Corynebacterium striatum was confirmed to be an important opportunistic pathogen, which could lead to multiple-site infections and presented high prevalence of multidrug resistance, particularly to quinolone antibiotics. This study aimed to investigate the mechanism underlying resistance to quinolones and the epidemiological features of 410 quinolone-resistant C. striatum clinical strains isolated from three tertiary hospitals in China.C. striatum clinical strains were isolated from different clinical samples of patients admitted to three tertiary teaching hospitals in China. Antibiotic susceptibility testing was performed using the microdilution broth method and pulsed-field gel electrophoresis (PFGE) was used for genotyping. Gene sequencing was used to identify possible mutations in the quinolone resistance-determining regions (QRDRs) of gyrA.A total of 410 C. striatum isolates were sensitive to vancomycin, linezolid, and daptomycin but resistant to ciprofloxacin. Depending on the antibiotic susceptibility testing results of 12 antimicrobial agents, the 410 C. striatum strains were classified into 12 resistant biotypes; of these, the three biotypes R1, R2, and R3 were dominant and accounted for 47.3% (194/410), 21.0% (86/410), and 23.2% (95/410) of the resistant biotypes, respectively. Mutations in the QRDRs ofgyrA were detected in all quinolone-resistant C. striatum isolates, and 97.3% of the isolates (399/410) showed double mutations in codons 87 and 91 of the QRDRs of gyrA. Ser-87 to Phe-87 and Asp-91 to Ala-91 double mutation in C. striatum was the most prevalent and accounted for 72.2% (296/410) of all mutations. Four new mutations in gyrA were identified in this study; these included Ser-87 to Tyr-87 and Asp-91 to Ala-91 ; Ser-87 to Val-87 and Asp-91 toGly-91 ; Ser-87 to Val-87 and Asp-91 to Ala-91 ; and Ser-87 to Ile-87 . The minimum inhibitory concentration of ciprofloxacin for isolates with double  and single  mutations was high (≥ 32 µg/mL). Based on the PFGE typing results, 101 randomly selected C. striatum strains were classified into 50 genotypes (T01-T50), including the three multidrug-resistant epidemic clones T02, T06, and T28; these accounted for 14.9% (15/101), 5.9% (6/101), and 11.9% (12/101) of all genotypes, respectively. The multidrug-resistant T02 clone was identified in hospitals A and C and persisted from 2016 to 2018. Three outbreaks resulting from the T02, T06, and T28 clones were observed among intensive care unit (ICU) patients in hospital C between April and May 2019.In total, 410 C. striatum isolates showed a high prevalence of multidrug resistance. Point mutations in the QRDRs of gyrA conferred quinolone resistance to C. striatum, and several mutations in gyrA were newly found in this study. The great clonal diversity, high-level quinolone resistance and increased prevalence among patients susceptible to C. striatum isolates deserve more attention in the future. Moreover, more thorough investigation of the relationship between quinolone exposure and resistance evolution in C. striatum is necessary.Quinolone-resistant  Corynebacterium striatum leads to multiple invasive infections [C. striatum isolates are multidrug resistant, particularly to quinolones [gyrA were believed to be majorly responsible for the resistance of C. striatum to quinolones [Recently, several reports have revealed that fections , 2, and inolones . Point minolones . Quinoloinolones .C. striatum was recently reported in China, the mechanism underlying the resistance of C. striatum to quinolones has been rarely reported in China [C. striatum [C. striatum is known to colonize multiple environmental and bodily surfaces [Although a high frequency of in China , 7. In ain China . Furtherstriatum . C. strisurfaces  and spresurfaces , 11.C. striatum to quinolones with a larger number of isolates collected from three tertiary hospitals in China and explored its genotypic characterization and prevalence potential.This study investigated the actual resistance mechanism of C. striatum strains isolated from aseptic sites were identified. The quality of the sputum samples was evaluated for qualification based on the number of leukocytes and epithelium via microscopy [C. striatum were routinely identified using VITEK-2 ANC card  and stored at − 80 °C. The isolates were further validated by MALDI-TOF microTyper  as well as 16S rRNA and rpoB sequencing technique [C. striatum strain from the same patient was selected in this study, whereas the repeated ones were excluded.The hospitals enrolled in this study included The Affiliated Hospital of Inner Mongolian Medical University , Shandong Provincial QianFuoShan Hospital , and Bayannaoer People’s Hospital , China, from March 2013 to May 2019. All echnique . Only onStreptococcus pneumoniae ATCC 49619 was used as the control.The antibiotic susceptibility test was performed using the broth microdilution method, and the antibiotics tested include penicillin (1–64 μg/mL), cefepime (1–64 μg/mL), imipenem (1–64 μg/mL), linezolid (0.5–4 μg/mL), erythromycin (0.5–64 μg/mL), clindamycin (1–32 μg/mL), gentamycin (1–32 μg/mL), tetracycline (1–64 μg/mL), vancomycin (0.5–4 μg/mL), sulfamethoxazole (0.5/9.5–8/152 μg/mL), daptomycin (0.06–1 μg/mL), ciprofloxacin (1–256 μg/mL), and moxifloxacin (0.06–32 μg/mL). The susceptibility test and result analysis were performed according to the 30th edition of the Clinical and Laboratory Standards Institute guidelines  and the C. striatum isolates was adjusted to 3.5–4.0 Mcf and digested with lysostaphin (1 mg/ml)  at 37 °C for 30 min. The bacterial chromosomal DNA of the isolates was extracted and cleaved using 40 U SwaI . The DNA of the S. Braenderup H9812 standard strain was extracted and cleaved using 40 U XbaI  and utilized as the molecular mass standard. Electrophoresis was performed on the CHEF-Mapper XA PFGE system , and PFGE profiles were analyzed using the Bionumerics v.7.6 software. The isolates with 100% similarity were considered indistinguishable [The bacterial density of the tested uishable , 16, andS. aureus isolates was extracted as per the instructions of the TIANamp Bacterial DNA kit . The pair of primers used for polymerase chain reaction (PCR) amplification and sequencing were Coryn-1 (GCG GCT ACG TAA AGT CC) and Coryn-2 (CCG CCG GAGCCG TTC AT). For PCR amplification, the protocol detailed by Sierra et al. was followed [C. striatum ATCC6940 was used as the control for sequence comparison among different clinical C. striatum isolates.The bacterial chromosomal DNA of the tested followed . The PCRC. striatum strains  were isolated from sputum  were collected from hospital A, 10.0% (41/410) from hospital B, and 12.2% (50/410) from hospital C. Most of the C. striatum isolates to imipenem, erythromycin, clindamycin, tetracycline, gentamycin, sulfamethoxazole, and trimethoprim were 90.7% (372/410), 98.8 (405/410), 98.5 (404/410), 70.5 (289/410), 53.2 (218/410), and 98.0 (402/410), respectively  were identified, which were resistant or intermediate to most antibiotics tested in this study, except for vancomycin, linezolid, and daptomycin. The resistance features of the 12 resistant biotypes diversely changed  of the isolates. Meanwhile, four new mutations in gyrA were found in this study, including Ser-87 to Tyr-87 and Asp-91 to Ala-91 , Ser-87 to Val-87 and Asp-91 to Gly-91 , Ser-87 to Val-87 and Asp-91 to Ala-91 , and Ser-87 to Ile-87 . Isolates with double mutations  in gyrA showed high minimum inhibitory concentration (MIC) of ≥ 32 µg/mL to ciprofloxacin, whereas 72.7% (8/11) of the C. striatum strains with a single mutation in gyrA presented with high MIC of ≥ 32 µg/mL to ciprofloxacin. For five quinolone-sensitive C. striatum clinical isolates collected in this study, no mutation in gyrA was identified.Single-site and double-site mutations within the QRDRs of C. striatum strains with different resistance biotypes were randomly selected for PFGE typing, and 50 PFGE types were identified, among which types T02 (15 isolates), T06 (6 isolates), and T28 (12 isolates) were dominant clones that accounted for 14.9% (15/101), 5.9% (6/101), and 11.9% (12/101), respectively. The isolates belonged to the clones with similar antibiotic resistance features and were multidrug resistant. These three clones were mainly isolated from patients admitted to the neurosurgery unit and intensive care unit (ICU)  and 91 (D → A) of gyrA were identified in these three dominant clones  of the patients died. The average age of patients and length of hospital stay were 58 years and 30 d, respectively. Moreover, 97% (32/33) of the patients were exposed to different antibiotics 2 weeks before C. striatum isolation, and most of these patients had different comorbidities, including cerebrovascular events , chronic obstructive pulmonary disease , and malignant diseases  . Meanwhile, five C. striatum strains were isolated from whole blood samples and one strain was repeatedly isolated from an immunosuppressed patient with infective endocarditis. Consistent with the results of previous investigations [C. striatum strains detected in this study were sensitive to vancomycin and linezolid. By contrast, all isolates were ciprofloxacin-resistant, and most had high MICs (≥ 32 µg/mL) to ciprofloxacin. Three dominant resistance biotypes were identified in this study, which accounted for 91.5% (375/410), and the strains were resistant to penicillin, cefepime, ciprofloxacin, and erythromycin. Although the isolates from the three hospitals were categorized into 12 resistant biotypes, no significant difference in geographical distribution was observed. Daptomycin resistance in C. striatum has been reported in previous studies [In this study, most of the 410 igations , 16, the studies , 18; howC. striatum isolates were confirmed to have double mutations in gyrA (codons 87 and 91). However, not all isolates presented with MIC of ≥ 32 µg/mL to ciprofloxacin. By contrast, a single mutation in gyrA (codon 87) was detected in 11 C. striatum isolates, and the MICs of 72.7% (8/11) isolates to ciprofloxacin were ≥ 32 µg/mL. The significant relationship between amino mutation patterns and resistance levels to quinolones was not observed in this study. Furthermore, four new mutation patterns in C. striatum were found in this study, including three double mutations in codons 87 and 91 of gyrA and a single mutation in codon 87 of gyrA. Two kinds of new mutations, the double mutation pattern  and the single mutation pattern (87-S → I), were previously found in C. urealyticum and C. jeikeium [C. striatum have not been reported previously, which had double mutations in codon 87 and 91. In particular, isolates with the double mutation pattern  accounted for 24.6% (101/410). Further, Sierra et al. [C. striatum strains with a single mutation in codon 87 were ciprofloxacin-resistant but sensitive to moxifloxacin, suggesting that other resistance mechanisms contribute to moxifloxacin resistance in C. striatum. However, no moxifloxacin-sensitive strain was detected in this study, and a higher level of resistance of C. striatum to moxifloxacin was observed. The MIC90 of the 410 C. striatum isolates to moxifloxacin was high, i.e., up to 8 µg/mL, indicating severe quinolone resistance in C. striatum in these hospitals. Unfortunately, quinolones appear to exert double effects on C. striatum, which could both induce its resistance to quinolones and promote C. striatum acquisition among susceptible patients [C. striatum infection or prevent the development of resistance to quinolones.The level of quinolone resistance depended on the type of amino substitution; isolates with double mutations on codons 87 and 91 showed a higher level of quinolone resistance than those with a single mutation on codon 87 , 16. In jeikeium . To the a et al.  found thC. striatum is associated with specific PFGE types [To further verify whether some dominant clones could prevail in a hospital environment, the PFGE typing method was employed, which is considered the gold standard method for the epidemiological investigation of important hospital-acquired bacterial pathogens . A previGE types . HoweverGE types , consistC. striatum clones could rapidly spread among inpatients and that C. striatum could transmit from person to person via healthcare personnel or the medical environment [C. striatum among susceptible patients within the hospital environment. Unfortunately, based on the current data, it was difficult to confirm whether the dominant clones belonged to the same origin in these three hospitals as they are located at long distances. Further, three outbreaks were observed among patients admitted to the ICU of hospital C in 2019, resulting from the T02, T06, and T28 clones, respectively. Of note, the three clones coexisted among different patients in the ICU of hospital C from April to June 2019, indicating a multisource feature and rapid spread of C. striatum. Therefore, more efficient measures for infection control should be implemented to better controlling the transmission of C. striatum. Moreover, 33 patients were confirmed to be involved in these three nosocomial outbreaks; most of these patients had at least one comorbidity and 97% (32/33) were exposed to at least one broad-spectrum antibiotic 2 weeks before C. striatum isolation. This finding implies that for special hospital units with critically ill patients, more attention should be paid when C. striatum strains are isolated.Consistent with the findings of this study, Baio et al.  reportedironment , 22. ThrC. striatum clones showed resistance to some widely used biocides at different levels, including high-level disinfectants  [C. striatum isolates from different hospitals to commonly used biocides should be further evaluated, and the selective use of effective biocides should be considered. In this study, 50 PFGE genotypes were identified among 101 C. striatum isolates, and high genotyping diversity was observed in the strains isolated from the same hospital, particularly those isolated from hospitals A and B. This suggests that multidrug C. striatum isolates originate from diverse origins and more dominant clones are selected under suitable circumstances, such as an increasing number of susceptible patients and nonrational application of broad-spectrum antibiotics.A recent report revealed that some dominant ldehyde) . TherefoC. striatum isolates were genotyped using the PFGE method owing to the limitation of the expenditure. The actual classification and distribution of different clones among different wards or patients may lack accuracy owing to selection bias. Therefore, dominant clones and nosocomial outbreaks may have been underestimated based on these data. Furthermore, a limited number of isolates were obtained from hospitals B and C, and only the isolates from 2019 were eventually analyzed in this study. Therefore, the actual resistant phenotypes of C. striatum isolates and prevalence potential of dominant clones within these two hospitals need to be further explored.The limitations of this study are indicated as follows. Only 101 C. striatum clinical strains analyzed in this study showed multidrug resistance, particularly to quinolones. The diversity of mutation in the gyrA of C. striatum is reported for the first time in this study. The strict restriction of quinolone use and prevention of its selective pressure on C. striatum may be the most effective way to minimize C. striatum colonization or infection and suppress the development of its resistance to quinolones. Dominant clones should be paid more attention in future infection control strategies as they can persist in hospitals for a long period and widely spread among susceptible patients.Most


The treatment of myopericarditis is different than that of acute myocardial infarction (AMI). However, since their clinical presentation is frequently similar it may be difficult to distinguish between these entities despite a disparate underlying pathogenesis. Myopericarditis is primarily an inflammatory disease associated with high C-reactive protein (CRP) and relatively low elevated troponin concentrations, while AMI is characterized by the opposite. We hypothesized that evaluation of the CRP/troponin ratio on presentation to the emergency department could improve the differentiation between these two related clinical entities whose therapy is different. Such differentiation should facilitate triage to appropriate and expeditious therapy.We evaluated the CRP/troponin ratio on presentation among patients consecutively included in a large single center registry that included 1898 consecutive patients comprising 1025 ST-elevation myocardial infarction (STEMI) patients, 518 Non-STEMI (NSTEMI) patients, and 355 patients diagnosed on discharge as myopericarditis. CRP and troponin were sampled on admission in all patients and their ratio was assessed against discharge diagnosis. ROC analysis of the CRP/troponin ratios evaluated the diagnostic accuracy of myopericarditis against all AMI, STEMI, and NSTEMI patients.Median admission CRP/troponin ratios were 84, 65, and 436 mg×ml/liter×ng in STEMI, NSTEMI and myopericarditis groups, respectively (p<0.001) demonstrating good differentiating capability. The Receiver-operator-curve of admission CRP/troponin ratio for diagnosis of myopericarditis against all AMI, STEMI, and NSTEMI patients yielded an area-under-the curve of 0.74, 0.73, and 0.765, respectively. CRP/troponin ratio>500 resulted in specificity exceeding 85%, and for a ratio>1000, specificity>92%.The CRP/troponin ratio can serve as an effective tool to differentiate between myopericarditis and AMI. In the appropriate clinical context, the CRP/troponin ratio may preclude further evaluation. It is frequently difficult to differentiate on clinical grounds between myopericarditis and acute myocardial infarction (AMI) as the cause of an acute cardiac event  since boTroponin and C-reactive protein (CRP), the universal inflammatory factor , are genThe current study was based on our local heart institute registry that included all consecutive patients admitted between January 2011 and April 2017 with any type of chest pain with electrocardiographic changes and discharged after evaluation and therapy with either the diagnosis of AMI or myopericarditis. Patients with the discharge diagnosis of pulmonary embolism, decompensated heart failure, aortic dissection, and sepsis were excluded.According to hospital routine, CRP and troponin are sampled on presentation to the emergency department in all patients. Admission CRP/troponin ratio was calculated for all patients and used for analysis of its ability to differentiate between patients diagnosed with myopericarditis and all AMI patients or between STEMI or NSTEMI patients only. The local institutional review board approved this retrospective registry-based analysis of prospectively collected data. Informed consent was waived due to the retrospective and diagnostic nature of the study. The study required no external funding.Diagnosis myopericarditis is an inflammatory disease involving both the pericardium and the myocardium Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is a single-center, retrospective study evaluating the C-reactive protein (CRP) to troponin ratio on presentation in 1898 patients admitted to the Emergency Department for chest pain. The Authors evaluated the performance of the CRP/troponin ratio alone to distinguish between myocardial infarction  and myopericarditis (19%). Median ratio values were much higher in patients with myopericarditis (p<0.001), with a fair performance at receiver operating characteristic analysis (area under the curve of 0.74), and a specificity >85% for a CRP/troponin ratio >500 (identified through the Youden method). The Authors conclude that the CRP/troponin ratio may allow to effectively discriminate between myopericarditis and MI. This conclusion is very reasonable, and may be seen as a way to reappraise the current diagnostic workup of chest pain, which includes the search for a dynamic troponin elevation and the assessment of inflammatory biomarkers, including CRP, as part of a broader evaluation. The identification of a rule-out cut-off for MI is particularly interesting in order to avoid further testing including urgent coronary angiography. In my opinion, this last point should be explored in a greater detail. Please find some other comments below.The negative predictive value (NPV) and negative likelihood ratio (LR-) are important parameters for rule-out cut-off. Please provide their values for the 500 cut-off and check if you can find other cut-offs that maximize the NPV or with a LR- <0.1.After identifying a rule-out cut-off in the whole population, please assess its performance across different age categories, given that patients with MI are usually older than those with myopericarditis (as confirmed in your cohort).You should also assess the diagnostic performance of the troponin/CRP ratio to distinguish myopericarditis from NSTEMI alone.Please evaluate also the diagnostic performance of your ratio across categories of renal function, which is known to influence troponin values.Troponin values should be better expressed as ng/L. Furthermore, please specify throughout the paper that you are evaluating high-sensitivity troponin T.To better quantify the added value of the troponin/CRP ratio, you may consider measuring the added value of the troponin/CRP ratio over troponin or CRP alone, or a combination of variables  through discrimination analysis.Please specify if the diagnosis of MI was based on European Society of Cardiology guidelines.Reviewer #2: This is an interesting study aimed to assess the diagnostic role of CRP/troponin ratio to differentiate between myopericarditis and acute myocardial infarction. The Authors observed that high values of CRP/troponin ratio may support the diagnosis of myopericarditis in patients presenting with an acute cardiac syndrome. Data are convincing and correctly analyzed and discussed, however since the echocardiographic pattern of global hypokinesia is a strong predictor of myocarditis, it should be more useful to include in multivariate analysis also the presence of global hypokinesia. It is possible to hypotesize that CRP/Troponin ratio may be more useful to discriinate between ACS and myocarditis only in the subset of patients presenting with regional LV dysfunction and without pericarditis**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  18 Feb 2021Reviewer #1: This is a single-center, retrospective study evaluating the C-reactive protein (CRP) to troponin ratio on presentation in 1898 patients admitted to the Emergency Department for chest pain. The Authors evaluated the performance of the CRP/troponin ratio alone to distinguish between myocardial infarction  and myopericarditis (19%). Median ratio values were much higher in patients with myopericarditis (p<0.001), with a fair performance at receiver operating characteristic analysis (area under the curve of 0.74), and a specificity >85% for a CRP/troponin ratio >500 (identified through the Youden method). The Authors conclude that the CRP/troponin ratio may allow effective discrimination between myopericarditis and MI. This conclusion is very reasonable, and may be seen as a way to reappraise the current diagnostic workup of chest pain, which includes the search for a dynamic troponin elevation and the assessment of inflammatory biomarkers, including CRP, as part of a broader evaluation. The identification of a rule-out cut-off for MI is particularly interesting in order to avoid further testing including urgent coronary angiography. In my opinion, this last point should be explored in a greater detail. Please find some other comments below.The negative predictive value (NPV) and negative likelihood ratio (LR-) are important parameters for rule-out cut-off. Please provide their values for the 500 cut-off and check if you can find other cut-offs that maximize the NPV or with a LR- <0.1.We have calculated the positive and negative likelihood ratios for the different subgroups as requested by the reviewer and have incorporated these calculations at the end of the results section. However, our opinion is that the indicators that should be optimized are the specificity, PPV, and possibly the positive LR. In other words, we should strive to minimize FP diagnosis of myopericarditis. This is because the consequence of a false-positive diagnosis is that an ischemic patient is considered to have myopericarditis, the result of which is inappropriate therapy .Calculation of the positive and negative likelihood ratios of the CRP/troponin ratio>500 to yield a true diagnosis of myopericarditis among all study patients yielded 3.8 and 0.5, respectively. A similar calculation of positive and negative likelihood ratios at CRP/troponin≥500 for myopericarditis against NSTEMI only yielded 4.96 and 0.56, and against STEMI only yielded 3.4 and 0.58, respectively. By the same token, the positive and negative likelihoods for a CRP/troponin ratio>1000 to diagnose myopericarditis against AMI were 6.6 and 0.64, against STEMI these were 5.1 and 0.65, and against NSTEMI- 14.7 and 0.62, respectively. Finally, we have realized that myopericarditis most resembled NSTEMI by clinical presentation and electrocardiographic pattern and, therefore as remarked by the reviewer, the most important aspect was to compare the diagnostic yield of the CRP/troponin ratio to distinguish between these entities. We have added all relevant statistical indicators  and found very high specificity and positive likelihood values for a ratio>500-1000, which are the important indicators as mentioned above (minimizing the incidence of FP). After identifying a rule-out cut-off in the whole population, please assess its performance across different age categories, given that patients with MI are usually older than those with myopericarditis (as confirmed in your cohort).As a response to the comment by the reviewer a multivariate logistic regression model was performed in order to predict myopericarditis disease with adjustment to the independent parameters of age, CRP/troponin ratio, and creatinine. Patient age and CRP/troponin ratio were assessed in the model by their quartiles: <50, 50-59, 59-60, >69 years for age, and 29, 29-117, 117-455, >455 for the CRP/troponin ratio.The second to the fourth age quartiles compared with the first quartile were negatively associated with myopericarditis . The younger the patient, the more likely was a diagnosis of myopericarditis. The third and fourth quartiles of CRP/troponin ratio compared with the first quartile were statistically significantly associated with myopericarditis . There was no correlation between creatinine concentration and myopericarditis (p=0.52).You should also assess the diagnostic performance of the troponin/CRP ratio to distinguish myopericarditis from NSTEMI alone.We must admit that this requirement, as suggested by the reviewer, fits very well the character of the findings in this study, since the vast majority of myopericarditis patients presented with an ECG pattern and echocardiographic findings compatible with a NSTEMI and not as STEMI patients. We have, therefore, evaluated the accuracy of CRP/troponin  to differentiate myopericarditis from NSTEMI (revised Table 2). We have also added to the revised paper the appropriate ROC figure not presented in the original version of the paper. In addition, we have evaluated the positive and negative likelihood ratios for CRP/troponin ratio=500 and 1000 to differentiate between myopericarditis and NSTEMI yielding the ratios of 4.96 and 0.56, respectively. These values are frequently encountered in these patients with myopericarditis . We have added to the conclusion section this consideration as well as the conversion factor for the sake of those centers who express troponin as ng/liter. At any rate, it is the novel concept that we describe and matters while every center can choose its ratio and to be applied in the clinical arena.Please evaluate also the diagnostic performance of your ratio across categories of renal function, which is known to influence troponin values.This is a good point and the comment makes sense, as the reviewer has probably assumed, that increased creatinine level representing lower renal function would lead to increased troponin level in the absence of ischemia. This could attenuate the accuracy of the CRP/troponin ratio to diagnose myopericarditis by spuriously decreasing its value. We thank the reviewer for this comment, since mildly elevated troponin levels in the presence of decreased renal function could certainly reduce the diagnostic accuracy of the CRP/troponin ratio even in the absence of ischemia. However, we assumed that such an effect would be minor since troponin usually demonstrates a 2-3-fold increase in patients with mild to moderate kidney failure whereas CRP increases 10 to 20 fold in myopericarditis. So, this effect was not expected to invalidate the diagnostic value of the CRP/troponin ratio. Anyway, as a response to the reviewer's comments, we have performed a logistic regression analysis to evaluate the relative effect of age, renal function and CRP/troponin ratio on the diagnosis of myopericarditis . The probability of pericarditis decreased by 10% for every 1-year increase in age (p<0.001), while creatinine level did not affect the accuracy of the CRP/ratio to diagnose myopericarditis. There was no correlation between creatinine concentration and myopericarditis (p=0.52). We incorporated this analysis in the revised manuscript.Troponin values should be better expressed as ng/L. Furthermore, please specify throughout the paper that you are evaluating high-sensitivity troponin T.We have, indeed, switched in our medical center to the ng/L dimensions for troponin values several years ago. However, much of the data was expressed as ng/ml when we have initially assessed the value of CRP/troponin in the early stages of data analysis. In addition, we felt that it would be conceptually better to express the combined biomarker CRP/troponin as an integer or >1 values and not as a fraction if we would have used ng/liter dimensions. For example, in a typical case of an AMI, a CRP value of 3 mg/liter with a troponin value of 100 ng/liter would yield a CRP/troponin value of 0.03, or 0.003 if measured troponin value is 1000 ng/liter. We felt that the CRP/troponin values of 30 and 3 obtained if ng/ml values are used, would be better mentally assimilated in routine practice. Following reviewer’s remark we added to paper this consideration that obtained CRP/troponin biomarker ratio should be divided by 1000 if used in the clinical field when troponin is measured in ng/liter. To better quantify the added value of the troponin/CRP ratio, you may consider measuring the added value of the troponin/CRP ratio over troponin or CRP alone, or a combination of variables  through discrimination analysis.In response to the recommendation of the reviewer, we have performed a similar analysis ROC analysis to assess the accuracy of CRP or troponin to differentiate between myopericarditis and AMI. Using Troponin yielded an AUC of 0.41 (CI: 0.363-0.454). This represents a low differentiating power, in some way, a chance finding. CRP findings were different. For the whole study population the obtained AUC was 0.747, almost identical to performance of the CRP/troponin ratio. This makes sense since a patient that presents with considerable CRP elevation  and a slightly elevated troponin concentration probably is afflicted by myopericarditis, and not AMI that presents with a normal or minimally elevated CRP. However, at the lower CRP levels the CRP/troponin performs better to differentiate myopericarditis from myocardial infarction.When evaluating the diagnostic accuracy of the CRP/troponin ratio to identify myopericarditis against acute myocardial infarction it performed similarly to the diagnostic yield of high-level CRP. However, at low but higher than normal concentrations of CRP, the ratio was diagnostic and CRP levels were not. Therefore, the CRP/troponin ratio should be used across all CRP levels as advocated in the paper. The diagnostic accuracy of the CRP/troponin ratio to diagnose myopericarditis against AMI yielded an AUC of 0.723 . The diagnostic accuracy of the CRP/troponin ratio to diagnose myopericarditis against NSTEMI yielded an AUC of 0.752 . Whereas, the diagnostic accuracy of the CRP to diagnose myopericarditis against NSTEMI yielded an AUC of 0. 56  and was not statistically significant at all.Please specify if the diagnosis of MI was based on European Society of Cardiology guidelines.Yes, the diagnosis of MI was based on European Society of Cardiology guidelines. We assumed this fact as granted and, therefore, did not mention it explicitly. We thank the reviewer for his comment and added this premise to the manuscript. Reviewer #2: This is an interesting study aimed to assess the diagnostic role of CRP/troponin ratio to differentiate between myopericarditis and acute myocardial infarction. The Authors observed that high values of CRP/troponin ratio may support the diagnosis of myopericarditis in patients presenting with an acute cardiac syndrome. Data are convincing and correctly analyzed and discussed, however since the echocardiographic pattern of global hypokinesia is a strong predictor of myopericarditis, it should be more useful to include in multivariate analysis also the presence of global hypokinesia. It is possible to hypothesize that CRP/Troponin ratio may be more useful to discriminate between ACS and myocarditis only in the subset of patients presenting with regional LV dysfunction and without pericarditisFollowing analysis of the data, or the echocardiographic data in particular, we have observed that the classic pattern of diffuse hypokinesis is rare, especially if myopericarditis (and not myocarditis) patients are evaluated. In agreement with the reviewer we must admit that most myopericarditis patients present, with regard to their ECG pattern and echocardiographic findings and frequently also clinically, as NSTEMI and not as STEMI patients. Hence, as the reviewer stated, the diagnostic challenge is to differentiate myopericarditis patients with electrocardiographic STT changes and regional wall motion abnormalities resembling acute myocardial infarction from NSTEMI patients. Hence, we have reanalyzed the data after excluding the patients with no wall motion abnormalities or those with uniform wall motion abnormality. Seventy-seven patients (21.7%) of the myopericarditis group compared to 18% among the NSTEMI group and 8% among the STEMI group did not manifest regional wall motion abnormalities on echocardiography. Re-calculation of ROC after excluding this population yielded an AUC of 0.745, very similar to the result obtained for the entire study population regardless of their echocardiographic pattern. In other words, including the only patients, that resemble AMI patients, with regional motion abnormalities, did not significantly alter the accuracy of the CRP/troponin ratio for differentiating myopericarditis from AMI. 25 Feb 2021Differentiation between Myopericarditis and Acute Myocardial Infarction on Presentation in the Emergency Department Using the Admission C-reactive Protein to Troponin RatioPONE-D-20-40200R1Dear Dr. Meisel,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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Faced with the increasing demand for blood and greater restrictions on ensuring the safety of transfusions, voluntary donation is currently the only and best alternative for the health system to have a sustainable and safe blood supply. In this context, one of the primary strategies of blood transfusion centres is to increase the satisfaction of their active donors so that they maintain their intention to donate in the future and, in turn, make the necessary recommendations so that third parties can become new donors. That is why this paper raises a question for research concerning, what should the premises be to guide the management of blood transfusion centres to maintain and even increase the satisfaction and loyalty of their active donors? In order to respond to this issue, a change in paradigm is proposed based on a management model supported by donor orientation and service quality as basic pillars, as well as on the development of a number of key social capital resources that explain this orientation toward the donor. In both approaches, the donor becomes the cornerstone of decision-making, moving away from the traditional model which is focused more on achieving targets related to the collection of blood bags. Through the empirical analysis of a sample of 126 employees from various transfusion centres and 26,833 active donors in nine Spanish autonomous communities, we have been able to validate this proposed management model as a useful tool to blood promotion agents in their decision-making processes. Blood transfusions help save millions of lives every year and ensure that the health system works well, improving patient quality of life and life expectancy by increasing complex interventions in emergency or routine situations. The importance of blood donation is fundamental because 1) blood production in the laboratory is currently impossible, (2) population decline and aging are two factors that negatively affect the balance between supply and demand, 3) the increasing implementation of new donor exclusion measures to ensure safe transfusion and, (4) the voluntary donation system represents the best alternative for the safety and sustainability of the blood supply, since systems based on ‘donation payments’ can present threats to the health and safety of recipients, as well as to the donors themselves . Results in β = 0.538, β = 0.126 and β = 0.325), thus accepting H8a, H8b and H8c; (2) both Donor orientation and Service quality management are key elements for Satisfaction and Donation Intention, with Service quality being of greater relevance (β = 0.482 and β = 0.152 versus β = 0.015 and β = 0.037), thus giving empirical support to hypothesis H1, H2, H6 and H7; and (3) Satisfaction is a direct antecedent of Donation Intention (β = 0.200), thus accepting H3, and both are direct antecedents of Recommendation (β = 0.065 and β = 0.230), so hypotheses H4 and H5 also obtained empirical support.To this end, structural equation modelling was used, which makes it possible to know the linear relations between the different constructs included in the model. The results of the same and its adjustment levels are shown in The validation of the proposed multilevel model has made it possible to contrast the suitability and usefulness of a new management paradigm in the CTS, after demonstrating the relationships between the constructs analysed. Thus, it brings to the forefront the importance of proper internal management of CTS focused on donor satisfaction and loyalty. This new management model requires, on the one hand, the enhancement of social capital resources, coordination, shared values and trust, which enable the CTS to achieve the capacity to effectively target donor satisfaction; and, on the other hand, perform their functions with quality levels perceived by donors, satisfying them and motivating them to increase the much-needed loyalty toward donation. Consequently, it is clear that those responsible for CTS management must focus their actions toward a dual objective, which would imply, on the one hand, improve the CTS internal behaviour dynamics by focusing on employees, so that they in turn focus on the donors. On the other hand, it involves improving the tangible and intangible aspects of the donation process  to improve the perception of quality in the donor’s experience. This need to properly manage the CTS human resources may require that their officers  receive complementary training in the area of people management. This would include such areas as leadership, motivation and commitment, as well as communication and conflict management dynamics, among others.If we focus on the two pillars—Donor orientation and Service quality—that, according to the results of this research, support donor satisfaction and loyalty, a number of additional recommendations arise for the CTS. Firstly, and with regard to the dimensions that make up donor orientation, we should consider the lowest mean score of the dimension ‘Intelligence generation’ that, based on the scale proposed in this study to measure the donor orientation, would lead us to recommend that centres update data on: donation evolution, factors that caused inactive donors to stop donating blood, factors that prevent non-donors from donating blood, and actions taken by the blood transfusion centres/services from other Spanish autonomous communities.Moreover, given the important difference between the centres participating in the study in terms of the efforts that they devote to the dissemination of intelligence, it would be advisable for certain centres to strengthen that dissemination internally and between each other. To this end, the staff responsible for marketing (or similar) activities can hold regular meetings to share and discuss data collected on donors with other divisions or departments, thus ensuring that donor information can be accessed by the staff. Also, they can share information about blood transfusion centres/services from other Spanish autonomous communities. This, in turn would strengthen coordination between areas of the centres and even among centres. This Interfunctional coordination–structural dimension of social capital—has also shown a very low score in this study compared to that of the cognitive and relational dimensions, in which case, structural dimension has been the one that shows great disparity among autonomous communities. Thus, and based on the scale proposed to measure this structural dimension of social capital, members of different areas or departments should interact frequently in order to improve donor recruitment and loyalty, and coordinate activities so as to provide donors with a satisfactory service.Secondly, the results of this paper show the need for optimal quality in service management in order to achieve satisfaction, donor repetition, and their recommendation to third parties, especially given that the latter has more disparate scores depending on the transfusion centre to which the donor participating in the study belongs. Thus, it is convenient to establish a “culture of service” among the CTS staff . As the As stated before, the current situation of the CTS indicates that there is room for improvement with regard to donor orientation, the provision of social capital resources, and the quality of service offered. This improvement would require a significant temporary and economic investment that involves various internal divisions in order to achieve, as indicated in the previous paragraphs: a) periodic generation and dissemination of valid and reliable information, b) a strengthening of coordination between areas of the centres and even among the centres, c) an improvement in the post-donation phase, and d) the upgrading of the facilities in which the donation process is carried out. All this will imply at the same time a challenge and great opportunity for the operation of the transfusion centres, allowing them to achieve the necessary donation figures and rates. In line with the proposal of Jaworski and Kohli , the tooFinally, and with the aim of improving the overall effectiveness of the national blood donation system, it may be advisable to encourage the development of mechanisms for communication and sharing of knowledge, experiences and problems among the CTS officials in the different autonomous communities, so that a common pool of best practices can be developed. In particular, sharing successful marketing experiences that have been developed at certain CTS may be beneficial to the national blood donation system as a whole by reducing the current differences among the various autonomous communities in terms of: dynamics of internal social capital and donor orientation perceived by the CTS employees, experience of donors , and donors’ satisfaction and loyalty -expressed in terms of intention to donate again and to recommend the donation to third parties-.Faced with the need to analyse the evolution toward market orientation within the organisations and shed some light on the cultural change process implied thereby , a longiThis research has some limitations. First, it has not delved into the causes behind the differences found among CTS in Spain in relation to each of the constructs in the management model proposed. In depth qualitative studies should be carried out that reveal the cultural causes or other causes of such differences that cannot be detected through a transversal quantitative methodology. Secondly, the generalisation of its results is not possible. Cultural aspects, such as those related with religion or conscience, or institutional aspects, such as the type of organisation that CTS conform in different countries and the regulative environment that dictates the international or regional standards on blood donation could condition the orientation of the CTS to the donors as well as their donors’ satisfaction and loyalty. Therefore, additional research in other cultural or institutional contexts could enhance the generalisation of the results achieved in this research.S1 Table(DOCX)Click here for additional data file.


Polyorchidism is a rare congenital urologic abnormality. It is usually diagnosed incidentally by imaging or during surgery. We report a case of triorchidism, in a 2 years old boy which was discovered during elective orchidopexy. The testes was localized in the inguinal region and was of normal size. We opted for orchidopexy of this supernumeray testis due to its size and its ability to reach the scrotum. The preservation of the supernumerary testis has been a controversial issue. This case report offers brief discussion of polyorchidism with review of similar literature. Polyorchidism is a rare congenial anomaly catheterized by the presence of more than two testes which can be either intra- or extra-scrotal in location. The most common variant of polyorchidism reported is triorchidism. To date, around 200 cases have been reported.Although the exact etiology of polyorchidism is not known, the proposed mechanism is considered to be longitudinal or transverse division of the genital ridge possibly by development of peritoneal bands during early embryogenesis. The most commonly used classification system for polyorchidism is the Leung Classification which is based on the probable embryologic variation.Majority of patients are asymptomatic and are incidentally diagnosed either via imaging or during surgical explorations for undescended testis, inguinal hernia, testicular torsion, hydrocele and malignancy. The management of polyorchidism is controversial but generally depends on multiple factors like the location of testis, the reproductive potential, size of the testicle and age.We report a rare case of triorchidism in a two years old patient who was incidentally diagnosed during elective orchidopexy with review of similar cases in attempt to shade further light into the subject matter.A 2-year-old boy was diagnosed to have left undescended testis. On examination, right testis was palpable in the right hemi-scrotum but the left hemi-scrotum was empty with a palpable testis at the inguinal region. Since it was palpable, ultrasound was not done and patient was directly scheduled for orchidopexy. He was explored through left inguinal incision and the intra operative finding was, two non-atrophic testicles located in the inguinal region sharing the same vas and with two separate epididymis . In thisPolyorchidism is a rare congenital urogenital abnormality. It has been identified in variable age group ranging from the neonatal age to 8th decade of life but most patients are reported to be in their early adulthood.According to the Leung classification, there are 4 types of polyorchidism which are described as follows:Type 1-Supernumerary testis that lacks an epididymis or vas and has got no attachment to the usual testis.Type 2- the supernumerary testis draining into the epididymis of usual testis and sharing a common vas.Type 3- the supernumerary testis has its own epididymis and both epididymis of ipsilateral testis drains into common vas.Type 4- complete duplication of testis, epididymis and vas.Type 2 is considered to be most common and together with type 3, they compromise 90% of polyorchidism.Most patients with polyorchidism are diagnosed incidentally on imaging or during surgical exploration. Polyorchidism is associated with undescended testicles in up to 40% of the cases.The most common type of polyorchidism is triorchidism and is mostly located on the left side (66%) which was also the case in our patient.The management of polyorchidism has been subjected to much debate. Due to current advances in imaging modalities, several authors recommend conservative management for patients with orthotopic polyorchidism while for patients with non-scrotal polyorchidism exploration is warranted. The other debatable issue is to either do orchidopexy or orchidectomy for the supernumerary testis. Those that are for orchidopexy, argue that preserving the supernumerary testis provides a better fertility potential and with the current advanced imaging modalities which are considered to be safe and highly sensitive for early detection of testicular malignancy, this mode of management is justified. The other authors claim that majority of the supernumerary testis have reduced or absent spermatogenesis and have increased risk of malignancy that warrants removal particularly in those which are atrophic.In addition, the incidence of testicular malignancy in polyorchidism in most reviews is said to be between 5.7 to 7%. This incidence rate is much higher than normal population (0.004%) and even higher than the incidence of cancer in undescended testis (0.045%). However, in all reported cancer cases, malignancy was found only in a non-scrotal  supernumerary testis.In our case, we have opted to preserve the non-atrophic testis which was placed in the scrotum. Moreover, we counseled the parents on the need for long-term surveillance.This case illustrates the unexpected presentation of polyorchidism during elective orchidopexy and the management controversies regarding the supernumerary testis. Hence, surgeons doing groin exploration should be aware of this condition.Ethics approval and consent to participate:Written informed consent was obtained from the parents for publication of this case series and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.The datasets with more images and patient data are available from the corresponding author on reasonable request.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.HGA and HAG were primarily involved in the management of the patient. AMS was also involved in the management of the patient and follow up. HAG wrote the case summary and the case report with review of the literature was written jointly by HAG and HGA. All authors have read and approved the final case report.Registry not required as this is not a first-in-man case report.Dr. Hiwote Girma Assefa.Department of Surgery.St. Paul's Hospital Millennium Medical Collage, Ethiopia.Not commissioned, externally peer-reviewed.The authors declare that they have no competing interests.


H3F3A K27M mutation using digital droplet PCR. In 10 patients, CSF was obtained by lumbar puncture at presentation. A definitive detection of H3F3A K27M mutation was achieved in only one case (10%); H3F3A K27M mutation was suspected in three other cases (30%). H3F3A K27M mutation was detected in two patients in CSF obtained by ventricular tap during a ventriculo-peritoneal shunt for obstructive hydrocephalus. Cases in which a definitive assessment was possible (definite H3F3A K27M or definite H3F3A wildtype) tended to be younger  and have a higher concentration of CSF protein  compared to nondefinite cases. Low proliferation and apoptotic rates seemed to be characteristics of DMG unfavorable for liquid biopsy. More advanced lesions with necrosis and evidence of dissemination were unlikely to be candidates for lumbar puncture due to the fear of exacerbating obstructive hydrocephalus. Methods to safely sample CSF and a more sensitive detection of ctDNA are necessary for reliable liquid biopsy of DMG at presentation.Recent studies have suggested the feasibility of detecting H3K27M mutations in the cerebrospinal fluid of diffuse midline glioma (DMG) patients. However, cerebrospinal fluid from patients in these studies were collected mainly during biopsy, ventriculo-peritoneal shunt procedures or postmortem. We assessed circulating tumor DNA (ctDNA) extracted from cerebrospinal fluid (CSF) and plasma in a series of 12 radiographically suspected and/or pathologically confirmed diffuse midline glioma patients and assessed for  HIST1H3B (H3.1K27M) or H3F3A (H3.3K27M) have been reported in 50 to 80% of diffuse intrinsic pontine gliomas and thalamic gliomas [Recurrent H3B H3.1K7M or H3F gliomas ,5,6; thuMYD88 mutation status in circulating tumor DNA (ctDNA) extracted from cerebrospinal fluid (CSF) and biopsied tissue in 21 primary and secondary central nervous system lymphoma patients using droplet digital PCR (ddPCR) [We have previously reported a 100% match of the  (ddPCR) ,10 or ne (ddPCR)  of ctDNAg for 10 minutes to remove cells, and the supernatant was stored at −80 °C within 30 minutes of collection. ctDNA was extracted using the Maxwell RSC ccfDNA Plasma Kit , according to the manufacturer’s instructions. For all samples, DNA was dissolved in 60 μL of elution buffer and stored at −20 °C until further use. ddPCR reagents and primer/probe mix for H3F3A K27M were purchased from Bio-Rad . A 20 μL PCR mix, composed of 10 μL 2× ddPCR Supermix for Probes , 1 μL ddPCR Mutation Assay (Bio-Rad) and 9 μL ctDNA, was loaded into sample wells of an eight-channel disposable droplet generator cartridge (Bio-Rad). An additional 70 μL of droplet generation oil (Bio-Rad) was loaded into the oil well for each channel. After droplet generation, the droplets were transferred into a 96-well PCR plate and subjected to thermal cycling. Amplification was carried out on the 20 μL reaction mixture on the QX-200 ddPCR system (Bio-Rad). After PCR, the 96-well PCR plate was subjected to the QX-200 droplet reader (Bio-Rad), and data were analyzed by QuantaSoft analysis software (Bio-Rad). H3F3A K27M-mutation specific signals were generated in the hexachloro-fluorescein channel. We considered definite mutant cases to have a fractional abundance of 0.1% or more and to have three or more mutant droplets and/or wildtype droplets detected. Cases in which only one or two mutant droplets were detected were considered H3K27M-mutant-suspect. Cases where two or fewer wildtype droplets were detected were considered unreliable. ddPCR was performed in at least two separate wells for each case whenever possible. Surgical removal or biopsy was performed in two cases (cases #7 and #11) with genetic confirmation of H3F3A K27M.Twelve patients with radiographically suspected diffuse midline glioma, treated at the Department of Neurosurgery, Niigata University Hospital between November 2017 and January 2021, who underwent liquid biopsy, were enrolled in the study. Written consent was obtained from patients and families for liquid biopsy after approval from the institutional review board #G2018-0008). Detailed methods of ddPCR have been previously published [-0008. DeU test and tests for associations between different parameters by the Fisher’s exact test for 2 × 2 contingency tables. Statistical analyses were performed using GraphPad Prism 9 software . p < 0.05 was considered significant.Comparisons between two groups were carried out using the Mann–Whitney Lesions were located at the pons in eight patients, medulla oblongata in one patient, thalamus in two patients and temporal lobe in one patient . A liquiH3F3A K27M mutation was obtained in only one (10%) patient showing diffuse hyperintensity of the pons on T2-weighted MR images (H3F3A K27M was suspected in three (30%) other patients at presentation, but only one or two mutant droplets were detected from each well in these patients  cases, the ddPCR results were considered unreliable, with a sufficient number of mutant and wildtype droplets not being detected.Lumbar puncture was performed in 10 patients at presentation . HoweverR images A and an R images B. A highR images C. Exophypatients . Two casH3F3A K27M was obtained at recurrence from two patients, both in CSF obtained from ventricular tap during ventriculo-peritoneal shunting for obstructive hydrocephalus in a recurrent DMG case with heterogeneously enhancing residual lesion of the right thalamus  versus nondefinite (defined as H3F3A K27M-suspect or unreliable) cases  and to have a higher concentration of CSF protein .We compared the clinical, radiographical and CSF characteristics of cases in which there was a definite diagnosis (defined as either a definite e) cases . Due to HIST1H3B (H3.1K27M) or H3F3A (H3.3K27M) mutations have been reported in 50 to 80% of diffuse intrinsic pontine gliomas and thalamic gliomas [Recurrent H3B H3.1K7M or H3F gliomas ,5,6. ThuHIST1H3B K27M mutations in four out of six (66.7%) diffuse midline gliomas. In that study, CSF was either obtained from the ventricles by external ventricular drainage at tumor biopsy (n = 2), at reservoir placement (n = 2) or by reservoir tap during treatment (n = 2). Thus, CSF was not collected by lumbar tap before treatment in any of the cases. Subsequently, two large studies were reported. Pan et al. reported the detection of H3F3A K27M mutations (n = 27) or HIST1H3B K27M mutations (n = 5) in 32 out of 57 (56%) DMG patients by next generation sequencing (NGS). CSF was obtained from the cisterns during biopsy in 52 (91.2%) cases, from during ventriculo-peritoneal shunt procedures in two (3.5%) cases, and by lumbar puncture in only three (5.7%) cases [Three previous reports highlight the feasibility of detecting H3K27M mutations in DMG patients by ctDNA analysis in CSF and plasma ,10,11. H2 vs. 373 mm2) [R technology [An important study by Miller et al. detected ctDNA in CSF obtained by lumbar puncture in 42 out of 85 (49.5%) patients harboring WHO grade 2, 3 and 4 gliomas using NGS. ctDNA was detected from a higher percentage of grade 4 tumors (64% to 44%), in patients with a positive CSF cytology (18% vs. 0%), higher CSF protein concentration (72 vs. 56), higher number of prior resections (two vs. one), in recurrent cases (55% vs. 19%) and in larger-volume tumors (1553 mm373 mm2) . Additiochnology .MYD88 L265P mutations in CSF in primary and secondary CNS lymphoma patients using ddPCR, with a 100% match of the MYD88 L265P mutation status between tumor and CSF in 21 cases with available tumor samples [MYD88 L265P mutations from the plasma and urine of PCNSL patients [We have previously reported a high detection rate of patients . Since cpatients , reflectpatients , DMGs arpatients ,18, freqpatients .HIST1H3B K27M mutations [MYD88 L265P and a 100% match of the MYD88 mutation status between CSF and tumor. Therefore, we can safely say that, at present, detecting H3F3A K27M in CSF obtained by lumbar puncture in DMG patients is difficult. Using the Maxwell RSC ccfDNA Plasma Kit, up to 4 mL of ctDNA can be extracted in CSF instead of the 1 mL used in the present study, potentially improving the detection of small amounts of ctDNA. However, obtaining large amounts of CSF can be challenging and possibly harmful in these patients. The development of more sensitive methods, including next generation sequencing or more sensitive PCR methods, is necessary for the reliable detection of H3F3A K27M in DMG patients before the commencement of treatment.The limitations of the present study include the small sample size, low number of pathologically confirmed cases and inability to detect H3F3A K27M by ddPCR in CSF obtained by lumbar puncture in DMG patients at presentation is low. Diagnosis by liquid biopsy at presentation may be possible in younger patients with a high protein concentration in CSF or evidence of leptomeningeal disease. Liquid biopsy is an exciting alternative to biopsy in DMGs, but both case selection and the development of more sensitive methods are vital.In the present report, we found that the detection rate of


Tetrahedral DNA nanostructures (TDNs) are molecules with a pyramidal structure formed by folding four single strands of DNA based on the principle of base pairing. Although DNA has polyanionic properties, the special spatial structure of TDNs allows them to penetrate the cell membrane without the aid of transfection agents in a caveolin-dependent manner and enables them to participate in the regulation of cellular processes without obvious toxic side effects. Because of their stable spatial structure, TDNs resist the limitations imposed by nuclease activity and innate immune responses to DNA. In addition, TDNs have good editability and biocompatibility, giving them great advantages for biomedical applications. Previous studies have found that TDNs have a variety of biological properties, including promoting cell migration, proliferation and differentiation, as well as having anti-inflammatory, antioxidant, anti-infective and immune regulation capabilities. Moreover, we confirmed that TDNs can promote the regeneration and repair of skin, blood vessels, muscles and bone tissues. Based on these findings, we believe that TDNs have broad prospects for application in wound repair and regeneration. This article reviews recent progress in TDN research and its applications. HighlightsWith advantages of unsurpassed structural stability, excellent material editability, good biocompatibility and individual endocytic pathway TDNs have been used extensively.in vitro and in vivo are illustrated.Different biological effects and mechanism that TDNs play Multiple clinical applications in repair and regeneration of tissues and organs are described.The anionic property of DNA hinders its transport through cell membranes ,2. MeanwTDNs have excellent biocompatibility, programmability, higher density and better serum and structural stability than native DNA ,13. TheyTissue wound repair and regeneration are complex and highly coordinated processes that depend on individual health conditions and other physiological factors ,23. VariThe development of DNA nanomaterials has undergone several stages ,6,30. DNA TDN is composed of four equidistant single-stranded DNA molecules , with ea2, 10 nM HCl, pH = 8.0), heating to 95°C for 10 min and then cooling to 4°C for 30 min , IL-6 and tumor necrosis factor α (TNF-α) . TDNs caTDNs have been found to have anti-infective effects and regulate innate and adaptive immunity ,77 Tabl. For exaTherefore, because of their 3D structure and cell membrane permeability, especially their effects in promoting cell proliferation, cell migration and anti-apoptotic, anti-inflammatory, antioxidant, anti-infective and immune regulation functions , we systin vitro and rat trauma models in vivo. We found that TDNs stimulate the production of vascular endothelial growth factor and basic fibroblast growth factor in human skin fibroblast cells. Meanwhile, TDNs regulate the inflammatory immune response by activating the AKT signaling pathway, which inhibits the secretion of TNF-α and IL-1β in vitro. We also found that after TDN treatment, the healing rate of skin wounds was significantly improved, fibrosis and inflammation in the scar area were significantly reduced and scar formation was ultimately reduced [Wound healing involves a series of processes involving wound tissue regeneration, granulation tissue hyperplasia and skin scar tissue formation. Corneal epithelial wound healing is more complicated and includes cell proliferation, death and migration ,84. Poor reduced . These r reduced .in vivo Matrigel plug assay demonstrated the excellent angiogenesis-promoting effects of TDNs. Compared with the control group, TDNs could notably induce neo-vessel formation 7 days after a mixture of human umbilical vein endothelial cells and Matrigel was injected into the right ventral side of nude mice for 7 days [Vascularisation is critical for the survival of tissue engineering grafts. Researchers have made a series of explorations in this field, including the use of growth factors and endothelial cells to form composite scaffolding materials or microsurgery techniques to promote angiogenesis . Neverthr 7 days . In addir 7 days . These fMuscles may deteriorate after injury, which affects their physical appearance and motor function . Muscle in vivo [As an editable and stable nanomaterial ,35, TDNsin vivo . This inAcute myocardial infarction is the most serious ischemic heart disease of the coronary arteries, and timely reperfusion can help reverse myocardial ischemia . NeverthBone tissue defects are common in clinical practice and are usually caused by trauma and infection. Reconstruction of the defective bone tissue remains a challenge for surgeons. Adipose-derived mesenchymal stem cells (ADSCs) have attracted attention in the clinical application and development of orthopedic regenerative medicine, especially because they are rich in resources, easy to isolate and cause minimal trauma. They are found to be an excellent source of bone marrow mesenchymal stem cells for clinical application in bone tissue regeneration . To dateAccumulating evidence has shown that TDNs are important regulators of cell proliferation ,15. In aIn addition to promoting the proliferation, migration, differentiation and osteogenesis of ADSCs, other effects of TDNs at the epigenetic level have also aroused great interest. We further explored the changes in stem cell DNA methylation and gene expression after ADSCs were treated with TDNs. We found that TDNs could also regulate DNA methylation levels, thereby promoting ADSC proliferation and inhibiting apoptosis . Our resOsteoarthritis is a common degenerative pathological process of articular cartilage that is accompanied by excessive chondrocyte apoptosis. Cartilage tissue is a non-vascular connective tissue covering the articular surface and plays an important role in the maintenance of normal biological functions. However, owing to their poor self-repair ability, it is of great importance to use cells, biological materials and other stimuli to jointly regulate the regeneration of cartilage cells, thereby promoting the repair of damaged cartilage tissue.Based on the anti-apoptotic and antioxidant effects of TDNs in a variety of diseases, we further explored their effect on chondrocytes. We found that TDNs can be internalized by chondrocytes without other auxiliary agents, and are mainly concentrated in the cytoplasm. In addition, chondrocyte autophagy is enhanced by the activation of the PI3K/AKT/mTOR signaling pathway by TDNs . FurtherAs a common inflammatory oral disease, periodontitis significantly affects the quality of life of patients . A previIn vitro, we found that the MAPK/ERK signaling pathway, which contributes to PDLSC inflammation, was inhibited by TDN treatment. Compared with the group treated with lipopolysaccharides (LPS) alone, the group treated with both LPS and TDNs showed elevated protein levels of ERK, JNK and P38. Meanwhile, even under inflammatory conditions, TDNs can promote the osteogenic differentiation of PDLSCs. The gene expression of ALP and RUNX2 in PDLSCs was enhanced and the synthesis of osteogenic factors such as OPN and RUNX2 was increased, suggesting that TDNs can improve osteogenic capacity. An animal model of periodontitis was established using the second molar of Sprague-Dawley (SD) rats. Except for the influence of height or density, alveolar bone tissue in the inflammatory model remained almost the same as in the control group after treatment. The number of osteoclasts, the level of pro-inflammatory factors IL-1β and IL-6 and the infiltration of inflammatory cells all decreased significantly in the periodontium after TDN treatment. The alveolar bone was greatly repaired to its normal form by TDNs [ by TDNs .These studies suggest that TDNs have great potential for bone tissue repair and regeneration.Stem cell therapy has been considered a promising method for repairing damaged nerve tissues . HoweverDespite these findings, it is still difficult to determine the effect of TDNs in promoting NSC migration, which is a complex biological process . The keyAlzheimer’s disease (AD) is a common neurodegenerative disease that usually manifests as progressive neurocognitive dysfunction and neuronal damage . ClinicaIn addition, Parkinson’s disease (PD), a neurodegenerative disease with an incidence rate second to AD, has attracted our attention . The maiTDNs have multiple biological efficacies, unsurpassed structural stability, excellent material editability, good biocompatibility and individual endocytic pathways. Because of these traits, TDNs have been used extensively in disease treatment research, especially in the repair and regeneration of tissues and organs. New opportunities and challenges have been introduced in this promising field of nanomedicine.in vitro or in mice, rats and rabbits, which differ in histomorphology and pathophysiological mechanisms from humans. These differences make it necessary to conduct similar studies on large animals, such as pigs or primates, to further verify the clinical effects and application of TDNs in tissue repair and regeneration. Third, long-term cytotoxicity is a concern. Oligonucleotides are biodegradable and biocompatible; however, their properties may change when DNA is designed as nanostructures. There are unknown risks and benefits, especially in terms of the long-term cytotoxicity and biological availability of TDNs under physiological conditions. Therefore, dynamic and longitudinal follow-up and in-depth research should be conducted. At the same time, the use of DNA origami technology can promote diverse designs and modifications to the structure and function of TDNs, which may remedy these limitations. Hopefully, these challenges can be addressed through multidisciplinary collaboration among clinical doctors, biologists and material engineers.However, the development of TDN materials faces some obstacles. First, the high cost of synthesis may limit mass production and large-scale clinical application of TDNs in tissue repair and regeneration. To solve this problem, the mirror structure of TDNs is obtained by DNA origami technology, which maintains the TDN structure to guarantee long-lasting efficacy, stronger serum stability and longer physical retention times. Second, most studies were conducted In summary, the special spatial structure and small size of TDNs enable them to enter cells without the aid of transfection agents and participate in mediating cellular functions. TDNs can promote cell migration, proliferation and differentiation, and they possess anti-inflammatory, antioxidant, anti-infective and immune regulation capabilities. These properties can promote skin wound healing, reduce scar formation and promote the regeneration and repair of vascular, musculoskeletal and nerve tissues. We believe that TDNs have great potential for application in wound repair and regeneration. Further research is urgently required in this promising field.YD, WC, XY, YL, XM and XC wrote the manuscript. All authors have read and approved the final manuscript.This research was supported by the National Key R&D Program of China (No. 2019YFA0110600), National Natural Science Foundation of China , China Postdoctoral Science Foundation  and West China Hospital Postdoctoral Science Foundation (No. 2020HXBH104).None declared.β: Interleukin-1β; LPS: Lipopolysaccharides; MIRI: Myocardial ischemia–reperfusion injury; NSCs: Neural stem cells; PD: Parkinson’s disease; PDLSCs: Periodontal ligament stem cells; ROS: Reactive oxygen species; TDNs: Tetrahedral deoxyribonucleic acid nanostructures; TNF-α: Tumor necrosis factor α.AD: Alzheimer’s disease; ADSCs: Adipose-derived mesenchymal stem cells; EPS: Extracellular polysaccharides; HO-1: Hemeoxygenase-1; IL-1


TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca2+ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies.  The pathogenicity of the variants was determined in experiments with myofibers in which the phenotype was [TNNC2 variants occur and cause a mild congenital myopathy phenotype with patients surviving into adulthood. Screening of patients with congenital myopathy for variants in TNNC2 is warranted. The lack of more reports on disease-causing variants in TNNC1 and TNNC2 underscores the importance of TnC for skeletal muscle structure and function.Until recently, the gene encoding fast skeletal troponin C,  disease . In 2021amilies.  One famiy domain B. The oty domain B. The pader age.  Moleculatype was  by replaTNNI1 ), which is located on chromosome 1q32.1, encodes for slow skeletal troponin I (ssTnI), and consists of 187 amino acids from nine exons ), which is located on 11p15.5, encodes for fast skeletal troponin I, and consists of 182 amino acids from ten exons ), which is located on 19q13.42, encodes for cardiac troponin I, and consists of 210 amino acids from eight exons. The 30 extra amino acids are located at the N-terminal side of the protein. As cardiac troponins exceed the scope of this review, we here focus on TNNI1 and TNNI2. TnI and TnT are structurally connected in the troponin core domain with the IT arm, a rigid interface consisting of coiled-coil α-helices  TNen exons B; and  [TNNI2 and TNNT3, respectively [TNNI2 and TNNT3 variants that lead to DA cause an increased Ca2+ sensitivity in fast skeletal myofibers. This could result in increased muscle stiffness and therefore increased joint stiffness [TNNI2 expression has been found in osteoblasts, chondrocytes of long bone growth plates, and cartilage [TNNI2 expression in osteoblasts remains to be studied and the role of TNNI2 in these tissues is still unclear. However, it might have implications for the distal limb joints. Zhu et al. suggested that DA could be caused by TNNI2 variants that impair bone development [To date, sis (DA) ,58. Bamsectively ,60. DA iectively ,63,64,65tiffness . Howeverartilage ,68,69. Telopment .TNNT1, which is located on chromosome 19q13.4 and encodes slow skeletal troponin T containing 278 amino acids , TNNT2, which is located on chromosome 1q32 and encodes cardiac troponin T containing 298 amino acids ); and (3) TNNT3, which is located on chromosome 11p15.5 and encodes fast skeletal troponin T (fsTnT) containing 269 amino acids , TNNT2 is mainly expressed in cardiac fibers, embryonic and neonatal skeletal muscles also express TNNT2 [2+ sensitivity of the MgATPase, resulting in a different contractile response to Ca2+, because of phosphorylation and N-terminal charge differences [2+ dependent [2+ is bound to TnC. The interaction of T2 and tropomyosin is thought to be stronger at lower Ca2+ concentrations [TnT is the troponin subunit that anchors the troponin complex on tropomyosin ,71,72,73ss TNNT2 ,75,76,77ferences ,80,81. Tferences . The rodferences ,83,84,85ependent . The T2 TNNT1 cause nemaline myopathy 5 (NEM5). Initially, NEM5 was confined to the Amish and therefore called Amish nemaline myopathy [TNNT1 is one of at least 12 genes reported to be implicated in NEM, a congenital myopathy [Variants in myopathy ,71,72,86myopathy ,88. TNNTmyopathy ,72,89,90myopathy ,63. NEM In 2000, Johnston et al. were the first to describe a variant in ssTnT in the Old Order Amish community leading to NEM5.  PatientsThe first case of NEM5 outside the Amish community was described by van der Pol et al. . Two chiTNNT1 variants described above all result in truncation of ssTnT, which might impair the function of one or both of the tropomyosin binding sites, possibly resulting in an altered localization of the troponin complex relative to tropomyosin. Also, truncation of ssTnT could lead to impaired function of the troponin I and/or troponin C binding site(s), resulting in the inability to form the troponin complex. Furthermore, the absence of healthy and mutated ssTnT in slow-twitch fibers could result in atrophy of slow-twitch myofibers [The yofibers ,87. The TNNT1 leading to congenital myopathy were described [TNNT1 can manifest into a congenital myopathy.Recently, two novel recessive variants in escribed ,100. Pelescribed  The variTNNT3 was implicated only in DA, similar to TNNI2 [TNNT3 variant c.681 + 1G > A results in exon 14 skipping, leading to a very severe phenotype of NEM and DA.Until recently, to TNNI2 ,62,101. to TNNI2  The patito TNNI2 . Thus, tHere, we focus on potential therapeutic strategies that directly target the sarcomere. For other therapeutic strategies, such as physical therapy, orthotic support, respiratory support, orthopedic surgery, and drug therapies that do not specifically target the sarcomere, but instead target downstream effects of variants in sarcomere genes, we refer to the reviews of Jungbluth et al. and Wang et al. ,102.2+ sensitivity of force in patients’ myofibers, i.e., a lower force production at submaximal Ca2+ levels. Particularly in patients with these variants, troponin activators might pose a therapeutic strategy [Skeletal troponin variants often cause a decreased Castrategy ,104,105.2+ sensitivity of force by 20–30% [NEB (NEM2), who were shown to have a reduced Ca2+ sensitivity of force [TNNC1 or TNNI1 variants leading to a skeletal muscle defect have been reported. In addition, variants in TNNT1 lead to a lack of ssTnT protein, and importantly, levosimendan affects cardiac TnC, leading to undesired effects on cardiac function.To target slow skeletal myofibers, levosimendan, a slow-twitch/cardiac-specific troponin C activator, is available ,107. Levy 20–30% ,110,111.of force . The myotirasemtiv was used to repair the Ca2+ sensitivity of force in myofibers isolated from biopsies of patients with a TNNC2 variant [Tirasemtiv is a fast skeletal muscle troponin activator, which selectively increases Ca2+ sensitivity in fast-twitch myofibers. After treatment with tirasemtiv, the patients’ myofibers produced significantly more force at submaximal Ca2+ levels, restoring the Ca2+ sensitivity of force to levels of myofibers isolated from healthy subjects. De Winter et al. used tirasemtiv, in vivo and in vitro, to improve muscle performance in transgenic NEM mice by increasing the Ca2+ sensitivity of force and increasing fatigue tolerance [tirasemtiv did not meet its primary endpoint in a recent phase 3 clinical trial in amyotrophic lateral sclerosis patients in part because of issues with tolerability  [Reldesemtiv (formerly CK-2127107), a second generation fast skeletal muscle troponin activator that is structurally distinct from tirasemtiv, has a lower side effect profile and is currently under clinical trial investigation [To target fast skeletal myofibers,  variant . Tirasemolerance . Howevernsomnia) . Reldesetigation ,116.It is important to highlight that many patients with congenital myopathies, including those with variants in genes encoding skeletal troponin, experience difficulties with breathing due to impaired functioning of the respiratory muscles. During daily life activities, slow-twitch myofibers activated by small motor neuron cell bodies are recruited before fast-twitch myofibers activated by large motor neuron cell bodies are recruited (Henneman’s size principle) . Thus, t2+ sensitivity of force. Future therapeutic approaches to combat skeletal troponin-related myopathies include the introduction of healthy skeletal troponin protein in patients’ muscles by adenoassociated viral vector (AAV) gene therapy. The introduced, healthy, protein competes with the mutated protein that causes the myopathy. Incorporation of the healthy protein could lead to significant improvement in muscle function, as shown by experiments in which endogenous TnC in myofibers isolated from patients with a TNNC2 variant was replaced by recombinant, healthy TnC. This restored the Ca2+ sensitivity of force and thus increased force at submaximal Ca2+ levels [Variants in genes encoding skeletal troponins cause a sequence of events, starting at the transcription of the mutated gene, that lead to translation of altered transcript and the generation of mutant protein and subsequently to muscle dysfunction in the form of myofiber atrophy or altered Ca+ levels . Further+ levels . Recent + levels ,121,122.+ levels . HoweverActa1 His40Tyr knock-in mice, Lindqvist et al. cloned a cDNA construct encoding the cardiac alkali myosin light chain (MYL4) into an AAV expression plasmid and performed intramuscular injections of rAAV harboring a myosin transgene [An alternative approach to combat muscle weakness due to skeletal troponin variants could be to target myosin and improve the efficiency of ATPase activity. To improve muscle contraction in ransgene . Introduransgene . Thus, MMYH7), which is also expressed in slow skeletal muscle, and thus, it could potentially increase muscle contractility of slow-twitch myofibers. Slow-twitch myofibers isolated from Neb cKO animals showed an increased Ca2+ sensitivity of force upon Omecamtiv Mecarbil administration [The effect of Omecamtiv Mecarbil (formerly known as CK-1827452) on skeletal muscle was recently studied. Omecamtiv Mecarbil was originally developed to treat systolic heart failure . It targstration . HoweverTNNC1, the skeletal muscle phenotype being overshadowed by a cardiomyopathy phenotype. Reported variants in skeletal troponin encoding genes predominantly lead to changes in the Ca2+ sensitivity of force, myofiber atrophy, and/or a lack of protein (Troponin is a key regulator of muscle contraction. To date, only a few variants in skeletal troponin genes have been reported A–C. This protein D. Severa