Tribulus terrestris L. (TT) ingredients have anti-inflammatory and antioxidant activities, but their effects on exercise-induced muscle damage (EIMD) in trained athletes are uncertain. The purpose of this single-blind placebo-controlled trial, in accordance with CONSORT guidelines, was to examine the effect of 6 weeks of TT supplementation on muscle metabolism, inflammation biomarkers, and oxidant status. Thirty trained male CrossFit® athletes were randomly assigned to be supplemented with 770 mg/day of TT (intervention group (IG)) or receive a placebo daily (control group (CG)) for 6 weeks. Muscle damage enzymes, inflammation biomarkers, and Total Antioxidant Status (TAS) were assessed at baseline (T1), 21 days after baseline (T2), and after 42 days (T3). Grace, a Workout of the Day, was measured in T1 and T3. Statistical significance (p < 0.05) was found between IG and CG in Lactate Dehydrogenase (LDH), C-reactive protein (CRP), and TAS levels at the end of the follow-up. Furthermore, TAS levels were significantly (p < 0.05) lower at T2 and T3 relative to baseline in the IG, also LDH and CRP increased significantly (p < 0.05) at T2 and T3 relative to baseline in the CG. No significant (p > 0.05) decreases in muscle damage or inflammation biomarkers were observed, although a slight downward trend was observed after 6 weeks for supplemented athletes. TT supplementation could attenuate the CrossFit® training program-induced oxidative stress, muscle damage, and inflammation which could be due to the natural antioxidant and anti-inflammatory properties of TT.  At the beginning of the 6-week intervention period, individuals randomly received pre-packed capsules which included either a placebo (PL) or the TT supplement and labeled with the day of consumption. The randomization sequence was statistically designed to assign the participants to the two study groups (control group (CG) and intervention group (IG)) using a stratified block design. The IG received 2 TT capsules from Quamtrax® Pharmaceuticals  with 770 mg daily; TT extract 72.64% (385 mg/cap) at 40% of saponins. The CG received 2 placebo capsules of 100 mg, formulated using maltodextrin capsules to simulate the color and texture of the TT capsules, to generate a highly reliable blinding. The placebo was manufactured at the Magistral Formulation Laboratory of a Pharmacy . Participants were instructed to ingest the capsules orally with 200 mL of water, in 1 daily dosage, taken 30 min after CrossFit® workout.The sample size estimation was performed using the G* Power 3.1.97 statistical power analysis program  . The num® athletes were instructed using 2 diet tracking methods previously used in our studies [−1 for each CrossFit® athlete [To standardize diet and antioxidant status, participants were advised to have the same standard diet  prescribed by a registered professional dietitian-nutritionist. This dietitian-nutritionist carefully documented in detail the daily food and fluid intake of the subjects throughout the trial to ensure that they had followed the diet they were given. Participants were instructed on how to complete the diet-recall questionnaires and determine food servings and sizes by a registered professional dietitian. The CrossFit studies ,21: (i)  athlete .® athletes. Bioelectrical impedance analysis (BIA)  was employed to calculate body mass and fat mass; a stadiometer was used to measure height. An internationally certified anthropometrist (ISAK level 3 with certificate number: #63673929292503670742) performed the anthropometric measurements of all CrossFit® athletes arrived at the laboratory at 8:30 am and upon arrival sat comfortably for 30 min. The collection and transport of blood samples was performed following the World Anti-Doping Agency (WADA) standards [All participants attended the laboratory for blood collection at 3 specific times: at baseline (T1), 21 days after baseline (T2), and at the end of the study after 42 days (T3) . Blood standards . The pretandards ,21. Serutandards . All biotandards .® training sessions was between 6:00 p.m. and 8:00 p.m. Each 1.5 h CrossFit® training session consisted of 3 sections such as specific warm-up, WODs, and cool-down. Each workout was performed in the participants’ own gym, supervised by a certified CrossFit® Degree 1- or 2-certified instructor (each). All subjects completed the same physical activity routines to ensure that they performed the same prescribed workout during the study. In addition, CrossFit® athletes were specifically asked not to perform CrossFit routines outside of the structured training program.Participants trained in 3 sessions on alternate weekdays  for a total of 4.5 h/week. The start of all CrossFit® athletes do to monitor their performance improvements. Thanks to its great scalability, Fran has become famous as a key exercise to know the athlete’s progress and many CrossFit® athletes use it as a unique yardstick to measure their physical capacity and analyze physical progress [® competitions, and it is considered an advanced level of CrossFit® with times over 4 min [Fran’s inclusion WODs consisted of three rounds of thrusters and pull-ups for 21, 15, and 9 repetitions [progress . Furtherprogress  have repprogress  verified® protocols [® athletes performed 30 clean and jerk repetitions, and the time in seconds (s) to complete them was measured. The male athletes were required to use weights of 61.4 kg [TT supplementation had not yet begun, and at the final day of the study (T3) after 6 weeks. We have controlled the fatigue level using the Grace test (WODs) following the international CrossFitrotocols ,2. For Gn = 30) completed a form intended to assess the effectiveness of the single-blinding process in the study. The results of the questionnaire indicated that 90% (n = 27) of the CrossFit® athletes did not identify the group to which they were assigned. Only 1 participant stated and correctly knew which group in the study he belonged to. These results could indicate that the single blinding was fruitful. The participants (https://apps.apple.com/es/app/random-number-generator-app/id1476396989) (accessed on 15 July 2021). The statistical analysis was carried out using STATA version 15.0 , SPSS software version 24.0 , and Microsoft Excel (Microsoft Excel version 19). The results were contained as means and standard deviations. Statistical significance was established at a value of p < 0.05. The Shapiro–Wilk test was used to determine the normality of the variables. Parametric tests were used because the data followed a normal distribution. To determine the differences between the groups (IG and CG) at baseline and the end of the study, a t-test for independent variables was used for the sample characteristics, dietary assessments, and performance tests. In addition, a t-test for dependent variables was used to determine the existence of significant differences between the performance tests at T1 and T3. An analysis of variance (ANOVA) with repeated measures was performed to examine the effects of the time × TT supplementation interaction on the different groups (IG and CG) and the muscle damage , inflammation (IL-6 and CRP), and oxidant response (TAS) of the groups. The differences between the biomarkers at different times during the study were determined using the Scheffé test. The percent changes in the WODs test studied in the CG and IG between T1 and T3 were calculated through the following formula: Δ% = ((T3 − T1)/T1) ∗ 100; to determine whether the differences in these percentages between groups were significant, the t-test for independent samples was used.For the random assignment of participants to both study groups (CG and IG), we used the sequence created by the “Random Sequence Generator” software (based on stratified block design) based on study entry date , and 6 athletes were discarded for presenting results, time to complete the test > 250 s. In addition, two participants were rejected because they chronically consumed anti-inflammatory drugs that could alter the biomarkers of the study. Finally, 30 CrossFit® athletes were involved in the study. Thirty athletes were randomly assigned into two groups  and intervention group ). None of the 30 participants dropped out of the study or interrupted the intervention, so all participants were tested at T1, T2, and T3  were obtained in the anthropometric and exercise performance profile between CG and IG, as described in No significant differences (p > 0.05) for the dietary components reported in During the study, there was no significant difference between CG and IG  between the time points  nor between CG and IG were detected for ALT, AST, Mb, aldolase, CK, CK-MB. Significant differences (p < 0.05) were observed between the three study points in the CG for LDH. In addition, for LDH there were significant differences (p < 0.05) in Condition × Time between both groups (CG and IG). For the CG, a significant increase (p < 0.05) in LDH was observed at T2 (353.11 ± 9.12 UI/L) and T3 (366.50 ± 10.71) compared to T1 (312.13 ± 10.81 UI/L). However, LDH had a downward trend in the IG from baseline (328.59 ± 5.49 UI/L) to T3 (314.14 ± 4.70 UI/L) (No significant differences (70 UI/L) .p < 0.05) were observed between the three study points  in the CG for CRP. In addition, for CRP there were significant differences (p < 0.05) in Condition × Time between both groups (CG and IG). In addition, for the CG, a significant increase (p < 0.05) in CRP was observed at T2 (1.51 ± 0.22 mg/L) and T3 (1.55 ± 0.22 mg/L) compared to T1 (1.21 ± 0.30 mg/L) (p >0.05) decrease was observed throughout the three points evaluated in the study  (Significant differences (30 mg/L) . However and T3) .p < 0.05) at T2 (1.46 ± 0.02 mmol/L) and T3 (1.48 ± 0.05 mmol/L) compared to T1 (1.34 ± 0.07 mmol/L). In the CG, TAS remained practically unchanged from T1 (1.31 ± 0.05) to T3 (1.33 ± 0.06). Significant differences (p < 0.05) between both groups, IG and CG, were observed for TAS  between the time points nor between the groups were reported for hematological biomarkers (No significant differences (omarkers .p > 0.05) in Grace, but there was a slight trend of improvement in the IG (IG −25.00 ± 10.74 %Δ) compared to the CG (−24.73 ± 10.58%Δ).TT (770 mg/day) in trained male CrossFit® athletes were significantly (p < 0.05) different compared to CG, throughout the three points of the study. In addition, TAS levels were significantly (p < 0.05) lower at T2 and T3 relative to baseline in the IG. No significant (p > 0.05) decreases in muscle damage or inflammation biomarkers were observed, although a slight downward trend was observed after 6 weeks of the study for supplemented athletes. For a better understanding, we have divided the discussion into several sections.Our study’s results showed that LDH, CRP, and TAS levels after 6 weeks of supplementation with TT’s supplementation for 2 weeks with 500 mg/day had no effect on IL-6 or hs-CRP in healthy non-athletic men after resistance exercise. According to these findings, we did not observe any effect on IL-6. However, we showed a significant difference regarding between-group changes in CRP (p < 0.05) and a significant (p < 0.05) increase in the CG, with a moderate downward trend in the IG. These differences could be due to the supplementation time, dose, or composition of TT´s supplement.WODs are recognized as one of the modalities of very-high-intensity functional training with minimal or no rest between bouts, making WODs a strenuous workout . In respTribulus may be due to the modulating action on inflammatory pathways of TT components [TT lacks immunomodulatory properties, which regulate inflammatory states for a longer duration. However, an in vitro study in RAW 264.7 cells [Tribulusamide D (phenolic amides from the fruits of TT) treatment reduced the expression of inflammatory cytokines IL-6, IL-10, and TNF-α [The potential anti-inflammatory effect of mponents . The polmponents ,32, but mponents . Based omponents ,32, it cp < 0.05) decrease in plasma total antioxidant capacity after vigorous WODs. A CrossFit®-based metabolic conditioning session increases oxidative stress in proportion to the intensity or fatigue induced by the duration of the training session [® would trigger different major sources of reactive oxygen species (ROS) contribution during and after WODs [TT´s polyphenols and flavonoids act as antioxidants [TT. In addition, in cadmium-intoxicated rats supplemented with TT extract (5 mg/kg) that reduced the elevation of thiobarbituric acid reactive substances (TBARS) [p < 0.05) increased TAS. TAS evaluates the dynamic balance between the antioxidant system and prooxidants [TT, we could hypothesize that long-term supplementation of TT  could be effective in increasing TAS in the CrossFit® training program.A recent study  showed a session . The higter WODs . TT´s pooxidants ,35 whichoxidants  by neutroxidants . Da silvoxidants  reported (TBARS) , thiobaroxidants . Regardi® training program, a process of muscle tissue breakdown and dumping of intramyocellular contents into the systemic circulation is initiated [TT supplemented athletes, especially on LDH plasma levels, where their behavior was statistically significant (p < 0.05) compared to the non-supplemented. Overall, muscle biomarker levels of the participants in the IG were lower than those of the CG after the exercise, but this decrease was not statistically significant. Supplementation of TT at higher doses or for a longer period could lead to a significant improvement. Three previous studies examined muscle damage biomarkers [TT supplementation demonstrated a significant reduction for CK activity (during high-intensity training) [TT powder and extract [TT. These differences may be due to bioactive components of TT supplementation [TT´s muscle protective action via flavonoids, alkaloids, phenols, and saponins [TT might effectively lessen the muscle damage because of a CrossFit® intense training-based regimen. After a high-intensity CrossFitnitiated . Sustainnitiated . These bnitiated , which nnitiated . EIMD innitiated ; similaromarkers ,39,40 inraining) . In addi extract . Howeverentation . TT´s musaponins ,39 are nsaponins ,42. Thersaponins . TherefoTT treatment such as: gastrointestinal problems [TT (770 mg/day), and no alterations on hematological biomarkers have been reported. The TT´s dose administered was within the safety range (250 mg–9000 mg) proposed by the Scientific Committee of the Spanish Agency for Consumer Affairs, Food Safety and Nutrition (AECOSAN) [Human clinical trials reported several side effects after problems ,44, gyneproblems , priapisproblems , nephrotproblems ,48, hepaproblems , sleep dproblems ,48. Our AECOSAN) .® training program was performed looking for athletes to perform movements with the highest possible intensity to generate fatigue and induce a certain degree of muscle damage and oxidative stress [® training program performed with athletes suggests that the results could be considered accurate, as the evaluated results showed alterations in muscle metabolism and biomarkers of inflammation and oxidative status. In addition, the CrossFit® training program increased athletic performance in the Grace test, which could imply suitability as a physical training program. In addition, prolonged periods of intensive exercise training  can reduce immunity and decrease red blood cells, hematocrit, and hemoglobin levels [® training program and 6-week supplementation regimen with TT (770 mg/day) could be appropriate. A CrossFite stress ,51. Our n levels . In our TT supplement administered was not determined. The main limitation was the small sample size, which limits the interpretation of the results. Gender may be a factor influencing the severity of muscle damage, oxidative stress, and inflammation responses in humans . In addition, the percentage of components of the ® experience, no differences between the two study groups (IG and CG), and the control of dietary intake. On the other hand, the strengths of our study were the randomized single-blind placebo-controlled design, the application of CONSORT guidelines, the inclusion of athletic subjects with previous CrossFitTT supplementation of 770 mg/day in CrossFit® trained men could slightly attenuate the exercise-induced oxidative stress, muscle damage, and inflammation which could be due to the natural antioxidant and anti-inflammatory properties of TT. Therefore, further studies are needed in this field to corroborate the effects of TT on muscle performance/behavior in athletes training at a high intensity.Oral TT supplementation could be used during seasons of high physical demand, to intensify skeletal muscle recovery and counteract oxidative damage and inflammation generated by EIMD. It is necessary to consider that the supplementation strategy implemented on athletes should be done considering the physiological effects of TT.


The levels of circulating troponin are principally required in addition to electrocardiograms for the effective diagnosis of acute coronary syndrome. Current standard-of-care troponin assays provide a snapshot or momentary view of the levels due to the requirement of a blood draw. This modality further restricts the number of measurements given the clinical context of the patient. In this communication, we present the development and early validation of non-invasive transdermal monitoring of cardiac troponin-I to detect its elevated state.Our device relies on infrared spectroscopic detection of troponin-I through the dermis and is tested in stepwise laboratory, benchtop, and clinical studies. Patients were recruited with suspected acute coronary syndrome.r = 0.7774, P < 0.001, n = 52 biologically independent samples) between optically-derived data and blood-based immunoassay measurements with and an area under receiver operator characteristics of 0.895, sensitivity of 96.3%, and specificity of 60% for predicting a clinically meaningful threshold for defining elevated Troponin I.We demonstrate a significant correlation (This preliminary work introduces the potential of a bloodless transdermal measurement of troponin-I based on molecular spectroscopy. Further, potential pitfalls associated with infrared spectroscopic mode of inquiry are outlined including requisite steps needed for improving the precision and overall diagnostic value of the device in future studies. The number one cause of death in the US is heart disease. With 10 million patients visiting the emergency departments in a year with chest pain, 8 million are unrelated to cardiac issues. This places a burden on hospitals leading to suboptimal patient outcomes. In patients with cardiac issues, the time clinicians take to intervene dictates reversible or irreversible heart damage. However, current markers used to test for cardiac issues require blood sampling, limiting access to and frequency of testing. This study introduces a non-invasive cardiac marker measurement device without any form of blood draw, based on measurements taken by a wearable device through the skin. Preliminary studies show high conformance to the standard of care technologies, indicating that the technology has potential to enable more rapid, frequent, accessible and non-invasive detection of cardiac issues such as heart attacks. Titus et al. develop a technological platform for the non-invasive transdermal measurement of cardiac troponin-I, a marker of myocardial injury. Preliminary testing of their device, which works via infrared spectroscopy, indicates that troponin can be detected with reasonable performance, in the absence of a blood draw. Over 80% of these are due to non-cardiac causes, resulting in an unnecessary burden in the ED, revealing the need for an instant non-invasive screening technique that can streamline the ED workflows1. Furthermore, 1 out of 5 myocardial infarctions (MI) is asymptomatic (silent), leading to nearly 200,000 silent MIs each year in the US2. Therefore, the development of new technologies that can allow early non-invasive detection of myocardial injury is imperative.Over 10 million patients present with chest pain3 to assess cardiac injury has been around since the 1990s. The state-of-the-art4 troponin quantitation mechanism is based on immunoassays5 involving the use of two or more antibodies, one of which is labeled, typically with a chemiluminescent tag, which adds another level of complexity6 in the analysis. While immunoassays are highly developed and accurate, extensive sample preparation including cumbersome blood7 draws are required. Furthermore, it mandates the logistics between physician and laboratory. Point-of-care (POC) assays are becoming increasingly available such as Abbott iSTAT8 but suffer from low sensitivity making them incompatible with rapid rule-out algorithms9. High sensitivity immunoassays have advanced state of art possibilities for point of care and home healthcare modalities with innovations by startups like Luminostics10. Similarly, recent developments with microneedle patches11 further demonstrate promise for longitudinal monitoring of the levels of inflammatory biomarkers. While the recent POC12 solutions reduce time to test results, there still remains a dependency on blood draw coupled with lower analytical sensitivity compared to central laboratory testing, hence limiting their application toward effective discharge from the ED. Posited in this letter as a solution is infrared spectroscopy13, a widely used and applied characterization technique due to its ability to probe into the material at the molecular level and hence an inherently sensitive mode of interrogation. The most appealing aspects include the benefit of minimal or no sample preparation required. There are innovative wearable devices based on functional-near-infrared light interrogation14 such as, cortical hemodynamics15, blood oxygenation16, blood glucose monitoring17, etc. However, these methodologies are not sensitive to the molecular composition of materials and therefore require labeling or tagging the molecule of interest for its detection. Molecular spectroscopy is one of the cornerstones of analytical tools used to study structural and compositional chemistry18. This technology typically involves the study of the interaction between mid-infrared (MIR) radiation with matter for which, as a diagnostic tool in a wearable format, there is no device yet to-date that has shown potential. There have been some endeavors leveraging the diagnostic capabilities of MIR with analytes such as in serum19, urine20, breath21, skin22 etc. Being well established as an indispensable tool in material science23, infrared spectroscopy has been since applied in food, drug, environmental, forensics disciplines and importantly in the biomedical field such as cancer detection24, and even cardiac care26. Another advantage27 is that, testing using non-invasive devices like pulse oximeter, breathalyzers, and bilirubinometers, are exempt from CLIA regulations because a sample is not taken from the body. Recently, complex MIR spectrometers have been scaled down in footprint to fit into portable-sized benchtop devices such as the MZ5 by OceanInsight. However, there is no non-invasive wearable diagnostic device based on MIR spectroscopy. IR spectroscopy has been elusive29 as an alternative to current POC solutions due to the following reasons30: (a) Signal to noise ratio strongly dictates the minimum detectable limit (b) Since all matters are sources of infrared radiation, efficiencies of IR-based devices can be confounded by stray light (c) The most sensitive mode of operation which is Fourier Transform IR spectroscopy requires a large footprint and is highly sensitive to mechanical vibrations, due to moving components thus often confined to ex vivo modalities. This correspondence outlines steps taken to circumvent or mitigate the mentioned challenges leading to an efficacious non-invasive device capable of risk-stratifying31 ACS32 patients based on Troponin-I (cTnI) levels.Detection of cardiac troponinsThe bloodless transdermal infrared spectroscopic device to assess elevated troponin-I was developed over four research phases.First, an investigational study was conducted to determine the spectral features that are unique to cardiac markers such as cardiac Troponin I , creatine kinase-MB , with the optical characterization of these substances in their pure form. A Nicolet ThermoFisher IS50 infrared spectrometer  employing a (single-bounce) diamond IRE, is used to identify a spectral signature for cardiac troponin. This allows one to optically detect and quantify the presence of that biomarker in a host substrate such as whole blood. De-identified healthy whole blood was procured and characterized to determine if there are any confounding overlaps in the absorbance peaks of blood and cardiac biomarkers.33 Siemens hs-cTnI. The blood samples were optically characterized ex vivo with the modality of total internal reflection using Nicolet ThermoFisher IS50 Fourier Transform infrared spectrometer  employing a (single-bounce) diamond IRE. Each blood sample to be characterized is one microliter in volume as deposited on the IRE. Each sample was measured in triplicates, with every repeat being an average of 32 co-added scans at a resolution of 4 cm−1.Consequently, to confirm the efficacy of the ATR mode of interrogation, 30 biologically independent de-identified blood samples with the corresponding measurement of high sensitivity-cTnI values were procured in collaboration with the department of laboratory medicine at UCSF upon executing a Human Material Transfer Agreement. These blood samples that had already been collected prior to this study in accordance with applicable laws, regulations, patient consent forms and authorizations pursuant to Institutional Review Board. Further ethics approval and consent were not required as the samples were de-identified and anonymized in accordance with the Health Insurance Portability and Accountability Act. Blood samples included were those identified with a spectrum of Troponin-I values between the limit of detection (LOD) and upper reporting limit of the SOC assay namely, Advia CentaurA benchtop Attenuated Total Reflectance (ATR) based spectrometer by OceanInsight was implemented in a Cardiac care setting in collaboration with West Virginia University, Heart & Vascular Institute. It was hypothesized that transdermal measurement in the indirect correlation with myocardial injury would provide insights toward possible troponin measurement capability. An optical measurement was performed on the thumb of 4 normal and 5 cardiac patients. A multi-variate cluster analysis was performed on this data employing Ward’s algorithm with squared Euclidean method focused on the wavelength range including some of the unique absorbance features relating to Cardiac Troponin-I with an interrogation window of 1.7 µm. The specificity of this window to cTnI as opposed to cTnT and other troponin isoforms is to be investigated in future studies. This unbiased heterogeneity analysis classifies data based on similarity.ATR-FTIR although proven efficacious, is challenging as a candidate for POC devices due to its large size and susceptibility to mechanical vibrations. Typically with FTIR, a wavelength sweep is done by moving the mirrors of an interferometer and consequently transforming the data from a spatial to frequency domain. However, there isn’t a need for a wavelength sweep as the wavelength ranges of interest, based on the ex vivo characterization of cardiac biomarkers, are previously determined. This allows for the selective sensitization of the infrared detector to the wavelengths corresponding to cardiac troponin-I by means of interferometric optical filters. As the next developmental step, an ambulatory non-invasive trans-dermal wearable device was deployed toward a pilot study conducted in the cardiac observation unit by recruiting patients with suspicion of ACS upon obtaining proper consent to obtain 24 biologically independent samples. The wrist wearable was installed on the patient’s wrist within 10 min of a SOC blood draw, with the cTnI levels reported using the CE certified Snibe Maglumi-1000 high sensitivity assay. This version of the optical sensor was designed to be portable  by using a broadband infrared light source, germanium IRE and, thermopile detector with specifically chosen filters sensitive to two optical ranges: the first is representative of the Amide II band which is used as an internal normalization reference, the second range such that cTnI would have the largest contribution to the absorbance. Due to the absence of moving parts and complex optical components, the small form factor was achieved while minimally affected by mechanical vibrations. The confounding effect of stray light was negated by pulsing the emitter at 4 Hz while polling the thermopile at 8 Hz thus recording both the on and off state of the emitter. A differential of these two states accounted for the extraneous light captured by the detector.The pipeline used for the data analysis schema toward establishing a correlation between the proposed noninvasive device and standard-of-care data is illustrated in Fig. g for 10 min. At ZSFG, the plasma was tested for cTnI using a high sensitivity assay on the Siemens Centaur Analyzer34 . This assay has a LOD of 2.5 ng/L and a 99th percentile of 47 ng/L. In some cases, results from routine troponin testing were used and when that was not available, permission from the patient was granted to collect a fresh blood sample through an intravenous line or venipuncture. At SHRI, CE certified Snibe Maglumi-1000 high sensitivity cTnI assay was employed with an LOD of 0.01 ng/L and a 99th percentile of 19 ng/L. Within 20 min of blood collection for hs-cTnI testing, the 4 channel sensor wearable was installed on the underside of the patient’s wrist and left unattended for 5 min. Infrared readings along with accelerometer data were obtained on a continuous basis. When completed, the sensor was removed, and a file containing these readings were sent through a WiFi connection directly to RCE for data processing.In two concurrent follow-on pilot studies, the performance of such a 4 channel sensor was evaluated on subjects recruited at the Zuckerberg San Francisco General (ZSFG) Hospital emergency department and Sengupta Hospital and Research Institute (SHRI), yielding 29 and 23 biologically independent samples respectively. These patients over the age of 18, were recruited agnostic of sex, ethnicity etc., strictly based only on the presentation of chest pain under suspicion of Acute Coronary Syndrome. The protocols were reviewed and approved by the corresponding Institutional Review Boards, and all participants signed a written consent form to participate. The patients were selected to represent a wide range of troponin values. Blood was collected in tubes containing lithium heparin, centrifuged at 1000 × 35 was used to obtain these ROC plots. In this library, trapezoids are used to compute the AUC indicating an average AUC of 0.853 for a bootstrapped model with sensitivity of 100% and specificity of 70.59%. Confidence intervals are computed with Delong’s method with bootstrap resampling36. Pearson’s correlation coefficient was used to indicate significant linear relationship among quantitative variables and regression analysis was done. A value <0.05 was considered as significant. All statistical tests were performed using Python, R and JMP.Categorical variables were presented as counts and percentages, and continuous variables as means and standard deviations. Receiver operator characteristics (ROC) plot displays performance of a binary classification method with discrete output. Elevated or non-elevated state of troponin was used as the binary output and optical device values as the input for the binary classification method. An R packageFurther information on research design is available in the 37 of the material, light is absorbed leading to an active vibrational mode. This results in certain energies or wavelengths of light being absorbed by the material that are unique to the material. Thus, the material can be compositionally characterized by performing a differential measurement of the light before and after it passes through the material. Traditional configurations involve directing the infrared radiation through the sample to be measured and detecting the light on the opposite side using a thermal sensor. This restricts the mode of interrogation to in vitro or ex vivo measurement. To overcome this issue, an ATR38 configuration is adopted where light is totally internally reflected inside an internal reflection element (IRE) or prism of a higher refractive index than the material to be characterized  was observed between optical and SOC-derived troponin-I data within the range of 2.5–250 ng/L.First research step was an ex vivo exploratory phase where the aim was to determine of the ATR configuration was capable of detecting cTnI in blood. This phase involved the testing of blood samples of patients with various cTnI levels on a research grade ATR based infrared spectrometer. The optical readouts representing the signature cTnI absorbance peaks were investigated for correlation Fig.  with thaAs the next step, the transdermal modality was tested by having patients recruited for coronary angiograms (CAGs) place their thumbs on the surface of a commercial ATR infrared spectrometer. A classification analysis Fig.  based on39, as informed by the 99th percentile of troponin-I distribution in a reference population, a decision threshold of 19 ng/L is established for elevated troponin-I indicating acute myocardial injury. Upon categorizing the population into two class labels divided by the threshold, a logistical regression analysis  was observed with a sensitivity of 100% and specificity of 70.59%.As the next step, the feasibility of an ATR spectrometer to be miniaturized into a hand-held point of care device. Such a device was designed and 24 chest pain patients were tested by mounting the device on their wrists. The SOC cTnI concentration served as the ground truth for correlation analysis with the non-invasively obtained optical data. Based on the fourth universal definition of myocardial infarctionThe reason for the lower specificity is thought to be influenced by at least one known factor. As witnessed by the principal investigator, three patients presented with tremors or restlessness which significantly deteriorated the data quality. To prevent this in future studies, the device has been outfitted with a tension-adjustable band to ensure consistent contact between the wrist and the sensing area. Also, an accelerometer was incorporated which will inform the data processing algorithm allowing recovery of some noisy data. Further, there is a possibility that the optical absorbance range primarily used to detect cTnI could have contributions from other biomaterials. The effect of these optical confounders will be reduced in future studies by employing multi-channel optical filters with a narrower range of transmission to the detector thereby improving specificity.r = 0.7774 was achieved. A very wide range of hs-cTnI concentrations among the patients were analyzed to determine if sensor exhibited some limitation in the reportable range. While test accuracy for high cTnI values are not required in the diagnosis and management of AMI, it was important to demonstrate that the sensor does not suffer from the “hook effect,”, i.e., readings declining with increasing concentrations above a threshold. Typically, the clinical laboratory dilutes samples with an appropriate diluent for analyte concentrations exceeding the upper reportable range of the assay. No inaccurate results were observed for a cTnI concentration up to at least 20,000 ng/L without the need for any dilutions.Based on the learnings from the previous phases, a high fidelity POC device was tested for validation in a clinical setting. Figure 27. This is not a new concept, as sensors have been used to measure oxygen saturation (pulse oximeter) and total bilirubin (bilirubinometer). Both of these techniques measure pigmented analytes using absorbance within the visible spectrum of light. The troponin sensor uses infrared absorbance and has been shown to have more analytical sensitivity.In contrast to in vitro, in vivo, ex vivo measurements and implantable devices, the term “on vivo” testing can refer to the use of non-invasive wearable devices41. At this point in the development of the sensor, it is unclear which forms of circulating troponin are being detected. It is likely that the infrared absorbance signatures will somewhat differ between the various forms affecting the absolute accuracy of the sensor. For this reason, the best medical application for this sensor may be in the early rule out of AMI. Upon clinical consensus, AMI rule-out algorithms have been developed using hs-cTnT42 and hs-cTnI assays4. Accordingly, a diagnosis of AMI can be excluded with either a baseline measurement or after collection of a second sample at 1 h, if a patient presents to the ED with a 3 h history of chest pain or longer. The current turnaround time for reporting hs-cTn results from the central laboratory is about 1 h. Point-of-care (POC) testing can reduce the turnaround time to under 30 min, from the time of collection to reporting of results. While there is great interest in developing POC tests for hs-cTn, currently there are no hand-held hs-cTn devices approved for clinical use by the FDA. The use of a sensitive on vivo sensor can produce results within 5 min, without the need of collecting blood and transferring the sample to the testing device. It may be possible to provide continuous readings of cTnI, e.g., at 5 min intervals, if the device is left on the patient. This would enable a caregiver to determine if troponin values are increasing in time. The RCE sensor is portable and self-contained, and therefore testing can be conducted before the patient arrives at the hospital. It remains to be determined if the sensor is sufficiently robust enough to enable accurate testing in other clinical scenarios such as, within an emergency vehicle. When the ATR scheme is employed to interrogate the presence of cardiac biomarkers using infrared light, it is not entirely well understood up to what depth the evanescent light penetrates. However, as made evident by the correlation results, it is clear that the returning evanescent light from the skin back into the IRE surface contains information that is indicative of the presence or absence of cardiac biomarkers. Thus the nature of the measurand or the component of the skin that contains the cardiac biomarkers being interrogated by the light is still unclear. Interstitial space is one of the potential possibilities discussed here. Interstitial fluid is a thin layer of fluid that bathes the cells. This fluid serves as a vehicle for nutrients for the cells. More importantly, they also serve as a conduit for extracellular signaling43. It has already been shown that glucose levels can be monitored by sampling the interstitial fluid. Mid-infrared (MIR) spectroscopy is exquisitely sensitive to the chemical composition of the biological materials in the so-called fingerprint region (2000–800 cm−1). The penetration depth via the epidermis is too shallow to interrogate the blood capillaries. However, the penetration depth is sufficient to interact with the interstitial fluid. For human skin, the approximate penetration depth is about 1–10 micrometer indicating that the light could potentially interact with the epidermis and the interstitial fluid with the optical sensor surface placed under the wrist . Another possibility for the local source of cardiac proteins is the superficial glands present in the epidermis. Sweat glands comprise eccrine, apocrine, and apoeccrine glands44. It is well understood that a classical symptom of myocardial infarction is profuse sweating or diaphoresis45. Sweat comprises many proteins, cytokines, and amino acids. The directed infrared light could interrogate the sweat glands through the epidermis. Sebaceous glands could also contain cardiac proteins diffused via capillaries as they are present directly under the stratum corneum near the hair follicles. Further investigational efforts are needed to establish a source of origin for these biomarkers.After myocardial injury, troponin is released in a variety of forms including ternary complexes (troponin T-I-C), binary complexes (I-C), free submits (T and C), and fragments thereof46 optical device is demonstrated in Fig. Owing to the modality of optical data collection, the device is hardly susceptible to light pollution and ambient conditions. This is largely mitigated by pulsed measurement and dark background measurement. Considering the fact that the patients recruited irrespective of co-presenting symptoms did not affect the correlation, indicates the potential for the high specificity of the optical technique. The evolution of the transdermalIn conclusion, this letter posits a patient-centric modality for troponin-I monitoring that can inform efficient triaging and timely intervention in the current cardiac clinical workflow. This remote-monitoring-capable technology can be envisioned to empower clinicians in determining the timely clinical course of action to prevent any unnecessary myocardial injury. Furthermore, the use of such non-invasive devices can be extended upstream to facilities such as urgent cares and EMS where blood draws don’t often occur.Peer Review FileReporting Summary


The adverse effects of neuromuscular junction dysfunctions caused by immune checkpoint inhibitor (ICI) drugs have not been thoroughly assessed in the clinics.To assess the neuromuscular junction dysfunctions in cancer patients with adverse events caused by ICI therapy by searching the Food and Drug Administration Adverse Event Reporting System (FAERS) database.The FAERS data from January 2004 to December 2020 were collected to analyze the association between neuromuscular connection dysfunction and ICI use. Disproportionate analysis and Bayesian analysis were used to quantify the association between the neuromuscular junction dysfunctions and ICIs. The onset time and outcome of neuromuscular junction dysfunctions in different ICI regimens were also compared.Out of 88,617 adverse event reports, 557 neuromuscular junction dysfunction reports (0.63%) were analyzed. Marketed ICI drugs, including ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, cemiplimab, avelumab, as well as their combinations, showed positive associations with four detection methods. Most of the adverse event reports were associated with the use of nivolumab (53.32%) and pembrolizumab (31.96%). However, nivolumab-related neuromuscular junction dysfunctions were similar with pembrolizumab . The onset time of neuromuscular junction dysfunctions showed no significant difference among different ICIs (p > 0.05).Analysis of FAERS data identified that over 30% (32.85%) of reports of neuromuscular junction dysfunctions resulted in death. Ongoing monitoring, risk evaluations, and further comparative studies of ICIs should be considered. The development of immune checkpoint inhibitors (ICIs) is a revolutionary milestone in immune-oncology. ICIs revive the anti-tumor immune responses by interrupting the co-inhibitory signaling pathways and promoting immune-mediated tumor cell clearance . Immune-ICI-related neurological adverse events are relatively infrequent. However, pooled analyses on these events show severe morbidity and mortality , 9. Theshttps://fis.fda.gov/extensions/FPD-QDE-FAERS/FPD-QDE-FAERS.html) covering the period from January 2004 to December 2020. We conducted a retrospective pharmacovigilance survey. FAERS is a public, voluntary reporting database that provides information on adverse events and drug error reports submitted by healthcare professionals, consumers, or pharmaceutical companies. The data is not only from the United States but also from other countries. The FAERS data file contains demographic and administrative details, drug information, the Medical Dictionary for Regulatory Activities’ (MedDRA) preferred terminology for adverse event (REAC) coding, patient outcomes, reporting sources, initiation of treatment that includes date and end date reports, and indications for use. A deduplication procedure was performed according to the FDA’s recommendations of selecting the latest FDA_DT when the CASEIDs were the same and selecting the higher PRIMARYID when the CASEID and FDA_DT were the same. A total of 88,617 reports related to ICIs were mined from the FAERS database, and 557 reports (0.63%) were associated with neuromuscular junction dysfunction.We conducted a retrospective pharmacovigilance study using data from the FAERS database (® (Index Nominum) was used as a dictionary for ICIs. Reports involving the seven immunotherapies for cancer treatment  were identified using text string searches for each drug by generic names, brand names, and abbreviations. Reports listing one or more of therapies mentioned above as the primary suspect (PS) or secondary suspect (SS) agent were included. We excluded drugs reported with the eligible drug listed as interacting or concomitant, as well as those without cancer-related indications.Adverse events were identified using the MedDRA terms “Recurrence of neuromuscular blockade (10068408)”, “Ocular myasthenia (10049168)”, “Neuromuscular blockade (10029315)”, “Neuromuscular block prolonged (10029314)”, “Myasthenic syndrome (10028424)”, “Myasthenia gravis neonatal (10028419)”, “Myasthenia gravis crisis (10062758)”, “Myasthenia gravis (10028417)”, in the REAC file. The drugs in the FAERS database can be registered using different conventions; therefore, the MICROMEDEXBased on disproportionality analysis and Bayesian analysis, 4 algorithms were applied: reporting odds ratio (ROR), the proportional reporting ratio (PRR), the Bayesian confidence propagation neural network, and the multi-item gamma Poisson shrinker. In combination, these algorithms were used to identify the association between drugs and adverse events. A deduplication procedure was performed as described by firstly linking CASE number with relevant ISRs, secondly through the 4-key fields used in missing data handling  and thirdly revising drug and event reported and the single imputation approach was performed to manage missing data .Descriptive analyses were used to summarize the clinical features of the patients with neuromuscular junction dysfunctions collected from the FAERS database. Time to onset of neuromuscular junction dysfunctions between different ICI regimens was compared using nonparametric tests when the data were not normally distributed . Pearson’s chi-square test or Fisher’s exact test was used to comparing outcome events and fatality rates between different ICI regimens. Statistical significance was determined with 95% CI and p<0.05. Data mining and statistical analyses were performed with SAS, version 9.4 .Out of 88,617 adverse reports, 557 neuromuscular junction dysfunction reports (0.63%) were documented in the FAERS database with ICIs Table 2.The number of neuromuscular junction dysfunction events was the highest for nivolumab (n=201), followed by pembrolizumab (n=175). The mean age of these patients was slightly over 70 years. Most of the cases of neuromuscular junction dysfunctions involved with ICIs were reported from North America, Asia, and Europe . Most of the cases were submitted by physicians for nivolumab (44.28%) and by consumers for pembrolizumab (45.14%), and the cases appeared more often in females than in males .All ICI were associated with neuromuscular junction dysfunction, and signal detection was positive among all four methods Table 4.p=0.006) and durvalumab (p=0.019); similar results were found in nivolumab . However, when combined with ipilimumab, nivolumab led to a significantly faster onset of events than atezolizumab (p= 0.037)  Table 5.To determine the prognosis of neuromuscular junction dysfunctions after ICI treatments, we assessed neuromuscular junction dysfunctions following usage of ICIs Table 6.Our study concludes that though quite rare , ICIs lead to severe adverse reactions Table 6 Drugs that target the PD-L1/PD-1 pathway may exhibit a more favorable toxicity profile than CTLA4 blockade and conventional chemotherapy . HoweverCTLA4 is an immune checkpoint molecule expressed on immune cells that contains both CD4+ and CD8+ T cell subsets and B cells. CTLA4 antibody (ipilimumab) can restore T cells priming and activation by tumor antigens and promote antitumor T cell response. The PD-L1/PD-1 interaction is another checkpoint for anti-cancer immunity that functions downstream of T cell priming and activation. PD-1 is expressed on the surface of activated T cells, but its specific ligand PD-L1 is present in multiple tissue types, including many other different cancer cells. When PD-L1 binds to PD-1, it inhibits T cell proliferation and activity and reduces cytokine production, helping malignant cells evade the host’s immune response. Therapeutic antibodies designed to target PD-L1/PD-1 interactions include antibodies that inhibit PD-1 (nivolumab and pembrolizumab) or PD-L1 (atezolizumab).The clinical presentation of neuromuscular junction dysfunctions is often atypical, with considerable overlap between myasthenia gravis and myopathy, as well as cardiac/respiratory complications. However, mortality was high in these patients, despite adequate treatment strategies including corticosteroid. Several pathogenic mechanisms, including neuronal damage by T cells and autoantibodies and/or cytokine-mediated inflammation processes, have been hypothesized. However, the pathogenesis of these ICI-related complications is not completely understood . ClinicaChoosing a fast and effective signal detection method provides a valuable signal for drug risk management, which can severely impact human health and minimize harm to humans. However, risks are difficult to detect only through experimental studies with limited sample size. Therefore, this study provides a reference to confirm the occurrence of neuromuscular junction dysfunctions associated with ICIs through post-marketing surveillance studies.Healthcare professionals treating cancer patients are advised to be aware of Guillain-Barre Syndrome (GBS) symptoms. These patients need immediate attention and should be given a low threshold for hospitalization to facilitate work-up and monitor severe or life-threatening symptoms . CurrentWhile the data mining techniques used in this study have many advantages as a tool for detecting signs of adverse drug reaction, it should also be noted that this technology does not solve all problems in detecting and analyzing signs of adverse drug reaction. The signals from FAERS were used only for qualitative research.This study has certain limitations. First of all, data mining technology cannot completely reflect all clinical information from patients. Detailed information from clinical follow-ups and other studies are also needed to validate the signals detected. Secondly, data mining technology cannot address ADR reporting system issues such as inaccuracy, false reporting, incomplete reporting, underreporting, and arbitrariness. Thirdly, qualitative research makes it difficult to quantify the side effect signals of neuromuscular junction dysfunctions from a few ADRs. Finally, the restrictions of limited sample size cannot be overlooked.Immune checkpoint inhibitors need to be prescribed more cautiously. Analysis of FAERS data showed that over 30% of reports of neuromuscular junction dysfunctions resulted in death, emphasizing the importance of constant monitoring, risk assessment, and more comparative studies.The original contributions presented in the study are included in the Article/supplementary material. Further inquiries can be directed to the corresponding authors.All contributors are listed as co-authors. BY and DL: conceptualization. QD and XY: methodology. BZ: data retrieving. DL: formal analysis and investigation. HC: writing original draft preparation. PZ: writing. QZ: review and editing. BY: supervision. All authors contributed to the article and approved the submitted version.This study was supported by the Chinese Pharmaceutical Association-Servier Youth Hospital Pharmaceutical Innovation Research Funding Project and Hospital Development Center of Shanghai Municipality (SHDC2020CR3085B).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


BackgroundChiropractors often use manual thrust cervical spinal manipulative therapy (thrust-cSMT) to treat musculoskeletal neck conditions. We hypothesized <50% of surveyed Asia-Pacific chiropractors would report using thrust-cSMT given potential contraindications, and secondarily explored predictors of thrust-cSMT use.Materials and methodsWe designed, validated, achieved sufficient reliability, and disseminated a survey to explore thrust-cSMT use. The survey queried chiropractors’ characteristics , and use of thrust-cSMT for uncomplicated neck pain and vignettes describing vertebral artery disorders, Arnold-Chiari malformation, and anterior cervical discectomy and fusion (ACDF). We performed logistic regression for each vignette with thrust-cSMT as the dependent variable and chiropractor characteristics as covariates.ResultsThere were 241 respondents, having 12.8±10.9 years in practice, representing >15 countries. Less than 50% of chiropractors reported the use of thrust-cSMT for each vignette, including vertebral artery insufficiency (14%) and stenosis (17%), Arnold-Chiari type I (18%) and type II (5%), C5/6 ACDF (39%) and C3-6 ACDF (27%). Regressions identified significant predictors of increased or decreased use of thrust-cSMT including time spent with new patients, focus on subluxation, degree, group practice environment, use of thrust-cSMT on a healthy patient, and hours reading scientific literature (P<.05 for each).ConclusionsThis study was the first to chiropractors’ use of thrust-cSMT for complicated neck pain and found that most Asia-Pacific chiropractors reported avoiding this treatment in the presence of a potential treatment contraindication. The use of thrust-cSMT in complicated neck pain may be related to practice characteristics. However, further research is needed to identify specific reasons why chiropractors use or avoid thrust-cSMT. Chiropractors are portal-of-entry healthcare providers that frequently manage musculoskeletal conditions affecting the spine such as low back and neck pain . ChiroprSpinal manipulative therapies include a range of manual therapies directed to the joints of the spine . HoweverVertebrobasilar insufficiency (VBI), also called posterior circulation insufficiency, is a condition involving transient ischemia of the posterior brain circulation often caused by vertebral or basilar artery atherosclerosis . PatientArnold-Chiari malformation describes an inferior displacement of the cerebellar tonsils through the foramen magnum and may present with varied symptoms including headache, sensory changes, vertigo, and ataxia . The typAnterior cervical discectomy and fusion (ACDF) is one of the most common cervical spine surgeries and is often performed for degenerative cervical spine conditions such as stenosis with myelopathy . PatientPrevious studies have suggested that chiropractors have different viewpoints with regard to the role of spinal manipulation and the scope of chiropractic care. For example, one recent United States survey identified three distinct subgroups of chiropractors based on their focus being: (1) spine and neuromusculoskeletal care, (2) primary care, or (3) vertebral subluxation detection and removal . StudiesConsidering there are complex conditions affecting the neck which may prompt patients to seek chiropractic care, yet at the same time present a precaution or contraindication to thrust-cSMT, our primary aim was to develop and conduct a survey to explore chiropractors’ approaches to these conditions with respect to use or avoidance of thrust-cSMT. Our hypothesis was that manual thrust-cSMT would be used by less than 50% of respondents for each clinical scenario  describing a hypothetical precaution or contraindication. Secondarily, we aimed to explore if any practice characteristics independently predicted chiropractors’ choice to use thrust-cSMT.Study designThis study was a cross-sectional survey of chiropractors in the Asia-Pacific region. The Ethics Committee of the Chiropractic Doctors Association of Hong Kong approved this study  and granted a waiver of written patient consent, as consent was implied by return of the completed survey. The survey was sent via Google Forms link to participants starting in October 2022, and the response window concluded in December 2022. The survey included details regarding consent to participate, and the respondents’ names and other identifying information were not collected so the responses remained anonymous. The study co-authors developed the survey and then conducted validity and reliability testing before disseminating it to respondents. The survey and information sheet sent to respondents are available in an open-access repository .Setting and participantsThe survey targeted practicing chiropractors in the Asia-Pacific region who were fluent in English, as English is an official language in several countries in this region including Australia, New Zealand, and Hong Kong Special Administrative Region. Asia-Pacific chiropractors receive their chiropractic education in a variety of chiropractic colleges internationally , thus poTo our knowledge, two Asia-Pacific chiropractic associations have been described in the scientific literature previously. In 2021, the Australian Chiropractors Association was reported to have over 3,000 registered chiropractors . Also inVariablesSurvey items describing respondents’ practice characteristics were adapted from previous studies which included already-validated questions when possible. Chiropractors’ country of practice included a list of countries in the Asia-Pacific region with a chiropractic association. This variable was considered separately from the country of education as not all Asia-Pacific countries have chiropractic educational institutions . We omitMultiple survey items addressed chiropractors’ educational background. Years in practice was included as chiropractic curricula have changed over time , thus chRespondents were also asked if they would perform manual thrust-cSMT for an otherwise healthy 45-year-old female patient with localized neck pain who had no headache or neurologic deficits on exam. This question served as a control for the other clinical responses, to gauge whether a chiropractor would use cSMT in the absence of any stated complex conditions or comorbidities. Thrust-cSMT was defined in the survey as any form of spinal manipulation involving a manual impulse or thrust directed to the cervical spine.The subsequent clinical vignettes attached a comorbidity prompt which mentioned the presence of: (1a) unilateral vertebral artery stenosis causing symptoms of posterior circulation insufficiency, including dizziness and headache, (1b) 50% stenosis of the vertebral artery unilaterally as identified by computed tomographic angiography, (2a) Arnold-Chiari malformation type I with an inferior position of the cerebellar tonsils 4 millimeters caudal to the foramen magnum, (2b) Arnold-Chiari malformation type II with an inferior position of the cerebellar tonsils 13 millimeters caudal to the foramen magnum, (3a) anterior cervical discectomy and fusion at C5/6 , performed two years previously, and (3b) anterior cervical discectomy and fusion from C3 to C6 , performed two years previously.Respondents were asked if they would use manual thrust-cSMT for each clinical vignette, with response options including “yes,” “no,” and “don’t know/unsure.” The indeterminate response was included to allow for knowledge uncertainty, as clinicians could be unfamiliar with the question’s content or could be unable to provide a yes/no answer without additional information .Data sourcesThe survey was the sole source of data for this study. Data were stored within the Google Forms platform and downloaded into a Microsoft Excel spreadsheet upon completion of each phase of validity testing, reliability testing, and results.BiasPilot testing was used to diminish the likelihood of potential biases related to the wording of the clinical prompts. Study co-authors sent the survey to eight expert chiropractors and two expert physical therapists to determine face validity. Three of these experts also had a PhD degree. These individuals provided open-ended feedback, for example, to clarify confusing or leading questions. During piloting several changes were made to the questionnaire including omitting a question about gender, which was unnecessary to our study objectives and adding a “don’t know/unsure” response for the clinical vignettes.To limit the likelihood that respondents would favor socially desirable responses , the surAfter pilot testing, the clinical questionnaire was disseminated to a smaller group of 38 Asia-Pacific chiropractors employed in Hong Kong and India. Considering the survey was deemed reliable , it was further disseminated without additional changes.Study sizeThe sample size for reliability testing for the clinical questions was calculated using a rule of thumb estimate which requires five respondents per survey item, or in this case 35 respondents . The samStatistical methodsDescriptive statistics were calculated in Microsoft Excel while regression models were conducted using IBM Statistical Package for the Social Sciences . The reliability of the clinical questionnaire was examined using Guttman’s lambda six, rather than Cronbach’s alpha, as Guttman’s lambda is more applicable to smaller samples . This vaA multiple binary logistic regression model was performed for each clinical vignette prompt, with the dependent variable being the decision to perform thrust-cSMT and the independent variables being the practitioner characteristics. Prior to multiple regression, we conducted bivariate correlation testing using a two-tailed Pearson correlation matrix to test for multicollinearity between covariates. Country of education and degree had a statistically significant (P<.001) correlation coefficient of 0.5, likely explained as certain countries only offer specific types of chiropractic degrees. Accordingly, the country of education was discarded from the regression models. Country of practice was also discarded as a variable, considering certain countries only had a single respondent, which led to cell counts of zero and instability of the regression model. “Don’t know / unsure” responses to the clinical vignettes were eliminated from these regression models such that the dependent variable could be analyzed as a binary response via regression.In the regression models, the following variables were considered to represent a nominal level of measurement: responses to each clinical vignette, country of chiropractic education and practice, practice environment, level of education, and subluxation focus. The following variables were considered a scale level of measurement: years in practice, time spent with new patients, rate of imaging, and hours spent reading scientific literature.The reporting of our results adheres to the guidelines of Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) for cross-sectional studies .RespondentsRespondents had on average [SD] 12.8±10.9 years in practice, spent 2.8±4.5 hours reading the scientific literature per week, and spent 38.3±17.5 minutes with new patients, and 53.4±36.8% reported obtaining imaging during the first six weeks of care. The country of education was most often the United States (34%) followed by Australia (27%), New Zealand (15%), Malaysia (11%), and other countries (13%). The country of practice was most often Hong Kong Special Administrative Region (27%) followed by Australia and New Zealand (17% each), Taiwan (8%), and other countries (30%)  reporting the use of thrust-cSMT was less than 50% for all vignettes, including vertebral artery insufficiency , vertebral artery stenosis 17%; 13-22%), Arnold-Chiari malformation type I , Arnold-Chiari malformation type II , C5/6 ACDF , and C3-6 ACDF . These percentages as well as the percentage of respondents indicating “no” or “don’t know/unsure” are reported along with 95% CIs in Figure 2%, ArnolRegression modelsResults of the regression models are shown in Table For the vignette describing VBI, greater hours per week reading scientific literature was associated with an increased likelihood of reported use of thrust-cSMT . Fewer minutes spent with new patients was associated with a reduced likelihood of reporting thrust-cSMT . Chiropractors who agreed that the primary focus of chiropractic is to detect and treat vertebral subluxation were more likely to report the use of thrust-cSMT .For the vignette describing 50% stenosis of the vertebral artery, chiropractors with a master’s degree were less likely to report the use of thrust-cSMT . Chiropractors in a chiropractic group practice were also less likely to report the use of thrust-cSMT . Decreased minutes spent with new patients was associated with a reduced likelihood of reporting the use of thrust-cSMT .For the vignette describing Arnold-Chiari malformation type I, reduced minutes spent with new patients was associated with a reduced likelihood of reporting the use of thrust-cSMT . For the vignette describing C5/6 ACDF, chiropractors reporting the use of thrust-cSMT on the healthy control patient were also more likely to report the use of thrust-cSMT in this scenario .This cross-sectional survey of Asia-Pacific chiropractors was the first study to our knowledge that explored chiropractors’ use of cSMT in patients with neck pain and precautions or contraindications to this treatment. Our findings supported our hypothesis as greater than half of the respondents indicated that they would not use thrust-cSMT in each clinical vignette of precaution/contraindication, suggesting that chiropractors are cautious with the use of thrust-CSMT in these scenarios. Further, we identified several significant independent predictors of the use of thrust-cSMT. The most frequent predictor was reduced minutes spent with new patients, which predicted a reduced likelihood of thrust-cSMT in three vignettes. Other predictors were significant in one vignette each including a focus on subluxation, degree, practice environment, use of thrust-cSMT on a healthy patient, and hours reading scientific literature.To our knowledge, there are few survey studies that have examined chiropractors’ use of thrust spinal manipulation in cases of precaution or contraindication to this therapy. In one study of United States’ chiropractors, 54% of respondents indicated that they would either “rarely” or “never” use thrust manipulation in the lumbar spine of a patient with a surgical lumbar fusion . Our stuOne previous survey study of Australasian chiropractors found that only 38% would use thrust-cSMT for patients with cervical spinal stenosis . While sWe are unaware of any survey studies that examined chiropractors’ use of cSMT in patients with vertebral artery disorders or Arnold-Chiari syndrome. Until now, the use of cSMT in these patients could only be ascertained from case reports, which may have an inherent publication bias. To our knowledge, there are only five published cases in which a chiropractor managed a patient with VBI, and thrust-cSMT was applied in three of these patients . LikewisWhile the most frequent predictor of avoiding thrust-cSMT was a reduced number of minutes spent with new patients, it is unclear why this was the case. New patient examinations often consist of history taking and physical examination procedures to characterize the source of the patient’s chief complaint. Therefore, it is possible that chiropractors who spend less time during examination have less information to base their treatment and are accordingly more cautious. Further research is needed to explore factors related to thrust-cSMT use and time spent with patients.While most respondents indicated they would use thrust-cSMT on a healthy patient , a minority indicated they would not, or were unsure. We suspect that the reason for this finding is that certain chiropractors tend to avoid thrust-cSMT altogether. There is a range of techniques of spinal manipulation, some of which do not involve a manual thrust. One of the most common non-thrust techniques is instrument-assisted manipulation . In a survey of British chiropractors, 2% of respondents indicated they typically used this method. In another survey of Australasian chiropractors 36% indicated they would use instrument-assisted manipulation on a patient with myofascial pain  [A master’s degree (compared to a diploma) independently predicted a reduced likelihood of use of thrust-cSMT in the vignette describing 50% stenosis of the vertebral artery. While it is unclear why educational level impacted thrust-cSMT use in this vignette, we suggest that different educational curricula may lead to different practice behaviors. Chiropractic educational requirements and scope of practice vary across the Asia-Pacific region, however, any individual completing a formal chiropractic educational program is referred to as a “chiropractor.” One recent survey of the chiropractic profession in Australia, which has its own chiropractic educational institutions, reported that most chiropractors had a bachelor’s degree (35%) followed by master’s 33%) and doctoral (30%) degree, with the remaining percentage having a diploma [% and docThe chiropractic profession is officially recognized and regulated in several Asia-Pacific countries, including Australia, Hong Kong Special Administrative Region, Japan, Malaysia, New Zealand, The Philippines, Singapore, and Thailand . The legIn the present study, only a small percentage of chiropractors received a chiropractic diploma (3%), all of which were obtained in a country where chiropractic is legally regulated. These degrees were therefore likely formal graduate diplomas. This also suggests that the present study did not include respondents with a diploma obtained through an abbreviated program in a country where chiropractic is not regulated. The duration of study required to obtain a bachelor’s, master’s, or doctoral degree of chiropractic varies per country and program. Beyond the diploma level, chiropractic programs typically require a minimum of four to five years of education , in addiThese results may inform future studies including prospective cohort studies involving the treatment of patients with these conditions. For example, the clinical scenarios listed  could be listed as exclusions in the eligibility criteria of experimental study designs, as chiropractors tend to avoid thrust-cSMT in these patients.This study has certain limitations. Not all Asia-Pacific chiropractors are fluent in English, which effectively reduced the available sample size. While the sample size was sufficient for our regression models, certain countries were poorly represented in relation to the number of practicing chiropractors. The clinical vignettes may be oversimplified, whereas in a real-world setting chiropractors could perform additional examination procedures, order further testing, or consult with a medical specialist before deciding to use thrust-cSMT. As Arnold-Chiari malformation type I may be identified incidentally via imaging, the source of the patient’s neck pain or reason for chiropractic treatment in this vignette could have been made clearer. As a survey study, respondents may opt to provide socially desirable responses . This coAlthough this study identified key predictors of cSMT use, it cannot answer the question of which conditions represent an absolute contraindication to cSMT, or which cSMT approaches would lead to improved clinical outcomes with regard to neck pain or other outcomes. Further studies regarding the safety and effectiveness of cSMT in these patient populations are needed. However, as the clinical presentations described in our survey may be uncommon and could represent contraindications to cSMT, such research may be challenging or unethical to conduct.Follow-up surveys also could examine each of the conditions of precaution/contraindication in greater depth. An individual survey for each condition  would allow for more detailed questions about spinal manipulation technique selection. For example, it is not only important to know if thrust-cSMT is avoided, but what treatments are provided in place of this treatment . Further research is also needed to explore the influence of several other risk factors on chiropractors’ decision to use cSMT, such as hypertension, osteoporosis, connective tissue disorders, and smoking . In addiThis study was the first to explore chiropractors’ use of thrust-cSMT for complicated neck pain and identified that Asia-Pacific chiropractors typically avoid this treatment in the presence of potential contraindications including vertebral artery disorders, Arnold-Chiari malformation, and ACDF. Chiropractors’ use of thrust-cSMT in the presence of complicated neck pain may depend on providers’ characteristics, including time spent with new patients, focus on subluxation, degree, group practice environment, use of thrust-cSMT on a healthy patient, and weekly hours reading scientific literature. Further research is needed to explore reasons why chiropractors use or avoid thrust-cSMT in specific clinical scenarios.


Aspergillus fumigatus infection in a 22-year-old woman immunocompromised due to steroid and anti-TNF therapy in the context of ulcerative colitis and interferon gamma deficiency. A 4-year treatment course requiring neurosurgical intervention on four occasions and prolonged antifungal therapy, including isavuconazole, resulted in clinical cure with a corresponding decrease in CSF beta-d-glucan to <30 pg/mL. Serum and CSF galactomannan levels were not elevated at any point during the clinical course.This case highlights the use of -beta-d glucan to direct treatment of a cervical spinal cord  Aspergillus fumigatus is the offending agent in over 70% of cases, but other subspecies, including A. niger, A. flavus, and A. terreus, have been described [Invasive aspergillosis (IA) infections of the central nervous system (CNS) are most common in immunocompromised patients and can present as endophthalmitis, meningitis, ventriculitis, encephalitis, and brain abscess. escribed . Infectiescribed ,3. As a escribed ,5. Rare escribed ,7,8.Aspergillus infection include abscess formation, thrombosis, and hemorrhage. Prognosis is generally dismal. In the past, mortality of 86–100% has been reported [Aspergillus azoles, and survival was shown to be 31% in a landmark paper describing therapy with a combination of voriconazole and neurosurgery [Aspergillus infections and accurately reflects response to treatment [Complications of CNS reported ,10. Outcosurgery . In a reosurgery . Treatmeosurgery . Furtherreatment . The tesreatment ,16,17. TIn August 2017, a 22-year-old woman was admitted with a seemingly unprovoked reduced level of consciousness, having been found by her mother at home. Magnetic Resonance Imaging (MRI) with venography revealed severe cerebral venous sinus thrombosis of the straight sinus, and emergency thrombectomy and catheter-directed thrombolysis were performed. On day five of admission, a VP shunt was inserted due to obstructive hydrocephalus. With improvement in level of consciousness around day 42, the patient revealed that she had been having low-grade headaches and neck pain in the weeks preceding admission, which were ongoing. There were no other symptoms at the time, and a recent trip to the Greek islands was uneventful. The patient had a background history of ulcerative colitis, requiring multiple courses of corticosteroids in the preceding 6 months and the commencement of adalimumab in the months preceding admission. Other medications included the combined oral contraceptive pill. The patient was having daily isolated fevers >38.5 °C. There were no focal clinical signs on examination at this time apart from limited neck range of motion. A timeline of subsequent events is given in 3 , raised protein 122 mg/dL (CSF range: 15–45 mg/dL), and low glucose 1.6 mmol/L (range 3.5–7.7 mmol/L); no organisms were seen or cultured. A CSF ® MEFILMARRAY Panel  was negative. Other CSF tests, including West Nile Virus, JC virus, GM , Aspergillus PCR, Candida PCR, 16 s ribosome, 18 s ribosome, AFB, GeneXpert, TB cultures, and Toxoplasma PCR were also negative. CSF BDG was >500 pg/mL.Initial laboratory findings were as follows: QuantiFERON, EBV serology, CMV serology, and cryptococcal antigen testing were all negative, as was serum galactomannan (GM) index . Serum BDG was positive . Initial lumbar puncture results showed leukocytes 465 cells/mm®), which was subsequently switched to voriconazole due to infusion reaction. With the development of weakness of the left upper limb on day 97, repeat imaging showed significant enlargement of the cervical mass and emergency C2–C4 laminectomy and decompressive surgery was performed. Histopathology displayed chronic inflammation only and no other diagnostics for TB or bacterial or fungal infection yielded a diagnosis. Ambisome® was reintroduced at this time in place of voriconazole. Unfortunately, the patient experienced further weakness in both upper limbs, ataxia, and upper motor signs of her right lower limb on day 194 and required further C1 laminectomy and mass excision. At this time, the patient was on amikacin, vancomycin, and Ambisome®, in addition to empiric TB treatment. Necrotizing granulomas, septate hyphae, and A. fumigatus PCR positivity were findings from the histopathological samples and therapy was rationalized to voriconazole monotherapy.An MRI brain scan was performed in the context of the neck pain, which showed hyperintensity of the cord at C1–C2 level, ®  service) and oral isavuconazole. Follow-up CSF testing at that time showed raised WCC 253 cells/mm3 , protein 3331 mg/dL (CSF range 35–45 mg/dL), and BDG 103 pg/mL. CSF GM was negative. An UC flare in January 2019 prompted the use of steroids for over 1 month. Cognizance of the ongoing risk of immunosuppression in the context of invasive fungal CNS infection resulted in the decision to perform total colectomy in February 2019. The patient was admitted again in April 2019 with new neurology in the form of leg weakness and loss of anal tone. MRI findings suggestive of progression of infection from skull base to T11, new leptomeningeal nodular enhancement, and a rise in CSF BDG > 500 pg/mL prompted the addition of high-dose steroids and an increase in Ambisome from 5 to 10 mg/kg dose. CSF GM was not performed at this time, but serum CSF was negative. Short-interval MRI at 9 days showed further progression and intrathecal (IT) amphotericin B was commenced, guided by IDSA coccidioidomycosis protocol [Initially, the patient improved clinically and radiologically. The development of hypertension attributed to voriconazole, despite normal trough levels 1.65 mcg/mL (range 1.0–5.5 mcg/mL), prompted a change in therapy in July 2018 (day 332) to Ambisome® 3 mg/kg daily with attendance to hospital three times weekly for IT amphotericin B with a staged increase in IT amphotericin B up to 1.5 mg over a 15-week period. Worsening left hand strength and paresthesia in the C7 dermatome prompted an MRI in September 2019, which revealed progression of the dural mass with impingement of C7 nerve root. CSF analysis showed WCC <1 cells/mm3 (range 0–5 cell/mm3), protein >600 mg/dL (range 35–45 mg/dL), glucose of 4.2 mmol/L (range 3.5–7.7 mmol/L), and BDG 202 pg/mL. CSF and serum GM testing were not performed at this time. A C6 laminectomy and mass excision were performed. Necrotizing granulomas were seen histologically, but no diagnostics confirmed Aspergillus infection. New intracordal enhancement in October 2019 was found on interval MRI, voriconazole was added to peripheral Ambisome® and IT amphotericin B. Immune reconstitution inflammatory syndrome (IRIS) was also considered at this time as a possible cause of new cord changes, but CSF BDG (171 pg/mL) was still raised at this time with paired normal serum BDG (51 pg/mL). Both serum and CSF GM were negative. Further anterior cervical discectomy was performed to mitigate cord-stretch myelopathy in January 2020. Derangement in liver blood tests prompted a switch from voriconazole to isavuconazole. In addition, renal impairment occurred at this time, which required regular IV fluids and sodium bicarbonate, and a reduction in IV Ambisome® to twice weekly. Isavuconazole and Ambisome® were continued in the outpatient setting. Over the following months, the patient had extensive rehabilitation but required the use of a wheelchair. CSF BDG performed in August 2020 was negative (<30 pg/mL). Interval MRI in September 2020 showed persistent but stable abnormality from C1–4 and adjacent dura. Similar findings were seen on further MRI in April 2021; the patient remained on twice-weekly Ambisome® and isavuconazole throughout this period. By June 2021 CSF WCC was <5 cells/mm3, Aspergillus PCR was negative, and CSF GM and BDG (<30 pg/mL) were within normal range. Antifungals were discontinued at this time. The patient is currently undergoing rehabilitation with close follow-up.In July 2019, interval imaging showed abnormality confined to the cervicothoracic cord and adjacent dura. The patient was discharged with a plan for self-administered OPAT of AmbisomeAspergillus infections. Treatment in these patients can be complicated due to several factors: underlying immunosuppression or the ongoing need for immunosuppressants ; interactions of antifungals with co-medications, particularly warfarin, rifampicin, and antiepileptics; adverse drug events; acquisition of secondary nosocomial infections; and importantly, the assessment of antifungal therapy effectiveness in real time. In this patient’s treatment course, there were multiple episodes of neurological deterioration with corresponding worsening MRI findings. Discerning whether these changes in clinical status represent worsening fungal infection, post-operative inflammation, IRIS, or secondary infection can be difficult and can result in additional antimicrobials, corticosteroids, lumbar punctures, and even neurosurgical intervention. At pivotal times of clinical deterioration in this case, raised CSF BDG pointed towards active fungal infection and directed therapeutics towards escalation of antifungals and appropriate debulking surgery. As serum and CSF GM and serum BDG tests were persistently negative throughout this patient’s course, CSF BDG was the only marker for active fungal disease. Furthermore, a persistently normal BDG for over a 10-month period in the context of a clinically stable course despite persistent change on neuroimaging was supportive of cessation of antifungal therapy.This case highlights the convoluted clinical course that can be seen with complex CNS Exserohilum rostratum infection (>95%), and cryptococcal meningitis (89%) [Aspergillus meningitis and ventriculitis in case reports and appears to have greater dynamic analytical range than CSF GM for this purpose [Aspergillus meningitis or encephalitis showed a median index of 6.58 (range 0–1.0) and CSF levels were always higher than serum [Coccidoides spp., Candida spp., Cryptococcus spp., Pneumocystis, and Fusarium. Conversely, this case also highlights that despite severe infection with abscess formation, dural involvement and the need for multiple debulking procedures, GM was never raised in CSF or serum. In this setting of negative GM testing, use of CSF BDG can be useful to guide treatment progress and has been described in another case of otitis with meningeal extension [The use of BDG, a fungal cell wall polysaccharide, in CSF analysis as a reliable marker of fungal CNS infection is gaining traction . High seis (89%) ,19. One is (89%) . CSF BDG purpose ,17. In oan serum . GM doesxtension .n = 8) [n = 1) or adverse effects (n = 8) and had improvement or stabilization of disease between 138 and 810 days of therapy [Aspergillus medullary spinal abscess using isavuconazole in conjunction with low-dose Ambisome®. The reduced adverse effect profile and the need for prolonged treatment courses in CNS infections may see this triazole preferentially used if efficacy can be shown in future studies. This case also highlights the role of CSF BDG in diagnosing and managing such a complex disease.This case also supports the use of isavuconazole for fungal CNS infections. Although isavuconazole is non-inferior to voriconazole for non-CNS IA, data for CNS infection are lacking . Althougn = 8) . Additio therapy . Two cas therapy ,28. As f