The 2018 Global Initiative for Chronic Obstructive Lung Disease (GOLD) recommends combination long-acting muscarinic antagonists/long-acting beta2-agonists (LAMA + LABA) as preferred maintenance therapy for patients with symptomatic chronic obstructive lung disease (COPD) after monotherapy and stepping up to triple therapy  in case of further exacerbations. Restrictions on TT recommendations have primarily been driven by higher pneumonia risk associated with regular ICS use. Evidence suggests that TT is overprescribed, which may affect economic and clinical outcomes.To compare health plan-paid costs, COPD exacerbations, and pneumonia diagnoses among patients newly treated with a LAMA + LABA regimen composed of tiotropium (TIO) + olodaterol (OLO) in a fixed-dose combination inhaler (TIO + OLO) or TT in a U.S. Medicare Advantage Part D insured population.This retrospective study identified COPD patients aged ≥ 40 years who were initiating TIO + OLO or TT (index regimen) between January 1, 2014, and March 31, 2018, from a national administrative claims database. Continuous insurance coverage for 12 months pretreatment (baseline) and ≥ 30 days posttreatment (follow-up) was required. Patients were followed until the earliest of study end , discontinuation of index regimen (≥ 60-day gap in index regimen coverage), switch to a different regimen, or health plan disenrollment. Before analysis of outcomes, TIO + OLO and TT patients were 1:1 propensity score-matched on baseline demographics, comorbidities, COPD medication use, medical resource use, and costs. Cohort differences in post-match outcomes were assessed by Wald Z-test  and Kaplan-Meier method (time to first COPD exacerbation and pneumonia diagnosis).P < 0.001); cohort differences in the medical component  were driven by lower mean acute inpatient costs . Combined mean COPD plus pneumonia-related medical costs were $5,212 (39.0%) lower for TIO + OLO versus TT , and total mean all-cause costs were $9,221 (30.4%) lower for TIO + OLO versus TT . Kaplan-Meier analysis found longer time to first severe COPD exacerbation (P = 0.020) and first pneumonia diagnosis (P = 0.002) for TIO + OLO versus TT and a lower percentage of TIO + OLO patients experiencing these events . A secondary analysis, which expanded the TIO + OLO cohort to include any LAMA + LABA regimen, had similar findings for all outcomes.After matching, each cohort had 1,454 patients who were well balanced on baseline characteristics. Compared with TT, the TIO + OLO cohort incurred $7,041 (41.1%) lower mean COPD-related total costs ($10,094 vs. $17,135; COPD patients initiating TIO + OLO incurred lower costs to health plans and experienced fewer COPD exacerbation and pneumonia events relative to TT. These findings provide important real-world economic and clinical insight into the GOLD recommendations for TIO + OLO and LAMA + LABA therapy. The study findings also indicate the continued inconsistency between the recommendations and real-world clinical practices pertaining to TT. What is already known about this subjectCombination long-acting muscarinic antagonists (LAMA) plus long-acting beta2-agonists (LABA) are guideline-recommended as maintenance therapy for patients with symptomatic chronic obstructive pulmonary disease (COPD) after monotherapy.Given the higher risk of pneumonia with regular inhaled corticosteroid (ICS) use, the addition of ICS to LAMA + LABA, thereby stepping up to triple therapy (TT), is recommended only for the most severe patients who continue to experience exacerbations.TT is commonly overprescribed to less severe patients in clinical practice, which may affect economic and clinical outcomes; however, previous research comparing LAMA + LABA versus TT in a real-world setting in a robust manner is limited.What this study addsThis analysis compared outcomes for patients newly treated with tiotropium (TIO) + olodaterol (OLO), a fixed-dose combination of LAMA + LABA in a single inhaler, with TT in a U.S. Medicare Advantage Part D insured population.This study adds to previous research comparing LAMA + LABA in a real-world setting by examining both health and economic outcomes in the same study and focusing on the period when patients remained on treatment.Compared with TT, TIO + OLO-treated patients incurred 41.1% lower annualized health plan-paid costs for COPD-related services and had a lower percentage of patients with both severe COPD exacerbations and pneumonia diagnoses while taking a significantly longer time to their respective first occurrences.4 The Global Initiative for Chronic Obstructive Lung Disease (GOLD) report categorizes patients with COPD for pharmacologic management according to exacerbation risk and symptom burden: Group A (low exacerbation risk/low symptom burden), Group B (low exacerbation risk/high symptom burden), Group C (high exacerbation risk/low symptom burden), and Group D (high exacerbation risk/high symptom burden).5 The 2018 GOLD report (referenced during the current study execution) recommends dual therapy with long-acting muscarinic antagonists (LAMAs) plus long-acting beta2-agonists (LABAs) for patients in Groups B-D who have persistent symptoms and/or exacerbations on LAMA or LABA monotherapy. Among patients in Group C who continue to experience exacerbations, the addition of inhaled corticosteroids (ICSs) to LABA (LABA + ICS) may be also be considered.5 Finally, triple therapy (TT), consisting of LAMA + LABA + ICS, is only recommended for Group D patients and as an escalation step from LAMA + LABA (or LABA + ICS).5Chronic obstructive pulmonary disease (COPD) has been diagnosed in 5.9% of the U.S. adult population  and imposes significant clinical and economic burdens on patients, payers, and health care systems.6; a meta-analysis of clinical trials found a 53% higher risk of pneumonia for TT versus LAMA + LABA.7 The latest (2020) GOLD report further limits recommendations for ICS-containing regimens as an initial therapy to Group D patients with a history of or with concomitant asthma or for the minority with elevated eosinophil blood counts, a potential but unconfirmed biomarker for ICS responsiveness in patients with persistent exacerbations.11 Despite these recommendations, several studies have reported widespread use of TT among patients in GOLD Groups A-C, thereby unnecessarily exposing patients to an elevated risk of pneumonia, wasting scarce health care resources, and contributing to the ever-escalating cost burden.16Recommendations for TT use have been restricted due to risk/benefit considerations given the substantially higher risk of pneumonia associated with ICS use in COPD patients17 Studies examining economic outcomes found substantial cost savings among patients initiating LAMA + LABA, specifically as tiotropium (TIO) + olodaterol (OLO)18 in a fixed-dose combination inhaler, relative to TT.21 Our previous study examined clinical and economic outcomes for TIO + OLO versus TT and found that initiating TIO + OLO relative to TT was associated with 35% lower annual COPD-related health plan-paid costs and a smaller proportion of patients experiencing severe COPD exacerbations and pneumonia occurrences.21 While these results provided important insight into the differential burden of TIO + OLO versus TT, they were limited by the intent-to-treat approach, which observed patients for the entire first year after therapy initiation, irrespective of subsequent therapy changes that are relatively common in clinical practice.23 Further, requiring 12 months of follow-up may lead to selection of healthier cohorts, increasing the survival bias if a treatment is associated with survival.24 This study sought to address these limitations while expanding our previous findings by examining outcomes for the duration that patients remained on their initial regimen. We also leveraged the availability of additional data and refined the assessment of the burden attributable to pneumonia.Emerging real-world evidence suggests advantages of LAMA + LABA over TT. A study of patients in clinical practice reported a reduced rate of exacerbations with LAMA + LABA versus TT.25The primary objective of this study was to compare health care costs, exacerbations, and pneumonia outcomes among patients newly treated with TIO + OLO versus TT in a real-world setting. The secondary objective extended the TIO + OLO cohort to patients treated with any LAMA + LABA versus TT. Consistent with our previous study, we focused on patients with managed Medicare insurance because Medicare incurs the largest share of COPD-related costs in the United States.International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) and International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) diagnosis and procedure codes and site of service codes. Pharmacy claims include National Drug Code numbers for medications dispensed with quantity, dose, and number of days supply. The ORD is geographically diverse in coverage and represented approximately 21%  of the U.S. population, with Medicare Advantage prescription drug (MAPD) plan benefits in 2018. No identifiable protected health information was extracted or accessed during the course of the study, and all data were accessed in compliance with the Health Insurance Portability and Accountability Act’s rules; therefore, institutional review board approval or waiver of authorization was not required.This retrospective study used administrative claims data from January 1, 2013-May 31, 2018 from the Optum Research Database (ORD). The ORD contains enrollment information and medical and pharmacy claims submitted to each patient’s health plans for reimbursement. Medical claims include 26 Index regimen was the first of the above 4 regimens observed for ≥ 30 consecutive days, without other overlapping nonindex maintenance regimens . Patients initiating LAMA + LABA or TT were retained for this analysis. The index date for patients initiating single inhaler regimens, such as TIO + OLO, was the first pharmacy claim during the identification period. For multiple inhaler regimens, including open LAMA + LABA and most patients initiating on TT, the index date was defined as the start of ≥ 30 consecutive days of overlapping supply of LAMA and LABA—and for TT, ICS— components (free or fixed-dose combinations).The first step in the selection process was to identify patients initiating a COPD maintenance therapy  between January 1, 2014, and March 31, 2018 (identification period) using pharmacy claims. LABA monotherapy was not considered because of its infrequent usage (< 3% of monotherapy patients).21The diagnosis of COPD was established by ICD-9-CM (491.xx-493.2x and 496.xx) or ICD-10-CM (J41-J44) codes on ≥ 2 medical claims on separate dates of service during the study period . Additional inclusion criteria were patients aged ≥ 40 years as of the index date, complete demographic information availability, and ≥ 13 months of continuous medical and pharmacy enrollment in an MAPD health plan involving 12 months of pre-index (baseline period) and ≥ 1 month of post-index (follow-up period) data. Patients with ≥ 2 medical claims on separate dates of service involving a diagnosis of asthma, lung cancer, or cystic fibrosis during the study period and evidence of the index regimen during the baseline period were excluded.The primary analysis included COPD patients who were initiating treatment with only TIO + OLO (and no other LAMA + LABA) or TT. The secondary analysis, comparing LAMA + LABA versus TT, used the same patient selection criteria as the primary analysis but expanded the TIO + OLO cohort to include patients whose index regimen was any LAMA + LABA .Patients were followed until the earliest occurrence of index regimen discontinuation, switch to a nonindex regimen, health plan disenrollment, or end of the study period . Discontinuation was defined as a gap of ≥ 60 days between pharmacy fills of the index regimen. The discontinuation date was the last prescription runout date  prior to the occurrence of ≥ 60-day gap. Switch was defined as a change to a nonindex regimen for ≥ 30 consecutive days.28 COPD maintenance and rescue medication use was ascertained from pharmacy claims. Severe COPD exacerbations were defined as an inpatient admission with a COPD diagnosis code in the primary position. Moderate exacerbations required either an emergency department (ED) visit with a COPD primary diagnosis or an ambulatory visit with a COPD diagnosis code in any position plus a pharmacy claim for an oral corticosteroid (OCS) or COPD guideline-recommended antibiotic prescription within 7 days following the ambulatory visit. Exacerbations occurring within 14 days of each other were considered a single exacerbation episode and were classified as per the highest severity. Patients meeting GOLD C/D criteria  during the baseline period were flagged for further examination.5Demographic characteristics were determined on the index date. Clinical characteristics including Quan-Charlson comorbidity and Elixhauser scores were calculated during the 12-month baseline period.plus all outpatient pharmacy costs. COPD-related total costs included the sum of costs of all medical claims with a diagnosis of COPD in any position plus outpatient pharmacy claims for COPD medications . COPD-attributable total costs, a subset of COPD-related total costs, were the sum of medical claims with a COPD diagnosis in the primary position only plus outpatient pharmacy claims for COPD-related treatment. Pneumonia-related medical costs included medical claims with a diagnosis code for pneumonia and/or acute bronchitis/bronchiolitis in any position (codes previously reported).21 COPD/pneumonia-related medical cost burden included the costs of claims involving a diagnosis for COPD, pneumonia, and/or acute bronchitis/bronchiolitis.Follow-up costs were determined from medical and pharmacy claims beginning on the index date. Medical costs included costs for medical service use and were categorized as acute inpatient, ED, ambulatory (office and outpatient), and other . All-cause total costs were the sum of all medical costs for any medical service 29 Additional outcomes, including assessment of pneumonia diagnoses and exacerbations (as defined above) during follow-up, started on the day after the index date.To examine the incremental burden of pneumonia to COPD-related cost, non-COPD pneumonia-related costs were estimated by accounting for pneumonia-related claims and excluding those with a COPD diagnosis on the same claim. To avoid skewing of means by patients with extremely short or long follow-ups, all cost outcomes captured during the variable follow-up period were annualized at the cohort level by summing the total costs for all patients and dividing by the sum total days on treatment for all patients and then multiplying the result by 365. All costs measures were computed from health plan-paid costs and inflation-adjusted to 2017 U.S. dollars.Patients in the TIO + OLO and TT cohorts were matched 1:1 on baseline characteristics using propensity score nearest neighbor matching (PSM), within a caliper of 0.001 of the standard deviation of the estimated logit of the propensity score. Iterative modeling was conducted to identify the logistic model that met the caliper of 0.001. Pre-PSM cohort differences in baseline characteristics were analyzed by Pearson Chisquare test and 2-sample t-test for categorical and continuous measures, respectively. Post-PSM baseline characteristics and cost outcomes were analyzed by Rao-Scott test  and Wald Z-test using robust standard errors in an ordinary least squares regression (continuous measures). Kaplan-Meier analyses were conducted to examine time to first COPD exacerbation and first pneumonia diagnosis; cohort differences were analyzed with the log-rank test. For key outcomes, the number needed to treat (NNT) of TT patients who would instead need to be treated with TIO/OLO to avoid 1 event was calculated. Rates were derived from the annualized Kaplan-Meier analyses.The secondary analysis (LAMA + LABA vs. TT) repeated the same PSM process and statistical analysis methods used in the primary analysis.A total of 1,592 TIO + OLO and 7,310 TT patients initiated their index regimen from January 1, 2014-March 31, 2018 . After matching, 1,454 patients remained in each cohort.P > 0.05. The only remaining significant differences between cohorts after matching were a slightly higher percentage of patients in the TT cohort with a high exacerbation risk (standardized difference = 6.9%), any exacerbation history (standardized difference = 9.0%), and counts of any exacerbation in the TT cohort, which were driven by the rate of moderate exacerbations (standardized difference = 10.5%). The mean time on index treatment was longer for TIO + OLO versus TT . The most common index TT regimen was a LABA/ICS combination (95.9%) with LAMA monotherapy (95.5%).Before matching, the TIO + OLO and TT cohorts were similar in mean age (72.3 vs. 72.5 years) but differed significantly for most other characteristics . The TT P < 0.001; P = 0.002) and was driven primarily by lower mean acute inpatient costs . Lower mean COPD-related pharmacy costs in the TIO + OLO cohort accounted for the balance of the costs savings .Patients treated with TIO + OLO incurred $7,041 (41.1%) lower mean annualized COPD-related total costs  and $3,374 for pharmacy costs , leading to a difference of $5,253 in total costs . Mean annualized all-cause total costs were lower by $9,221 (30.4%) for TIO + OLO versus TT .After accounting for pneumonia, COPD/pneumonia-related medical costs were lower by $5,212 for TIO + OLO versus TT, averaging $8,209 and $13,421 for the TIO + OLO and TT cohorts, respectively . Thus, tP = 0.020; P = 0.162).The Kaplan-Meier analysis revealed that time to first severe COPD exacerbation was longer for TIO + OLO versus TT (P = 0.002). At 1 year, 14.5% of TIO + OLO vs. 19.3% of TT patients were diagnosed with pneumonia (NNT = 21). There was also a longer time to first diagnosis of pneumonia or acute bronchitis/bronchiolitis . After PSM, standardized differences were < 10% for all baseline characteristics, and the only significant difference between cohorts was a slightly higher percentage of LAMA + LABA patients treated with rescue medications or OCSs at baseline .P < 0.001).After matching, the LAMA + LABA cohort primarily represented patients initiating umeclidinium + vilanterol (69.4%) followed by TIO + OLO (23.3%); the most common index TT regimen was an ICS/LABA combination (95.9%) + LAMA monotherapy (95.6%). Duration of treatment averaged 140 versus 103 days for the LAMA + LABA and TT cohorts, respectively (P < 0.001).Consistent with the primary analysis, the LAMA + LABA cohort had lower mean annualized costs versus TT, with differences between cohorts averaging $6,412 for COPD-related total costs , $4,258 for COPD/pneumonia-related medical costs, $5,250 for COPD-attributable total costs , and $8,071 for all-cause total costs , with a lesser proportion experiencing these events .The Kaplan-Meier analysis of time to first severe COPD exacerbation, any COPD exacerbation, pneumonia, and pneumonia or acute bronchitis/bronchiolitis diagnosis also favored the LAMA + LABA cohort  observed in the current study likely are due to the on-treatment methodology that provides a more sensitive comparison of therapies.Among rigorously matched cohorts who were followed for the duration of patients who remained on their index treatment, mean annualized cost savings to health plans for TIO + OLO averaged $7,041 for total COPD-related costs and $9,221 for all-cause costs compared with TT. When the TIO + OLO cohort was expanded to include any LAMA + LABA regimen, similar, albeit slightly lower, annualized total cost savings were observed for LAMA + LABA over TT, with differences of $6,412 for COPD-related costs and $8,071 for all-cause costs. The current study builds on our recent real-world study that compared TIO + OLO with TT among newly treated MAPD plan beneficiaries for 12 months using an intent-to-treat approach and found mean annual plan savings of $4,118 and $5,384 for COPD-related costs and all-cause total costs, respectively, among patients on TIO + OLO.21 The pattern of lower acute inpatient costs and longer time to first COPD exacerbation  was also observed for LABA + LAMA versus TT. While there was a trend for reduced COPD exacerbations of any severity for TIO + OLO versus TT, the results did not achieve statistical significance, likely due to the TIO + OLO cohort’s smaller sample size.Savings in COPD-related medical costs for TIO + OLO contributed 52.1%  of the difference in COPD-related total costs between cohorts. This was primarily driven by reduced acute inpatient costs (83.3%) and aligns with the cohort differences for severe COPD exacerbations. The time to first severe COPD exacerbation was longer for TIO + OLO, with fewer patients diagnosed (9.0% for TIO + OLO vs. 16.1% for TT); these results also confirm our previous findings.30 This may be attributable to differences in the patient populations—a direct function of the trials’ inclusion criteria.31 All clinical trial participants had symptomatic COPD, with the majority having a history of exacerbations.30 Although we could not assess symptom burden in our study, only 41.7% and 46.2% of the TIO + OLO and TT patients, respectively, had baseline exacerbations, indicating that disease severity differences may account for the disparity in exacerbation results observed between real-world versus clinical trial settings.These exacerbation findings differ from a recent meta-analysis of clinical trials that compared TT with LAMA + LABA  and found a reduced rate of exacerbations (moderate or severe) with TT.COPD-related pharmacy costs accounted for 47.9%  of the savings in total COPD-related costs for TIO + OLO vs TT. This may be driven by the use of a single inhaler to deliver TIO + OLO while > 99% of TT patients used multiple inhalers. COPD-related pharmacy cost savings were similar for LAMA + LABA , with 97% of LAMA + LABA patients on fixed-dose single inhalers versus < 1% of the TT cohort. Nevertheless, COPD-related pharmacy costs encompass other COPD medications , and the potential impact of the new single inhaler TT entrant on pharmacy costs will depend on its usage, pricing, and other factors.32 Furthermore, patients with COPD who develop pneumonia are more likely to die as a result of pneumonia than their non-COPD counterparts.33 Thus, the risk of pneumonia should be a key consideration in selecting appropriate COPD maintenance therapy. When accounting for pneumonia/bronchitis, the annualized COPD-related medical costs increased by 27.3% in the TIO + OLO cohort and 32.7% in the TT cohort, resulting in additional cost savings of 22.0%  for TIO + OLO. The lesser pneumonia burden for TIO + OLO is supported by the longer time to first pneumonia/bronchitis diagnosis and a smaller percentage of patients diagnosed (22.8% vs. 28.4%). Results were very similar for LAMA + LABA compared with TT and are consistent with the increased risk of pneumonia observed in clinical trials.30Lower respiratory tract infections such as pneumonia not only add to the economic burden of COPD but also precipitate the majority of COPD exacerbations.15 The 2018 GOLD recommendations reserve TT for the most severe patients: those in Group D who experience further exacerbations and/or inadequate symptom control after dual bronchodilator therapy or, in some cases, LABA + ICS.5 Nevertheless, only 46.2% (59.4% prematch) of patients in our TT cohort had a baseline history of exacerbations (of any type), and only 21.5% (38.7% before matching) could be considered at high risk for exacerbations, placing them in GOLD Groups C or D. While we could not differentiate Groups C and D, even assuming that all patients with a high exacerbation risk were also highly symptomatic , then roughly 60%-80% of patients received GOLD-discordant TT. This is consistent with previously reported ranges of TT overprescribing (64.0%-65.9%) observed in a similar COPD MAPD plan population.21 After applying the per-patient per-year cost differential in COPD/pneumonia-related burden between the cohorts  for the subset of TT patients with low exacerbation risk , we estimate that switching these TT patients to TIO + OLO could save health plans annually ~$12.5 million  in total COPD-related expenditures.Our results are consistent with prior research documenting overprescribing of TT in real-world clinical practice.8 Nevertheless, 90.1% (60.0% prematch) of TT-treated patients in this study had no baseline exposure to LAMAs and/or LABAs. The reasons for overprescribing of TT in this study and others are unknown, but possible explanations for noncompliance with GOLD recommendations include lack of clinician awareness or understanding of GOLD recommendations.35 Further, asthma and COPD share some common features, and ICS is the mainstay of asthma pharmacotherapy36; although we excluded patients with asthma from our study, misclassification of COPD as asthma in general practice or a consideration of asthma/COPD coexistence could result in overuse of ICS.37 Previous research in COPD populations has shown that treatment with ICS-containing regimens, most commonly LABA + ICS, is the most common precursor to TT and that overuse of ICS-containing regimens is a major cause of the deviation from GOLD recommendations.39 The GOLD 2019/2020 reports provide the strongest limitations on ICS-containing therapies to date: LABA + ICS as an initial therapy is considered only for patients in Group D with a history of/with concomitant asthma or elevated blood eosinophils, and TT is not recommended for initial management.40 Further, their follow-up recommendations limit TT to only those with persistent dyspnea post-LABA + ICS therapy or patients with further exacerbations and elevated blood eosinophil counts post-LAMA + LABA therapy.40 These results underscore the need for additional research to understand the rationale behind overprescribing of TT and a renewed and urgent emphasis on clinician education to align real-world prescribing patterns with GOLD recommendations, especially given the recent availability of single-inhaler TT.GOLD recommendations limiting TT to patients with high exacerbation risk have been relatively constant over the years, and both GOLD 2018 and 2020 recommend TT only as an escalation step from LAMA + LABA or LABA + ICS.8Claims-based studies have inherent limitations, including miscoding and omissions. They also lack some clinically relevant characteristics, such as symptom burden, lung function, smoking status, and blood eosinophil count. We did not distinguish between initial treatment at COPD diagnosis and escalation pathway. Similar to TT overprescribing, a majority of TIO + OLO and LAMA + LABA patients (90% prematch and > 83% postmatch) had no baseline exposure to LAMA or LABA. This suggests potential overprescribing of dual therapy as per GOLD recommendations.41 We also note that the most pronounced differences in Kaplan-Meier severe COPD exacerbation curves between TIO + OLO (and LAMA + LABA) versus TT began after ~7 months on therapy, at which point 23% and 15% of the at-risk TIO + OLO and TT cohorts were retained. This may reflect the benefit of dual therapy’s longer treatment duration or worsening outcomes with TT, but additional research is warranted into this subset.Although we matched patients 1:1 using a restrictive standard for matching within narrow caliper bands, modest but significant cohort differences for 4 exacerbation characteristics remained between TIO + OLO and TT post-PSM. However, the standardized difference for these characteristics was < 11%, indicating a rigorous match, given that 20% is the accepted threshold.42 real-world results may differ. Next, our definition of therapy switch and discontinuation relied on outpatient pharmacy claims. Any patients who may have switched to a nebulized delivery method would have prompted premature censoring of the patient’s follow-up.43 Finally, our results may not be generalizable to nonmanaged Medicare beneficiaries, patients with other forms of insurance, or the uninsured.Further, we did not assess index regimen adherence. Most TT patients used two inhalers while the TIO+OLO cohort (and 97% of LAMA+LABA) used a single inhaler. Although a clinical trial found no difference in exacerbations for TT delivered by 1 versus 2 inhalers,Annualized costs savings to health plans for patients initiating TIO + OLO averaged $7,041 and $8,586 for COPD-related and COPD/pneumonia-related costs compared with TT. The first occurrence of severe COPD exacerbation and pneumonia diagnosis during follow-up took a significantly longer time for the TIO + OLO cohort. Broadening the TIO + OLO cohort to include any LAMA + LABA regimen resulted in slightly lower but similar costs savings and exacerbation and pneumonia outcome benefits. These real-world findings highlight the lower economic and clinical burden of TIO + OLO (or LAMA + LABA) relative to TT and provide new context for prescribing according to GOLD recommendations while also presenting evidence indicating the persistent inconsistency between the recommendations and real-world clinical practices pertaining to TT.


With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states.TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study.Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic . We identified a panel of six proteins  as potential indicators for microglial activation. Moreover, we confirmed three of these proteins  to be significantly elevated in the CSF of Alzheimer’s (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals.We identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer’s Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates  also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders.The online version contains supplementary material available at 10.1186/s13024-023-00657-w. During the last decade, genome-wide association studies (GWAS) and whole genome sequencing have identified microglial expressed risk variants for late onset Alzheimer’s disease (LOAD) . TriggerTREM2 or the lysosomal protein progranulin (GRN) providing us with two completely opposite microglial activation phenotypes. In TREM2 knock-out (ko) models, microglia are locked in a homeostatic state, while microglia from GRN ko models are hyperactivated , midazolam [5 mg/kg], and fentanyl [0.05 mg/kg] (MMF). The MMF-mix was dosed according to bodyweight of each mouse and administrated via intraperitoneal injection. CSF was collected from the cisterna magna by a single stereotactic puncture of the dura using a glass capillary, following a previously described procedure . The colImmediately after CSF sampling, brains were harvested and microglia were isolated using the Magnetic-Activated Cell Sorting (MACS) technique, following the manufactures instructions , MACS Miltenyi Biotec), with slight modifications similarly to a previously published protocol . In brieGRN ko line [TREM2 ko line, while the MwoI enzyme was used for the GRN ko line. We further explain parameters for the generation of TREM2 ko iPSCs in the Appendix Figure All experiments including iPSCs were performed in compliance with all applicable guidelines and regulations. iPSC culture of the female iPSC line A18944  based on E8 Flex/VTN , CRISPR/Cas9-based genome editing, iPSC quality control measures were performed as described previously for the  ko line , with thGRN ko, and TREM2 ko) were used. The cells were carefully dissociated from the plate using a cell scraper, washed with their media, and subsequently transferred to centrifugation tubes (15 ml), pooled pairwise, yielding n = 6 for each experimental group. The tubes were centrifuged at 3000 x g for 6 min at 4 °C and the conditioned media was transferred to a Protein LoBind tube (Eppendorf) (media fraction). Each well of the plates was washed with 0.5 ml PBS and pooled following the same order as the pooling of the collected cells in the same 15 ml centrifugation tube and the pellet resuspended, resulting in a total volume of 1 ml and n = 6 per experimental group. The 1 ml of PBS with the resuspended cells was transferred to a 1.5 ml Protein LoBind tube (Eppendorf), centrifuged at 3000 x g for 6 min at 4 °C, and PBS aspirated, leaving the pellet. The conditioned media was aliquoted and stored at -80 °C (media fraction). Each cell pellet was stored at -80 °C (microglia fraction).Throughout the procedure, cells and PBS were kept on ice. In total, two 6-well plates from each experimental group  and 12 non-symptomatic non-carriers (CON) were analyzed. Data from one GRN mutation carrier was excluded as this participant was not fulfilling the criteria of symptomatic FTD, resulting in a comparison of 11 FTD-GRN and 12 CON. All participants were enrolled in ARTFL (U54NS092089) or LEFFTDS (U01AG045390), together ALLFTD, which is a North American research consortium to study sporadic and familiar FTD. ALLFTD has 27 sites in North America. Participants are between the ages of 18–85. Inclusion criteria for ALLFTD are clinical diagnosis of an FTD syndrome, or being a member of a family with a known FTD-associated genetic mutation or a strong family history (modified Goldman score of 1 or 2) of FTD syndromes. For the study presented here, the group defined as FTD-GRN was defined as symptomatic GRN-mutation carriers. All CSF from the ALLFTD consortium were approved and provided by the National Centralized Repository for Alzheimer’s Disease and Related Dementias (NCRAD). CSF obtained from ALLFTD was collected following NCRAD standard operating procedure. In brief, CSF was collected in the morning between 8 am – 10 am, preferably fasted or following a low-fat diet. Lumbar puncture was performed in a sterile field using an atraumatic technique. Within 15 min of collection, CSF samples are centrifuged at 2000 x g for 10 min at room temperature. Post centrifugation CSF is aliquoted into pre-cooled aliquot tubes and within 60 min of CSF collection aliquots are snap frozen on dry ice and stored at -80 °C. Data from the European Medical Information Framework Alzheimer’s Disease Multimodal Biomarker Discovery (EMIF-AD MBD) cohort are publicly available [1−42[+) and amyloid-negative (A−). For correlation analysis CSF levels of TREM2, GFAP and CHI3L1, as included in the publicly available data [In total, CSF of 12 symptomatic FTD heterozygous vailable . Proteomble [1−42, which aGrn ko, and Trem2 ko – all sacrificed when 12 months old. In total, we analyzed CSF from 14 mice (n(Wt) = 6; n(Grn ko) = 4; n(Trem2 ko) = 4) and microglia from 19 mice (n(Wt) = 7; n(Grn ko) = 4; n(Trem2 ko) = 8). The hiMGL samples included both cell lysates and conditioned media, each collected from three different iPSC lines: WT, GRN ko, and TREM2 ko, with a sample size of n = 6 for each sample type and line. For human CSF, the CSF of 11 symptomatic FTD heterozygous GRN mutation carriers (FTD-GRN) and 12 non-symptomatic non-carriers (CON) was analyzed. All samples were prepared and analyzed together according to sample type; mouse CSF, mouse microglia, hiMGL media, hiMGL cell lysates, and hCSF. Mouse CSF was prepared using in-solution digestion. Mouse microglia, and hiMGL media was prepared using filter aided sample preparation (FASP) [In total, 14 sets of samples have been collected from mice, hiMGL and participants of the ALLFTD study. Samples were prepared and analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS) using a label-free quantification (LFQ) method. The mouse cohort consists out of three mouse lines: Wt, n (FASP) , while hn (FASP) .From each sample of mouse CSF, a volume of 5 µl was used for in-solution digestion in 0.1% (w/v) sodium deoxycholate according to a previously published protocol . Peptide15 µl of human CSF was mixed with 15 µl modified STET lysis buffer  to inactivate potential viruses. For hiMGL, cell pellets were lysed on ice in STET lysis buffer supplemented with protease inhibitors . The SP3 sample preparation was performed according to a previously published protocol  using 70Mouse microglia and hiMGL conditioned media were digested using filter aided sample preparation (FASP) as previously described . For micLiquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for label-free quantification (LFQ) of the proteolytic peptides. Two different setups were used. Mouse samples were analyzed on an Easy-nLC 1200 coupled to a Q-Exactive HF mass spectrometer  as previously described for microglia  using daFor human derived samples (hiMGL and hCSF), peptides were separated using a NanoElute nano HPLC equipped with either 30 cm (hCSF samples) or 15 cm (hiMGL samples) 75 μm ID columns packed with ReproSil-Pur 120 C18-AQ, 1.9 μm stationary phase  applying a 120 min gradient for lysates or a 70 min for hiMGL conditioned media and hCSF, which was online coupled to a TimsTOFpro mass spectrometer . Samples were analyzed using DIA parallel accumulation serial fragmentation. For hCSF, one scan cycle induced one MS1 full scan followed by 2 rows of 50 sequential DIA windows with 18 m/z width for peptide fragment ion spectra with an overlap of 1 m/z covering a scan range of 350 to 1200 m/z (hCSF). The ramp time was fixed to 166 ms and 5 windows were scanned per ramp. This resulted in a total cycle time of 3 s. For hiMGL samples, one scan cycle included one MS1 full scan followed by 2 rows of 40 sequential DIA windows with 22 m/z width for peptide fragment ion spectra with an overlap of 1 m/z covering a scan range of 350 to 1200 m/z. The ramp time was fixed to 100 ms and 4 windows were scanned per ramp. This resulted in a total cycle time of 2.1 s.https://github.com/vdemichev/DiaNN, Version 1.8) [https://github.com/vdemichev/DiaNN, Version 1.8) [Database search and label free quantification was performed using the MaxQuant software package   for DDA ion 1.8) . The anaion 1.8) , with whion 1.8)  was usedt-test was applied to evaluate the significance of proteins with changed abundance. In addition, multiple hypothesis correction was performed using a permutation-based FDR estimation [0 = 0.1) or the method of Benjamini, Krieger and Yekutieli [LFQ values were used for relative quantification, log2 transformed and statistically analyzed using Perseus (Version 1.6.15.0)  and Graptimation  (threshoekutieli  (threshoGrn ko and Trem2 ko mice and their corresponding age-matched control (Wt) mice  were significantly increased in lysates and media of GRN ko hiMGL  Fig. C-D. The MGL Fig. A and C.  WT Fig. A. Consisome Fig. A. In conate Fig. A [11, 12TREM2 ko hiMGL model are more subtle than those in GRN ko hiMGL. Comparison of the number of significantly upregulated proteins in cell lysates of GRN ko hiMGL  , of which 3 patients were diagnosed with mild cognitive impairment (MCI) at the timepoint of CSF sampling. In addition, the CSF of 12 non-symptomatic controls  without GRN mutations were included     FABP3 in cases of Parkinson’s disease with dementia (PDD), Dementia with Lewy bodies (DLB) , AD 48––48–50 anGPNMB has been reported as part of a DAM signature in mouse models with both, amyloid and tau pathology [Importantly, our data also revealed changes of the proteome that are not translatable across the mouse and human models, such as protein levels of MDH1, ENOPH1, CD84, and EPB41L3, proteins that are upregulated in the hiMGL model and in human CSF, but downregulated in mice , the raBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6


To explore the clinical characteristics of testicular torsion in children with non-scrotal initial symptoms who were misdiagnosed.A retrospective analysis of 73 cases children with testicular torsion and non-scrotal symptoms who were admitted to our department from October 2013 to December 2021 was performed. Patients were divided into misdiagnosis (27 cases) and clear diagnosis at first visit (46 cases) groups. Clinical data, including age at surgery, clinical presentation, physical examination, number of visits (≥2 times), affected side, time from initial symptoms to surgery, and surgical outcomes, were collected. The TWIST (Testicular Workup for Ischemia and Suspected Torsion) score was calculated and analyzed.P < 0.05). There were no statistically significant differences (P > 0.05) in age, affected side, TWIST score, guardian, direction of testicular torsion, intra-vaginal or extra-vaginal torsion, and Arda classification. Postoperative follow-up was 6–40 months. Of the 36 patients who required an orchiopexy, 1 had testicular atrophy at six months and 2 were lost to follow-up. The contralateral testis of the 37 children who underwent orchiectomies developed normally without torsion.Statistically significant differences between the misdiagnosis and clear diagnosis groups were seen in the time from initial symptoms to surgery, the number of visits, the degree of testicular torsion, and the rate of orchiectomy (The clinical manifestations of testicular torsion in children are diverse and can easily lead to misdiagnosis. Guardians should be aware of this pathology and seek timely medical attention. When the initial diagnosis and treatment of testicular torsion is difficult, the TWIST score during the physical examination may be useful, especially for patients with intermediate-to-high risk scores. Color Doppler ultrasound can assist in making the diagnosis, but when testicular torsion is highly suspected, routine ultrasound is not necessary as it may lead to delayed surgical treatment. It is g2.2.1.n = 27) with those who had a correct initial diagnosis (n = 46 cases) is shown in After approval by the ethics board of Anhui Provincial Children’s Hospital, a retrospective review of 73 pediatric patients diagnosed with TT on preoperative ultrasound and surgery was performed. Cases of epididymitis and torsion of the testicular appendages that presented with scrotal swelling were excluded. The age range of included patients was 8 months to 16 years, with an average age of 4.6 years. Patients with perinatal TT were excluded in our study. Left-sided TT was diagnosed in 48 patients, while 25 cases were on the right. The time from the onset of initial symptoms to surgery ranged from 5 h to 7 days, with an average of 2.9 days. Twenty-seven cases had non-scrotal initial symptoms, including 5 with lower abdominal pain, 8 with nausea and vomiting, 3 with periumbilical pain, 3 with crying and refusal to feed, 3 with inguinal pain, 4 with lumbar pain and discomfort, and 1 with a fever. A comparison of characteristics of patients with non-scrotal initial symptoms who were initially misdiagnosed . Continuous variables were presented as mean±standard deviation (SD) and categorical variables were presented as ratios(%). Comparisons were performed via chi-squared or Fisher’s exact test for categorical variables. 3.Of the 73 patients were included in our study, the degree of torsion ranged from 90° to 960°. The tunica vaginalis completely surrounded the testis, epididymis, and distal spermatic cord rather than connecting to the posterolateral portion of the testis and the base of the epididymis, confirming the bell clapper deformity. As a result, the testis and epididymis hung freely in the tunica vaginalis-created sac ①. An orcTwenty-seven patients were initially misdiagnosed, of which 8 were initially diagnosed as gastroenteritis or mesenteric lymphadenitis at other hospitals and treated with oral antibiotics with poor results. TT was found upon re-examination and emergency surgery was performed. Six patients with transferable right lower abdominal pain around the navel were initially diagnosed with appendicitis, but after ultrasound ruled out appendicitis, TT was confirmed. Four patients with lower back pain were initially diagnosed with urinary tract stones, but after ultrasound ruled out a stone, TT was found. One patient was initially diagnosed with constipation and treated with laxatives, but TT was diagnosed upon arrival at the hospital due to persistent pain. One patient who presented with a lump and pain in his groin was initially diagnosed with an incarcerated hernia with cryptorchidism, but intraoperative confirmation revealed that the testis was pulled and twisted to the groin.P > 0.05) (P < 0.05). Age, affected side, guardian, direction of TT, intra-scrotal or extra-scrotal torsion, and Arda grading were equivalent (P > 0.05) (The distribution of TWIST scores within the misdiagnosis group was as follows: 1 low risk 0–2 points), 10 intermediate risk (3–4 points), and 16 high risk (5–7 points). Children with an intermediate-to-high risk of TT accounted for 96.3% (26/27) of the total group. The distribution of TWIST scores within the clear diagnosis group was as follows: 2 low risk (0–2 points), 21 intermediate risk (3–4 points), and 23 with high risk (5–7 points). Children with an intermediate-to-high risk of TT accounted for 95.6% (44/46) of the total group. There was no statistically significant difference in the TWIST scores of the two groups ( > 0.05) . However > 0.05) , 2. points, Postoperative follow-up was 6–40 months. One patient of the 36 who underwent an orchiopexy had testicular atrophy after six months (his testis was found to be non-viable during surgery), 2 patients were lost to follow-up, and the remaining 33 did not have testicular atrophy. The contralateral testis of all 37 children who underwent orchiectomies developed normally without torsion.4.TT is a common pediatric urologic emergency that often occurs in neonates and adolescents, particularly after intense physical activity or cold weather conditions , 6. The The clinical presentation of TT can vary, but its typical symptoms include redness, swelling, and scrotal pain on the side of the torsion. It can also include nausea, vomiting, abdominal or groin pain, and fever. However, in cases where scrotal symptoms are not the initial manifestations of the torsion or when the torsion is incomplete, symptoms outside of the scrotum may be present. These include abdominal pain, nausea, vomiting, groin pain, fever, or a refusal to eat and irritability in younger children. In these patients, TT can be easily misdiagnosed or missed by the initial treating physician . The reaAt present, the diagnosis of TT mainly relies on a comprehensive physical examination by the initial doctor and a color Doppler ultrasound examination. A sonographer can diagnose TT based on decreased blood flow to the testicle. Although color Doppler ultrasound has a high accuracy, convenient implementation, simple detection, and a low cost, this mode of diagnosis has certain limitations. Color Doppler ultrasound examination has a sensitivity of 69%−91% and a specificity of 87%−100% for the diagnosis of TT –15. UltrTT can be diagnosed definitively based on its clinical features and ultrasound-assisted examination. As the likelihood of testicular salvage and the length of symptom persistence are inversely proportional in patients with TT, early testicular detorsion and surgical exploration are crucial . In caseIn summary, TT is a pediatric surgical emergency with a diverse clinical presentation. It is important to raise the awareness and attention of primary care physicians, emergency physicians, and urologists of the non-scrotal initial symptoms of TT in children. When ultrasound equipment or the experience of ultrasound technicians is limited, the TWIST scoring system can be used as part of a comprehensive physical examination. An intermediate-to-high risk TWIST score plays an important role in indicating TT. Therefore TWIST score is only important for surgeons when have a physical examination but not to those referring to surgery. Any suspicion of TT deserves being examined by pediatric urologist or surgeon. It is very important because of being associated with organ loss even 1 in 30 cases of low risk by scoring. When TT is highly suspected, timely surgical exploration or referral is necessary to avoid the consequences of delayed treatment. In addition, further cases need to be collected to summarise the experience of diagnosis and treatment of TT in children with non-scrotal initial symptoms, due to the small number of cases in this study and the short follow-up period.


Systemic infection has always been considered a relative contraindication to neuraxial anesthesia, despite the fact that infectious complications are relatively uncommon. Pregnancy-related physiological changes and coronavirus disease (COVID-19) neurotropic features may facilitate the virus’ entry into the central nervous system. The principal aim of this study was to test the safety of spinal anesthesia in “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2)-positive pregnant women and to examine cerebrospinal fluid (CSF) characteristics.We conducted a prospective observational single-center study in asymptomatic or paucisymptomatic consecutive pregnant SARS-CoV-2 patients who underwent spinal anesthesia for cesarean section. Women with severe infection were excluded because they underwent general anesthesia. At the time of spinal anesthesia, we collected CSF samples, and then we performed a chemical-physical analysis to look for signs of inflammation and for SARS-CoV-2 genome.IQR 0.5500 (0.6000–0.7100), and the average CSF protein concentration value was 23.2 mg/dl (SD 4.87). In all samples, genomes of SARS-CoV-2 and other neurotropic viruses were not detected.We included 26 women. No spinal anesthesia complications were reported in the perioperative period and after 2 months. All CSF samples were crystal clear, and all physical-chemical values were within physiological ranges: the median concentration of CSF/plasma glucose ratio was 0.66, Spinal anesthesia was safe in SARS-CoV-2 pregnant women with mild disease; no clinical maternal complications were detected, and no CSF changes indicative of inflammatory or infectious diseases that would compromise the safety of the procedure were found. Since the beginning of the pandemic, data on the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on pregnant women has been limited . AccordiTo date, no data are available about the safety of performing NRA in pregnant women positive for SARS-CoV-2, regardless of the presence of symptoms. Even though a preexisting infection has always been considered a relative contraindication to NRA, several studies have shown that major problems, like infectious complications, are rare after spinal and epidural anesthesia, even in pregnant women –6.Despite the fact that SARS-CoV-2 patients mostly developed respiratory symptoms, neurotropic features of the virus have been known since the beginning of the pandemic. In fact, SARS-CoV-2 may cause central neurological symptoms and complications by either directly accessing the central nervous system (CNS) or through an inflammatory-cytokine activation (cytokine storm) .Pregnancy leads to several anatomical and physiological changes, and the CNS is also involved . A decreThe CNS physiological changes during pregnancy and the presence of SARS-CoV-2 infection may reduce the safety of NRA.The principal aim of this study was to test the safety of NRA in SARS-CoV-2 pregnant women undergoing CS and to perform CSF analysis to evaluate the presence of the SARS-CoV-2 genome and chemical-physical signs of inflammation.We conducted a prospective observational single-center study in SARS-CoV-2 consecutive pregnant women without severe infection who underwent CS, to test the safety of NRA through the analysis of CSF.The study is designed and presented according to the STROBE guidelines .Data were collected at the Obstetric Department of ASST Papa Giovanni XXIII , a tertiary care referral center  for obstetric care in the North of Italy.Since the beginning, due to the exceptional pandemic period, the ethical committee approved all the studies on COVID-19, and the release of data was authorized in 2022 (REF: no. 61/2022).Patients were recruited between May 2020 and February 2022; the enrollment process is shown in Fig. Inclusion criteria were asymptomatic or paucisymptomatic SARS-CoV-2-positive pregnant women aged > 18 years who underwent CS under spinal anesthesia and who gave consent for both CSF collection and the subsequent analyses. Exclusion criteria were as follows: age < 18 years, women with severe symptoms  because they underwent CS under general anesthesia, women who underwent CS in epidural anesthesia after analgesia in labor, and women who did not give informed consent.All enrolled patients did not receive antiviral or corticosteroid therapy for the treatment of SARS-CoV-2 infection in the preoperative period since the symptoms were absent or mild SARS-CoV-2 positivity was identified by real-time reverse-transcriptase polymerase-chain reaction (RT-PCR) performed on nasopharyngeal specimens at the time of admission.For elective CS, admission was scheduled the evening before surgery. Urgent CS was indicated for labor dystocia, and the SARS-CoV-2 test was performed as soon as the mother entered the delivery room.We recorded demographic and clinical data  and the presence of one of the following symptoms and clinical signs at the moment of NRA: fever, headache, cough, anosmia, dysgeusia, rhinorrhea, diarrhea, and a preoperative chest radiograph (CXR) positive for pneumonia. In addition, a blood test was performed before CS to measure plasma glucose and blood C-reactive protein levels.A CSF sample was obtained when spinal anesthesia was performed at the time of the CS. In asepsis, a 25-gauge spinal needle was introduced into the lumbar subarachnoid space, and 2.5 ml of CSF was collected prior to intrathecal administration of the anesthetic drugs. Afterwards, each patient received 2.5 ml of levobupivacaine, 0.5% isobaric with sufentanil 2.5 μg in 0.5 ml, and morphine 100 μg in 1 ml for a total volume of 4 ml.After spinal anesthesia, an intravenous bolus of ephedrine (6 mg) and phenylephrine (50 mcg) was administered to prevent hypotension. This was repeated if necessary.CSF samples were stored at −80 °C in the hospital biobank , and latWe looked for complications of spinal anesthesia in the perioperative period and, through a phone inquiry, 2 months after CS. We assessed the presence of orthostatic headache, paresthesias, dysesthesias, and myalgias in the lower limbs.D = 600 nm). Glucose levels were determined through the enzymatic hexokinase method. Afterwards, we calculated the CSF/plasma glucose ratio as previously indicated by Bernardi et al. [CSF chemical-physical analysis was performed on the Atellica Siemens platform (CH module). The total protein assay was performed with the colorimetric biuret technique . Additionally, each sample was analyzed in order to target specific genomic regions of the following viruses: herpes simplex virus 1, herpes simplex virus 2, Epstein-Barr virus, Nonnormal distributed data are presented as median and interquartile range (IQR) . Normal distribution was assessed using the Shapiro-Wilk test, and qualitative variables are presented as frequencies and percentages. Statistical analysis and plot modeling were performed using SPSS (Version 28).Between March 2020 and February 2022, 87 patients scheduled for CS tested positive for SARS-CoV-2. Of these, 66 underwent CS with spinal anesthesia, and 26 were included in the final analysis, according to Fig. In our population, elective CS was scheduled in 80.8% of the cases, while 19.2% of the women underwent CS in urgent regime. The gestational age at delivery was > 37 weeks in 22 women and between 32 and 37 weeks in 4 patients; 61.5% of women were asymptomatic, and 38.5% were symptomatic.Detailed demographic and clinical data are summarized in Tables IQR 0.5500 (0.6000–0.7100), the average CSF protein concentration value was 23.2 mg/dl, and standard deviation (SD) was 4.87. Data are summarized in Fig. All CSF samples were crystal clear, the median concentration of CSF/plasma glucose ratio was 0.66 with SD 0.21). All blood and CSF values obtained are summarized in Table All CSF C-reactive protein values resulted < 0.05 mg/dl, while the blood C-reactive protein concentration resulted > 1 mg/dl in 20 patients. Only 8 samples were sufficient to perform lactate levels detection; CSF lactate average value was 1.7 mmol/l  were not detected.In each CSF sample, the presence of SARS-CoV-2 and other neurotropic viral genomes , and the average CSF protein concentration value was 23.2 mg/dl (physiologic range 15–45 mg/dl) (SD 4.87). All physiologic ranges used as a parameter in our study referred to the healthy general population. There is only one study from 1979 that investigated CSF physiologic ranges during physiological pregnancy, but it did not evaluate glucose levels [In our analysis, all the CSF samples were crystal clear, and all physical-chemical values were within physiological ranges, even in pauci-symptomatic women. The median concentration of CSF/plasma glucose ratio was 0.66 , e levels .CSF/plasma glucose ratio and CSF lactate levels are known to be age, but not gender dependent . There aCSF lactate concentration does not depend on blood lactate levels; the physiologic range is between 1.2 and 2.1 mmol/l in the adult population . In the The fact that all physical-chemical values were within physiological ranges, despite elevated systemic blood C-reactive protein values, could potentially suggest that the systemic inflammatory process is not predictive of an inflammatory response in CSF. Furthermore, we emphasized that none of the patients received antiviral or anti-inflammatory medication prior to surgery, which may have affected the results of these analyses.However, it was not possible to assess white blood cells (WBC) count in the CSF, because of cellular lysis due to the sample defrosting procedure.All CSF samples analyzed were negative for the detection of SARS-CoV-2 RNA. This finding agrees with two previous smaller studies that investigated the presence of viral RNA in the CSF of SARS-CoV-2 pregnant women , 17. In Nonetheless, in the literature, the presence of viral RNA in CSF was found only in patients with severe neurological symptoms; in 2021, a review reported an incidence of 6% of SARS-CoV-2 RNA in CSF in patients with severe neurological manifestations . FurtherIn our population, none of the patients had intraoperative hypotension, and ephedrine and phenylephrine therapy was effective.In addition, none had the typical orthostatic CSF headache, despite the collection of CSF, and no complications were reported.This study has some limitations. Our analyzed cohort was small, and CSF samples were stored; therefore, immediate analysis could not be performed, and cellularity could not be assessed.Maybe it is too early to demonstrate that NRA is a completely safe procedure in SARS-CoV-2 pauci or asymptomatic pregnant women, although in this study we found that all CSF samples were negative for detection of virus, and also in literature it seems that neurological complications of systemic infections are uncommon.Currently, CSF analysis appears to be the only clinically acceptable invasive method to evaluate CNS responses to infection. However, the detection rate of SARS-CoV-2 in the CSF is highly dependent on the type of disease and the time of sample collection. The titers of viruses in the CSF may change over the course of a patient’s illness due to possible CSF clearance.Therefore, CSF testing may fail to give positive results due to delayed sampling. Although some patients showed negative results for SARS-CoV-2 in the CSF, the possibility of CNS infection cannot be completely excluded in these patients, as demonstrated in some autopsy studies. There is a “window of time” for the virus to enter the CNS that needs to be considered .Otherwise, this study contains new ideas that deserve to be explored by other larger sample sizes in multicenter studies and highlights how few references exist for the typical range of CSF values in healthy pregnant patients; as a result, all comparisons were made with reference to the healthy general population.Despite the small sample size in our investigation and the limits of the CSF analysis, we discovered no clinical and CSF alterations consistent with inflammatory or infectious conditions that may otherwise compromise the safety of neuraxial procedures, even in SARS-CoV-2 pregnant women.Certainly, further investigations of CSF features in obstetric patients are necessary. Identification of CSF physiological values in this population is essential for detecting any alterations or biomarkers indicative of obstetrical disorders or systemic pathologies.