In silico Mechanics of Stem Cells Intramyocardially Transplanted with a Biomaterial Injectate for Treatment of Myocardial Infarction.

PURPOSE: Biomaterial and stem cell delivery are promising approaches to treating myocardial infarction. However, the mechanical and biochemical mechanisms underlying the therapeutic benefits require further clarification. This study aimed to assess the deformation of stem cells injected with the biomaterial into the infarcted heart. METHODS: A microstructural finite element model of a mid-wall infarcted myocardial region was developed from ex vivo microcomputed tomography data of a rat heart with left ventricular infarct and intramyocardial biomaterial injectate. Nine cells were numerically seeded in the injectate of the microstructural model. The microstructural and a previously developed biventricular finite element model of the same rat heart were used to quantify the deformation of the cells during a cardiac cycle for a biomaterial elastic modulus (Einj) ranging between 4.1 and 405,900 kPa. RESULTS: The transplanted cells' deformation was largest for Einj = 7.4 kPa, matching that of the cells, and decreased for an increase and decrease in Einj. The cell deformation was more sensitive to Einj changes for softer (Einj ≤ 738 kPa) than stiffer biomaterials. CONCLUSIONS: Combining the microstructural and biventricular finite element models enables quantifying micromechanics of transplanted cells in the heart. The approach offers a broader scope for in silico investigations of biomaterial and cell therapies for myocardial infarction and other cardiac pathologies.

Multiscale characterization of heart failure.

Dilated cardiomyopathy is a progressive irreversible disease associated with contractile dysfunction and heart failure. During dilated cardiomyopathy, elevated diastolic wall strains trigger mechanotransduction pathways that initiate the addition of sarcomeres in series and an overall increase in myocyte length. At the whole organ level, this results in a chronic dilation of the ventricles, an increase in end diastolic and end systolic volumes, and a decrease in ejection fraction. However, how exactly changes in sarcomere number translate into changes in myocyte morphology, and how these cellular changes translate into ventricular dilation remains incompletely understood. Here we combined a chronic animal study, continuum growth modeling, and machine learning to quantify correlations between sarcomere dynamics, myocyte morphology, and ventricular dilation. In an eight-week long volume overload study of six pigs, we found that the average sarcomere number increased by +3.8%/week, from 47 to 62, resulting in a myocyte lengthening of +3.3%/week, from 85 to 108 µm, while the sarcomere length and myocyte width remained unchanged. At the same time, the average end diastolic volume increased by +6.0%/week. Using continuum growth modeling and Bayesian inference, we correlated alterations on the subcellular, cellular, and organ scales and found that the serial sarcomere number explained 88% of myocyte lengthening, which, in turn, explained 54% of cardiac dilation. Our results demonstrate that sarcomere number and myocyte length are closely correlated and constitute the major determinants of dilated heart failure. We anticipate our study to be a starting point for more sophisticated multiscale models of heart failure. Our study suggests that altering sarcomere turnover-and with it myocyte morphology and ventricular dimensions-could be a potential therapeutic target to attenuate or reverse the progression of heart failure. STATEMENT OF SIGNIFICANCE: Heart failure is a significant global health problem that affects more than 25 million people worldwide and increases in prevalence as the population ages. Heart failure has been studied excessively at various scales; yet, there is no compelling concept to connect knowledge from the subcellular, cellular, and organ level across the scales. Here we combined a chronic animal study, continuum growth modeling, and machine learning to quantify correlations between sarcomere dynamics, myocyte morphology, and ventricular dilation. We found that the serial sarcomere number explained 88% of myocyte lengthening, which, in turn, explained 54% of cardiac dilation. Our results show that sarcomere number and myocyte length are closely correlated and constitute the major determinants of dilated heart failure. This suggests that altering the sarcomere turnover-and with it myocyte morphology and ventricular dimensions-could be a potential therapeutic target to attenuate or reverse heart failure.

Using machine learning to characterize heart failure across the scales.

Heart failure is a progressive chronic condition in which the heart undergoes detrimental changes in structure and function across multiple scales in time and space. Multiscale models of cardiac growth can provide a patient-specific window into the progression of heart failure and guide personalized treatment planning. Yet, the predictive potential of cardiac growth models remains poorly understood. Here, we quantify predictive power of a stretch-driven growth model using a chronic porcine heart failure model, subject-specific multiscale simulation, and machine learning techniques. We combine hierarchical modeling, Bayesian inference, and Gaussian process regression to quantify the uncertainty of our experimental measurements during an 8-week long study of volume overload in six pigs. We then propagate the experimental uncertainties from the organ scale through our computational growth model and quantify the agreement between experimentally measured and computationally predicted alterations on the cellular scale. Our study suggests that stretch is the major stimulus for myocyte lengthening and demonstrates that a stretch-driven growth model alone can explain [Formula: see text] of the observed changes in myocyte morphology. We anticipate that our approach will allow us to design, calibrate, and validate a new generation of multiscale cardiac growth models to explore the interplay of various subcellular-, cellular-, and organ-level contributors to heart failure. Using machine learning in heart failure research has the potential to combine information from different sources, subjects, and scales to provide a more holistic picture of the failing heart and point toward new treatment strategies.

Endothelial cells on an aged subendothelial matrix display heterogeneous strain profiles in silico.

Within the artery intima, endothelial cells respond to mechanical cues and changes in subendothelial matrix stiffness. Recently, we found that the aging subendothelial matrix stiffens heterogeneously and that stiffness heterogeneities are present on the scale of one cell length. However, the impacts of these complex mechanical micro-heterogeneities on endothelial cells have not been fully understood. Here, we simulate the effects of matrices that mimic young and aged vessels on single- and multi-cell endothelial cell models and examine the resulting cell basal strain profiles. Although there are limitations to the model which prohibit the prediction of intracellular strain distributions in alive cells, this model does introduce mechanical complexities to the subendothelial matrix material. More heterogeneous basal strain distributions are present in the single- and multi-cell models on the matrix mimicking an aged artery over those exhibited on the young artery. Overall, our data indicate that increased heterogeneous strain profiles in endothelial cells are displayed in silico when there is an increased presence of microscale arterial mechanical heterogeneities in the matrix.

Effect of intra-myocardial Algisyl-LVR™ injectates on fibre structure in porcine heart failure.

Recent preclinical trials have shown that alginate injections are a promising treatment for ischemic heart disease. Although improvements in heart function and global structure have been reported following alginate implants, the underlying structure is poorly understood. Using high resolution ex vivo MRI and DT-MRI of the hearts of normal control swine (n = 8), swine with induced heart failure (n = 5), and swine with heart failure and alginate injection treatment (n = 6), we visualized and quantified the fibre distribution and implant material geometry. Our findings show that the alginate injectates form solid ellipsoids with a retention rate of 68.7% ±â€¯21.3% (mean ±â€¯SD) and a sphericity index of 0.37 ±â€¯0.03. These ellipsoidal shapes solidified predominantly at the mid-wall position with an inclination of -4.9°â€¯±â€¯31.4° relative to the local circumferential direction. Overall, the change to left ventricular wall thickness and myofiber orientation was minor and was associated with heart failure and not the presence of injectates. These results show that alginate injectates conform to the pre-existing tissue structure, likely expanding along directions of least resistance as mass is added to the injection sites. The alginate displaces the myocardial tissue predominantly in the longitudinal direction, causing minimal disruption to the surrounding myofiber orientations.

Monarthritis: differential diagnosis.

Acute monarthritis should be regarded as infectious until proved otherwise. Early evaluation is crucial because of the capacity of some infectious agents to destroy cartilage rapidly. The history and physical examination can provide highly suggestive clues, but a definitive diagnosis may depend on arthrocentesis and analysis of synovial fluid. The diagnosis of acute monarthritis is rarely established by radiography. The most common cause of bacterial arthritis is Neisseria gonorrhoeae. Staphylococcus aureus and streptococci are the organisms most frequently implicated in nongonococcal bacterial arthritis, although the possibility of Gram-negative bacteria or anaerobes should not be overlooked in intravenous drug users or immunocompromised patients. Inflammation in a large joint, particularly the knee, might arouse suspicion of Lyme disease. Other, less frequently encountered infectious causes of acute monarthritis include tuberculosis and other mycobacteria, fungi, and viruses. Arthroscopic examination and synovial tissue biopsy may be necessary to diagnose such processes. Microscopic examination of the synovial fluid may reveal a crystalline etiology for monarthritis. Monosodium urate crystals induce gout, usually in the toe, ankle, or midfoot, while calcium pyrophosphate crystals cause pseudogout, most often in the knee or wrist. Acute monarthritis is sometimes a manifestation of osteoarthritis or an early sign of a systemic arthritis such as rheumatoid or reactive arthritis. Processes underlying acute monarthritis can also evolve into a more chronic clinical picture as exemplified by the spondyloarthropathies.

Methotrexate and histologic hepatic abnormalities: a meta-analysis.

STUDY OBJECTIVE: To determine the risk of liver toxicity from the long-term administration of methotrexate in patients with rheumatoid arthritis or psoriatic arthritis. DESIGN: A meta-analysis of 15 studies examining the relationship between long-term, low-dose methotrexate administration and biopsy evidence of liver fibrosis. PATIENTS: A total of 636 patients from 15 studies. RESULTS: The incidence of progression of liver disease (defined as worsening of at least one grade on the histologic classification of Roenigk) among 636 patients was 27.9% (95% confidence intervals 24.3 to 31.6). The rate of progression of liver disease in the 15 studies was associated with the cumulative dose of methotrexate (p = 0.01). Patients on average had a 6.7% (95% confidence intervals 2.1 to 11.4) chance of progressing at least one histologic grade on liver biopsy for each gram of methotrexate taken. The overall incidence of advanced pathologic changes on liver biopsy (grades IIIB or IV) among 636 patients was 5.0% (95% confidence intervals 3.5 to 7.0). The development of advanced histologic changes was not associated with the cumulative dose of methotrexate (p = 0.08). Patients who according to their history were heavy drinkers (at least 100 g of alcohol per week) were more likely to have advanced changes on liver biopsy (17.8% versus 4.5%, p = 0.0003) and to show histologic progression (73.3% versus 25.9%, p = 0.0002). Patients with psoriasis were more likely than patients with rheumatoid arthritis to have advanced changes (7.7% versus 2.7%, p = 0.003) and histologic progression (33.1% versus 24.3%, p = 0.02). CONCLUSIONS: The risk of liver toxicity in patients undergoing long-term, low-dose methotrexate therapy is substantial, and that risk increases with the total cumulative dose and with heavy consumption of alcohol. Heavy users of alcohol should not receive long-term methotrexate therapy. For most patients who are not heavy users of alcohol, liver biopsies should be done periodically to monitor for the occurrence of liver toxicity.

The long persistence of CMV DNA in the blood of renal transplant patients after recovery from CMV infection.

A total of 30-50% of all renal transplant recipients undergo infections caused by human cytomegalovirus. With the introduction of ganciclovir and foscarnet for specific antiviral therapy there is an increasing demand for diagnostic tools that allow the early and rapid identification of CMV as the causative agent of the observed disease. We and others previously showed the direct detection of pp65 antigen in peripheral blood leukocytes to be an excellent marker for active cytomegalovirus infection. In order to establish whether the detection of CMV DNA by the polymerase chain reaction (PCR) supplies further information in this regard, we compared both methods. In 41 renal transplant patients the PCR assay yielded a sensitivity of 100% compared with 87.5% of the antigenemia assay. Specificities reached 67% and 92.5%, respectively. In 5 patients without both serological signs of infection and antigenemia, CMV DNA was also found. The duration of CMV DNA detection in PBL during active infection was significantly longer than antigenemia. Even after successful treatment of symptomatic CMV disease, DNA was present for a period of weeks without any relapse of disease. In contrast, antigenemia disappeared after antiviral therapy and reappeared only in one patient with relapse of CMV disease. We conclude that PCR offers no advantages over antigen detection in monitoring for CMV infections after renal transplantation.

Renal graft rejection or urinary tract infection? The value of myeloperoxidase, C-reactive protein, and alpha2-macroglobulin in the urine.

Previous investigations have shown that the determination of two acute-phase proteins in the urine, C-reactive protein (CRPu) and alpha2-macroglobulin (alpha2-MGu), allows a noninvasive diagnosis of acute renal graft dysfunction. A reliable differentiation between rejection and urinary tract infection can be made only when considering the C-reactive protein in serum and urine at the same time (CRPs:CRPu ratio). Therefore, a diagnostic procedure independent of parameters other than urinary proteins is needed. As granulocytes play only a minor role in graft rejection but are a common feature in urinary tract infection, we determined a marker of granulocytes (myeloperoxidase) in urine (MPOu). Eighty-nine renal transplant recipients were included in the study. In normal courses, CRPu, alpha2-MGu, and MPOu were within the normal range. In 15 cases of acute interstitial rejection, an increased excretion of CRPu and alpha2-MGu could be confirmed, but MPOu could not be detected. On the occasion of acute vascular rejection (n=6), with the exception of one case, MPOu could not be observed. The pattern of the three urinary proteins differed in urinary tract infections (n=40): MPOu could be detected in all cases, CRPu in 50% of cases, and alpha2-MGu in 73% of cases. In patients with cytomegalovirus infection (n=7), no MPOu, CRPu, or alpha2-MGu was found. In conclusion, the simultaneous measurement of the three proteins allows a complete, noninvasive, differential diagnostic procedure of renal graft dysfunction.

Treatment of posttransplant hypertension by laparoscopic bilateral nephrectomy?

BACKGROUND: Hypertension is an important risk factor for the development of chronic graft failure and decreased graft and patient survival after renal transplantation. METHODS: Between September 1994 and August 1996, 14 patients underwent laparoscopic bilateral nephrectomy for treatment of drug-resistant hypertension after successful renal transplantation. Common causes of hypertension were largely excluded before bilateral nephrectomy. A scoring system was developed for comparison of different antihypertensive regimes. In this system, points were given according to type and dosage of each antihypertensive drug. RESULTS: At 6-month follow-up, all patients showed well-controlled blood pressure (median of mean arterial pressure: 104 vs. 130 mmHg preoperatively, P<0.001, n=14), and significantly fewer antihypertensive drugs were needed according to the scoring system (48.9+/-20.9 points vs. 105.9+/-23.5 points preoperatively, P<0.001, n=14). During laparoscopy, three conversions to open surgery were necessary. Postoperatively, four complications occurred. After laparoscopy, immunosuppression and other oral medication were given continuously. The hospital stay ranged between 3 and 6 days (median: 5 days). CONCLUSIONS: The results indicate that bilateral nephrectomy using the laparoscopic technique can be an effective alternative method for a selected group of patients with severe hypertension, which is unresponsive to conservative management after successful renal transplantation with regard to improving the long-term graft survival.

Pulmonary manifestations of the eosinophilia-myalgia syndrome associated with tryptophan ingestion.

Pulmonary manifestations are not infrequent in the L-tryptophan-induced eosinophilia-myalgia syndrome (EMS). However, previous reports have not described the results of longitudinal pulmonary function, exercise testing, high-resolution computerized tomographic (HRCT) scanning of the chest, or detailed bronchoalveolar lavage (BAL) analysis. We report six patients with EMS who had dyspnea. The diffusing capacity for carbon monoxide was decreased in five patients tested. Exercise testing with arterial blood gas sampling in three patients was consistent with pulmonary vascular or parenchymal disease. Serial exercise testing in two of these patients demonstrated marked improvement temporally associated with corticosteroid treatment. In four patients, HRCT scanning of the chest was abnormal. One of these patients showed no abnormality on routine chest roentgenogram. Two patients undergoing BAL exhibited increased eosinophils in the lavage fluid; a third had elevated lymphocytes. Serial measurements of fibroblast proliferation-stimulating-activity in samples of BAL fluid obtained from serial examinations in two patients exhibited heightened pretreatment activity that returned to the normal range following corticosteroid therapy. In these two patients, increased proportions of T-suppressor/cytolytic (CD8+) cells were observed in the BAL fluid. Despite aggressive immunosuppressive therapy, one of the patients died of respiratory failure. Another remains markedly dyspneic with pulmonary hypertension. Of the remaining four patients, two exhibited resolution of pulmonary symptoms after systemic corticosteroid therapy, and two experienced partial improvement.

Serum erythropoietin and creatinine concentrations as predictive factors for response to recombinant human erythropoietin treatment in anaemic tumour patients on chemotherapy.

Recent studies have shown that recombinant human erythropoietin (rHuEPO) is effective in correcting anaemia in about 50% of tumour patients. Predictive parameters for the response to rHuEPO still need to be established. In the present prospective study, rHuEPO therapy was scheduled in 22 patients with solid tumours for 12 weeks (3x10,000 U rHuEPO/week s.c.). If response was not achieved within 4 weeks, the dose was increased to 3x20,000 U rHuEPO/week. All patients received combined chemotherapy (ifosfamide, carboplatin, etoposide) before and during rHuEPO therapy. 10 of the 22 patients responded to rHuEPO and did no longer need blood transfusions. In 8 of the 10 responders and in 2 of the 12 non-responders serum creatinine concentration was increased before rHuEPO therapy was started. In addition, the endogenous serum EPO concentrations were significantly lower in the responders versus the non-responders. We conclude that rHuEPO is primarily effective in patients with chemotherapy-induced renal impairment. The rate of the response to rHuEPO is high when the baseline serum EPO level is <75 U/l and the serum creatinine concentration is greater than normal (or the estimated creatinine clearance <60 ml/min).

Glomerular proteinuria as an early sign of renal-transplant rejection.

The introduction of cyclosporin gave rise to an additional problem in the surveillance of renal transplant patients, namely the differentiation between cyclosporin toxicity and acute transplant rejection. The development of assays for specific proteins in urine has produced a non-invasive solution to this problem. In 55 renal transplant patients the following proteins were determined daily in 24 h-urine samples: IgG, transferrin (TF), albumin, beta 2-microglobulin (beta 2-MG), retinol binding protein (RBP), alpha 1-microglobulin (alpha 1-MG) and alpha 1-antitrypsin (alpha 1-AT). All proteins were determined quantitatively using immunoluminometric assays and 10 microliters urine in dilutions from 1:1-1:100. The urinary protein excretion was related to the actual creatinine clearance as this index gave the best differentiation between normal and abnormal status. In 24 h-urine, intraindividual peaks of IgG, TF and albumin were seen regularly in acute rejection episodes. However, a peak in the "tubular" proteins (RBP, beta 2-MG, alpha 1-MG) could not be detected. After effective treatment of the rejection episode, the renal function improved and the protein excretion returned to prerejection episode levels. In bacterial infection of the urogenital tract, urinary alpha1-AT levels rose. They returned to normal after successful antibiotic treatment. In two cases of cyclosporin toxicity neither glomerular nor tubular proteins were excreted in abnormal amounts when compared with transplant patients without complications, the only changes being an increase in serum creatinine as a result of reduced renal function.

The difficulties of differentiating vasculitis from its mimics.

The signs and symptoms of vasculitis are not specific, and tests for confirming the diagnosis can be misleading. Thus, when considering a diagnosis of vasculitis, physicians need to keep an open mind. With a case vignette, the author illustrates some of the difficulties in diagnosing "vasculitis."

Update on NSAIDS in the elderly.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can effectively reduce pain and inflammation. Although these agents are potent inhibitors of prostaglandin synthesis, they probably have other effects on the inflammatory process. NSAIDs produce a number of side effects, particularly in elderly patients. Understanding the effects of aging on drug pharmacokinetics will help minimize these complications.

When to try COX-2-specific inhibitors. Safer than standard NSAIDs in some situations.

COX-2-specific inhibitors, by sparing COX-1 enzyme and its physiologic functions, are a safer option than regular NSAIDs in patients who are at risk for gastrointestinal bleeding (e.g., patients with a history of peptic ulcer disease, gastritis, alcoholism, concomitant corticosteroid or anticoagulant use). They have been approved for use in arthritis, and their efficacy is comparable to that of other NSAIDs. Further clinical data are needed to establish the long-term safety profile of these newly introduced drugs.

[Necrotizing enterocolitis: a historical and current review]. / Darmbrand (Enteritis necroticans). Eine historische und aktuelle Ubersicht.

Enteritis necroticans, locally called "Darmbrand", is a severe and life threatening infectious disease which was epidemic in Northern Germany after World War II. Darmbrand had a limited appearance, occurring only for a few years. In Lübeck many cases were diagnosed in 1946/1948 and the book "Darmbrand, Enteritis necroticans" was published in 1949 by clinicians and pathologists. Enteritis necroticans is also known as a tropical cause of bloody diarrhea and is caused by Clostridium perfringens Type C (type beta-toxin). The disease is related to pig feasts in Papua New Guinea. Although necrotizing enterocolitis is now a rather rare disease we must be aware of the appearance of this fulminant entity. This paper represents a review on the historic and current aspects of enteritis necroticans and discusses the epidemiology, pathogenesis and treatment of this disease.

[POEMS syndrome with chronic renal failure]. / POEMS-Syndrom mit Niereninsuffizienz.

CASE HISTORY AND CLINICAL FINDINGS: A 43-year-old male was admitted to the hospital because of subfebrile temperatures since 3 months, weight loss of 9 kilogramms and lateral foot pains with bilateral leg edema. Over the last years, the patient had consulted a doctor several times for upper respiratory infections, orchitis and a spinal neck syndrome. At the physical examination, we found a temperature of 37.4 degrees C, no rales, no crackles at auscultation of the lung, bilateral ankle edema and livid, pressure-dolent skin changes at the lateral margins of both feet. The blood pressure was 170/100 mmHg. EXAMINATIONS: The following pathologically laboratory results were found: erythrocyte sedimentation rale (ESR) 35/55 mm n. W., C-reactive protein (CRP) up to 39 mg/l, leucocytes up to 13.5/nl, LH and FHS were elevated corresponding to hypergonadotropic hypogonadism, renal failure with at the beginning a selective glomerular proteinuria, as well as a monoclonal IgG-gammopathy. The bone marrow aspiration as well as the bone marrow biopsy revealed neither plasmocytoma nor a malignant systemic disease. The ultrasound examination showed enlarged liver, spleen, and kidneys. TREATMENT AND FOLLOW-UP: After excluding a connective tissue disease and an infection and with the missing proof of a malignant tumor treatment was started with parenteral methylprednisolon 500 mg on 3 consecutive days under the hypothesis of classic panarteriitis nodosa, even when multiple biopsies were negative. Under the treatment, the elevated inflammatory parameters and renal failure improved, but deteriorated quickly after discontinuation of the corticosteroid medication. The second kidney biopsy showed a chronic scaring glomerulopathy of the hemolytic uremic syndrome type. In the follow-up the renal insufficiency was improved by corticosteroids. With the manifestation of a bilateral sensory polyneuropathy in January 1994, the diagnosis of a POEMS syndrome was most likely. CONCLUSION: According to the literature, up to 50% of the cases with POEMS syndrome reveal renal failure. Most times a glomerular microangiopathy is shown histologically. A treatment trial with corticosteroids is justified.

Nocardial infection in a renal transplant recipient--a case report.

Report is given about a 50 year old renal transplant recipient who developed signs of a severe pneumonia 37 days post transplantation. The diagnosis following chest X-ray and physical examination was multifocal nodular pneumonia of unknown origin in an immunosuppressed patient. Although a varying antibiotic chemotherapy was administered at high doses he died 4 weeks later without identification of the infective agent. Post-mortem and microbiological examinations revealed a systemic suppurative infection caused by Nocardia asteroides. Percutaneous or open lung biopsy within the first 10 days after onset of clinical symptoms has to be recommended to secure the diagnosis and treatment with sulphonamides.

American College of Rheumatology classification criteria for Sjögren's syndrome: a data-driven, expert consensus approach in the Sjögren's International Collaborative Clinical Alliance cohort.

OBJECTIVE: We propose new classification criteria for Sjögren's syndrome (SS), which are needed considering the emergence of biologic agents as potential treatments and their associated comorbidity. These criteria target individuals with signs/symptoms suggestive of SS. METHODS: Criteria are based on expert opinion elicited using the nominal group technique and analyses of data from the Sjögren's International Collaborative Clinical Alliance. Preliminary criteria validation included comparisons with classifications based on the American­European Consensus Group (AECG) criteria, a model-based "gold standard"obtained from latent class analysis (LCA) of data from a range of diagnostic tests, and a comparison with cases and controls collected from sources external to the population used for criteria development. RESULTS: Validation results indicate high levels of sensitivity and specificity for the criteria. Case definition requires at least 2 of the following 3: 1) positive serum anti-SSA and/or anti-SSB or (positive rheumatoid factor and antinuclear antibody titer >1:320), 2) ocular staining score >3, or 3) presence of focal lymphocytic sialadenitis with a focus score >1 focus/4 mm2 in labial salivary gland biopsy samples. Observed agreement with the AECG criteria is high when these are applied using all objective tests. However, AECG classification based on allowable substitutions of symptoms for objective tests results in poor agreement with the proposed and LCA-derived classifications. CONCLUSION: These classification criteria developed from registry data collected using standardized measures are based on objective tests. Validation indicates improved classification performance relative to existing alternatives, making them more suitable for application in situations where misclassification may present health risks.