Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Streptococcus milleri in intraabdominal abscesses in children after appendectomy: incidence and course

Intraabdominal abscesses are a common complication after appendectomy, especially in children. In this study, we describe the incidence and course of this complication in relation to the cultured pathogens found in intraabdominal abscesses. The charts of all patients between 1 and 18 years of age undergoing appendectomy in 3 hospitals between January 2006, and July 2009, were retrospectively reviewed. Presence of an intraabdominal abscess was confirmed with abdominal ultrasound examination. We collected all details concerning the appendectomy, pus cultures, and postoperative course in these patients. Two hundred fifty-nine patients underwent appendectomy during the study period. Subsequently, abdominal ultrasound studies showed an intraabdominal abscess in 18 (7%) patients. Intraabdominal abscesses developed more frequently after perforated appendicitis (23%) than after simple appendicitis (2%). The incidence of postoperative abscesses did not differ significantly between open (5.6%) or laparoscopic (6.3%) appendectomy. However, the rate was high (38%) in the patients in whom the appendectomy was converted from laparoscopic to open. In 15 out of the 18 patients with a postoperative abscess drainage was performed. In pus cultures of the drained abscesses Streptococcus milleri and Escherichia coli were the most commonly isolated pathogens. Presence of S milleri was associated with prolonged hospital stay (13.9 versus 9.0 days, P = .105) and prolonged antibiotic treatment (11.3 versus 4.8 days, P = .203). The incidence of intraabdominal abscesses is high after perforated appendicitis in children (23%). Our data suggest that the presence of S milleri correlates with a more complicated postoperative course after appendectomy in childre

A Systematic Review and Meta-Analysis of Diagnostic Performance of Imaging in Acute Cholecystitis

Purpose: To update previously summarized estimates of diagnostic accuracy for acute cholecystitis and to obtain summary estimates for more recently introduced modalities. Materials and Methods: A systematic search was performed in MEDLINE, EMBASE, Cochrane Library, and CINAHL databases up to March 2011 to identify studies about evaluation of imaging modalities in patients who were suspected of having acute cholecystitis. Inclusion criteria were explicit criteria for a positive test result, surgery and/or follow-up as the reference standard, and sufficient data to construct a 2 3 2 table. Studies about evaluation of predominantly acalculous cholecystitis in intensive care unit patients were excluded. Bivariate random-effects modeling was used to obtain summary estimates of sensitivity and specificity. Results: Fifty-seven studies were included, with evaluation of 5859 patients. Sensitivity of cholescintigraphy (96%; 95% confidence interval [CI]: 94%, 97%) was significantly higher than sensitivity of ultrasonography (US) (81%; 95% CI: 75%, 87%) and magnetic resonance (MR) imaging (85%; 95% CI: 66%, 95%). There were no significant differences in specificity among cholescintigraphy (90%; 95% CI: 86%, 93%), US (83%; 95% CI: 74%, 89%) and MR imaging (81%; 95% CI: 69%, 90%). Only one study about evaluation of computed tomography (CT) met the inclusion criteria; the reported sensitivity was 94% (95% CI: 73%, 99%) at a specificity of 59% (95% CI: 42%, 74%). Conclusion: Cholescintigraphy has the highest diagnostic accuracy of all imaging modalities in detection of acute cholecystitis. The diagnostic accuracy of US has a substantial margin of error, comparable to that of MR imaging, while CT is still underevaluated. (C) RSNA, 201

Minder onterechte appendectomie door echografie en CT

To evaluate the effect of the use of ultrasonography (US) and optional computed tomography (CT) or diagnostic laparoscopy on the percentage of unnecessary appendectomies in patients with suspected acute appendicitis. Prospective and comparison with a historical control group. Following the introduction of ultrasound imaging as an initial step, the outcomes in all patients presenting with suspected appendicitis in the emergency department were prospectively collected during a period of 18 months (July 2006-December 2007). Results were compared to retrospectively collected data on all patients who had undergone appendectomy for acute appendicitis in 2001, before the introduction of this imaging investigation. Of the 312 consecutive patients in the emergency department with suspected acute appendicitis, the condition was excluded in 51 patients following clinical and laboratory investigation. The diagnostic algorithm was applied in 239 of the 261 patients (92%). All of them had initial US, followed by additional CT in 75 patients (31%) and diagnostic laparoscopy in 12 patients (5%). Appendectomy was performed in 130 patients, and 8 (6%) of the appendices were shown to be healthy following pathological investigation. Before the implementation of preoperative imaging 36 of the 170 appendices (21%) were healthy. Following the introduction of imaging techniques in accordance with the guideline there was a significant reduction in the percentage of unnecessary appendectomies (21% versus 6%; p <0,001). The complete supplementary diagnostic algorithm had a positive and negative predictive value of respectively 90% and 98% for acute appendicitis. Structural implementation of US with optional CT and diagnostic laparoscopy in patients with suspected acute appendicitis resulted in a lower percentage of unnecessary appendectomie

Acute appendicitis on abdominal MR images: training readers to improve diagnostic accuracy

PURPOSE: To determine if training with direct feedback helps to improve the diagnostic performance of inexperienced readers in the detection of appendicitis on magnetic resonance (MR) images. MATERIALS AND METHODS: The institutional review board approved this retrospective study and waived the requirement for informed consent. Nine radiologists and eight residents without experience in evaluating MR images for acute abdominal conditions evaluated a training set of images from 100 MR imaging examinations of patients suspected of having appendicitis and received direct feedback after each evaluation. An expert panel made a diagnosis of appendicitis in 45 patients and an alternative diagnosis in 55 patients on the basis of histopathologic examination and follow-up. Readers recorded two diagnoses: the first after viewing images from conventional MR sequences (half-Fourier rapid acquisition with relaxation enhancement and true fast imaging with steady-state precession) and the second after viewing diffusion-weighted (DW) MR images. Reader sensitivity and specificity were calculated per set of 25 cases. RESULTS: The average reader sensitivity for detecting appendicitis improved significantly after training (0.82 vs 0.92, P = .003); the average specificity improved nonsignificantly (0.82 vs 0.88, P = .10). Sensitivity for radiologists increased from 0.81 in the first set of 25 cases to 0.91 in the last set, and specificity improved from 0.82 to 0.85. For residents, sensitivity increased from 0.82 to 0.94, and specificity increased from 0.82 to 0.91. Sensitivity improved from 0.80 to 0.87 (P < .001) in all readings combined when DW images were read in addition to conventional MR images. CONCLUSION: Diagnostic accuracy of inexperienced readers in the evaluation of abdominal MR images for acute appendicitis improved after training with direct feedback, and the addition of DW images improved reader sensitivity

Acute Appendicitis on Abdominal MR Images: Training Readers to Improve Diagnostic Accuracy

Purpose: To determine if training with direct feedback helps to improve the diagnostic performance of inexperienced readers in the detection of appendicitis on magnetic resonance (MR) images. Materials and Methods: The institutional review board approved this retrospective study and waived the requirement for informed consent. Nine radiologists and eight residents without experience in evaluating MR images for acute abdominal conditions evaluated a training set of images from 100 MR imaging examinations of patients suspected of having appendicitis and received direct feedback after each evaluation. An expert panel made a diagnosis of appendicitis in 45 patients and an alternative diagnosis in 55 patients on the basis of histopathologic examination and follow-up. Readers recorded two diagnoses: the first after viewing images from conventional MR sequences (half-Fourier rapid acquisition with relaxation enhancement and true fast imaging with steady-state precession) and the second after viewing diffusion-weighted (DW) MR images. Reader sensitivity and specificity were calculated per set of 25 cases. Results: The average reader sensitivity for detecting appendicitis improved significantly after training (0.82 vs 0.92, P = .003); the average specificity improved nonsignificantly (0.82 vs 0.88, P = .10). Sensitivity for radiologists increased from 0.81 in the first set of 25 cases to 0.91 in the last set, and specificity improved from 0.82 to 0.85. For residents, sensitivity increased from 0.82 to 0.94, and specificity increased from 0.82 to 0.91. Sensitivity improved from 0.80 to 0.87 (P <.001) in all readings combined when DW images were read in addition to conventional MR images. Conclusion: Diagnostic accuracy of inexperienced readers in the evaluation of abdominal MR images for acute appendicitis improved after training with direct feedback, and the addition of DW images improved reader sensitivity. (c) RSNA, 201

Comparison of imaging strategies with conditional contrast-enhanced CT and unenhanced MR imaging in patients suspected of having appendicitis: a multicenter diagnostic performance study

To compare the diagnostic performance of imaging strategies with magnetic resonance (MR) imaging and computed tomographic (CT) imaging in adult patients suspected of having appendicitis. Institutional review board approval was obtained prior to study initiation, and patients gave written informed consent. In a multicenter diagnostic performance study, adults suspected of having appendicitis were prospectively identified in the emergency department. Consenting patients underwent ultrasonography (US) and subsequent contrast-enhanced CT if US imaging yielded negative or inconclusive results. Additionally, all patients underwent unenhanced MR imaging, with the reader blinded to other findings. An expert panel assigned final diagnosis after 3 months. Diagnostic performance of three imaging strategies was evaluated: conditional CT after US, conditional MR imaging after US, and immediate MR imaging. Sensitivity and specificity were calculated by comparing findings with final diagnosis. Between March and September 2010, 229 US, 115 CT, and 223 MR examinations were performed in 230 patients (median age, 35 years; 40% men). Appendicitis was the final diagnosis in 118 cases. Conditional and immediate MR imaging had sensitivity and specificity comparable to that of conditional CT, which resulted in 3% (three of 118; 95% confidence interval [CI]: 1%, 7%) missed appendicitis, and 8% (10 of 125; 95% CI: 4%, 14%) false-positives. Conditional MR missed appendicitis in 2% (two of 118; 95% CI: 0%, 6%) and generated 10% (13 of 129; 95% CI: 6%, 16%) false-positives. Immediate MR missed 3% (four of 117; 95% CI: 1%, 8%) appendicitis with 6% (seven of 120; 95% CI: 3%, 12%) false-positives. Conditional strategies resulted in more false-positives in women than in men (conditional CT, 17% vs 0%; P = .03; conditional MR, 19% vs 1%; P = .04), wherease immediate MR imaging did not. The accuracy of conditional or immediate MR imaging was similar to that of conditional CT in patients suspected of having appendicitis, which implied that strategies with MR imaging may replace conditional CT for appendicitis detectio

Diagnostic accuracy and patient acceptance of MRI in children with suspected appendicitis

To compare magnetic resonance imaging (MRI) and ultrasound in children with suspected appendicitis. In a single-centre diagnostic accuracy study, children with suspected appendicitis were prospectively identified at the emergency department. All underwent abdominal ultrasound and MRI within 2 h, with the reader blinded to other imaging findings. An expert panel established the final diagnosis after 3 months. We evaluated the diagnostic accuracy of three imaging strategies: ultrasound only, conditional MRI after negative or inconclusive ultrasound, and MRI only. Significance between sensitivity and specificity was calculated using McNemar's test statistic. Between April and December 2009 we included 104 consecutive children (47 male, mean age 12). According to the expert panel, 58 patients had appendicitis. The sensitivity of MRI only and conditional MRI was 100% (95% confidence interval 92-100), that of ultrasound was significantly lower (76%; 63-85, P  < 0.001). Specificity was comparable among the three investigated strategies; ultrasound only 89% (77-95), conditional MRI 80% (67-89), MRI only 89% (77-95) (P values 0.13, 0.13 and 1.00). In children with suspected appendicitis, strategies with MRI (MRI only, conditional MRI) had a higher sensitivity for appendicitis compared with a strategy with ultrasound only, while specificity was comparable. • In children, MRI has a higher sensitivity for appendicitis than ultrasound. • Ultrasound followed by MRI in negative or inconclusive findings is accurate. • The tolerance for ultrasound and MRI in children is comparable. • MRI can be performed in children in an emergency settin

MRI features associated with acute appendicitis

To identify MRI features associated with appendicitis. Features expected to be associated with appendicitis were recorded in consensus by two expert radiologists on 223 abdominal MRIs in patients with suspected appendicitis. Nine MRI features were studied: appendix diameter >7 mm, appendicolith, peri-appendiceal fat infiltration, peri-appendiceal fluid, absence of gas in the appendix, appendiceal wall destruction, restricted diffusion of the appendiceal wall, lumen or focal fluid collections. Appendicitis was assigned as the final diagnosis in 117/223 patients. Associations between imaging features and appendicitis were evaluated with logistic regression analysis. All investigated features were significantly associated with appendicitis in univariate analysis. Combinations of two and three features were associated with a probability of appendicitis of 88 % and 92 %, respectively. In patients without any of the nine features, appendicitis was present in 2 % of cases. After multivariate analysis, only an appendix diameter >7 mm, peri-appendiceal fat infiltration and restricted diffusion of the appendiceal wall were significantly associated with appendicitis. The probability of appendicitis was 96 % in their presence and 2 % in their absence. An appendix diameter >7 mm, peri-appendiceal fat infiltration and restricted diffusion of the appendiceal wall have the strongest association with appendicitis on MRI. • An enlarged appendix, fat infiltration and restricted diffusion are associated with appendicitis. • One such feature on MRI gives an 88 % probability of appendicitis. • Two features in combination give a probability of appendicitis of 94 %. • Combinations of three features give a probability of appendicitis of 96 %. • The absence of these features almost rules out appendicitis (2 %