In multiple sclerosis (MS), iron rim lesions (IRLs) are associated with pronounced tissue damage, higher disease severity and have been suggested as an imaging marker of chronic active inflammation behind the blood–brain barrier indicating progression. Furthermore, chronic intrathecal compartmentalized inflammation has been suggested to be a mediator of a cerebrospinal fluid (CSF)–related tissue damage.To investigate CSF markers of intrathecal inflammation in patients with at least one IRL compared to patients without IRLs and to investigate tissue damage in lesions and normal-appearing white matter (NAWM) with proximity to CSF spaces.A total of 102 patients (51 with at least 1 IRL and 51 age-/sex-matched patients without IRL) scanned with the same 3T magnetic resonance imaging (MRI) and having CSF analysis data were included.Patients with at least one IRL had higher disability scores, higher lesion volumes, lower brain volumes and a higher intrathecal immunoglobulin G (IgG) synthesis. Apparent diffusion coefficient (ADC) values in IRLs were higher compared to non-IRLs. We observed a negative linear correlation of ADC values in all tissue classes and distance to CSF, which was stronger in patients with high IgG quotients.IRLs are associated with higher intrathecal IgG synthesis. CSF-mediated intrathecal smouldering inflammation could explain a CSF-related gradient of tissue damage. Compartmentalized inflammation proceeds behind a nearly closed blood–brain barrier in the brain parenchyma as well as in the meninges, where lymphoid follicle-like structures of predominantly B-cells and plasma cells activate mainly microglial cells in the cortex via soluble substances like interferon gamma (INF-γ) and tumour necrosis factor (TNF)-alpha, leading to pronounced mainly subpial cortical demyelination. Furthermore, the permanent low-burning inflammation is presented by chronic active MS lesions, which are characterized by a peripheral rim of iron-laden proinflammatory microglia/macrophages associated with ongoing slow degree of demyelination and axonal loss, thus reflecting a compartmentalized inflammation.4In multiple sclerosis (MS), chronic compartmentalized inflammation represents a fundamental driver of clinical progression.5 Previous studies demonstrated that the presence of IRLs is associated with more severe clinical disability, progressive disease course and central nervous system atrophy.58 Furthermore, there is increasing evidence that chronic, intrathecal compartmentalized inflammation represents an important mediator of a cerebrospinal fluid (CSF)–related tissue damage.10 Indeed, previous studies have demonstrated a CSF-related gradient with a more severe tissue matrix damage in the normal-appearing white matter (NAWM) and chronic lesions in proximity to the inner and outer surfaces of the brain.1116In vivo, chronic active lesions can be identified on iron-sensitive magnetic resonance imaging (MRI) sequences as paramagnetic or iron rim lesions .Against this background, the aims of this study were (a) to evaluate CSF markers of intrathecal inflammation in MS patients with at least one IRL compared to patients without IRLs and (b) to investigate their relationship with tissue damage in lesions (both IRLs and non-IRLs) and the NAWM, as well as with distance to the adjacent CSF spaces. (2) at least 18 years of age, (3) MRI acquired on the same 3T MRI system using identical imaging parameters and (4) availability of CSF-related data (obtained as part of diagnostic purposes). In cases CSF was obtained more than once, the data closest to the time of MRI were used for further analysis as suggested previously.19 Signs of acute inflammatory activity (relapses and/or presence of acute contrast-enhancing lesions) at the time point of lumbar puncture (LP) were manually recorded from medical and radiology reports. Exclusion criteria were the presence of neurological diseases other than MS, cardiovascular or respiratory disease and missing or insufficient MRI data. On the days of MRI examinations, age, sex, disease duration, disease phenotype, disease-modifying therapies (DMTs), Expanded Disability Status Scale (EDSS) including the pyramidal functional system score, relapses and number of contrast-enhancing lesions were assessed.We retrospectively screened our database to identify two groups of patients (MS patients with at least one IRL on susceptibility-weighted imaging (SWI) and an age- and sex-matched group without IRLs) who met the following inclusion criteria: (1) diagnosis of definite MS according to the 2010 criteria,b = 0/1000 s/mm2, FOV = 220 mm, slice thickness 4 mm, resolution = 0.98 × 0.98 × 4.0 mm) including apparent diffusion coefficient (ADC) calculations; SWI  = 1.5 mm, voxel-size = 0.9 × 0.9 × 1.5 mm  and T1-weighted images  acquired 10 minutes after contrast-injection (single dose gadoterate meglumine). SWIs were generated automatically by the scanner software provided by the manufacturer.MRI was performed on a 3T MR system , 20-channel head coil) including the following sequences: three-dimensional (3D) magnetization-prepared rapid acquisition gradient-echo  = 2.49 ms, repetition time (TR) = 1900 ms, inversion time (TI) = 900 ms, field-of-view (FOV) = 240 mm, spatial resolution = 0.9 × 0.9 × 0.9 mm); 3D fluid-attenuated inversion recovery ; diffusion-weighted echo planar images , percentage and absolute values of the intrathecally synthesized immunoglobulins and oligoclonal bands. Blood–CSF barrier dysfunction  and intrathecal Ig synthesis were determined based on the method of Reiber. Since the upper limit of the reference range for an intact blood–CSF barrier function is age-dependent, pathological cutoffs for Qalb were defined by an age-adjusted equation as described previously.All CSF analyses were performed at the Institute for Clinical Chemistry, Medical Faculty Mannheim, Heidelberg University, including the following parameters: leukocyte count, CSF protein concentration, CSF glucose and lactate, CSF and serum of albumin, immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) including the corresponding ratios , blinded to clinical and CSF data, evaluated MR images jointly. If uncertainty remained regarding potential lesion classification, an additional reviewer (with 30 years’ experience) was consulted for final determination of lesion classification. SWIs were evaluated for the presence of chronic lesions with a characteristic hypointense rim as described previously.https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/SIENA), automatic segmentation of deep grey matter (DGM) volumes was performed with FSL FIRST (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FIRST).Brain volumes, normalized for subject head size, were calculated on lesion-filled 3D MPRAGE data sets using SIENAX (https://www.fil.ion.ucl.ac.ukon), 3D MPRAGE and 3D-FLAIR data sets were co-registered to the quantitative ADC maps applying a rigid body transformation. Lesion segmentation was performed semi-automatically on 3D FLAIR images using the lesion prediction algorithm of the Lesion Segmentation Toolbox . An experienced reader manually corrected lesion masks using MRIcroGL (https://nitrc.org/projects/mricrogl). According to their appearance on SWI, an IRL- and a non-IRL-mask was generated. To investigate voxel-wise CSF-related tissue damage in lesions and the NAWM, tissue segmentation was achieved using the segmentation algorithm of SPM12 on MPRAGE images. Lesion masks were subtracted from the white matter masks to produce an NAWM mask. The CSF tissue mask was then binarized using FSL (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSL) with a probability threshold of 0.95. We used a conservative threshold of 0.95 since lower thresholds tend to overestimate CSF spaces. The binary CSF mask was then overlaid with a distance transformation map created with Convert 3D software (http://www.itksnap.org/pmwiki/pmwiki.php?n=Convert3D.Doc2). To account for partial volume effects, the first 2 mm around the borders of the CSF space were excluded from further analysis. Furthermore, we excluded all lesion voxels <1.5 mm. Non-zero voxels were converted to ASCII values, resulting in a two-dimensional (2D) matrix of distance and ADC values for each voxel in the lesion and NAWM mask.Using SPM12  and a CSF-related gradient of tissue damage, the whole study cohort was dichotomized according to the median IgG quotient (MS patients with ‘high’ IgG quotient and MS patients with ‘low’ IgG quotient). Voxel-wise analysis of tissue damage in this supplementary analysis was performed analogously to the analysis already described above.Statistical analyses were conducted using R (version 4.0.2) and IBM SPSS Statistics . Correlational analyses of imaging data were conducted using the Spearman rank correlations. Descriptive and exploratory analyses, as well as confirmatory multivariate models, were conducted using the appropriate tests depending on the distribution of the variable data. Normality was confirmed or rejected by calculation of Kolmogorov–Smirnov test and inspection of the histograms and Q–Q plots. As most of our data was non-normally distributed, non-parametric statistics were used. In the description of the analyses, we chose to report medians and interquartile range (IQR) accordingly.The local ethics committee approved this study. Patient consent was waived by our institutional review board due to the retrospective nature of the study and the lack of patient interaction.Characteristics of the study population according to clinical and radiological data are summarized in MS patients with at least one IRL had significantly higher disability scores , a significantly higher T2 lesion volume and significantly lower brain volumes compared to patients without IRLs. We found no statistically significant differences between both groups regarding DMT, high efficacy DMT, disease duration, relapses and number of contrast-enhancing lesions.The results of the CSF analysis are reported in −3 mm2/s vs 1.04 (IQR = 0.97–1.13) × 10−3 mm2/s; p = 0.007, Wilcoxon test). Median ADC values in non-IRLs did not statistically differ in both patient groups (1.04 (IQR = 0.97–1.13) × 10−3 mm2/s vs 1.03 (IQR = 0.97–1.1) × 10−3 mm2/s; p = 0.468, Mann–Whitney U test). ADC values in lesions (both IRLs and non-IRLs) were significantly higher compared to the NAWM , whereas we observed no statistically significant differences in ADC values in the NAWM in patients with at least one IRL (0.77 (IQR = 0.75–0.79) × 10−3 mm2/s) compared to patients without IRLs (0.76 (IQR = 0.74–0.78) × 10−3 mm2/s; p = 0.088, Mann–Whitney U test).In patients with at least one IRL on MRI, median ADC values in IRLs were significantly higher compared to non-IRLs (1.09 (IQR) = 1.03–1.24) × 10n = 32; MS patients without IRLs, n = 19) compared to the ‘low’ IgG group . The corresponding scatterplots are demonstrated in Z-matrices for comparison-of-correlations of linear correlations of ADC values and distance from CSF are provided in Supplemental Tables S1 and S2.Voxel-wise ADC values of lesions and the NAWM with regard to the distance from the CSF space in patients with ⩾1 IRL and patients without IRLs are plotted in  Besides an association of IRLs with worse disease severity and lower brain volumes, the authors also found a pathologically elevated albumin quotient as a marker of blood–CSF barrier disruption in MS patients with IRLs. We now provide some additional observations regarding the association between IRLs, pathogenic CSF profiles and a CSF-related tissue injury.In MS, IRLs have been suggested as an imaging marker for chronic active inflammation behind the blood–brain barrier, indicating progression. A recent study also investigated the relationship between IRLs and clinical, MRI and CSF profiles.58 In addition, IRL patients had significantly higher levels of intrathecal IgG production as represented by Reibergram (IgG quotient), as well as by percentage and absolute values of intrathecally synthesized fractions. On quantitative ADC maps, a sensitive MRI method to quantify the severity of tissue damage in lesions and normal-appearing brain tissue,24 we found significantly higher ADC values in IRLs compared to non-IRLs, emphasizing their destructive nature.46 Furthermore, we observed an inversely proportional tissue damage in lesions (both IRLs and non-IRLs) and the NAWM to the distance from the CSF space. This result is in line with previous studies that demonstrated a periventricular-related tissue injury of T2-hyperintense MS lesions.1116 Of note, patients with ‘high’ IgG quotients showed a greater magnitude of negative correlation with ADC values compared to patients with ‘low’ IgG quotients.In this study, MS patients with at least one IRL also showed more aggressive clinical and MRI features, indicating their relevance as a novel imaging marker of disease severity and progression.10 The results of our CSF analysis support this assumption. Indeed, we found significantly higher levels of intrathecal IgG synthesis indicating a pronounced compartmentalized inflammation in MS patients with at least one IRL compared to patients without IRLs. Interestingly, a previous study demonstrated that MS patients with intrathecal IgG synthesis had a higher risk and shorter time to EDSS worsening during follow-up and patients with IRLs ultimately showed more severe clinical disability and a progressive disease course.58 Of note, some studies also reported that intrathecal IgM synthesis is associated with a more severe disease course,26 whereas in this study, intrathecal IgM synthesis did not statistically differ between both groups. The lack of correlation with IgM could be interpreted to mean that the presence of IRLs is pathoimmunologically mediated by IgG rather than IgM.One possible hypothesis for this CSF-related tissue injury contains a chronic compartmentalized intrathecal inflammation consisting of B-lymphocyte activation and activated microglia. Furthermore, activated astrocytes and microglia  produce B-cell activating factors, supporting in turn B-cell survival and thus resulting in a potentially vicious cycle of a self-sustained chronic compartmentalized neuroinflammation. It seems conceivable that, through diffusion into the brain parenchyma, these neurotoxic factors lead to demyelination, axonal degeneration and eventually disability progression.Ongoing tissue damage induced by B-lymphocytes, specifically located in the meninges, includes complement-mediated injury and release of immune activating molecules and proinflammatory cytokines. IRLs are mainly located in periventricular regions,22 where the vein density is the highest. Interestingly, a previous study demonstrated the iron rim at the lesion edge is characterized by an extensive presence of CD68-positive demyelinating inflammatory infiltrates  that distribute along veins crossing the lesion edge. Furthermore, the authors observed CD8-positive T-lymphocytes in the perivascular space of veins and capillaries, suggesting that both T- and B-lymphocyte-mediated factors play a deleterious role in tissue damage near the inner surface of the brain. Besides tissue damage observed near the ventricles, previous studies also found significantly lower volumes in the distant cortical grey matter in patients with IRLs.18 One possible explanation for this would be that tissue damage in periventricular regions is linked to that of the cortex, suggesting a shared mechanism of injury.13CSF-mediated damage in proximity to the ventricles might not only spread from ventricular ependyma but also from the venous perivascular ‘Virchow–Robin spaces’, which are connected to the subarachnoid space. Patients with acute relapses have higher CSF leukocyte counts than patients with chronic progressive or inactive disease, and previous studies demonstrated that the CSF leukocyte count and the albumin CSF/serum quotient correlate with proximity of lesions to the lumbar CSF space and the temporal latency of the LP at the time of relapse.31 While CSF leukocyte counts might decrease under DMTs, the humoral response (IgG synthesis) usually remains unchanged.33 However, some studies also reported a reduced intrathecal IgG synthesis or a disappearance of oligoclonal IgG bands during DMT. Furthermore, recent studies suggested that DMTs also might have a beneficial, but delayed effect on tissue damage in IRLs.35 With regard to the time interval between CSF analysis and MRI assessment, it is also important to mention that previous studies demonstrated that in a small percentage of IRLs, the rim even wanes and finally disappears during follow-up.37 Finally, our results are somewhat in contrast to a recent retrospective study in that we found a significantly higher IgG quotient, percentage and absolute intrathecal IgG synthesis in IRL patients, whereas this was not observed by the other group. Therefore, a prospective study including more patients would be highly interesting.This study has potential limitations. First, this was a retrospective evaluation of an existing patient population. This is particularly important with regard to the interpretation of the CSF results. However, the average interval between CSF analysis and time point of MRI in this study was similar to that in a previous study.In conclusion, IRLs are associated with higher intrathecal IgG synthesis and thus both point in the same direction, indicating progression. Whether IRLs can be used as an imaging marker of intrathecal compartmentalized inflammation can only be speculated at present and requires larger studies to further distinguish a temporal from causal relationship. Furthermore, our results suggest that tissue damage in lesions and the NAWM is related to distance to CSF. Whether this CSF-related gradient of tissue damage is related to intrathecal inflammation or other factors needs to be investigated in future studies.Click here for additional data file.Supplemental material, sj-doc-1-msj-10.1177_13524585231155639 for Cerebrospinal fluid–related tissue damage in multiple sclerosis patients with iron rim lesions by Matthias Wittayer, Claudia E Weber, Maximilian Kittel, Michael Platten, Lucas Schirmer, Hayrettin Tumani, Achim Gass and Philipp Eisele in Multiple Sclerosis JournalClick here for additional data file.Supplemental material, sj-docx-2-msj-10.1177_13524585231155639 for Cerebrospinal fluid–related tissue damage in multiple sclerosis patients with iron rim lesions by Matthias Wittayer, Claudia E Weber, Maximilian Kittel, Michael Platten, Lucas Schirmer, Hayrettin Tumani, Achim Gass and Philipp Eisele in Multiple Sclerosis Journal


Furthermore, we conducted molecular detection of orthohantavirus in host animals. The total orthohantavirus infection rate of rodents was 2.72% (9/331); the specific infection rate of specific animal species was 6.10% (5/82) for the Apodemus chevrieri, 100% (1/1) for the Rattus nitidus, 3.77% (2/53) for the Rattus norvegicus, and 12.50% (1/8) for the Crocidura dracula. In this study, a total of 21 strains of orthohantavirus were detected in patients and rodents. The 12 orthohantavirus strains from patients showed a closer relationship with Seoul orthohantavirus (SEOOV) L0199, DLR2, and GZRn60 strains; the six orthohantavirus strains from Rattus norvegicus and Apodemus chevrieri were closely related to SEOOV GZRn60 strain. One strain (XYRn163) from Rattus norvegicus and one strain (XYR.nitidus97) from Rattus nitidus were closely related to SEOOV DLR2 strain; the orthohantavirus strain from Crocidura dracula was closely related to the Luxi orthohantavirus (LUXV) LX309 strain. In conclusion, patients with HFRS in Xuangyun County of Dali Prefecture are predominantly affected by SEOOV, with multiple genotypes of orthohantavirus in host animals, and, most importantly, these orthohantavirus strains constantly demonstrated zoonotic risk in humans.Hemorrhagic fever with renal syndrome (HFRS) is a zoonotic disease transmitted by several rodent species. We obtained clinical data of HFRS patients from the medical records of the People’s Hospital of Xiangyun County in Dali Prefecture from July 2019 to August 2021. We collected epidemiological data of HFRS patients through interviews and investigated host animals using the night clip or night cage method. We systematically performed epidemiological analyses of patients and host animals. The differences in the presence of rodent activity at home (χ Hantavirus to the family Hantaviridae with four subfamilies, seven genera, and fifty-three species [Mammantavirinae are hosted by bats, moles, shrews, and rodents, and are divided into four genera: Loanvirus, Mobatvirus, Thottimvirus, and Orthohantavirus, among which only Orthohantavirus with rodents as hosts causes human diseases. Thus far, 10 of the total 38 species in the genus Orthohantavirus lead to human infections, causing two diseases: hemorrhagic fever with renal syndrome (HFRS), mainly distributed in Eurasia, and hantavirus pulmonary syndrome (HPS), mainly distributed in the American continent [The latest report of the International Committee on Taxonomy of Viruses (ICTV) elevates the original genus  species ,2. The hontinent ; in contontinent ,6,7,8,9.Apodemus agrarius) and Norway rat (Rattus norvegicus), respectively [R. norvegicus to A. agrarius, and HTNOV was also detected in R. norvegicus in the Shandong Province of China [R. norvegicus and A. agrarius) in Jiangsu Province, and SEOOV was also present in voles in Zhejiang Province, suggesting host spillover and host switching between different genotypes of orthohantavirus. Genetic variation in orthohantavirus is a result of host adaptation to environmental changes, and genetic diversity in orthohantavirus is the result of cross-species transmission, virus-host coevolution, and host migration, which may be an essential driver of orthohantavirus evolution and species formation [Eothenomys miletus) in Luxi County of Yunnan Province (designated LUXV), which now has been assigned as the new orthohantavirus species Luxi orthohantavirus [Genetically diversified orthohantavirus strains isolated from rodents, humans, and other hosts revealed the presence of at least 10 HTNOV subtypes and 6 SEOOV subtypes with significant geographic aggregation in different regions of China . Previouectively . Moreoveof China ,15. In aormation ,16. In ontavirus .Apodemus agrarius, which tends to live in agricultural areas, waterside meadows, forests, and logging [Rattus norvegicus is considered the primary host [Orthohantaviruses can cause two types of human infections: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In China, HFRS is mainly induced by HTNOV and SEOOV . Among t logging ,20; SEOOary host . Europe ary host . Notablyary host . In thisThe relevant materials involving human and animal biomedical research were reviewed by the Medical Ethics Committee of Dali University, and it is considered that the project conforms to medical ethics (ethics certificate number: DLDXLL2020007), and it agreed to collect relevant samples and data.2 mL anticoagulated venous blood was collected from patients. It is necessary to use sterile blood collection needles and correct disinfection during blood collection. Then the collected blood samples were automatically analyzed with a hematology analyzer , including red blood cell count, white blood cell count, platelet count, red blood cell specific volume, average red blood cell volume, average red blood cell hemoglobin content, and other indicators.In this study, qualitative urine protein testing was used, and the dry chemical method was used to detect the pH of urine in the initial test. Sulfosalicylic acid method was used to retest for positive results in the initial test. According to the results of the test strip and the different degrees of turbidity, sedimentation, and coagulation of the reaction liquid, the urine protein content is screened and roughly estimated.Orthohantavirus IgM/IgG antibodies were detected by colloidal gold kit . Laboratory operations were performed according to the instructions, which are as follows: leave the test card for 30 min, balance to room temperature, tear the aluminum foil bag, take out the test card, and lay it flat on the operating table. The control line C in the colloidal gold detection card is mouse anti-human Orthohantavirus polyclonal antibody, T1 is anti-human Orthohantavirus IgM antibody, and T2 is anti-human Orthohantavirus IgG antibody. Use a sampler to add 10 µL of the serum to be tested into the sampling hole, and after the specimen has penetrated the sample pad, add 2 drops (or 100 µL) of the specimen diluent. The test card only shows negative C control line; C control line and T1 IgM antibody line were IgM positive. C control line and T2 IgG antibody line were both IgG positive. If the C control line does not appear, this experiment is invalid. Test results within 15–20 min; more than 30 min is required to determine if the effect is invalid.The HFRS is divided into clinically diagnosed cases (CDC) and laboratory-diagnosed cases (LDC). CDC is defined according to Chinese “WS 278-2008 Diagnostic Criteria for Epidemic Hemorrhagic Fever”. LDC is ascertained with the positive IgM/IgG against orthohantavirus by colloidal gold immunoassay of the patients with HFRS who were admitted to the Department of Infectious Diseases of the 9th Affiliated Hospital of Dali University  from July 2019 to August 2021. The cases were classified into four clinical types: (1) Light: body temperature below 39 °C, mild symptoms of poisoning, no other bleeding phenomena except bleeding points, light kidney damage, no shock and oliguria; (2) Medium: body temperature 39–40 °C, severe symptoms of poisoning, obvious bulbar conjunctival edema, systolic blood pressure less than 90 mmHg or pulse pressure less than 30 mmHg during the course of the disease, with obvious bleeding and oliguria, urine protein (+++); (3) Severe: body temperature > 40 °C, severe poisoning symptoms and exudation signs, toxic psychiatric symptoms, and shock, skin ecchymosis and luminal bleeding, shock and kidney damage severe, oliguria, lasting less than 5 days or anuria within 2 days; (4) Critical disease: one of the conditions in which refractory shock, bleeding from vital organs, oliguria for more than 5 days or anuria for more than 2 days, heart failure, pulmonary edema, cerebral edema, cerebral hemorrhage, or cerebral herniation occurs on the basis of severe secondary infection.The whole blood and sera of patients diagnosed with HFRS from July 2019 to August 2021 were collected in the 9th Affiliated Hospital of Dali University. Whole blood samples were collected using vacuum blood collection tubes without anticoagulant substances. The blood samples were centrifuged, the upper serum was stored in cryopreservation tubes, and the liquid nitrogen preservation was returned to the laboratory and stored in a refrigerator at −80 °C for examination.Clinical data of the cases included general information about the patients, i.e., name, gender, age, ethnicity, occupation, and home address (current address); onset of illness, including date of onset, date of admission, admission diagnosis, date of discharge, discharge diagnosis, and clinical outcome; and clinical signs of the patients on admission and laboratory test results. According to the case investigation data in the “Surveillance Program of Hemorrhagic Fever with Renal Syndrome” of the Chinese Center for Diseases Control and Prevention, the epidemiological investigation was conducted on HFRS patients.From October 2020 to August 2021, the host animal surveys were conducted in nine residential areas and farming fields in Xiangyun County of Dali Prefecture, Yunnan Province. A host animal survey was performed by the night clip or night cage methods, using rat cage or rat clip as tools and homemade fried ham sausage as bait. Five rat cages were placed in each villager’s house, and a total of 100 rat cages were placed every day; rat traps were placed at five-meter intervals on the cultivated land. After the rodents captured at the scene were numbered, classified, and identified by morphology, two copies of liver and lung tissues were dissected, placed in cryopreservation tubes, stored with liquid nitrogen, transported in liquid nitrogen tanks, and returned to the laboratory for orthohantavirus detection.Orthohantavirus nucleic acids were extracted from host animal liver and lung tissues as well as from patient blood samples using the QIAamp RNA kit  according to the manufacturer’s instructions. The viral RNA is stored in a −80 °C freezer until use. RT-PCR amplification of the L-gene fragment of orthohantavirus was performed on the extracted RNA with the orthohantavirus universal primer: HAN-L-F1: 5′-ATGTAYGTBAGTGCWGATGC-3′, HAN-L-R1: 5′-AACCADTCWGTYCCRTCAT-C-3′; HAN-L-F2: 5′-TGCWGATGCHACIAARTGGTC-3′, and HAN-L-R2: 5′-GCRTCRTCWGARTGRTGDGCAA-3′ , using tNucleic acids extracted from the liver and lung tissues of host animals were taken. The cytochrome b (Cyt b) gene was amplified using universal primers MamCybF: 5′-ATGATATGAAAAACCATCGTTG-3′ and MamCybR: 5′-TTTCCNTTTCTGGTTTACAAGAC-3′ as described previously ,26. The https://www.r-project.org/ (accessed on 8 March 2023)), select the corresponding operating system ), download R Integrated Development Environment (IDE) RStudio. Modify the heat map information using parameters: ”color”, “columnme”, “rownanme”, “cellwidth”, “cellheight”, “cluster_row/col”, “labels_row/col”, etc. included in the package “pheatmap”. The phylogenetic analysis of representative orthohantavirus strains was performed using Molecular Evolutionary Genetics Analysis (MEGA) software version 11 by the maximum-likelihood (ML) method and the corresponding optimal model T92 + G + I with the bootstrap value set as 1000 replicates. Orthohantavirus reference sequences used for the comparative analysis: MN022852.1 (Seoul virus), MN022865.1 (Seoul virus), AF288297.1 (Seoul virus), KM948595.1 (Seoul virus), KX079474.1 (Seoul virus), KF977219.1 (Seoul virus), HQ992814.1 (Seoul virus), AF336826.1 (Hantaan virus), KT885047.1 (Hantaan virus), JN831949.1 , HE801635.1 , MN639746.1 , HQ404253.1 (Luxi virus), MK386154.1 (Tula virus), MT514299.1 (Tula virus), MK386155.1 (Tula virus), and DQ825770.1 .The positive products from orthohantavirus-specific RT-PCR were purified by gel cutting using MinElute Gel Extraction Kit  and sent to the company  for sequencing with both forward and reverse primers. The sequencing results were assembled and analyzed using the Seqman and EditSeq models in DNASTAR Lasergene Bioinformatics Software version 17, and then the orthohantavirus sequences were analyzed for identity comparison using the Megalign model in the software. Afterward, the newly detected orthohantavirus sequences in this study were compared with the existing orthohantavirus sequences in the GenBank database, and the sequence alignment was performed using Clustal X. Sequence similarity heatmaps are plotted using R and its package. On the official website of R  and clinically diagnosed cases accounted for 19.47% (37/190). The age of onset ranged from 7 to 85 years, with a mean age of 48.5 ± 16.7 years, mainly in the age group of 40–59 years, and the occupation was mainly farmers. Duration of hospitalization (in one year): 95 cases of HFRS were reported from February 2020 to January 2021, including 24.21% (23/95) in spring (January to March); 37.89% (36/95) in summer (April to June); 22.11% (21/95) in autumn (July to September); and 18.95% (18/95) in winter (October to December). Cases were reported throughout the year, and most patients with HFRS were admitted in spring and summer. Distribution of cases in Xiangyun County of Dali Prefecture: Xiangcheng Town accounted for 23.68% (45/190), Yunnan Yi Town accounted for 15.26% (29/190), Xiazhuang Town accounted for 14.74% (28/190), Midian Town accounted for 9.47.% (18/190), Shalong Town accounted for 9.47% (18/190), Pupeng Town accounted for 9.47% (18/190), Liuchang Town accounted for 6.32% (12/190), Hedian Town accounted for 4.74% (9/190), Luming Town accounted for 2.11% (4/190), Dongshan Town accounted for 2.11% (4/190), see Rattus tanezumi, 24.77% (82/331) of Apodemus chevrieri, 16.01% (53/331) of Rattus norvegicus, 8.46% (28/331) of Eothenomys miletus, 6.34% (21/331) of Crocidura attenuata, 2.11% (7/331) of Tupaia belangeri, 2.42% (8/331) of Crocidura dracula, 1.81% (6/331) of Mus musculus, 0.60% (2/331) of Rattus niviventer, 0.60% (2/331) of Eothenomys eleusis, 0.60% (2/331) of Mus pahari, and 0.30% (1/331) of Rattus nitidus. The dominant species in residential areas were Rattus tanezumi and Rattus norvegicus, and the dominant species in field cultivation areas was Apodemus chevrieri. After orthohantavirus nucleic acid detection, the total orthohantavirus infection rate in host animals was 2.72% (9/331), among which 6.10% (5/82) were Apodemus chevrieri, 3.77% (2/53) were Rattus norvegicus, 12.50% (1/8) were Crocidura dracula, and 100% (1/1) were Rattus nitidus , 73.7% (140/190), and 67.89% (129/190), respectively, which were the most common clinical manifestations among hospitalized patients. HFRS has the specific clinical manifestation of “three reds and three pains” , among which one of three reds accounted for 7.89% (15/190), two of them accounted for 14.21% (27/190), and all three reds accounted for 13.68% (26/190). None of the three reds accounted for 64.2% (122/190). One, two, three, and none in three pains were 36.84% (70/190), 21.58% (41/190), 9.47% (18/190), and 32.11% (61/190), respectively.2 = 8.71, p = 0.033 < 0.05), fever , neck redness , generalized pain , abdominal pain , hypotension,  and shock , in the four clinical subtypes , 37.37% (71/190), 14.21% (27/190), and 12.11% (23/190), respectively. There were statistically significant differences for the clinical manifestations, fatigue , and 53.16% (101/190) of cases showed normal white blood cell counts. Decreased platelet count cases accounted for 65.79% (125/190), and positive urine protein cases accounted for 68.95% (131/190).p = 1.000 > 0.05).The orthohantavirus RNA was detected in 6 of 98 (6.12%) cases with anti-orthohantavirus IgM (+) and IgG (+) patients; 3 of 49 (6.12%) cases with anti-orthohantavirus IgM (+) and IgG (−) patients; 2 of 28 (7.14%) cases with anti-orthohantavirus IgM (−) and IgG (+) patients; and 1 of 15 (6.67%) cases with anti-orthohantavirus IgM (−) and IgG (−) patients. There was no statistically significant difference in the detection rate of orthohantavirus RNA among these four groups , the presence of rodent-proof equipment in food , and the presence of rodents or rodent excrement in the workplace  , medium cases were 38.89% (42/108), severe cases were 9.26% (10/108), and critical cases were 10.19% (11/108). There were statistically significant differences between the four clinical subtypes with the presence of rodent activity at home (χ < 0.05) .The 21 orthohantavirus-positive samples  were sequenced after RT-PCR amplification. The L fragment size was 400–440 bp, among which primers were designed for the specimen of patient number XYPatient5, and the final sequence of spliced L fragment size was 2089 bp.Apodemus chevrieri in this study shared highest sequence identity, ranging from 94.92% to 99.49%, to the SEOOV GZRn60 strain; orthohantavirus strains of XYPatient35 (OP326509), XYPatient53 (OP326511) from HFRS cases, XYRn5 (OP326533) and XYRn163 (OP326531) from Rattus norvegicus, and XYR.nitidus97 (OP326530) from Rattus nitidus had highest sequence identity, ranging from 89.00% to 97.46%, to the SEOOV DLR2 strain; orthohantavirus strains of XYPatient7 (OP326503), XYPatient16 (OP326506), XYPatient13 (OP326505), XYPatient62 (OP326512), and XYPatient36 (OP326510) from HFRS cases shared highest sequence identity (58.27–88.47%)to the SEOOV L0199 strain , XYPatient17 (OP326507), XYPatient75 (OP326513) XYPatient10 (OP326504), XYPatient22 (OP326508), from HFRS cases and XYAc15 (OP326534), XYAc16 (OP326535), XYAc17 (OP326536), XYAc19 (OP326537) XYAc20 (OP326538) from 9 strain .Crocidura dracula) and LUXV LX309 strain from the red-backed vole (Eothenomys miletus) was (82.35%) [The nucleotide sequence identity between XYCd93 (OP326532) from the White-tipped shrew ((82.35%) . At the Of the 12 orthohantavirus strains detected in the HFRS patient samples, five orthohantavirus strains  clustered in single clade and were closely related to SEOOV GZRn60 strain, two orthohantavirus strains (XYPatient35 and XYPatient53) were closely related to SEOOV DLR2 strain, and the other five orthohantavirus strains  clustered in a separate clade.Rattus norvegicus and five strains  from Apodemus chevrieri were closely related to SEOOV GZRn60 strain. One strain (XYRn163) from Rattus norvegicus and one strain (XYR.nitidus97) from Rattus nitidus were closely related to SEOOV DLR2 strain. Notably, the orthohantavirus strain (XYCd93) from Crocidura dracula and the LU309 strain from red-backed vole (Eothenomys miletus) showed a closer evolutionary relationship and clustered together to form a new branch  from w branch .Among the clinical manifestations of HFRS in this study, fatigue, fever, and redneck were more common in mild and medium cases. For severe and critical patients accompanied by some basic diseases or delayed treatment, which will lead to aggravation of the disease, the patient is more prone to systemic pain, diarrhea, abdominal pain, hypotension, and shock in severe cases. In addition, HFRS has specific “three reds and three pains” in the clinic; 122 of 190 cases did not have “three reds” of clinical manifestations, and 61 cases did not have “three pains” of clinical manifestations. Therefore, the specific clinical manifestations of HFRS for diagnosis were not comprehensive, meaning that it is easy to miss the diagnosis and that further laboratory testing is warranted. Clinical laboratory tests are usually considered the leading indicators, such as increased white blood cell count, decreased platelet count, and positive urine protein in patients with HFRS. In this survey, 53.15% of patients with HFRS had normal white blood cell counts; therefore, white blood cell counts, platelet counts, and urine protein do not directly reflect whether a patient has HFRS, and the accuracy of laboratory indicators for HFRS can be affected by the presence of certain underlying diseases in patients. Studies have shown that the levels of procalcitonin (PCT) and C-reactive protein (CRP) in HFRS patients are significantly increased and are positively associated with the clinical severity of HFRS ,28. TherOur present study indicated that fewer people in the natural foci had been vaccinated against HFRS in the past. For residents in residential areas, rodent control facilities with rodent-infested food at home are inadequate, or they often work in wild farms or fields, and have direct or indirect contact with rodent excreta or rodent-contaminated food that are more likely to increase the risk of HFRS. Due to the limited sample size of HFRS patients, there may be some mild patients not hospitalized and treated, or patients choose other hospitals far away. The investigation of symptomatic cases of 108 hospitalized HFRS patients could not accurately conclude the overall epidemiology of HFRS in Xiangyun County of Dali Prefecture, and further investigation is required.HFRS in China is mainly caused by HTNOV, SEOOV, and PUUOV . Yunnan Rattus norvegicus, and the dominant species in the field is Apodemus chevrieri. XYCd93 is an orthohantavirus strain isolated from a Crocidura dracula captured in Dasongping Village, Midian Town, and its BLASTn results are most homologous to the PUUOV Osnabruck M34 strain in Germany [Rattus norvegicus carrying SEOOV. Humans have higher chances of contacting Rattus norvegicus and are more likely to be infected by SEOOV-causing HFRS.Among the orthohantavirus host animals captured from Xiangyun County of Dali Prefecture, the dominant species of rodents indoors is  Germany . The nucThe incidence of HFRS is influenced by various factors, including socioeconomic conditions, climatic factors, and rodent-human contact . For theIn conclusion, this study on the clinical and molecular epidemiology of HFRS in Dali Prefecture of Yunnan Province provides important scientific insights into the disease severity, environment correlation, and prevalence of orthohantavirus infection. Furthermore, we determined the infection rate and genetic diversity of orthohantaviruses in humans and various animal hosts. Our findings will contribute to the understanding of the interplay of orthohantavirus, humans, and animal hosts, highlighting the need for robust surveillance, improved sanitation conditions, and public health awareness to minimize the risk of zoonotic transmission of orthohantavirus in the HFRS natural foci area.


In recent years, advances in genetics and the integration of clinical-grade next-generation sequencing (NGS) assays into patient care have facilitated broader recognition of hereditary hematopoietic malignancy (HHM) among clinicians, in addition to the identification and characterization of novel HHM syndromes. Studies on genetic risk distribution within affected families and unique considerations of HHM biology represent exciting areas of translational research. More recently, data are now emerging pertaining to unique aspects of clinical management of malignancies arising in the context of pathogenic germline mutations, with particular emphasis on chemotherapy responsiveness. In this article, we explore considerations surrounding allogeneic transplantation in the context of HHMs. We review pre- and post-transplant patient implications, including genetic testing donor selection and donor-derived malignancies. Additionally, we consider the limited data that exist regarding the use of transplantation in HHMs and safeguards that might be pursued to mitigate transplant-related toxicities. RUNX1 Familial Platelet Disorder with Associated Myeloid Malignancies, RUNX1-FPDMM) was the first HHM to have its molecular basis elucidated, driven by heterozygous pathogenic RUNX1 variants  or azacitidine (n=11), response rates were 100% and 73%, respectively, and a median survival of 5.2 years was observed  during which patients with cytopenias were monitored before AML onset. When diagnosed, 69% of patients had acquired a second hit (somatic) DDX41 variant and most cases had a normal karyotype (75%) with bone marrow hypocellularity (93%)  may represent familial variants  and single CEBPA mutations (29%) , sometimes referred to as Thrombocytopenia 2, was first described in 1985 and molecularly characterized in 1999 as the result of pathogenic variants in the transcription factor RUNX1 . A wide  be ~35% , 22. RUNe status . Short te status –26. The e status , prematue status , 29. Bece status Figure 1e status .Unlike familial CEBPA-mutated AML, wherein AML development appears to be the principal clinical feature, other inherited cancer syndromes associated with HHMs and hematopoietic phenotypes also confer solid tumor risk. As mentioned above, the first hereditary cancer syndrome described by Li and Fraumeni detailed kindreds with sarcoma, leukemia, and cancers of the brain, breast, and adrenal gland . HypodipDDX41 as HHM-associated genes, 6/33 (18%) individuals with blood cancers had received prior solid tumor diagnoses, with prostate cancer being the most common  /33, 9%) . Table As mentioned above, proper HHM evaluation, ideally concurrent with initial workup of hematologic malignancy, provides both diagnostic and clinically actionable information that may prove highly relevant to the patient’s course. Expert protocols and recommendations guiding HHM evaluation have been described previously , 53. AltDonor cell leukemia (DCL) is a catastrophic yet often avoidable consequence of allogeneic transplant whereby pathogenic donor clones expand in the recipient and contribute to apparent “relapses.” This phenomenon may more accurately be described as donor-derived hematopoietic malignancy since the initial neoplasm is not truly relapsing from a dormant state, but rather, a new hematologic cancer is propagating. Prior to advent of commercially available NGS, cytogenetic testing was used to identify donor cell leukemias (DCL) as a complication of allogeneic HSCT . Early sSeveral mechanisms have been suspected to enable leukemic transformation of donor-derived cells , 69. InsThere are also ethical challenges arising from donor DNA sequencing after allogeneic HSCT. These considerations are of importance in cases of DCL, where donor germline variants can be detected and potentially carry significant clinical implications for the donor , 75. Morde novo disease often because the underlying causative germline variant is not discovered until after treatment has been initiated  acute graft-versus-host disease (GVHD) (38%) and moderate to severe chronic GVHD (33%) compared to those without any identified P/LP germline variant   . GVHD pranalysis . Though ansplant , 83.As mentioned above, GATA2 deficiency, stemming from pathogenic variants in GATA2, is comprised of a syndrome characterized by life-threatening opportunistic infections with non-tuberculous mycobacteria (NTM), fungal infections, or human papillomavirus (HPV) infections and a propensity to progress to MDS, CMML, and AML –19. BecaAlthough HCT is an effective means at mitigating, or even reversing clinical phenotypes associated of GATA2 deficiency, the optimal timing of HCT, and choice of conditioning regimen, donor sources, and GVHD prophylaxis remain areas of active investigation. With the potential to mitigate or avoid future syndromic disease manifestations, preemptive HCT in patients with GATA2 deficiency represents an attractive strategy. From 2013 to 2020, 59 patients underwent HCT with busulfan-based conditioning at the National Institutes of Health. Patients were eligible for transplant if they had at least one episode of a life-threatening opportunistic infection, with almost half of patients having received a transplant due to an established diagnosis of MDS or the presence of cytogenetic changes in the absence of overt morphologic disease, particularly monosomy 7. Overall survival (OS) and event-free survival (EFS) at 4 years were 85.1% and 82.1% respectively . It is iCEBPA-mutated AML. This syndrome is associated with high penetrance and a young age of AML onset without development of preceding myelodysplasia. Patients typically experience complete response to induction chemotherapy with standard regimens , but non-relapse mortality remains a serious obstacle towards more uniform deployment of potentially curative strategies to replace deleterious stem cell populations. Furthermore, as gene therapy approaches advance, less invasive means for correcting pathogenic variants may become feasible.GR conceived the article. TO’C, RS, RM-M, and GR researched and wrote the manuscript. All authors edited the manuscript. GR created figures included in publication. All authors contributed to the article and approved the submitted version.


ObjectiveThis cross-sectional survey aimed to investigate the utilization, challenges, and attitudes of chiropractors in Hong Kong towards laboratory testing and radiological imaging.Method 1, 2023. The target population for the survey was registered chiropractors in Hong Kong, which has a total of 325 practitioners. A total of 151 chiropractors participated in the survey, resulting in a response rate of 46.5%. The respondents provided information on their demographics, years of experience, practice settings, awareness of their right to request diagnostic tests, utilization of laboratory testing and imaging, and attitudes towards evidence-based practice. The survey data were analyzed using descriptive statistics.An online survey was conducted between May 1, 2023, and JuneResultThe survey findings revealed that a significant proportion of chiropractors in Hong Kong utilized laboratory testing and radiological imaging in their practice. Most respondents reported using laboratory testing to diagnose medical conditions and monitor existing conditions. Imaging is commonly used for diagnosing medical conditions and monitoring disease progression. However, a notable proportion of chiropractors reported facing rejection of their tests and imaging requests, which limited their ability to provide optimal care to their patients. The identified challenges included high rejection rates for specific imaging requests, leading to patient frustration, increased costs, and delayed diagnosis. Nevertheless, chiropractors in Hong Kong showed a strong belief in evidence-based practice and demonstrated a willingness to search for literature and incorporate it into their daily practice.By addressing this issue, our aim was to gain valuable insights into the status of laboratory testing and radiological imaging among chiropractors in Hong Kong. The findings of this study underscore the necessity for collaborative efforts among chiropractors, healthcare professionals, healthcare facilities, policymakers, and insurance companies to effectively address the challenges identified and improve patient care outcomes. Specifically, enhancing access to diagnostic tests and working towards reducing rejection rates will play a pivotal role in supporting chiropractors' role as primary healthcare providers in Hong Kong. Chiropractors are primary healthcare providers specializing in the diagnosis and management of neuromusculoskeletal complaints . Over thIn 1993, chiropractors in Hong Kong attained legal recognition and initiated the registration process under the Hong Kong Council . This reIn recent years, chiropractors have become integral members of Hong Kong’s primary healthcare system. They provide effective chiropractic interventions and offer holistic education and counseling to the general public, positively affecting the overall health of the population .Little is known about the use of diagnostic testing among chiropractors in Hong Kong. This study aimed to describe the utilization and attitudes towards laboratory and radiological testing among chiropractors in Hong Kong, as well as examine their attitudes toward EBP. By gaining insights into these aspects, we can further enhance the delivery of chiropractic care in this region.DesignThis study employed a cross-sectional survey design to investigate the utilization of laboratory testing, radiological imaging, and attitudes toward evidence-based practice (EBP) among chiropractors in Hong Kong. The study was approved by the ethics committee of the Chiropractic Doctors' Association of Hong Kong , and written consent was waived as consent was implied by the return of the completed survey. To ensure respondent anonymity, names, and other identifying information were not collected.SettingThe target population of this study consisted of practicing registered chiropractors in Hong Kong. Both full-time and part-time chiropractors were recruited to participate in the survey.InstrumentA comprehensive survey questionnaire was developed to gather data on the utilization of laboratory testing, radiological imaging, and attitudes towards evidence-based practice (EBP), drawing references from previous studies , 6. The Data sourcesThe data for this study were obtained through the distribution of a survey using the Google Forms platform. The survey was distributed to all registered chiropractors in Hong Kong through their respective chiropractic associations. Official invitations to participate were sent via email, utilizing existing communication channels within the chiropractic associations. The completed survey forms served as the sole source of data for this study. The data were extracted from the survey responses and input into a spreadsheet format for further analysis.BiasTo ensure the survey questions' clarity and estimate the time needed to complete the questionnaire, a pilot test was conducted with a small sample of chiropractors. All participants completed the survey within 10 minutes, and the pilot test revealed that the questions were clear and comprehensible. No adjustments were necessary.Sample sizeThe sample size was determined using a rule-of-thumb estimation, indicating that each survey item required five respondents. Thus, 115 respondents were required for the study. This sample size was considered feasible given that 325 chiropractors were registered under the Hong Kong Chiropractors Council . A surveAnalysisThe data extracted from the questionnaires were analyzed using Microsoft Excel. Frequency tables and descriptive statistics were employed to examine the current utilization of laboratory testing, radiological imaging, and attitudes toward EBP among chiropractors in Hong Kong.The survey was administered online through a Google Forms link between May 1 and June 1, 2023. A total of 151 responses were received, resulting in a 100% completion rate.RespondentsTable The study included a relatively balanced representation of both male and female participants. Males accounted for 53% of the respondents, whereas females accounted for 47%.The participants exhibited varying levels of experience in chiropractic practice. The largest group consisted of respondents with more than 10 years of experience, comprising 40.4% of the sample. The next most prevalent group had one to five years of experience, accounting for 27.2% of participants. Respondents with five to ten years and one year of experience represented 17.9% and 14.6% of the samples, respectively.The chiropractors in this study were engaged in different types of practice settings. The majority of participants (54.3%) were affiliated with group practices, while solo practitioners accounted for 45.7% of the respondents.A significant majority of respondents (95.4%) reported awareness of their right to request diagnostic imaging and laboratory examinations, while only a small percentage (4.6%) reported being unaware of this right.Laboratory investigationA significant majority of the respondents  reported requesting blood tests. Additionally, a high proportion of them  expressed confidence in their interpretation of these results.Figure The majority of respondents (92.7%) reported utilizing laboratory testing for diagnosing medical conditions. Furthermore, 81.5% of the participants reported utilizing laboratory testing to monitor their existing conditions, indicating its role in ongoing patient care. Laboratory testing was also frequently utilized for routine health checkups, as reported by 76.8% of respondents. Additionally, a significant proportion of chiropractors reported utilizing laboratory testing for screening purposes. This included both general screening (60.3%) and specific screenings, such as allergy and autoimmune testing (68.9%) and infection detection (62.9%). A noteworthy finding from this study was that a significant proportion of respondents (70.9%) reported that their blood test requests were rejected by medical laboratories. This study explored the situations experienced by chiropractors when test requests were rejected. The most common response by 81.2% of the respondents was to refer the patient to another health professional for co-management. Another common response by 71.1% of the respondents was to refer the patient to a specialist for a test referral, while 48.3% reported continuing care without undergoing the requested tests. Additionally, 35.6% of the respondents mentioned that patients gave up on care when their test requests were rejected. Figure ImagingAll the surveyed chiropractors reported having requested imaging for their patients and expressed confidence in interpreting the results.The study results revealed the purposes for which chiropractors utilized imaging in their clinical practice. Figure These findings demonstrated the diverse applications of imaging in chiropractic practice. Most of the respondents (92.7%) reported utilizing imaging to diagnose medical conditions and monitor the progression of diseases or conditions (81.5%). Additionally, a substantial proportion of chiropractors utilized imaging to guide treatment planning (79.3%), evaluate the effectiveness of treatment (77.9%), assess injuries (79.3%), and investigate unexplained symptoms (77.9%). Screening for certain conditions (67.5%) and evaluation of organ function (43.9%) were also identified as the purposes for which imaging was performed.A notable finding from this study was that a substantial proportion of respondents (80.1%) reported experiencing rejection of their imaging requests. The rejection rates of specific imaging requests were examined in detail. Radiography of the spine and the extremities revealed rejection rates of 23.3% and 82.9%, respectively.MRI imaging requests also faced rejection, with MRIs of the spine and the extremities demonstrating a rejection rate of 31.8% and 86.8%, respectively. These findings indicate the presence of notable obstacles in obtaining MRI images for both spinal and extremity evaluations.Brain MRI demonstrated a rejection rate of 77.5%, indicating the challenges in obtaining brain MRI scans for chiropractic purposes. PET, CT, ultrasonography, and mammography techniques demonstrated a rejection rate of 76%, 75.2%, 72.9%, and 69.8%, respectively. Figure This study demonstrated various situations when the test requests of the respondents were rejected by the imaging center. The findings indicate that a majority of chiropractors (83.6%) referred the patients to another health professional for co-management, whereas 72.6% referred the patients to a general practitioner for test referrals. Additionally, 56.2% continued care without the requested tests and 40.4% reported patients not returning for further care. Figure Practitioner attitudeAll respondents expressed a strong belief in the importance of having access to medical laboratory investigations and radiological imaging in their practice. They recognized the value of these diagnostic tools in enhancing patient care and treatment outcomes. However, when their requests for tests were rejected, they expressed significant concerns about their ability to provide optimal care to their patients. The high prevalence of patient frustration (90.5%), increased costs for patients (87.8%), and delayed diagnosis (91.9%) were acknowledged as key concerns resulting from rejected test requests. Additionally, the respondents also expressed concerns regarding inadequate patient care (85.1%) and the exacerbation of the patients' symptoms (75%), due to the unavailability of the requested tests. Notably, a small proportion of respondents (6.8%) reported no effect from rejected test requests. However, 60.8% of respondents reported that the rejection of test requests might lead to third-party complaints from insurance companies.Attitude towards evidence-based practiceTable A substantial number of respondents (81.5%) reported consistent incorporation of EBP into their daily chiropractic care practices. Furthermore, a significant proportion of respondents (78.5%) indicated their commitment to regularly updating their knowledge and skills regarding evidence-based chiropractic care practices.The present study emphasized the significance of diagnostic testing in the practice of chiropractors in Hong Kong, demonstrating their awareness of its importance in evaluating and addressing musculoskeletal complaints in patients . By incoThe survey findings indicate a high level of confidence among Hong Kong chiropractors in interpreting laboratory testing and radiological imaging results, reflecting their education and training that equip them with the necessary skills and knowledge to analyze diagnostic information , 9,10,11In practice, chiropractors encounter severe pathologies infrequently . A speciMoreover, the study indicated that chiropractors utilize laboratory testing and radiological imaging for diagnosing and monitoring medical conditions and performing routine checkups and screenings, thereby underscoring their expanding role in preventive care ,9.With the integration of diagnostic testing into their practice, chiropractors are assuming the role of primary healthcare providers, contributing to the overall management of patients' health. This expanded role highlights the importance of adopting a multidisciplinary approach to healthcare and fostering collaboration between chiropractors and other healthcare professionals to deliver comprehensive and integrated care ,10.However, chiropractors in Hong Kong face notable challenges due to the high rejection rate of test requests. Consequently, chiropractors often refer patients to other healthcare professionals for testing or co-management purposes. This limitation significantly hampers their ability to provide optimal care, leading to delayed diagnosis, increased patient frustration, and the potential for inadequate treatment.Laboratory testing and radiological imaging have become indispensable components of chiropractic practice. Addressing the barriers associated with communication and collaboration between chiropractors and medical laboratories or imaging centers is crucial to ensuring that they have the necessary tools to provide comprehensive and evidence-based care to their patients, ultimately improving overall healthcare outcomes. Overcoming these barriers is of paramount importance for optimizing the delivery of chiropractic services.Nevertheless, the survey findings demonstrated a positive attitude towards EBP among chiropractors in Hong Kong. This positive attitude aligns with that of other healthcare professionals, such as medical doctors in Hong Kong and other countries , 17-20. Moreover, a significant proportion of the respondents (78.8%) reported regularly updating their knowledge and skills in EBPs, indicating their commitment to ongoing professional development and staying updated with the latest advancements in chiropractic care. These findings underscore the importance of providing continued support and resources to facilitate EBPs among chiropractors. By fostering an environment that promotes EBP, chiropractors can enhance the quality of care for patients and contribute to advancements in the field.This study had some limitations. The online survey method and self-reported data would have introduced bias, and the study only adopted descriptive analysis, affecting the representativeness and generalizability of the findings. Also, this study did not explore the reasons behind test rejection or assess the level of knowledge regarding evidence-based practice among chiropractors. Therefore, further studies are required to address those limitations, explore the reasons behind test rejection, and assess the level of knowledge regarding EBP among chiropractors in Hong Kong.The present study provides valuable insights into the experiences, challenges, and attitudes of chiropractors in Hong Kong regarding medical laboratory investigations, radiological imaging, and EBP. These findings underscore the integration of laboratory testing and radiological imaging into chiropractic practice, indicating the potential for chiropractors to play a role as primary healthcare providers. However, the high rejection rate of test requests presents a significant obstacle for chiropractors in delivering comprehensive care to their patients. There is a need to improve access to diagnostic testing for chiropractors to optimize patient care. Collaboration between chiropractors and other health professionals is important for addressing barriers to comprehensive patient care. Furthermore, the positive attitude among chiropractors towards EBP provides a clear direction for enhancing the quality of care provided in this field; therefore, education on evidence-based practice should be integrated into chiropractic programs and continuing education.


Neurosarcodosis is one of the most frequent differential diagnoses of multiple sclerosis (MS) and requires central nervous system (CNS) biopsy to establish definite diagnosis according to the latest consensus diagnostic criteria. We here analyzed diagnostic values of basic cerebrospinal fluid (CSF) parameters to distinguish neurosarcoidosis from MS without CNS biopsy.alb), intrathecal production of immunoglobulins including oligoclonal bands (OCB), MRZ reaction, defined as a polyspecific intrathecal production of IgG reactive against ≥2 of 3 the viruses measles (M), rubella (R), and zoster (Z) virus, and CSF lactate levels. Additional inflammatory biomarkers in serum and/or CSF such as neopterin, soluble interleukin-2 receptor (sIL-2R) and C-reactive protein (CRP) were assessed.We retrospectively assessed clinical, radiological and laboratory data of 27 patients with neurosarcoidosis treated at our center and compared following CSF parameters with those of 138 patients with relapsing-remitting MS: CSF white cell count (WCC), CSF/serum albumin quotient  of single CSF parameters indicating neurosarcoidosis was highest, if (a) CSF WCC was >30/μl (PLR 7.2), (b) Qalb was >10 × 10−3 (PLR 66.4), (c) CSF-specific OCB were absent (PLR 11.5), (d) CSF lactate was elevated (PLR 23.0) or (e) sIL-2R was elevated (PLR>8.0). The combination of (a) one of three following basic CSF parameters, i.e., (a.1.) CSF WCC >30/ul, or (a.2.) QAlb >10 × 10−3, or (a.3.) absence of CSF-specific OCB, and (b) absence of positive MRZ reaction showed the best diagnostic accuracy .There was no significant difference in the frequency of CSF pleocytosis, but a CSF WCC > 30/μl was more frequent in patients with neurosarcoidosis. Compared to MS, patients with neurosarcoidosis showed more frequently an increased QAlb elevation and absence of intrathecal IgG synthesis as useful rule-in parameters and positive MRZ reaction as a rule-out parameter for neurosarcoidosis.Combined evaluation of basic CSF parameters and MRZ reaction is powerful in differentiating neurosarcoidosis from MS, with moderate to severe pleocytosis and Q Sarcoidosis is an inflammatory granulomatous disease, affecting the central nervous system (CNS) in 5–15% of cases, then referred to as neurosarcoidosis. The prevalence of neurosarcoidosis ~3–4/100'000) is much lower than multiple sclerosis (MS), which is by far the most frequent inflammatory CNS disease  and the most frequently evaluated differential diagnosis –10. Whil0'000 is Recent studies indicate absence of CSF-specific oligoclonal bands (OCB) as a good CSF marker to distinguish neurosarcoidosis from MS, which is the most frequently evaluated differential diagnosis . Howevern = 4), probable (n = 19), or definite (n = 4) neurosarcoidosis according to consensus diagnostic criteria (Alb), IgG, IgA and IgM CSF/serum ratios , frequency of intrathecal synthesis of IgG, IgA, and IgM according to Reiber's diagram , frequency of CSF-specific OCB, intrathecal production of IgG reactive against measles, rubella and varicella zoster virus (MRZ reaction), CSF lactate levels and CSF/serum glucose ratio. 25/27 (92.6%) neurosarcoidosis patients were untreated at the time of lumbar puncture, 1 patient with possible neursarcoidosis was under treatment with mycophenolate mofetil, 1 patient with definite neurosarcoidosis was under treatment with methotrexate. Informed consent was obtained from all patients. Since data of all patients were anonymized for this study, the local Cantonal Ethics Committee stated that the research project does not fall within the scope of the Human Reseach Act (HRA) and therefore, an authorization from the ethics committee is not required (BASEC Nr. Req-2022-01134).We retrospectively analyzed clinical, radiological and laboratory data from 27 patients with possible , serum interleukin 6 , serum interleukin-1 beta , serum angiotensin-converting enzyme , serum t et al. ] and C-rt et al. ] and theA CSF white cell count >4/μl was classified as “increased,” representing pleocytosis. WCC was further grouped into (i) 0-−4/μl (no pleocytosis), (ii) 5–30/μl (mild pleocytosis) and (iii) >30/μl (moderate pleocytosis) as an assessment of pleocytosis severity. CSF lactate was considered pathologic if outside of range 1.7–2.6 mmol/l. CSF/blood ratio of glucose was calculated, a ratio <0.5 was considered pathologic.Alb), with QAlb = CSF albumin concentration / serum albumin concentration. The upper reference limit of QAlb was calculated as QAlb = [4+(age/15)] × 10−3 according to Reiber et al.  and respective CSF/serum ratios were calculated. Function of the blood-CSF barrier (BCSFB) was assessed using CSF/serum albumin quotient  were, respectively calculated according to Reiber  on agarose gels and immunoblotting using IgG-specific antibodies in a semi-automated approach . OCB patterns were evaluated according to international consensus criteria , rubella- (R) and varicella zoster (Z) virus-specific IgG antibodies were measured in paired CSF and serum samples with commercial ELISA kits and fully automated ELISA processing. Serum  and CSF  were analyzed using commercial ELISA kits  from Euroimmun AG (Switzerland) and fully automated ELISA processing .spec) was calculated as previously described by Reiber (spec was assessed as CAIspec = Qspec/QIgG (if QLim (IgG) > QIgG), or CAIspec = Qspec/QLim (IgG), if QLim (IgG) < QIgG. The respective parameters were calculated as follows: Qspec = antigen-specific IgGCSF [AU]/antigen-specific IgGserum [AU]; QIgG = total IgGCSF [mg/l]/total IgGserum [mg/l]; QLim (IgG) = 0.93 × (−6)0.5 – 1.7 × 10−3; QLim (IgG) refers to the upper discrimination line of the hyperbolic reference range for the blood-derived IgG in CSF as absence of intrathecal IgG synthesis. CAIspec ≥1.5 indicated intrathecal synthesis of virus-specific antibodies. MRZ reaction (MRZR) was interpreted as positive, if polyclonal intrathecal production of antibodies against ≥2 of the 3 antigens measles (M)-, rubella (R)- and varicella zoster (Z) virus lysate, was detectable  and negative likelihood ratio (NLR) with 95%-confidence intervals (95%-CI) of single or combined basic CSF/serum parameters were analyzed to assess their value in distinguishing neurosarcoidosis from MS.Differences in age and disease duration were compared with the Kruskal-Wallis test. Differences in frequency of female gender, pleocytosis, elevated QFirst clinical symptoms of neurosarcoidosis patients included optic neuritis (14.8%), sensory and/or motoric dysfunction (22.2%), myelitis (22.2%), extraocular movement impairment (9.1%), gait disturbance (11.1%), cerebellar syndrome (3.7%), vestibular syndrome (9.1%), and meningoencephalitis (11.1%), strongly mimicking MS-like CNS syndromes in 81.5% of cases . 19/27 . 88.9% o2 microglobulin , elevated serum IL-6 , elevated serum ACE  and elevated serum CRP  in descending order, while no patient showed elevated levels of IL-1β   patients, elevated levels of sIL-2R in CSF or serum , elevated serum TNF-α , elevated serum β4, 0.0%) . Elevate4, 0.0%) .p < 0.001) and had a longer disease duration in months from first clinical presentation until lumbar puncture . Furthermore, female sex was more frequent in MS than in neurosarcoidosis  between patients with neurosarcoidosis and patients with MS, but a CSF WCC >30/μl was more frequent in patients with neurosarcoidosis  and a CSF WCC >100/μl was present in neurosarcoidosis patients only   . These f= 0.001)  18, 20,,p = 0.42= 0.001) , 30.Alb indicating BCSFB dysfunction showed a much stronger association with neurosarcoidosis than with MS  . A severe elevation of QAlb >20 × 10−3 was again present in neurosarcoidosis patients only . This finding matches well with the current literature, which reports increased CSF protein levels as alternative measure of BCSFB dysfunction in 45.5–76.4% (Elevated Q< 0.001) . This as.5–76.4%  , whereas no significant difference in intrathecal synthesis of IgA or IgM, respectively, was detected (p = 0.024) and IgA ratio , but not IgM ratio , than MS patients   . The lat< 0.001)  and Kidd< 0.001) ], but in< 0.001)  and in Z< 0.001) ] (Supple< 0.001) .p < 0.001) and pathological CSF/serum glucose ratio   .p < 0.001) (p = 0.004), rubella virus antigens  and varicella zoster virus antigens  were detected between neurosarcoidosis patients and MS patients  . Signifipatients . Not onlpatients . The rubpatients . These rpatients  31, 32), 32p < 0patients   and CSF  as well as elevated serum CRP  showed a strong association with neurosarcoidosis compared to MS  of single inflammatory markers and basic CSF parameters  and the Alb >10 × 10−3 (PLR 66.44 [95%-CI 9.07–486.92]), elevated CSF lactate (PLR 23.00 [95%-CI 5.26-100.6]) and absence of CSF-specific OCB (11.50 [95%-CI 5.58–23.71]) , but low sensitivity  and hence high values of respective NLRs  . Regardi–23.71]) . Lowest Alb >10 × 10−3, or (3) absence of CSF-specific OCB, showed more favorable values for sensitivity (92.6% [95%-CI 82.7–100.0%]), specificity (89.9% [95%-CI 84.8–94.9%]), PLR (9.13 [95%-CI 5.49–15.17]) and NLR (0.082 [95%-CI 0.02–0.31]) to distinguish neurosarcoidosis from MS  absence of CSF-specific OCB) led to an improvement in specificity from 89.9 to 92.8% (95%-CI 88.4–97.1%), in PLR from 9.13 to 12.79 (95%-CI 6.97–23.43) and in NLR from 0.082 to 0.080 (95%-CI 0.02–0.30) while high sensitivity of 92.6% (95%-CI 82.7–100.0) was preserved, marking the best available result regarding diagnostic accuracy in differentiating neurosarcoidosis from MS by CSF analysis.In the combined analysis of multiple parameters, the combination of (1) CSF WCC >30/ul, or (2) Q from MS  than the from MS . When inAlb >10 × 10−3 and/or absence of CSF-specific OCB together with absence of positive MRZ reaction are significantly more likely to indicate neurosarcoidosis than MS. As neurosarcoidosis is a serious and often devastating disease that requires therapeutic interventions that differ significantly from those for MS patients, it is of great importance to reliably distuingish between these two diseases and to diagnose the respective disease early and correctly.This retrospective single-center study demonstrates the value of analyzing basic CSF parameters and MRZ reaction to differentiate neurosarcoidosis from MS, which is most commonly considered as a differential diagnosis in routine clinical practice. We show that a CSF WCC >30/μl, QEarly work on neurosarcoidosis reported that it is the most common disease that can closely mimic MS and is initially misdiagnosed as MS . In our According to current literature, 38–69% of patients with neurosarcoidosis do not have a diagnosis of concomitant systemic sarcoidosis before the onset of neurologic symptoms or develop the neurological manifestations as the presenting syndrome of sarcoidosis , 20, 46,A very interesting and important observation in the routine clinical care of patients with relapsing or re-activated neurosarcoidosis is that neurological deficits often recur in the same way as in previous episodes of disease, due to re-inflammation of the same CNS structures that were previously affected, e.g., re-activation of the same myelitic lesion . In one Regarding radiological findings, our cohort of neurosarcoidosis patients showed MRI abnormalities in 89% of cases, most commonly leptomeningeal enhancement (59%). This again reflects typical frequencies reported in literature (69–83% of cases with MRI changes), with the exception, that leptomeningeal enhancement is reported a slightly less frequent  , 52. AltTo add another layer of complexity, there is an open debate in the field about coexistence of MS and sarcoidosis in individual patients –56. A stAlb or CSF protein) are in line with these studies. Conflicting results are reported regarding CSF-specific OCB as a sensitive measure of intrathecal production of IgG in neurosarcoidosis. Recent studies document a very low prevalence of CSF-specific OCB of 3–5%  absence of CSF-specific OCB, with or without additional parameters such as increased sIL-2R or negative MRZ reaction, yield PLR values near 10 or >10 and NLR values < 10. These sets of conditional combinations are therefore useful to differentiate between neurosarcoidosis and MS.Our study shows that these single parameters are not helpful to reliably distinguish neurosarcoidosis from MS, since either PLR was <10 or NLR was >0.1. Values of PLR >10 and NLR < 0.1 are usually regarded as useful , 37, as The MRZ reaction was first described in 1992 by Felgenhauer and Reiber. It is considered positive, if intrathecally produced IgG react against at least 2 out of 3 viral antigens, i.e., measles (M), rubella (R), and varicella zoster (Z) virus lysates. A positive MRZ reaction is found only in 63% of MS patients, but it is linked with a very high specificity for MS (98%) . ReportsWe found a low sensitivity of increased sIL-2R levels in CSF and/or serum for neurosarcoidosis (38–40%). Other studies reported increased levels in more than 50% of patients . AlthougSince CSF neopterin levels were elevated in 5/5 (100%) of our neurosarcoidosis patients, this may be an additional marker of interest to evaluate in the future for specificity and sensitivity analysis as well as PLR and NLR to distinguish neurosarcoidosis from MS. Unfortunately, neopterin values in CSF were only available in 5 neurosarcoidosis patients and in none of our MS patients, so that a representative analysis of sensitivity and specificity for neurosarcoidosis was not possible. Interestingly, an increase in CSF neopterin has so far been described not only in neurosarcoidosis –67 but an = 29) vs. MS (n = 12), and the sensitivity was 29% and the specificity 87% when comparing neurosarcoidosis with MS and other neuroinflammatory diseases (n = 29). Nordström et al.  neurosarcoidosis patients, and stood out as a sensitive marker among extraneuronal manifestations in our patients with neurosarcoidosis. Unfortunately, we did not have a sufficient number of cases with MS and FDG-PET or CT scan in our cohort to enable us to compare this parameter between neurosarcoidosis and MS patients, as this analysis is not part of the routine assessment in clinical practice. We did not find any studies in the literature that systematically reported the prevalence of lymphadenopathy in untreated MS as identified by means of FDG-PET or CT scan. We found only one observational study assessing signs of lymphadenopathy in MS patients by means of ultrasound . This stLimitations of this study are that the analysis was performed retrospectively and on a single-center level, that the overall number of patients with neurosarcoidosis was low, and that the minority of patients analyzed in this study had a histopathologically proven definite neurosarcoidosis. Analyzing the data retrospectively in principle depends on how accurately clinical, radiological, and laboratory findings are documented in the medical records. Strengths of this study are that we provide the community with robust likelihood ratios for combined sets of CSF parameters, which are useful for routine clinical practice.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.BV had a major role in the acquisition of the data, analyzed and interpreted the data, and drafted the manuscript for intellectual content. SN acquired, analyzed and interpreted the data, and revised the manuscript for intellectual content. MH analyzed and interpreted the data and revised the manuscript for intellectual content. MZ acquired and analyzed the data, and revised the manuscript for intellectual content. IJ designed and conceptualized the study, acquired, analyzed and interpreted the data, and drafted the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.


Pheropsophus occipitalis jessoensis (Coleoptera: Carabidae) and the assassin bug Sirthenea flavipes (Hemiptera: Reduviidae), using their potential predator, the pond frog Pelophylax nigromaculatus (Anura: Ranidae), which coexists with these insect species in the same habitat in Japan. We observed behavioural responses of this frog species (adults and juveniles) to adult Ph. occipitalis jessoensis and adult S. flavipes under laboratory conditions. Among the frogs, 100% and 75% rejected Ph. occipitalis jessoensis and S. flavipes, respectively, suggesting that, compared with the assassin bug S. flavipes, the bombardier beetle Ph. occipitalis jessoensis is more well-defended against frogs. An assassin bug or a bombardier beetle was provided to a frog that had encountered the other insect species. Frogs with a history of assassin bug encounter demonstrated a lower rate of attack toward bombardier beetles. Similarly, frogs with a history of bombardier beetle encounter demonstrated a lower rate of attack toward assassin bugs. Therefore, both the bombardier beetle Ph. occipitalis jessoensis and the assassin bug S. flavipes benefit from the mimetic interaction.In defensive mimicry, resemblance between unequally defended species can be parasitic; this phenomenon has been termed quasi-Batesian mimicry. Few studies have used real co-mimics and their predators to test whether the mimetic interactions were parasitic. Here, we investigated the mimetic interaction between two well-defended insect species, the bombardier beetle  Some weAnchomenus dorsalis (Pontoppidan) and the bombardier beetle species Brachinus sclopeta (Fabricius) have a similar body colour pattern (green-blue and red-brown) (Pheropsophus (Stenaptinus) agnatus (Chaudoir) (Sirthenea flavipes (Stål) (Hemiptera: Reduviidae: Peiratinae) has a black and yellow body colour similar to the colour of the bombardier beetle Pheropsophus (Stenaptinus) occipitalis jessoensis Morawitz  are chemically defended; their adults eject toxic chemicals at a temperature of 100 °C when they are attacked by predators . The disd-brown) . In Bornhaudoir) . In Japa mimicry , few stuPh. occipitalis jessoensis and the assassin bug S. flavipes) and their potential predator, the black-spotted pond frog Pelophylax nigromaculatus  (Anura: Ranidae), which coexists with these insects in the same habitat in Japan   and water (vapor) at a temperature of approximately 100 °C from the end of its abdomen (Pe. nigromaculatus (Lithobates catesbeianus (Shaw) (Anura: Ranidae)  . Adults alpidae) . The bom abdomen  to repelaculatus , the bulRanidae) , quails Ranidae) , and praRanidae) .i.e., labium) to inject them with painful venoms (S. flavipes (Sirthenea flavipes is found on the ground in grassland and farmland (Sirthenea carinata (Fabricius) (S. flavipes exclusively prey on mole crickets (S. flavipes uses a long proboscis (labium) to inject paralyzing venoms into prey, then feeds on the prey (Sirthenea flavipes aggressively stabs attackers with its proboscis when it is caught (S. flavipes reportedly causes severe pain to humans (S. flavipes shares the microhabitat (on the ground in grassland), prey (mole crickets), and body colour pattern (black and yellow colour) with the bombardier beetle Ph. occipitalis jessoensis  , juvenilcrickets ; S. flavthe prey ; Fig. 2.s caught . A bite o humans . The assssoensis . HoweverPe. nigromaculatus is distributed in East Asia, including Japan, Korea, and China  was compared with the rate of attack by frogs that had not encountered the indicated species. Finally, we discuss the adaptive significance of mimetic interactions between bombardier beetles and assassin bugs.To elucidate the nature of the mimetic interaction between bombardier beetles and assassin bugs, we observed behavioural responses of the frog Ph. occipitalis jessoensis were collected from Honshu  in July–September 2020 and May–October 2021. Adult beetles were housed separately in plastic cases  under laboratory conditions  (Lepidoptera: Noctuidae) (cf. Forty-three adults of the bombardier beetle dae) cf. . Prior tdae) cf. . The samS. flavipes were collected from Honshu (Hyogo and Shimane Prefectures) in August–October 2021. Adult bugs were housed separately in plastic cases  under laboratory conditions (25 °C). Prior to experiments, body length and weight were measured to the nearest 0.01 mm and 0.1 mg using electronic slide callipers and an electronic balance, respectively  in June–October 2021. Frogs were housed separately in plastic cages  in the laboratory at 25 °C (cf. Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae)—were provided as prey  between September 2021 and October 2021. We used frogs that had fed on mealworms >24 h before experiments. First, a frog was placed in a plastic cage . Next, a bombardier beetle (or an assassin bug) was transferred to the cage containing the frog. The behaviours of the frog and the bombardier beetle (or assassin bug) were recorded using a digital camera  and a digital video camera . The footage of recorded behaviour was reviewed to investigate how each insect could defend. The bombing sounds of bombardier beetles were checked to investigate whether bombing forced the frogs to reject the beetles. Stabbing by assassin bugs was investigated to determine whether stabbing forced the frogs to reject the bugs. When a frog did not attack a bombardier beetle or an assassin bug within 2 min, we considered it to have ignored the insect. When a frog swallowed a bombardier beetle or an assassin bug, we observed whether the frog subsequently vomited the insect . The same individuals of bombardier beetles, assassin bugs, and frogs were not used repeatedly in this experiment. The sample size was determined based on the number of assassin bugs collected in this study. Experiment 1 was part of the following experiment; specifically, the initial responses of 35 frogs observed in Experiment 2 were used as the data for Experiment 1.We used the predator sect cf. . When thPh. occipitalis jessoensis and the assassin bug S. flavipes via the potential predator Pe. nigromaculatus under laboratory conditions. Specifically, we investigated how a frog responded to a bombardier beetle or an assassin bug after the frog had encountered the other insect species . Only one pair of insect species was provided to each frog to minimise the negative impacts of well-defended insects on frogs. Healthy frogs were released after the experiments had been completed. No frogs were euthanised in this study.All analyses were performed using R version 3.5.2 .t-test was used to compare the body size (body length and weight) of bombardier beetles and assassin bugs; it was also used to compare the body size (snout–vent length and body weight) of pond frogs that attacked bombardier beetles and assassin bugs. A generalised linear mixed model (GLMM) with a binomial error distribution and a logit link was used to investigate the effects of insect species and frog encounter history on the rate of attack by frogs. The frog response  was used as a response variable. The insect species , frog encounter history , and the interaction between insect species and frog encounter history were used as fixed factors. Individual assassin bugs and frogs were used as random effects. The GLMM was conducted using the lme4 package version 1.1.19 in R  of 20 frogs successfully swallowed assassin bugs , althougP = 0.0471). The body size (body length and weight) significantly differed between bombardier beetles and assassin bugs; bombardier beetles were shorter but heavier than assassin bugs .The rate of bombardier beetle rejection by frogs (100.0%) significantly differed from the rate of assassin bug rejection by frogs ; five frogs (21.7%) attacked bombardier beetles (Twenty-one (91.3%) of 23 frogs attacked assassin bugs ; Table 2frogs 21.% attacken = 20); eight frogs (40.0%) attacked assassin bugs (Fifteen 75.0%) of 20 frogs attacked bombardier beetles 5.0% of 2. Assassisin bugs  and 7, wsin bugs .A history of assassin bug encounter reduced the rate of attack on bombardier beetles by frogs from 75.0% to 21.7% , althougn = 48), one (2.1%) died within 24 h (4 h) after swallowing an assassin bug; a dead assassin bug was found in the stomach of the dead frog. Other frogs (97.9%) were not harmed by our experiments.None of the bombardier beetles or assassin bugs that successfully defended against frogs died within 24 h after the experiments. Of the frogs  reportedly retains the memory of unpalatable prey for at least 1 day  catoirei (Dejean); adult Ph. catoirei individuals have a reddish head and pronotum  with S. flavipes in East and Southeast Asia  may be included in the mimetic complex; O. weisei adults share a similar microhabitat (on the ground in grassland), body colour pattern (black and yellow pattern), and movement pattern with Ph. occipitalis jessoensis and S. flavipes adults in Japan. Furthermore, the black and yellow body colour pattern of these insects is similar to the typical aposematic colour of stinging hymenopteran insects, such as paper wasps and bees (S. flavipes and Ph. occipitalis jessoensis with more robust protection from predators, compared with other colour patterns.Some aposematic species form ‘mimicry rings’ . Mimicryand bees . The pre10.7717/peerj.15380/supp-1Video S1The beetle ejected toxic chemicals at a temperature of 100 °C when it was stimulated by forceps. This is the video in Click here for additional data file.10.7717/peerj.15380/supp-2Video S2The assassin bug used its proboscis to attack, paralyze, and feed on the cricket. Video credit: Shinji Sugiura.Click here for additional data file.10.7717/peerj.15380/supp-3Video S3The frog captured the beetle in its mouth but spat out the beetle immediately after bombing had occurred. Video credit: Shinji Sugiura.Click here for additional data file.10.7717/peerj.15380/supp-4Video S4The frog captured the assassin bug in its mouth but spat out the bug immediately after stabbing had occurred. Video credit: Shinji Sugiura.Click here for additional data file.10.7717/peerj.15380/supp-5Supplemental Information 1Click here for additional data file.