to those patients for whom clinicians run out of therapeutic options. When used without administration/preparation errors, and with the exception of benzylpenicillin, the reported side effects are generally mild and reversible. As side effects have been reported after both IV and IT administration, and due to the lack of clear correlation between CSF concentrations and toxicity, it could be questioned whether the administration of extremely high IV doses puts patients at lower risk of side effects compared to IT administration. It is possible that the CSF concentration that is aimed for causes a toxic effect regardless of the route of administration. With the current limited knowledge of PK/PD in the CSF, it is not possible to choose an evidence-based efficacious dosing regimen, and also, routine TDM is not recommended in the literature. Generally, for lipophilic drugs with a molecular weight > 1000 g/mol and hydrophylic drugs with a molecular weight > 400 g/mol, once-daily IT dosing is usually performed . For antA search was conducted in PubMed using the name of the individual antibiotic in combination with ‘intrathecal’ or ‘intraventricular’. References from the papers included were also checked to find missing references. The antibiotics selected were based on those mentioned in the review paper of Nau et al.  as antib


Flow Cytometry - A Powerful Tool for Diagnosis and Therapy Monitoring in Hematology and Immunology, a Research Topic hosted by Frontiers in Medicine (Hematology), was launched in October 2021. The aim was to offer a platform to different research groups, that use flow cytometry for diagnosis and therapy monitoring in hematology and immunology, to present their valuable work and results. A total of five original research articles were published, together with two case reports and one review article. The topics were diverse, but connected by flow cytometry, which was the core method employed by all research teams.Hiza et al. used flow cytometry to measure the expression of CD38 or CD27 on CD4+ T cells producing interferon gamma and/or tumor necrosis factor alpha in patients with tuberculosis. This disease is still one of the main causes of morbidity and mortality due to an infectious agent, despite being treatable using a combination of drugs  is an intestinal illness with a significant economic effect on pig industry and is produced by Lawsonia intracellularis, an obligate intracellular bacterium  cell therapy coupled with granulocyte-macrophage colony-stimulating factor, in an attempt to improve neutropenia, uphold recovery of cellular immunity, and boost CAR T-cell expansion. CAR T cell therapy is currently being used as a treatment option in several hematological cancers. Nevertheless, its effectiveness is sometimes limited due to insufficient persistence within the host and multiple side effects  and T-lymphocyte activation  in the peripheral blood of children with Hodgkin's lymphoma (HL), to identify the potential prognostic factors for event-free survival. HL is one of the most frequent tumoral diseases diagnosed worldwide. Recent studies show that HLA-DR+/CD38 T cells may be related to relapse and refractoriness in pediatric HL  patients in terms of numbers, distribution across maturation stages or inhibitory receptors expression. They correlated this information with the genetic background offered by killer immunoglobulin-like receptors (KIRs) and the HLA-C genotypes. AML is a hematological malignancy that is heterogeneous, clinically, morphologically as well as genetically. It results from the clonal expansion of blasts of any of the myeloid lineages in the peripheral blood, bone marrow as well as other tissue (r tissue . Previour tissue . This stPopa et al. presented a case report concerning a pediatric patient with T-acute lymphoblastic leukemia in which flow cytometric immunophenotyping at diagnosis and during treatment was performed. Despite the various therapeutic measures that were used which included a stem cell allotransplantation, a chemoresistant clone persisted.Tadros et al. presented a case report where the early diagnosis of a hairy cell leukemia was possible by flow cytometric analysis of a small leukocyte population that exhibited a higher side scatter and brighter CD19/CD20. A total of 1,000,000 events were analyzed, which allowed a better study of the neoplastic subpopulation. Thus, flow cytometric analysis enabled the therapeutic decision and disease progression toward remission.Munteanu et al. reviewed the flow cytometric evaluation of the immunological effects of several vitamins, such as: the role of vitamins E in the prevention and treatment of different types of cancer, the properties of K vitamins in the development and maintenance of PC12 cells in Parkinson's disease, the effect of vitamin B5 on the loss of bone mass in low estrogen conditions, the anticancer role of vitamins B6, the role of Vitamin B9 in the regulation of Treg cells.Last, but not least, MC: Conceptualization, Writing—original draft. RG: Conceptualization, Formal analysis, Writing—review and editing. HB: Conceptualization, Formal analysis, Writing—review and editing. DS: Conceptualization, Formal analysis, Writing—review and editing. CT: Conceptualization, Formal analysis, Writing—review and editing.


Psoriasis (PSO) is a chronic skin condition that affects a variety of disorders, especially the cardiovascular system. This study investigated the association between PSO and peripheral arterial disease (PAOD).A retrospective cohort study design was carried out between 2000 and 2018. The exposure subject was a newly diagnosed PSO. The diagnosis of PSO was never elaborated as a comparison subject. Balanced heterogeneity of the two groups was used by propensity score matching. The cumulative incidence of PAOD between the two groups was performed using Kaplan-Meier analysis. The Cox proportional hazard model was used to measure the risk of PAOD risk hazard ratio.After matching the 1: 1 propensity score, 15,696 subjects with PSO and the same number of subjects without the diagnosis of PSO were recruited. The PSO subject had a higher risk of PAOD than the non-PSO subject . In the 40-64-year-old subgroup, the subject of PSO exhibited an increased risk of PAOD than the subject without PSO.Psoriasis is associated with an increased risk of peripheral arterial disease and curative care is necessary to reduce the risk of PAOD.. Psoriasis (PSO) is a chronic, recurrent, inflammatory disease. More than 125 million people, approximately 1%–2% of the general population worldwide, had PSO in 2020 . AlthougEvidence showed that PSO can contribute to cardiovascular risk. For example, chronic inflammation in PSO patients increased the accumulation of visceral adipose tissue and the noncalcified burden of the coronary artery . PSO patPeripheral arterial occlusive disease (PAOD) is an atherosclerosis that usually occurs in the lower limb artery with the formation of arterial thrombosis or embolism. Initially, PAOD is not easily recognized unless intermittent claudication is evident . Severe Not only can PAOD develop into serious diseases, but it also increases the risk of developing other cardiovascular diseases (CVDs), such as coronary heart disease and myocardial infarction (MI) , 16. PSOPathological changes in PSO and PAOD have certain common mechanisms based on the immune response. For example, significantly higher serum levels of cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin 4 (IL-4), and interleukin 6 (IL-6) are observed in both diseases , 21. A βConsidering the increased risk of CVD and common immunological markers for PSO and PAOD, we enrolled both patients with and without PSO from the 2000 Longitudinal Health Insurance Database (LHID 2000) and investigated whether PSO influences the risk of PAOD. We hypothesized that patients with PSO would have a significantly higher risk of PAOD.We retrieved a longitudinal health insurance database that was managed by the Health and Welfare Data Center (HWDC) of Taiwan. There are 2 million random sampling subjects from the registry of beneficiaries in the whole population for the year 2000. The database included data on outpatient, emergency, and hospitalization medical records, including medication, surgical operation, procedure, and expenditure data from 2000 to 2018. This study was certified by the institutional review board of Chung Shan Medical University Hospital (CS1-20056).We conducted a retrospective cohort study. The study included individuals who were newly diagnosed with PSO  at onset time from 2002 to 2017. To ensure accuracy, patients were included if any only if their diagnosis was given by a rheumatologist or dermatologist for ≥3 times outpatient visits or ≥1 inpatient stay. The index date was defined as the date of initial diagnosed PSO. In order to ensure that all instances of PAOD would be new onset PAOD, patients with a diagnosis of PAOD before the index date were excluded. The non-PSO group was defined as never diagnosed psoriasis and similar disorders  from 2000 to 2018.The primary outcome was a diagnosis of PAOD . All samples were traced until the outcome of interest, death, or the end of 2018, whichever came first.The demographic characteristics included in the analysis were age and sex and the comorbidities of hypertension , hyperlipidemia , chronic liver disease , chronic kidney disease , diabetes , chronic obstructive pulmonary disease , ischemic heart disease , stroke , and intracranial bleeding . An individual was defined as having these comorbidities if and only if they received a diagnosed within 1 year before the index date for ≥3 outpatient visits or ≥1 inpatient stay.First, a 1:4 age and sex matching was performed to ensure an identical index date for any given PSO participant with their non-PSO counterparts. If the non-PSO group had a previous diagnosis of PAOD when matched to the corresponding index date, that non-PSO group would be excluded and rematched with a new comparison group. Subsequently, the propensity score matching (PSM) was carried out by age, sex, and comorbidities, including the presence of hypertension, hyperlipidemia, chronic liver disease, chronic kidney disease, diabetes, chronic obstructive pulmonary disease, ischemic heart disease, stroke, and intracranial bleeding comorbidities. The propensity score was calculated using a binary logistic regression. The binary variable was the diagnosis of PAOD. PSM ensured the balanced heterogeneity of the two groups.The comparison of the PSO and non-PSO groups was performed using the absolute standardized difference (ASD). An ASD <0.1 indicated similarity in the characteristics of both groups . A PoissIn total, 16,054 patients with PSO and 1,887,483 patients who had never been diagnosed with PSO were recruited. After excluding patients with a diagnosis of PAOD before the index date, 15,677 patients were in the PSO cohort. Subsequently, 1:1 PSM by age, sex, and comorbidity was analyzed. Finally, 15,659 patients in the PSO cohort and 15,659 patients without PSO in the matched cohort were analyzed for their risk of PAOD . The meap = 0.017) showed that the cumulative incidence of PAOD was higher in the PSO group than in the non-PSO group  and 1.35 (95% C.I. = 1.17–1.55), respectively. The relative risk of developing PAOD in PSO group compared to non-PSO group was 1.26 (95% C.I. = 1.04–1.51) . The meaSO group .p for interaction < 0.001; The PSO group had an increased risk of PAOD . Patients aged 20 to 39 years , 40 to 64 years  and ≥65 years  had a higher risk of PAOD. Men had an increasing risk of PAOD than women . Furthermore, comorbidities such as hypertension, chronic kidney disease, diabetes, and stroke were also risk factors for PAOD . In the We enrolled 16,054 patients with newly diagnosed PSO from LHID 2000 from 2002 to 2017 in this study. We analyzed the association between PSO and the risk of PAOD using PSM. Our results showed that patients with PSO had a higher risk of acquiring PAOD than the non-PSO group. However, few studies have evaluated the risk association between PSO and PAOD. A case report described the co-occurrence of PSO with occlusive vascular disease and even gangrene on the lower limb in one patient . A case–PSO has been shown to increase the risk of CVD such as myocardial infarction, hypertension, valvular disease, and arrhythmia –34. PSO We also found that the PSO group had an increased risk of PAOD with increasing age using the Cox proportional hazards model. A subgroup analysis indicated that patients aged 40–64 years had an increased risk of PAOD in the PSO group. A cross-sectional study showed that, especially for symptomatic PAOD, prevalence rates were higher in elderly groups between 50 and >70 years . ElderlyIn addition to age, sex was a risk factor for PAOD. Male patients had a higher risk of PAOD than female patients. Sex-based differences in PAOD have been demonstrated in different studies. A study found a close relationship between visceral adiposity index and peripheral artery disease (PAD) in normal weight adults with hypertension among men but not among women . In contPSO may increase the risk of different diseases. The development of uveitis after PSO diagnosis has been reported . PSO incAlthough both diseases have common inflammatory pathways, the increased risk of PAOD with PSO may be due to common factors for both diseases, such as the overproduction of pro-inflammatory factors. PSO causes inflammation of the blood vessels, which is a critical step in initiating atherogenesis and the development of atherosclerosis . Pro-infRegardless of the actions of pro-inflammatory factors in blood vessels, serum markers can also be factors in PSO and PAOD. For example, galectin-3, which belongs to a member of galectins, a group of β-galactoside-binding proteins that exhibit biological functions such as cell–cell interaction, cell proliferation, and differentiation. Galectin-3 is postulated to be involved in the pathogenesis of a series of diseases, including the formation of psoriatic plaques and arthrosclerosis . FurtherThis study still has certain limitations. First, LHID does not provide information about the severity or different forms of PSO, which may affect the risk and onset of PAOD. Second, the database does not provide data on whether the lesion is located in the lower extremity. However, this could directly influence the risk of PAOD. Third, the database did not provide information on smoking, alcohol consumption, physical activity, diet, inflammatory markers, disease progression, disease markers, body mass index (BMI), and other areas were not provided by the database; these factors are potential confounders in this study. Regardless, by using the propensity score matching in the age, sex, and comorbidities, we can reduce the potential heterogeneity of the two groups.In conclusion, our study demonstrates that having PSO significantly increases the risk of PAOD. Patients with PSO should be especially careful to prevent PAOD and other cardiovascular diseases. Additionally, age is a risk factor for PAOD, especially in patients with PSO 40–64 years of age.


The adjusted logistic regression showed that participants with chronic non-communicable diseases (CNCDs) , diabetes , and obesity  were associated with a greater propensity to use polypharmacy. Conclusion: Our results show that older adults with CNCDs, diabetes, and obesity were more likely to use polypharmacy. The results reinforce the importance of using CGA in clinical practice in PHC.Background: With the aging population comes greater risks associated with polypharmacy, a significant public health problem. Objective: This study aimed to identify the prevalence of polypharmacy and its associated factors through Comprehensive Geriatric Assessment (CGA) among older adults treated in primary health care (PHC) in a large Brazilian urban center. Methods: We conducted a cross-sectional study with a random sampling of 400 older adults using primary health care. Polypharmacy was defined as the cumulative use of five or more daily medications. An assessment of a sociodemographic and health survey, fear of falling, and physical disabilities affecting activities of daily living and instrumental activities of daily living was conducted. Results: The mean age was 75.23 (SD: 8.53) years. The prevalence of polypharmacy and hyperpolypharmacy was 37% ( The world’s population is aging rapidly, especially in developing countries such as Brazil . With thThe indiscriminate use of polypharmacy is a significant public health challenge . There in = 2,967,952) identified that polypharmacy (≥5 drugs) increases the risk of death by 1.28 times, and hyper polypharmacy (≥10 drugs) increases the risk of death by 1.44 times [Several studies reinforce that the use of polypharmacy is associated with worse physical and mental health outcomes and increased mortality ,9,10. A 44 times , falls [44 times , dementi44 times , and dia44 times .According to the Pan American Health Organization, active aging should be able to maintain and improve health, and the indiscriminate use of polypharmacy can go against the concept of healthy aging as there is no maintenance of quality of life ,16. In aHealth professionals and researchers are very interested in exploring the impacts of polypharmacy on the health and quality of life of the older population. In 2019, polypharmacy was included as one of the three main areas of action of the third Global Patient Safety Challenge of the World Health Organization . In thisThis study is part of a longitudinal multidimensional research project with older adults in the city of São Paulo, SP, Brazil . This isp < 0.20 in the bivariate logistic regression), 13.5% prevalence of polypharmacy [p-value, α = 0.05, and 400 participants. The post hoc analysis showed a statistical power of 98%.The study was carried out at the Marcus Wolosker (Belenzinho) BHU by one of the authors, who has a Ph.D. in Collective Health and 10 years of experience as a nurse in primary health care (PHC). In 2018, Belenzinho BHU had around 5000 people aged 60 and over. We use the G*Power program 3.1.9.7  to calculate the statistical power of the analyses. Aiming to perform multivariate logistic regression models, with 11 independent variables  were carried out individually in a private environment during the nursing consultation. People aged 60 or over and registered at Belenzinho BHU participated in the survey. Participants with a medical diagnosis of dementia, Alzheimer’s, or severe cognitive or physical impairment did not participate in the research.Although there is no consensus on the concept of polypharmacy , we adopted the most used concept among health professionals and researchers, which is the use of 5 medications or more per day . The folRegarding sociodemographic variables, the following were evaluated: age group ; gender ; marital status (with or without a partner); knowing how to read or write (yes or no); years of study ; lives alone (yes or no); satisfaction with life (yes or no); health perception ; chronic illness  (yes or no); daily medication use (yes or no); smoking (yes or no) and history of falls (yes or no); last fall < 12 months (yes or no); basic activities of daily living  , developed in 2005 by Yardley and validated for Portuguese in 2010 . This scBasic activities of daily living (BADL): Assessed using the KATZ index—a tool developed in 1976 by Sidney Katz. It was validated for Portuguese in 2008 and used the degree of independence in the result of six functions . The resInstrumental activities of daily living (IADL): Lawton Scale—developed by Lawton and Brody in 1969. It was validated for Portuguese in 2008 and was used to assess the instrumental activities of daily living (IADL) . It is bData were analyzed using the Statistical Package for Social Sciences—SPSS 26 program . A descriptive analysis (absolute frequency and relative frequency) was performed to describe the sociodemographic and health characteristics of the participants.p < 0.10 in the unadjusted logistic regression analysis were included in the multivariate logistic regression model . The logistic regression model was run using the “stepwise forward selection” technique. A significance level of 5% was chosen for the test, with a 95% confidence interval.Hypothesis tests were performed as follows. First, a series of unadjusted logistic regression models were performed using the polypharmacy outcome (0 = no polypharmacy or 1 = with polypharmacy) and the independent variables. Second, the variables that presented n = 148), and of hyperpolypharmacy (≥10 per day) was 1% (n = 4). We identified that participants with a chronic illness were more likely to use polypharmacy (p < 0.001). We did not identify a significant difference in the other comparisons (p > 0.05).A total of 488 participants were invited to participate in the survey, of which, 400 (83.33%) completed all items in the questionnaire. p = 0.028).The description of CNCDs by systems is shown in p = 0.035), with CNCDs , with musculoskeletal disease , hypertension , diabetes , and obesity  were associated with a greater propensity to use polypharmacy.p = 0.003), diabetes , and obesity  were associated with a greater propensity to use polypharmacy.This study investigated the prevalence of polypharmacy among older adults assisted in PHC and, through a CGA, verified the factors associated with polypharmacy. Although the prevalence of polypharmacy found in the literature is heterogeneous, our results were similar to those in the literature compared with Brazilian and international evidence ,8,10,12.The prevalence of polypharmacy in the present study is in line with previous evidence ,29. DepeWe identified that the presence of CNCD was strongly associated with a greater propensity for polypharmacy. Several studies reinforce this relationship ,18,32. APolypharmacy in people with diabetes is associated with increased all-cause mortality, macrovascular complications, and hospitalization . With reThis study has some limitations that should be identified. First, the research was based on self-reported information based on memories, which may imply information bias. Second, the definition of polypharmacy was based on a drug cohort of ≥5 therapeutic classes; however, participants may be taking non-prescription medications. Third, the prevalence of polypharmacy estimated in our study may be underestimated and may be much higher. Fourth, the use of ORs from logistic regression may have overestimated the associations, and given the high prevalence of polypharmacy, we recommend future studies of these types of methods that can estimate prevalence ratios, such as log-binomial modeling or Poisson regression. Finally, this study was carried out with users of a BHU. Thus, the findings may be generalizable only to BHU users.Finally, this study has clinical implications that should be highlighted. The results of our study reinforce the need for greater surveillance of the use of prescribed medications and multidisciplinary interventions that optimize the balance between benefits and harm in the prescription of medications . ClinicaThis study shows that the prevalence of polypharmacy is high among older people in our study. Our results show that older adults with CNCDs, diabetes, and obesity were more likely to use polypharmacy. These results reinforce the importance of using Comprehensive Geriatric Assessment in clinical practice in PHC.


Mastitis is one of the most common diseases in the dairy industry in the world, and its treatment cost is also the most expensive of all diseases in the dairy industry. The research on the rapid diagnosis of cow mastitis is of great significance to the development of the dairy cow industry all over the world. In this study, we use microbiology and metabolomics techniques to explore the differential microbiota and metabolites in cow milk at different somatic levels, to find new biomarkers for the diagnosis of cow mastitis and provide a new plan for the diagnosis, prevention, and treatment of mastitis in the future.p < 0.05), and there was a significant correlation between microbiota and metabolites. Meanwhile, in this experiment, 75 differential metabolites were identified in the H group and L group, 40 differential metabolites were identified in the M group and L group, and six differential microorganisms with LDA scores more than four were found in the H group and L group. These differential metabolites and differential microorganisms may become new biomarkers for the diagnosis, prevention, and treatment of cow mastitis in the future.Dairy cow mastitis is one of the common diseases of dairy cows, which will not only endanger the health of dairy cows but also affect the quality of milk. Dairy cow mastitis is an inflammatory reaction caused by pathogenic microorganisms and physical and chemical factors in dairy cow mammary glands. The number of SCC in the milk of dairy cows with different degrees of mastitis will increase in varying degrees. The rapid diagnosis of dairy cow mastitis is of great significance for dairy cow health and farm economy. Based on the results of many studies on the relationship between mastitis and somatic cell count in dairy cows, microflora, and metabolites in the milk of Holstein cows with low somatic cell level , medium somatic cell level  and high somatic cell level  were analyzed by microbiome and metabolic group techniques. The results showed that there were significant differences in milk microbiota and metabolites among the three groups ( Milk is the most important source of dairy products for human beings. It is one of the ideal natural foods for human beings . In addiThe prominent feature of mastitis in cows is the presence of varying degrees of inflammation in the mammary glands, accompanied by a series of pathological changes. Among these changes, there is a profound relationship between the somatic cell count (SCC) in milk and mastitis in cows ,7,8. DaiExploring a more efficient and automatic method for the diagnosis of dairy cow mastitis was an area of intense research during the 1960s . With thStreptococcus agalactiae and Staphylococcus aureus is the most prevalent pathogen causing mastitis [Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli, are commonly isolated and cultured from milk samples of cows with clinical mastitis [Enterobacteriaceae, Streptococcus, Lactococcus, and Prototheca are often considered to be the pathogens of mastitis in modern dairy herds [In the late 1800s, there were studies that cow mastitis was caused by bacteria . After hmastitis . In addimastitis . At the ry herds ,31. Addiry herds . Studiesry herds . Therefory herds ,28.In recent years, with the continuous development of more and more new technologies, the search for biomarkers of disease has become a major research boom. Biomarkers are indicators to detect the biological process and pathological state of the body, which can reveal a variety of health and disease characteristics . AccordiIn this experiment, microbiology and metabolomics techniques were used to study the raw milk of dairy cows with different somatic cells, aiming to explore the differences and correlations between microorganisms and metabolites in the milk from different somatic cells. It is expected that this study will assist researchers in identifying the differences in microorganisms and metabolites between healthy cows and cows with different somatic milk. This analysis will enable the identification of biomarkers for cow mastitis, providing a foundation for researchers to further understand the overall metabolic mechanism of cow mastitis. Moreover, it will offer new ideas for the rapid diagnosis and effective prevention of mastitis, ultimately improving animal welfare. Improve the output and quality of milk and reduce the economic loss of the dairy industry. An in-depth study of important differential metabolites and microorganisms in different somatic levels of milk has important theoretical significance.In this experiment, based on the DHI records of a dairy farm in Guangdong Province, China over the past three months, 180 dairy cows were selected for routine milking. Before sampling, we first clean the cow’s udder using warm water at 40 °C; for the initial cleansing. Then, the cows’ four udders were washed with a 0.1% potassium permanganate solution. During the sampling process, the first three streams of milk were discarded, and then samples were taken from each of the cows’ four teats. Each milk sample collected per teat was 10 milliliters, and three samples were collected from each cow and placed in 50-milliliter sterile centrifuge tubes. After collecting the milk samples from each cow, they were quickly placed in a foam box containing dry ice for freezing. Once all the samples were collected, they were transported to the laboratory and frozen at −80 degrees Celsius. One of the samples collected from each cow was then taken out for SCC (somatic cell count) determination. Based on the SCC data, the cows’ previous history of mastitis, and their parity, 30 cows were selected for further analysis. Furthermore, 30 cows were selected and divided into 3 groups: 10 cows with low SCC (L group) ; 10 cows with medium SCC (M group) , and 10 cows with high SCC (H group) .® High-Fidelity PCR Master Mix with GC Buffer of New England Biolabs, and high efficiency and high-fidelity enzyme to ensure the efficiency and accuracy of amplification. The PCR products were detected by 2% agarose gel electrophoresis; the qualified PCR products were purified by magnetic beads, and the same number of samples were mixed according to the concentration of PCR products by enzyme labeling quantification. After being fully mixed, 2% agarose gel electrophoresis was used to detect PCR products, and the gel recovery kit provided by the Qiagen company was used to recover the products. Finally, the library was constructed by using TruSeq® DNA PCR-Free Sample Preparation Kit library construction kit, and the constructed library was quantified by Qubit and Q-PCR. After the library was qualified, NovaSeq6000 was used for computer sequencing.For the extraction and PCR amplification of microbial genomic DNA, the genomic DNA of the sample was extracted by CTAB or SDS method, and then the purity and concentration of DNA were detected by agarose gel electrophoresis. Take the selected 30 aliquots of DNA samples and transfer them into centrifuge tubes, then dilute the samples to a concentration of 1 ng/μL with sterile water. Using diluted genomic DNA as a template, according to the selection of sequencing region, PCR was performed using specific primers with Barcode, PhusionV/V) containing internal standard was added to a 50 microliter sample. Then, the sample was vortex for 3 min and centrifuged at 13,400 g for 10 min (4 °C). A 150 μL aliquot of the supernatant was collected and placed at −20 °C for 30 min, and then centrifuged at 13,400 g for 3 min (4 °C). 120 μL aliquots of supernatant were transferred for LC-MS analysis. All samples were acquired by the LC-MS system following machine orders. The analytical conditions were as follows, UPLC: column, Waters ACQUITY UPLC HSS T3 C18 ; column temperature, 40 °C; flow rate, 0.4 mL/min; injection volume, 2 μL; solvent system, water (0.1% formic acid): acetonitrile (0.1% formic acid); The column was eluted with 5% mobile phase B (0.1% formic acid in acetonitrile) at 0 min followed by a linear gradient to 90% mobile phase B (0.1% formic acid in acetonitrile) over 11minutes, held for 1 min, and then come back to 5% mobile phase B within 0.1 min, held for 1.9 min.A total of 30 samples were stored at −80 °C and were thawed on ice and vortexed for 10 s. A 150 μL extract solution  to assemble the reads of each sample. The splicing sequence obtained is the original Tags data (Raw Tags). The spliced Raw Tags needs to be strictly filtered to acquire high-quality Tags data (Clean Tags). Then, the Mathur method was used to analyze the species annotation with SILVA138.1 ‘s SSU rRNA database (the threshold was set to 0.8:1). The taxonomic information was obtained, and the community composition of each sample was calculated at each classification level: phylum, class, order, family, genus, and species. The phylogenetic relationships of all OTU/ASV representative sequences were obtained by fast multi-sequence alignment using MAFFT (v7.490) software. Finally, the data of each sample is homogenized, and the least amount of data in the sample is taken as the standard for homogenization processing. The subsequent Alpha diversity analysis and Beta diversity analysis are based on the homogenized data. The differences between the Alpha and Beta diversity index and dilution curve, PCA, PCoA, and NMDS diagrams were drawn by R software (Version 4.1.2), and functional annotation correlation analysis was carried out by Tax4Fun2.p-value . VIP values were extracted from OPLS-DA results, which also contain score plots and permutation plots, and were generated using the R package Metabo AnalystR. The data was log transform (log2) and mean centering before OPLS-DA. Identified metabolites were annotated using the KEGG Compound database , and annotated metabolites were then mapped to the KEGG Pathway database . Significantly enriched pathways are identified with a hypergeometric test’s p-value for a given list of metabolites.The original data file acquired by LC-MS was converted into mzML and formatted by Proteo Wizard software (v3.0). Peak extraction, peak alignment, and retention time correction were respectively performed by the XCMS program. The “SVR” method was used to correct the peak area. The peaks with a detection rate lower than 50% in each group of samples were discarded. After that, metabolic identification information was obtained by searching the laboratory’s self-built database, integrated public database, AI database, and met DNA. Unsupervised PCA  was performed by statistics function prcomp within R software. After that, metabolic identification information was obtained by searching the laboratory’s self-built database, integrated public database, AI database, and met DNA. Unsupervised PCA  was performed by statistics function prcomp within R software. For two-group analysis, differential metabolites were determined by VIP (VIP > 1) and Based on the DHI test results, we selected 30 cows for further experimentation. Then, 10 cows in group L, group M, and group H were selected for further experiments. The milk composition and somatic cell test results were as follows . In the The above 30 milk samples were extracted by DNA and amplified by PCR. One of the milk samples in group H was excluded after unqualified detection. A total of 2,280,991 original sequences were obtained from the remaining 29 milk samples by 16SrRNA gene sequencing. After quality control and filtration optimization, a total of 1,800,926 effective sequences were obtained. The average length read is 415 bp, and the details are shown in Alpha Diversity of 16SrRNA gene sequencing was used to detect the changes of microflora in milk samples at different somatic cell levels. Shannon and Simpson index can reflect the diversity of the microbial community, while Chao1 and ACE index can reflect the abundance of the microbial community. Shannon index and Simpson diversity index showed that compared with the M and L groups, the intestinal microbial diversity of the H group was higher, and there was no significant difference between M and L groups. The richness estimation factors Chao1 and ACE showed that the total number of species and the number of OTU in the community were the most in group L and the least in group H, and the number of OTU and the total number of species in the microbial community decreased with the increase of somatic cell number .Enterobacter, and with the increase of SCC, the relative abundance of Enterobacter decreased gradually, which was significantly lower than that of the H group, while the relative abundance of Methyloversatilis increased. In addition, compared with L group, the relative abundance of Pseudomonas in H group and M group increased significantly, while the relative abundance of Ralstonia decreased significantly. The relative abundance of Enterococcus in the H group was significantly lower than that in the L group and M group. The relative abundance of Sphingomonas and Staphylococcus increased significantly in H and M groups.According to the results of species annotation, the species with the highest abundance of each group in the rumen of postpartum dairy cows were selected to generate a species relative abundance histogram A. The rep-value = 0.049; M versus L:R = 0.0336, p-value = 0.222; H versus M:R = 0.0.2026, p-value = 0.325). In addition, in order to study the similarity between different samples, hierarchical cluster analysis using UPGMA showed that most of the samples in group L were clustered in their own group, while most of the samples in groups H and M were mixed together  is a nonlinear model based on the Bray-Curtis distance. According to the species information contained in the sample, it is reflected on the two-dimensional plane in the form of points. Using NMDS analysis, according to the species information contained in the sample, is reflected in the multi-dimensional space in the form of points, while the degree of difference between different samples is reflected by the distance between points, which can reflect the differences between groups and within groups of samples. As shown in together C, indicaRalstonia and Bifidobacterium_adolescentis in the L group, and Pseudomonas_aeruginosa, Aeromonas_veronii, Acinetobacter_johnsonii and Staphylococcus in group H. However, there were no biomarkers between group H and group M, group L and group M (t-test test was carried out to find out the species with significant differences (p < 0.05). It was found that there was also a significant difference in Bifidobacterium, Enterococcus, and Enterobacter abundance between H and L groups  through the t-test between groups. The results showed that a total of 19 grade 3 KEGG pathways were found to be differentially enriched between groups H and L. The abundance of nitrogen_respiration, nitrate_respiration, nitrite_respiration, aromatic_compound degradation, nitrous_oxide_denitrification, nitrite_denitrification, denitrification, nitrate_denitrification, dark_oxidation_of_sulfur_compounds, dark_thiosulfate_oxidation and nitrite_ammonification pathways of milk microbiome in group L was significantly lower than that in group H (p < 0.05)  B.p-value < 0.05; M versus L group: R2X = 0.335, R2Y = 0.989, Q2 = 0.686, p-value < 0.05) indicated that the OPLS-DA model possessed a satisfactory fit with effective predictive power , benzoyl-coenzyme A, 1-O-Hexadecyl-sn-glycero-3-phosphocholine, and significantly positively correlated with the differential metabolite Thr-Trp-Met. In addition, there was a significant negative correlation between Actinobacteriota and Pro-Ala-Leu, Glu-His-His. It was also significantly positively associated with MG /0:0), Dodecanedioic Aicd, MG(18:2/0:0/0:0), Glyceryl arachidonate and Hexadecanedioic acid. In addition, Verrucomicrobiae is also associated with Phenylacetylglycine, Lidocaine, prostaglandin E2 1-glyceryl ester, Ketoleucine, Prostaglandin h2, Isobutyryl CoA, and other differential metabolites showed a significant positive correlation. Meanwhile, there was a significant negative correlation between different microbiome Deferribacteres and different metabolites Pro-Gly-Val, NG-Amino-L-arginine, 5-Hydroxyisourate, 7-Hydroxywarfarin in group M and group L(Based on the Spearman correlation coefficient, we have found that the criteria for the significant correlation between differential microorganisms and metabolites were correlation |r| ≥ 0.7 and  follows . The resd group L.Staphylococci and Streptococci were the dominant bacteria in cow milk with clinical mastitis [Staphylococcus and Sphingomonas in the H group and M group was significantly higher than that in the L group. As a major foodborne pathogen, Staphylococcus is also one of the main pathogens of dairy cow mastitis. Studies have shown that in China and other countries, 10–40% of mastitis cases are caused by Staphylococcus [Enterobacter belongs to general non-pathogenic or conditional pathogenic bacteria, which may infect the body and lead to inflammation only when the body’s immunity is low [Enterobacter was the dominant bacteria in group L, rather than Lactobacillus and other beneficial bacteria as most studies thought [Bifidobacterium in group L was significantly higher than that in groups H and M. Bifidobacterium is an important beneficial microorganism, that can inhibit the growth of harmful bacteria, resist the infection of pathogenic bacteria, and improve the disease resistance of animals. The decrease in its relative abundance will further increase the incidence of mastitis in dairy cows [Previous studies have shown that the causes of mastitis infection in dairy cows are very complicated, and there are more than 150 pathogens, including bacteria, fungi, mycoplasma, and viruses. The changes in the structure and function of microflora in cow milk are considered to be related to the pathogenesis of many diseases. Due to the limitations of traditional microbial identification methods, about 25% of bacteria cannot be detected by routine culture in dairy cow mastitis cases ,45. Consmastitis . In thisy is low . Interes thought . In addiiry cows ,55.Ralstonia in group L was significantly higher than that in group H in the biomarkers of difference between group H and group L. Ralstonia is a widespread plant pathogen. So far, no research has shown that it is closely related to the occurrence of mastitis, which needs further study [Pseudomonas_aeruginosa, Aeromonas_veronii, and Acinetobacter_johnsonii in the H group was significantly higher than that in the L group. Acinetobacter_johnsonii is a gram-negative bacterium widely distributed in an aquatic environment, soil, and plants. It is mostly conditional pathogenic bacteria. It is easy to survive and reproduce in a moist environment, which leads to cow mastitis [Acinetobacter_johnsonii and Lactococcus, and Lactococcus could inhibit the adhesion of common bovine mastitis pathogens to mammary gland cells and reduce the histological changes of mammary glands, so the antagonism of Acinetobacter_johnsonii and Lactococcus may indirectly aggravate the occurrence of mastitis [Pseudomonas_aeruginosa is one of the pathogens of bovine mastitis. It is a kind of bacteria with low virulence, but it has the natural resistance to antibiotics and the ability to form biofilm, so it is difficult to treat this pathogen [Aeromonas_veronii is a gram-negative bacterium widely distributed in aquatic environments, and it is a common conditional pathogen of fish-human livestock. Some species can cause diseases in humans, fish, and other aquatic animals, which can cause disease, inflammation, and infection in animals. However, few studies have shown that it is related to the occurrence of cow mastitis. In addition, Aeromonas_veronii can reduce nitrate to nitrite and change the metabolites in milk [t-test results, the relative abundance of Lachnospira, Akkermansia, and Ruminococcus in group L was significantly higher than that in group M. These bacteria are recognized as beneficial bacteria. Lachnospira is a potentially beneficial bacteria that participates in the metabolism of a variety of carbohydrates and can increase the production of acetic acid and butyric acid, as well as water sister starch and other sugars to produce butyrate and short-chain fatty acids [Akkermansia is considered a candidate for the next generation of important beneficial bacteria, which can improve host metabolic function and enhance host immune function [Colidextribacter in group L was significantly higher than that in group M. Colidextribacter is a common pathogen of mastitis [Paeniclostridium, a highly pathogenic bacterium found in medium-level SCC milk, can also cause mastitis [In addition, LEfSe analysis showed that the relative abundance of er study . LEfSe amastitis ,58. Ma emastitis ,60. Pseupathogen . Aeromon in milk ,63. In aty acids ,65. Akkefunction ,67. Intemastitis . Low SCCmastitis . This stmastitis . HoweverStaphylococcus aureus. Similar to the findings of Grispoldi et al. [Actinobacteriota and Verrucomicrobiota in group H and group L and differential metabolites such as phosphocholine and Thr-Trp-Met in benzoyl-coenzyme. Verrucomicrobiota is a newly identified bacteria, including a few identified species, mainly found in aquatic and soil environments or human faces. Some studies have shown that in colder environments, the proportion of rumen Verrucomicrobiota in cattle is higher, which can help the host improve energy conversion efficiency [Akkermansia muciniphila of Verrucomicrobiota is an intestinal symbiote colonized in the mucous layer and is a promising candidate for probiotics, which is of great value in improving host metabolic function and immune response [Verrucomicrobiota, is an important amino acid in the growth of dairy cows and an important substance in protein synthesis in milk [Actinobacteriota is to produce antibiotics. Of the more than 2000 antibiotics found in the world, about 56% are produced by actinomycetes [Glycerylarachidonate, which has a significant positive correlation with Actinobacteriota, can increase the postpartum feed intake of most mammals and increase the metabolic rate of dairy cows [The metabolites in milk mainly come from the enzyme reaction in milk, the secretion of mammary epithelial cells, and the metabolism of different microorganisms in milk. Metabolic group data can provide further evidence for the changes in milk at different SCC levels . In thisi et al. . Dihydroi et al. . The 2,4i et al. . In addii et al. ,21,26,29ficiency . In addiresponse . The met in milk . Furtheromycetes . Glyceryiry cows . The sigIn summary, the results of this study showed that there were significant differences in microorganisms and metabolites in milk with different SCC levels. 75 differential metabolites were identified in H group and L group, and 40 differential metabolites were identified in M group and L group. Meanwhile, this study identified 6 distinct microorganisms in both the L group and H group, with LDA scores higher than 4. These microorganisms could potentially serve as diagnostic biomarkers for mastitis in dairy cows. However, further testing and validation are required to confirm their accuracy as biomarkers. These differential microorganisms and metabolites may be potential biomarkers for the diagnosis of mastitis in dairy cows, which can provide a new scheme for the diagnosis, prevention, and treatment of mastitis in the future. In addition, according to the differences between microorganisms and metabolites in different SCC levels, the Spearman correlation coefficient was calculated, and it was found that there was a significant correlation between some microorganisms and metabolites in milk. In future research, the above differential microorganisms and metabolites can be further studied to create a new method for rapid diagnosis of dairy cow mastitis.


Intubation-Surfactant-Extubation (InSurE) and less invasive surfactant administration (LISA) are alternative surfactant replacement therapy methods for reducing the complications associated with invasive mechanical ventilation. This study aimed to compare the Classic, InSurE, and LISA methods in Very-Low-Birth-Weight infants (VLBWIs) in South Korea.The Korean Neonatal Network (KNN) enrolled VLBWIs born between January 1, 2019 and December 31, 2020. They were analyzed retrospectively to compare the duration of respiratory support, length of hospitalization, mortality, and short-term outcomes of the three groups.p = 0.014, p < 0.01) and InSurE had the shortest hospitalization  although the results couldn't adjust for confounding factor because of irregular distribution. InSurE had the lower risk of intraventricular hemorrhage (IVH) grade II–IV [odds ratio (OR) 0.524 [95% confidence interval (CI): 0.287–0.956], p = 0.035] than in the Classic group. Mortality was lower in the InSurE  and LISA  groups than in the Classic group. There was a reduced risk of moderate to severe bronchopulmonary dysplasia (BPD) , pulmonary hypertension , periventricular leukomalacia (PVL) , and patent ductus arteriosus (PDA) with treatment  in the InSurE and LISA groups compared to the Classic group.The duration of invasive ventilator support was shorter in the following order: InSurE (3.99 ± 11.93 days), LISA (8.78 ± 29.32 days), and the Classic group (22.36 ± 29.94 days) (InSurE showed the lowest duration of invasive ventilator support, length of hospitalization. InSurE and LISA exhibited reduced mortality and decreased risks of moderate to severe BPD, pulmonary hypertension, PVL, and PDA with treatment compared to the Classic group. Respiratory distress syndrome (RDS) is a progressive disease characterized by a worsening state in the initial hours and days after birth, and is the leading cause of neonatal respiratory morbidity and mortality. In 1980s, the prognosis of RDS improved dramatically with the introduction of a combination of surfactant replacement therapy (SRT) with endotracheal intubation and application of mechanical ventilation . The ClaInSurE can help avoid the application of invasive mechanical ventilation in preterm infants who are initially managed with nasal continuous positive airway pressure (nCPAP), which is non-invasive form of ventilation. Studies have shown that InSurE reduces the need for invasive mechanical ventilation and the incidence of BPD . HoweverTo summarize, InSurE and LISA, especially LISA, have better outcomes than the Classic SRT. LISA has been performed in Korea since the mid-2010s. The Korean Neonatal Network (KNN), a database of Very-Low-Birth-Weight Infants (VLBWIs) at 22–34 weeks’ gestation admitted in the neonatal intensive care unit (NICU) in South Korea, began to investigate LISA as an SRT in 2019. The current study aims to investigate whether there is a difference in the duration of respiratory support, length of hospitalization, mortality, and short-term outcomes according to the SRT, including the Classic, InSurE, and LISA methods, in VLBWIs in South Korea.in vitro fertilization, birth weight, sex, 1- and 5-min Apgar scores, need for oxygen at birth, use of PPV at birth, chest compressions at birth, use of nCPAP at birth, number of surfactant administrations, premature rupture of membranes (PROM), outborn status, and postnatal steroid use. Perinatal risk factors included maternal age, diabetes mellitus (DM), hypertension, multiple birth, delivery type, use of antenatal steroids, chorioamnionitis, and use of antibiotics. The respiratory support and hospitalization durations were compared among the three methods. In the InSurE and LISA groups, invasive ventilator support was administered after tracheal intubation by clinician discretion when a fraction of inspired oxygen (FIO2) greater than 0.4 was still required after SRT, and when severe or recurrent apnea or persistent respiratory acidosis was observed. The duration of invasive ventilator support was measured in these cases within the InSurE and LISA groups. Additionally, the mortality and short-term outcomes of InSurE and LISA were compared with those of the Classic method, which is the most commonly used method. The following short-term outcomes were investigated: BPD  and characteristic findings of RDS in chest radiographs. Additionally, an FIOThe SRT procedure was carried out by skilled neonatal medical professionals specializing in neonatology, with the valuable assistance of neonatal intensive care unit nurses.VLBWIs were intubated with a 2.5–3 sized endotracheal tube, and surfactant was administered using a nasogastric catheter (3.5–5 Fr) inserted through the endotracheal tube. After surfactant administration, the endotracheal tube was removed from the infant. If necessary, the infants received nCPAP, nasal intermittent PPV (NIPPV), synchronized NIPPV (sNIPPV), or high-flow nasal cannula (HFNC) after extubation. Note that the KNN cohort data did not include premedication with atropine and sedation with fentanyl before intubation.A surfactant was instilled using a thin nasogastric catheter (3.5–5 Fr) with the aid of Magill forceps and under direct laryngoscopy. The catheter was inserted into the trachea through the vocal cords, and the surfactant was administered in 3–4 aliquots over 1–3 min, allowing time for airway clearance between each aliquot. Immediately following the surfactant administration, the nasogastric tube was removed. Throughout the procedure, infants with spontaneous breathing continued to receive noninvasive ventilation, such as nCPAP, NIPPV, sNIPPV, or HFNC. The KNN cohort data did not include premedication with atropine during LISA.p < 0.05 were considered statistically significant. Kruskal–Wallis tests were used to compare the duration of respiratory support and length of hospitalization in the NICU, and post hoc analysis was performed via pairwise comparison. A multivariate binary logistic regression model was used to compare the short-term outcomes and mortality of the Classic, InSurE, and LISA methods. The Classic method was designated the reference method. Confounding factors included clinical characteristics and perinatal risk factors that significantly differed among the three SRT groups. Adjusted survival curves for each SRT type were drawn using Cox proportional hazard analysis with the PROC PHREG process  for Windows. We analyzed clinical characteristics and perinatal risk factors with a Pearson's chi-square test or one-way ANOVA. Values with  process  in SAS VOf the 3,824 VLBWIs born during the study period, 1,157 were excluded for chromosomal anomalies, other congenital disorders, non-RDS, non-replacement of surfactant, or unknown SRT methods. The remaining 2,667 infants were enrolled, including 2,181 (81.77%) with the Classic method, 295 (11.06%) with InSurE, and 191 (7.16%) with LISA .The clinical characteristics and perinatal risk factors of the study population are as shown in p < 0.001) and LISA  groups. Moreover, the duration of invasive ventilator support was shorter in the LISA group than in the Classic group (p < 0.001). There was no significant difference between the three groups in the duration of noninvasive ventilator support (p = 0.271). The oxygen supply duration (days) in the Classic, InSurE, and LISA groups was 6.74 ± 11.97, 2.97 ± 8.83, and 5.98 ± 11.70, respectively. The oxygen supply duration was the shortest in the InSurE group, but there was only a significant difference between the InSurE and Classic methods (p < 0.001). There was no significant difference between the three groups in the duration of HFNC (p = 0.768).The duration of respiratory support and length of hospitalization are as shown in The length of hospitalization (days) in the Classic, InSurE, and LISA groups was 79.68 ± 48.87, 64.91 ± 24.07, and 75.91 ± 36.80, respectively. There was a significant difference between the InSurE and LISA groups and between the InSurE and Classic groups. The InSurE group had the shortest length of hospitalization.p = 0.045} and LISA  than with the Classic method. The survival curve for the three groups after adjusting for the same confounding factors is shown in p = 0.049] and LISA  groups than in the Classic group. The risk of IVH grade II–IV was lower in the InSurE group than in the Classic group . However, there was no significant difference in the risk of IVH grade II–IV between the LISA and Classic groups . The risk of pulmonary hypertension was lower with InSurE  and LISA  than with the Classic method. The PVL risk was lower in the InSurE  and LISA  groups than in the Classic group. The risk of PDA with treatment was lower in the InSurE  and LISA  groups than in the Classic group. There were no significant differences in risk of pulmonary hemorrhage, air leak syndrome, NEC grade II or higher, sepsis, and ROP with treatment between the InSurE and Classic groups and between the LISA and Classic groups.The mortality and short-term outcomes of the Classic group were compared with those of the InSurE and LISA groups; the Classic group was the reference. In the current study, the duration of invasive ventilator support and length of hospitalization were higher in the Classic group than in the InSurE and LISA groups as expected. However, InSurE had a shorter duration of invasive ventilator support and length of hospitalization of NICU than LISA, although LISA was introduced as an alternative to compensate for the complication of InSurE. In addition, the risk of IVH grade II–IV was lower in the InSurE group than in the Classic group while there was no significant difference in the risk of IVH grade II–IV between the LISA and Classic groups. LISA has shown better results than InSurE in reducing the incidence of BPD and the need for mechanical ventilation , 23–25. After adjusting for confounding factors, including clinical characteristics and perinatal risks that differed significantly among the three groups, both InSurE and LISA demonstrated better outcomes than the Classic method. These outcomes included lower risks of death and moderate to severe BPD, which are important outcomes of SRT, and lower risks of pulmonary hypertension, PVL, and PDA with treatment. Our data show that the Classic method remains the dominant SRT in South Korea, although it has the worst clinical characteristics and perinatal risk factors. It is possible that because it is the more familiar method, it is considered to be more stable and safer. However, the current study found that, after adjusting for confounding factors, mortality and the risk of short-term outcomes were lower in the LISA and InSurE groups than in the Classic group. New modes of surfactant delivery, such as InSurE and LISA, can decrease mechanical ventilation and BPD rates , 35, 36.This study has some limitations. First, it was a retrospective study despite using nationwide data. Second, due to the non-normal distribution of results, it was not possible to perform a linear regression analysis to adjust for perinatal risk factors and clinical data with statistically significant differences in order to compare the duration of respiratory support and length of hospitalization between the three SRT groups. Moreover, the infants who received the Classic method exhibited higher baseline severity compared to the group that underwent SRT using the InSurE and LISA methods. Consequently, the possibility that the results of Despite these limitations, to the best of our knowledge, this is the first study to compare the duration of respiratory support, length of hospitalization, mortality, and short-term outcomes among SRT methods, including the Classic, InSurE, and LISA methods, with nationwide data in South Korea. In addition, it is meaningful that the results were derived by correcting all confounding variables, such as clinical characteristics and perinatal risk factors, which showed statistically significant differences in mortality and short-term outcomes of three groups with differences in clinical characteristics and perinatal risk factors.The duration of respiratory support significantly differed among the Classic, InSurE, and LISA methods, with InSurE demonstrating the shortest duration for invasive ventilator support. InSurE also resulted in significantly shorter hospitalization compared to the Classic method and LISA. However, it's important to note that the aforementioned results could be influenced by the statistical limitations arising from the non-normal distribution of the data. Adjusting for confounding factors, such as clinical characteristics and perinatal risk factors that varied significantly among the three groups, revealed that InSurE and LISA were associated with significantly lower risks of death, moderate to severe BPD, pulmonary hypertension, PVL, and PDA with treatment compared to the Classic method.


Background: Changes in tryptophan metabolism through the kynurenine pathway (KP) are observed in several disorders and coupled with pathophysiological deviations. Methods: This study retrospectively compared the KP in serum in healthy subjects (108) with subjects with obesity (141), depression (49), and chronic obstructive pulmonary disease (COPD) (22) participating in four clinical studies and explored predictors of the changes in the KP metabolites. Results: Compared with the healthy group, the KP was upregulated in the disease groups with high kynurenine, quinolinic acid (QA), kynurenine/tryptophan-ratio and QA/xanthurenic acid-ratio and low kynurenic acid/QA-ratio. Tryptophan and xanthurenic acid were upregulated in the depressed group compared with the groups with obesity and COPD. The covariates BMI, smoking, diabetes, and C-reactive protein explained the significant differences between the healthy group and the group with obesity but not between the healthy group and the groups with depression and COPD, indicating that different pathophysiological conditions result in the same changes in the KP. Conclusions: The KP was significantly upregulated in the disease groups compared with the healthy group, and there were significant differences between the disease groups. Different pathophysiological abnormalities seemed to result in the same deviations in the KP. The kynurenine pathway (KP) is the main route for metabolising free tryptophan in humans. Deviating tryptophan metabolism is observed in various disorders with common pathophysiological characteristics, such as immune system activation ,2,3. ObeThe primary aim of this study was to compare the KP between healthy subjects and subjects with obesity, depression, and COPD. The secondary aims were to explore differences in the KP between the disorders, to study predictors of the changes in the KP metabolites, and to explore the pathophysiology.This study retrospectively combined data from four clinical studies at Innlandet Hospital Trust, Brumunddal, Norway and Inland Norway University of Applied Sciences, Lillehammer, Norway. Four groups were included in the study: Healthy volunteers  and subjects with obesity (the “Obesity” group), depression (the “Depressed” group), and COPD (the “COPD” group).2 or >35 kg/m2 with obesity-related complications) without severe not-obesity-related comorbidity referred to the obesity unit at Innlandet Hospital Trust, Gjøvik, Norway for evaluation of bariatric surgery were included in the “Morbid Obesity—Bio-Psycho-Social disorders” study (MO-BiPS study) [The Obesity group: Subjects aged 18–65 years old with morbid obesity (defined as BMI > 40 kg/mS study) . Data frThe Depressed group: Consecutive subjects above 18 years of age with depression according to ICD-10, F 32–34 spectre were included in a cross-sectional study at the Psychiatric division, Innlandet Hospital Trust, Norway and compared with subjects with unexplained neurological symptoms. Subjects with known infections or inflammatory disorders influencing the KP were not included. The background for the study with subject description is given in a previous paper by Hestad et al. . Depress3 supplementation and resistance training on a range of biological and health- and performance-related outcomes [The COPD group: In the Granheim COPD Study , subjects with a diagnosis of stable COPD  between 30–80%, and forced vital capacity (FVC) < 70% after reversibility testing) and healthy volunteers were included. Exclusion criteria were unstable cardiovascular disease, chronic granulomatous disease, known active malignancy in the last five years, serious psychiatric comorbidity, and musculoskeletal disorders preventing the participant from participating in resistance training. The study’s primary aim was to investigate the combined effects of vitamin Doutcomes . Only daThe Healthy group included subjects from two of the four studies. In addition to the healthy subjects in The Granheim COPD Study, volunteers from another randomised controlled trial conducted at Inland Norway University of Applied Sciences  were included in the Healthy group. This study aimed to study the effects of n-3 polyunsaturated fatty acids supplementation and resistance training on biological, health- and physical performance-related outcomes. Untrained (less than two sessions of resistance exercise per month and less than three hours of endurance exercise per week) was an inclusion criterion. Only data from the first visit in this study were used.2), daily smoking (no/yes), diabetes (no/yes) and C-reactive protein (CRP: mg/L).The following variables from the four studies were used in the analyses: Age (years), sex , body mass index (BMI: kg/mw/v) trichloroacetic acid (TCA) in water. After thorough mixing (8 min) and centrifugation , an aliquot of 5 µL was injected from the supernatant into the high-performance liquid chromatography (HPLC) system. HPLC was performed with an Agilent 1260 Infinity liquid chromatograph  with an Agilent 6460 Triple Quad LC/MS detector. Tryptophan and the metabolites were separated on a 3.0 mm × 100 mm, 2.6 µm, EVO C18 reversed-phase column from Phenomenex . The column temperature was 40 °C, and the gradient was 100% 0.1% formic acid in water to 100% 0.1% formic acid in acetonitrile. A seven-point calibration curve was created for each analyte for quantification of Trypt and the metabolites in test samples and control samples. VITAS AS, Oslo, Norway, performed the KP analyses.The Kynurenine pathway: Tryptophan (Trypt), kynurenine (Kyn), kynurenic acid (KA), quinolinic acid (QA), and xanthurenic acid (XA) were quantified in serum. The ratios Kyn/Trypt (K/T ratio × 1000), KA/Kyn × 1000, KA/QA × 1000, KA/XA and QA/XA were calculated. Protein precipitation of 20 µL human plasma after adding 20 µL internal standard solution  was performed using 60 µL 50% (Descriptive results are given as mean (SD) or number (%). One-Way ANOVA and Chi-square tests were used for unadjusted comparisons between the groups. Differences in the KP metabolites between the groups were analysed with a univariate general linear model with the KP metabolites as dependent variables adjusted for age and sex, and reported as unadjusted regression coefficients  with 95% confidence intervals.To study the effect of BMI, smoking, diabetes, and CRP on the KP metabolites, the variables were added one at a time to the first multivariable analyses.The last analytic step included all variables  at the same time in the multivariable analyses to study fully adjusted differences in the KP metabolites between the groups. Due to missing data, these analyses included 304 subjects. Diabetes as a variable was excluded from this step due to missing information in the COPD group.p-values ≤ 0.01 were judged as statistically significant.IBM SPSS Statistics for Windows, version 27.0 IBM Corp: Armonk, NY, USA, was used for the analyses. Due to the explorative design and multiple testing, Three hundred and twenty subjects were included in the analyses: 108 in the Healthy group, 141 in the Obesity group, 49 in the Depressed group, and 22 in the COPD group. The serum levels of the KP metabolites in the Healthy group and the differences in the KP metabolites between the Healthy group and the groups with diseases are given in p = 0.120. KA: 1.1 ; p = 0.448. QA: 20 ; p = 0.034. K/T ratio: 2.9 ; p = 0.229. QA/XA ratio: 2.2 ; p = 0.510.Obese compared with Healthy: Kyn: 49 ; p = 0.002.Depressed compared with Healthy: K/T ratio: 6.4 ; COPD compared with Healthy: No changes.p = 0.067. XA: 0.55 ; p = 0.086.Depressed compared with Obese: Trypt: 1312 ; p = 0.017.Depressed compared with COPD: QA/XA ratio: −10 ; p = 0.006.Obese compared with COPD: QA/XA ratio: −13 ; Below are given the changes in the differences in the KP between the groups of participants presented in The most important findings were the differences in the KP between the Healthy group and the groups with a disease, and the influence of the covariates BMI, smoking, diabetes, and CRP on the KP.p ≤ 0.01), which is far above the number expected to occur by chance. No KP metabolite showed significant differences between the Healthy group and all disease groups. Nevertheless, there were some overall tendencies: Kyn, QA, K/T-ratio, and QA/XA-ratio were high, and KA/QA ratio was low in the diseased groups compared with the Healthy group. These changes indicate an unfavourable upregulation of the KP in all diseased groups independent of disease. Kyn, K/T-ratio, QA and QA/XA are markers of inflammation, QA is neurotoxic and associated with depression and improves glucose control, KA/XA-ratio has been associated with mood disorders, and KA, QA, and XA regulate the carbohydrate metabolism [There were significant differences between the Healthy group and the groups with diseases. Eleven out of 30 comparisons of the KP metabolites between the Healthy group and the groups with diseases were statistically significant (defined as tabolism ,16,17,18The elevated levels of Kyn, KA, K/T ratio and QA in the Obesity group are in accordance with other studies ,9,19. AnThe changes in the Depressed group are in part identical to those in the Obese group with an upregulation of Trypt, Kyn, QA, and XA. The changes differ from the downregulation of Trypt and Kyn in subjects with depression reported in meta-analyses ,5,20. ThThe differences between the Healthy and COPD groups seem less impressive than between the Healthy group and the other groups. However, the findings in the COPD group should be interpreted cautiously because of the low number of subjects. Other studies have shown elevated levels of Kyn in subjects with COPD and hypothesised reduced Kyn clearance in the muscles as the pathophysiological mechanism ,8. If coThe increase in the KP metabolites with age and male sex and the lower levels in smokers are in accordance with other studies ,23.The differences between the groups with diseases were less distinct than between the Healthy group and the groups with diseases. The most unfavourable changes in the KP pathway were in the Depressed group. Trypt and XA were raised in the Depressed group compared with the Obese and COPD groups. The raised Trypt level was not expected ,5,20. XACRP was associated with five KP metabolites, BMI with two, and smoking and diabetes with one of the metabolites.CRP was associated with high Kyn, QA, K/T-ratio and QA/XA-ratio, and low KA/QA-ratio, confirming findings from other studies that these variables are markers of inflammation ,3. ProinThe association between Obesity and inflammation, as demonstrated in this study with high CRP values in the Obese group, is well known. CRP was associated with high levels of QA and low KA/QA ratio reflecting the proinflammatory and neurotoxic effects of QA and anti-inflammatory effects of KA . InflammThe negative association between smoking and most of the KP variables is known from other studies ,23. PosiMost KP metabolites are associated with carbohydrate metabolism . Kyn, KAAdjusting for age, sex BMI, smoking, and CRP at the same time unveiled exciting differences between the groups:Healthy versus Obese. When adjusting for all the covariates, there were no significant differences in KP metabolites between the Healthy and Obese groups. It is likely that inflammation measured as CRP, and to a lesser extent BMI, explain the differences between Healthy and Obese groups.Healthy versus Depressed. After adjusting for all the covariates, the significant differences in Trypt, Kyn, QA, and XA persisted. In addition, K/T ratio was statistically significant, indicating that these covariates were not crucial for the KP abnormalities in the Depressed group.Healthy versus COPD. Adjusting for all covariates did not significantly change the associations between the Healthy and COPD groups. They, therefore, did not explain the differences between the groups. In the COPD group, the effect of smoking is unreliable. The subjects were asked about actual smoking. Most subjects with COPD were previous and often heavy smokers who had recently stopped smoking.Before adjusting for BMI, smoking, and CRP, the differences in the KP between the Healthy group and the groups with diseases had the same pattern. After adjusting for the covariates, there were significant changes. BMI, smoking, diabetes, and CRP explained the KP abnormalities in the Obese group but not in the Depressed and COPD groups. The results might indicate that the pathophysiology behind similar KP abnormalities differs between the diseased groups. Inflammation measured as CRP seems to drive the difference between Healthy and Obese, but not between Healthy and the other disease groups.p-values ≤ 0.01 were used as statistically significant.The participants represented consecutive patients and healthy volunteers treated in a general hospital. Except for the differences in inclusion/exclusion criteria, there was no selection of participants in the four clinical studies. The Healthy group, which was a combination of two groups, might be an exception. One part of the Healthy group was healthy controls in the Granheim COPD study and older than the subjects in the Obese and Depressed groups. The other part of the Healthy group was untrained healthy volunteers in a study of the effects of n-3 polyunsaturated fatty acids supplementation and resistance training on skeletal muscle hypertrophy and therefore motivated for physical activity. All KP-metabolites were analysed in serum, kept in a biobank, and analysed in a specialised laboratory with the same method and at the same time. The comparisons of the KP and the pathophysiology in a single study with identical methods are superior to comparisons between independent studies. However, only a selection of the KP metabolites were analysed. Any effect of the unexpectedly high CRP values in the Healthy group is unknown. Other blood tests were analysed at different local laboratories at inclusion in the studies. There was no uniform way of collecting the clinical information, and differences might have occurred. Data on pharmacological therapy was judged as unreliable and not included in the analyses. To adjust for multiple testing, The study showed significant differences between the Healthy group and the Obese, Depressed and COPD groups. The KP pathway was upregulated in the disease groups compared with the Healthy group, and the differences between the Healthy group and the disease groups had more or less the same pattern. Adjusting for age, sex, BMI, smoking and CRP unveiled exciting differences. Inflammation, measured as CRP, and in part BMI explained the differences between the Healthy group and the Obese group, but not between the Healthy group and the Depressed and COPD groups. As discussed, the cause of elevated Kyn in subjects with COPD and Obesity might differ. The findings might indicate that pathophysiological differences cause similar changes in the KP.


Mollicutes have been associated with different pathologies of the urogenital tract in humans, with a high prevalence among adult men who have sex with men (MSM) and transgender women (TGW). However, few studies have been performed to investigate its prevalence among adolescents. In this study, we estimated the initial prevalence of Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP); the rate of misdiagnosis at different anatomical sites; and the associated factors with positive tests for Mollicutes among MSM and TGW aged 15 to 19 years enrolled in the PrEP1519 study.Some species of PrEP-1519 is the first study to investigate the effectiveness of pre-exposure prophylaxis for human immunodeficiency virus among adolescent MSM and TGW aged 15 to 19 in Latin America. Oral, anal, and urethral swabs were taken from 246 adolescents upon enrolment in the study to detect MG, MH, UU, and UP by quantitative polymerase chain reaction (qPCR). Bivariate and multivariate analyses were conducted by Poisson regression and 95% confidence intervals (95% CI) were estimated.Mollicutes was 32.1%. UU was the most prevalent species (20.7%), followed by MH (13.4%), MG (5.7%), and UP (3.2%); 67.3% of the positive samples would have been missed if only urethral samples had been taken. Receptive anal sex  and clinical suspicion of sexually transmitted infection  were factors associated with the detection of Mollicutes in general. Group sex  and receptive anal sex  were associated with the detection of Mycoplasma spp. No sociodemographic, clinical, or behavioural variable was significantly associated with the detection of Ureaplasma spp.The prevalence of Mollicutes was observed among adolescent MSM and TGW, especially at extragenital sites. Further research is required to understand the epidemiological profile of high-risk adolescents in different regions and contexts, and to investigate the pathogenesis of Mollicutes in the oral and anal mucosa before routine screening can be recommended in clinical practice.A high prevalence of The online version contains supplementary material available at 10.1186/s12879-023-08213-z. Sexually active adolescents constitute a population group that is vulnerable to sexually transmitted infections (STIs) due to several factors of psychosocial and biological nature –5. In adMollicutes species, the gap in data availability is even greater, since they rarely cause a symptomatic infection, which is one of the reasons why they are neglected in epidemiological surveillance and research. Despite this, this species has been associated with several pathologies of the urogenital tract, such as non-chlamydial non-gonococcal urethritis [Mollicutes may be present at different anatomical sites. The aim of this study was to estimate the prevalence of Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP) at different anatomical sites to assess the rate of missed detection and to analyse factors associated with the prevalence of Mollicutes among AMSM and ATGW.Data on the prevalence of STIs and associated factors, along with the risk of acquiring STIs, among adolescent MSM (AMSM) and TGW (ATGW) are extremely limited. Regarding the prevalence of ethritis –25, maleethritis –28, an iethritis , 30, andethritis . This spethritis –36, evenethritis , 38, andethritis –41. A miethritis –42. Furtethritis  suggestsA cross-sectional study based on baseline data obtained from the PrEP1519 cohort study was conducted in three Brazilian cities – Salvador, Belo Horizonte, and São Paulo – which aimed to analyse the effectiveness of HIV pre-exposure prophylaxis (PrEP) among adolescents from key populations who had a history of risk exposure to or vulnerability to HIV infection  to HIV in the previous 12 months, commercial sex work). Recruitment of participants was conducted by demand creation strategies on youth social venues and virtual networks , including dating apps . Also, public health services and non-governmental organizations (NGOs) could refer eligible participants to the study.In the present study, baseline data from April 2019 to February 2021 from the city of Salvador, the capital of the North-eastern Brazilian state of Bahia, was analysed. The inclusion criteria were AMSM and ATGW aged 15–19 years old (y/o), who had at least one act of sexual intercourse with another cisgender man or TGW in the past 12 months and had spent most of their time at the study site . Participants of the PrEP1519 study were offered PrEP and other combination prevention strategies, sexual health care, and STI testing, including those for bacterial STIs, with quarterly follow-ups conducted for up to 3 years.After checking the eligibility criteria and the information on the proposed steps for the study, individuals who agreed to participate in the study provided written informed consent or assent and were tested at the initial visit (baseline). Cotton swab samples from oral, urethral, and anal sites for MG, MH, UU, and UP were collected to perform real-time polymerase chain reaction (PCR) tests. A socio-behavioural questionnaire was provided at baseline. This study was approved by the Research Ethics Committee of the World Health Organization (Protocol ID: Fiotec-PrEP Adolescent study) and the Federal University of Bahia .A socio-behavioural questionnaire with questions about gender identity, access to health services, sexual practices, drug and alcohol use, and situations of violence was administered at baseline by trained investigators and was used for data analysis. The sociodemographic variables included: age (< 18 or ≥ 18 y/o), race/colour , schooling (elementary school/adult education or high school/higher education), population group (MSM or TGW), and sexual orientation . Behavioural variables were steady sexual partner in the last 3 months (yes or no), casual sexual partner in the last 3 months (yes or no), receptive anal sex (yes or no), insertive anal sex (yes or no), group sex (yes or no), interference of drugs or alcohol in condom use (yes or never), and condom use in the last 3 months (consistent or inconsistent). The clinical variable was clinical suspicion of STI , which consisted of signs and symptoms suggestive of STI, such as urethral discharge, presence of warts and lesions, dysuria, itching, and irritation, among others.Oropharyngeal, anorectal, and urethral cotton swabs were taken during the consultation with a doctor or nurse upon enrolment in the study. For the oropharyngeal swabs, the tongue was pressed down using a sterile tongue depressor to avoid contact with the tongue, cheeks, palate, and uvula, and the hydrophilic swab was then rubbed on the tonsils and behind the uvula. For the anorectal sample, the participant was asked to lie on their side with one leg slightly flexed. The swab was then introduced one to two centimetres beyond the rectal sphincter and rotated. For the urethral sample, the foreskin was retracted, exposing the glans, and a paediatric swab was carefully inserted about two centimetres into the urethral meatus and rotated. All samples were stored in 5 mL transport medium at 4ºC until processing . In the The DNA extracted from the samples was obtained using a boiling method with phosphate-buffered saline solution . After eMollicutes using primers to amplify a fragment with 280 bp [PCR tests were performed to screen for samples with h 280 bp , verifieh 280 bp , UU, andh 280 bp . The mic2), by the Shapiro–Wilks normality test, and the Mann–Whitney U test, because the data distribution was not normal.The bacterial load of the positive samples was estimated from the cycle threshold value using the StepOne™ system . To assess whether there was a difference in the load of the different species and at different anatomical sites, the comparisons made were determined by individual variance or error variance , and Fisher’s exact test was used to calculate statistical significance (p ≤ 0.05):To estimate the missed detection of Mycoplasma spp. and Ureaplasma spp.) or the class (Mollicutes) analysed individually. The bivariate analyses were conducted using Poisson regression with robust variance, estimating the prevalence ratio (PR) and respective 95% confidence interval (CI). The differences between the variables and the occurrence of the outcome were tested using Pearson’s chi-squared test or Fisher’s exact test , with significance set at 5% (p ≤ 0.05). For the multivariate analysis, backward stepwise regression was used. The adequate model was selected according to the Akaike information criterion and Bayesian information and was then assessed using the chi-squared test. Stata 15.0  was used for these analyses.The sociodemographic and behavioural variables were taken as independent variables for the bivariate analysis. The following dependent (outcome) variables were considered: presence (or not) of each species  of the genera , were high school or university students (89.3%), were self-identified as black (85.8%), MSM (93.9%), homosexual (64.2%), and had inconsistent condom use (60.7%)  was 32.1% (95% CI: 26.5–38.2). UU was the most prevalent species , followed by MH , MG , and UP . The two swab sites with the highest prevalence of infection were oral (19.6%) and anal (19.5%), followed by urethral (14.1%).The overall prevalence of A high rate of missed detection at the extragenital sites  was observed when the prevalence per site was compared with the prevalence at all three sites Table . A signiMollicutes at each swab site. Statistically significant differences were found for MG (p = 0.0008) and UU (p = 0.0087), which had lower Ct values at the anal and urethral sites, respectively, meaning that there was a greater bacterial load of MG at the anal site and a greater bacterial load of UU at the urethral site, thus, a greater risk of infection.Supplementary Figure SMollicutes infections, 69.6% (55/79) were mono-infections, with 10.9% (6/55) of infections by the same microorganism at more than one anatomical site. As for others, 31.6% (25/79) were co-infections (simultaneous infections. with two or more microorganisms), of which UU and MH were the most prevalent   and having had group sex  were associated with the detection of Mollicutes were receptive anal sex  and clinical suspicion of STI . Having had group sex  was associated with testing positive for Mycoplasma spp. No sociodemographic, behavioural, or clinical variable was significantly associated with Ureaplasma spp. As for the species, having had group sex  was associated with the detection of MH. No variable was statistically significantly associated with testing positive for MG and UU. As for UP, its low prevalence in our study population (3.2%) indicated that the confidence intervals of the variables were very large, compromising the validity of the results  [This is the first study to analyse the prevalence of . (2017)  that ana. (2017) .UU is possibly an etiological agent of non-gonococcal urethritis , 50, witThe prevalence of MG found in our study was 5.7% and we considered this to be high. It is similar to the prevalence of 5.1% and 6.6% found by Reinton et al. (2013)  and SoniMollicutes species studied, MG is regarded as an emerging STI in many countries, especially in Europe, where it contributes to 10% to 35% of non-chlamydial non-gonococcal urethritis in men [Of all the s in men . Furthers in men  and thats in men , 42. In s in men , 56, advs in men , since ss in men , 58–61.Mollicutes spp. should be carefully considered.One point worth highlighting is treatment failure, which not only induces resistance but can also contribute to the persistence of this pathogen and its transmission to sexual partners during sex without the use of condoms . TherefoMollicutes between the anatomical sites, there was a higher prevalence of oral and anal infections than urethral infections, which may be a reason why the missed detection rate would be high if only the urethral site had been screened (67.3%). Our findings corroborate those of other studies, which have found 53–85% of chlamydial infections, 64–77.9% of gonococcal infections, and 71.4% of MG infections in extragenital sites, meaning they would not be detected and treated if only urethral screening were performed [As for the distribution of erformed , 63, 64.erformed , 65, 66.Mollicutes had two or more simultaneous infections, 92% of which involved UU. A study with MSM who attended a genitourinary clinic observed that UU and UP only ever appeared in co-infections with Gardnerella vaginalis or other Mollicutes [Regarding the occurrence of co-infections, 31.6% of all the participants who tested positive for some species of llicutes . In our llicutes  in an inMollicutes can also co-occur with other microorganisms, such as gonorrhoea and chlamydia. Therefore, just as screening for Mollicutes is not recommended, screening for it only when the individual tests negative for other STIs may also be a severe problem. For instance, if patients are undiagnosed for MG, and treated for other diagnosed STIs, they may therefore be exposed to antibiotics that are resistant to or induce resistance in Mollicutes during such treatment [Mollicutes co-infection with other microorganisms and the clinical significance of these infections, including pathogenicity at extragenital sites. More assertive screening and treatment guidelines can be developed, reducing the risks of unnecessary routine screening and inappropriate treatment.Species of reatment , 68, 69.Mollicutes to be sexually transmitted, since variables of sexual behaviour, such as group sex and receptive anal sex, were associated with infection by Mollicutes, Mycoplasma spp., and MH. Group sex has already been identified as a risk factor for the acquisition of STIs [Our results indicate a strong tendency for species of  of STIs , as has  of STIs .Mollicutes may indicate their sexual transmission. MG has been considered an important agent of STIs for over ten years now [Mycoplasma and Ureaplasma that colonize the urogenital tract may be possible agents of STIs, which is also confirmed by the high bacterial load of MG in the anal swabs and UU in the urethral swabs taken for this study, indicating that these microorganisms may not just colonize these anatomical sites, but may also evolve to infections inducing symptoms. A study by Bissessor et al. (2016) [The association of these behaviours with the detection of ears now –74. The ears now . Our res. (2016)  corroborMollicutes. However, other microorganisms that were not investigated could be present and causing the symptoms; therefore, our results cannot be taken as evidence that the symptoms were (or were not) caused by Mollicutes. Thus, we emphasize the need for studies on the pathogenicity of these Mollicutes species in the urogenital tract, orally, and anorectally to determine to what extent and when they can be considered commensal, and when they should be regarded as etiological agents of STIs.As for the other variables, clinical suspicion of STI – namely, the presence of signs and/or symptoms of an STI – was also identified as a factor for the detection of Given the small sample size of ATGW in our study, the statistical analysis for this subgroup could not be properly conducted. However, according to the literature, identifying as a TGW is also associated with STI acquisition . SeveralMollicutes in the total population of adolescent MSM and TGW from Salvador, Brazil, let alone elsewhere, and particularly not in the case of TGW, given the very low sample size of this population. Also, given the overall low sample size of the study, the detection capacity of the statistical tests was limited and many results were not statistically significant. Furthermore, as the study was cross-sectional, it could not be ascertained whether any of the infections detected, symptomatic or not, were new or persistent. Also, we did not perform genotypic antibiotic resistance testing, limiting the selection of an adequate treatment strategy. Finally, the bacterial load was not quantified, limiting our ability to differentiate colonization from infection .Our study has some limitations. First of which is that our study population was based on convenience sampling. Our results may not represent the real prevalence of Mollicutes. Therefore, we stress the need to raise awareness in this population regarding the importance of taking preventive measures against the transmission of Mollicutes during sexual intercourse, including oral sex. Further research on adolescents is required to understand their epidemiological profile in other regions and sociodemographic contexts, investigate the pathogenesis of Mollicutes infections in the oropharynx and rectum, and evaluate the benefits of multisite screening for these microorganisms in high-risk adolescents before implementing it in clinical practice.A high prevalence of these microorganisms was found, especially in extragenital sites, and a significant association existed between sexual behaviour and detection of Additional file 1: Supplementary Table 1. Bivariate analysis of the prevalence of M. genitalium, M. hominis, U. urealyticum, and U. parvum among AMSM (N=231). PrEP1519, Salvador, Brazil, April 2019 to February 2021. Supplementary Table 2. Partial Socio-behavioural Questionnaire applied to participants on this study. Figure S1. Ct value  of samples that were positive for M. genitalium (MG), M. hominis (MH), U. parvum (UP), and U. urealyticum (UU) from oral, anal, and urethral swabs taken from AMSM and ATGW enrolled on the PrEP1519 study in Salvador, Brazil (N=246), April 2019 to February 2021.