Eosinophilic esophagitis: absolute eosinophilic count, peak eosinophilic count, and potential biomarkers of eosinophilic degranulation products-an in-depth systematic review.

Background: Eosinophilic esophagitis is a chronic inflammatory disorder, often relapsing. There is an increasing need to develop new alternative diagnostic and monitoring methods on a critical basis, which will provide samples through none or minimally invasive procedures. This study aims to identify and document the types and roles of potential biomarkers in eosinophilic esophagitis released by eosinophils as well as the potential relationship to the peak eosinophilic count and the degree of degranulation of in situ eosinophils (DGE/DGE + NDGE: degranulated eosinophils/degranulated eosinophils and non-degranulated eosinophils). Methods: This is the first in-depth systematic review study using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) parameters involving a literature search of academic databases (PubMed, Scopus, Medline, Google Scholar, and Cochrane Database, 2011-2022) targeting specifically the eosinophilic counts and ratio, and the eosinophilic degranulation products as potential biomarkers. Data were extracted from ten selected studies and presented on a spreadsheet. Results: The studies show the ability to detect eosinophilic and non-eosinophilic degranulation products, and absolute eosinophilic count in samples, including blood and urine, thereby serving as potential surrogates in making the diagnosis or monitoring disease progression in the future. There is an obvious paucity of studies that correlate potential biomarkers to the degree of degranulation of in situ eosinophils. Conclusions: A few minimally invasive methods and biomarkers may be suggested as alternative tools in diagnosing and monitoring eosinophilic esophagitis. While there is no consensus on the clinical usefulness of these biomarkers, our critical evaluation may suggest that the eosinophilic degranulation ratio (DGE/DGE + NDGE: degranulated eosinophils/degranulated eosinophils and non-degranulated eosinophils) in the esophagus may be critical for evaluating properly these biomarkers. An increasing trend may culminate in the potential clinical use of these biomarkers evaluated not only with the peak eosinophilic count, but also with the degranulation score upon regulatory bodies' approval to monitor eosinophilic esophagitis in the future. We strongly advocate for the necessity to score the esophageal biopsies with both a peak eosinophilic count and a score of the degranulated eosinophils.

Double Trouble: How Microbiome Dysbiosis and Mitochondrial Dysfunction Drive Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are closely related liver conditions that have become more prevalent globally. This review examines the intricate interplay between microbiome dysbiosis and mitochondrial dysfunction in the development of NAFLD and NASH. The combination of these two factors creates a synergistic situation referred to as "double trouble", which promotes the accumulation of lipids in the liver and the subsequent progression from simple steatosis (NAFLD) to inflammation (NASH). Microbiome dysbiosis, characterized by changes in the composition of gut microbes and increased intestinal permeability, contributes to the movement of bacterial products into the liver. It triggers metabolic disturbances and has anti-inflammatory effects. Understanding the complex relationship between microbiome dysbiosis and mitochondrial dysfunction in the development of NAFLD and NASH is crucial for advancing innovative therapeutic approaches that target these underlying mechanisms.

Liver Biopsy Handling of Metabolic-Associated Fatty Liver Disease (MAFLD): the Children's Hospital of Eastern Ontario grossing protocol.

Metabolic-(non-alcoholic) associated fatty liver disease (MAFLD/NAFLD) has increasingly become a worldwide epidemic. It has been suggested that renaming NAFLD to MAFLD is critical in identifying patients with advanced fibrosis and poor cardiovascular outcomes. There are concerns that the progression to non-alcoholic steatohepatitis (NASH) may become a constant drive in the future healthcare of children and adolescents. There is a necessity to tackle the emerging risk factors for NASH-associated hepatocellular carcinoma (HCC). In this narrative review, we present the current protocol of liver biopsy separated between pre-analytical, analytical, and post-analytical handling. Genetic association investigations have identified single nucleotide polymorphisms implicated in the progression of MAFLD-HCC, many of which seem to belong to the lipid metabolism pathways. PNPLA3 rs738409 variant, TM6SF2 rs58542926 variant, MBOAT7 rs641738 variant, and GCKR variants seem to be significantly associated with NAFLD disease susceptibility. In disclosing the current comprehensive protocol performed at the Children's Hospital of Eastern Ontario, Ottawa, ON, Canada, we support the most recent Kulkarni-Sarin's pledge to rename NAFLD to MAFLD. Grossing of the liver biopsy is key to identifying histologic, immunophenotypical, and ultrastructure data and properly preserving tissue for molecular genomics data.

Handling a biopsy with fatty liver disease Fatty liver disease is increasing worldwide. There is a necessity to tackle the emerging risk factors for the development of liver cancer following years of fatty liver disease. In this paper, we show our protocol at the Children's Hospital of Eastern Ontario, University of Ottawa, Ontario, Canada.

Investigating the Role of Non-Coding RNA in Non-Alcoholic Fatty Liver Disease.

Non-coding RNAs (ncRNAs) are RNA molecules that do not code for protein but play key roles in regulating cellular processes. NcRNAs globally affect gene expression in diverse physiological and pathological contexts. Functionally important ncRNAs act in chromatin modifications, in mRNA stabilization and translation, and in regulation of various signaling pathways. Non-alcoholic fatty liver disease (NAFLD) is a set of conditions caused by the accumulation of triacylglycerol in the liver. Studies of ncRNA in NAFLD are limited but have demonstrated that ncRNAs play a critical role in the pathogenesis of NAFLD. In this review, we summarize NAFLD's pathogenesis and clinical features, discuss current treatment options, and review the involvement of ncRNAs as regulatory molecules in NAFLD and its progression to non-alcoholic steatohepatitis (NASH). In addition, we highlight signaling pathways dysregulated in NAFLD and review their crosstalk with ncRNAs. Having a thorough understanding of the disease process's molecular mechanisms will facilitate development of highly effective diagnostic and therapeutic treatments. Such insights can also inform preventive strategies to minimize the disease's future development.

Microbial profile in bile from pancreatic and extra-pancreatic biliary tract cancer.

BACKGROUND: Dysbiotic biliary bacterial profile is reported in cancer patients and is associated with survival and comorbidities, raising the question of its effect on the influence of anticancer drugs and, recently, the suggestion of perichemotherapy antibiotics in pancreatic cancer patients colonized by the Escherichia coli and Klebsiella pneumoniae. OBJECTIVE: In this study, we investigated the microbial communities that colonize tumours and which bacteria could aid in diagnosing pancreatic and biliary cancer and managing bile-colonized patients. METHODS: A retrospective study on positive bile cultures of 145 Italian patients who underwent cholangiopancreatography with PC and EPC cancer hospitalized from January 2006 to December 2020 in a QA-certified academic surgical unit were investigated for aerobic/facultative-anaerobic bacteria and fungal organisms. RESULTS: We found that among Gram-negative bacteria, Escherichia coli and Pseudomonas spp were the most frequent in the EPC group, while Escherichia coli, Klebsiella spp, and Pseudomonas spp were the most frequent in the PC group. Enterococcus spp was the most frequent Gram-positive bacteria in both groups. Comparing the EPC and PC, we found a significant presence of patients with greater age in the PC compared to the EPC group. Regarding Candida spp, we found no significant but greater rate in the PC group compared to the EPC group (11.7% vs 1.96%). We found that Alcaligenes faecalis was the most frequent bacteria in EPC than the PC group, among Gram-negative bacterial species. CONCLUSIONS: Age differences in gut microbiota composition may affect biliary habitats in our cancer population, especially in patients with pancreatic cancer. Alcaligenes faecalis isolated in the culture of bile samples could represent potential microbial markers for a restricted follow-up to early diagnosis of extra-pancreatic cancer. Finally, the prevalence of Candida spp in pancreatic cancer seems to trigger new aspects about debate about the role of fungal microbiota into their relationship with pancreatic cancer.

Benign tumors of the chest wall.

Benign tumors of the chest wall are rare tumors that might arise from all the tissues of the chest: vessels, nerves, bones, cartilage, and soft tissues. Despite benign features, these tumors can have several histological characteristics and different behaviors. Even if they do not influence life expectancy, rarely they may have a potential risk of malignant transformation. They can cause several, oft, unspecific symptoms but more than 20% of affected patients are asymptomatic and are being diagnosed incidentally on chest radiograph or computed tomography scan. Pain is the most common described symptom. Together with a detailed medical history, a rigorous and meticulous clinical and radiological assessment is mandatory. If radiological features are unclear or in case surgery could not be performed, a biopsy should be indicated to establish a diagnosis. Radical surgical resection can often be offered to resect and cure these neoplasms, but this is might not be true for all types of tumors and, in some cases, their dimension or position might contra-indicate surgery. Given the rarity of these tumors, there is a lack of treatment's guidelines and prospective trials that include a significant number of patients. This review discusses, according to the latest evidence, the histological features and the best treatment of several chest wall benign tumors.

Sudden cardiac death and post-traumatic stress disorder: More research is needed.

Sudden cardiac death is an event which is traumatic for the individuals, who survive and their relatives. Very few research is concentrated on these survivals and the symptoms arising from post-traumatic stress disorders. In this journal, Birk et al. report on twelve eligible cardiac arrest survivors contacted, of which ten were enrolled. The authors report on heart rate variability biofeedback, which is, according to the authors, a promising non-pharmacologic approach for reducing anxiety. The intervention was comprised of daily sessions of diaphragmatic paced breathing and real-time monitoring of cardiac activity guided by a smartphone app and heart rate monitor. Ninety percent of the patients had good scores for intervention acceptability and feasibility, and 80 % reported good scores for its appropriateness and usability for reducing fear. Trait anxiety decreased significantly pre-to-post intervention. We comment on this finding highlighting other studies targeting sudden cardiac death and supporting that more research with very large randomized clinical trials is needed.

Carney complex: A clinicopathologic study on a single family from several Canadian provinces.

Carney syndrome is an autosomal dominant complex involving endocrinopathy, mucocutaneous hyperpigmentation, and different tumors, including cardiac myxomas. We report on a single family with several members affected with Carney syndrome. Family and individual medical histories were investigated in several Canadian provinces. The histology slides were also reviewed. Four family members (two young women, both sisters, their mother, and maternal grandmother) were found to harbor Carney syndrome. Everyone was presented with multiple and recurrent atrial myxomas of the heart, requiring multiple open cardiac surgeries. Breast myxomas and cutaneous hyperpigmentation were also revealed in one of the sisters and their mother. Interestingly, genetic testing was positive for the female family members and negative for the father and brother. We cannot rule out that the brother may have had a new mutation or harboring a mosaic. The young woman's brother did not have cardiac myxoma but developed a unilateral Sertoli cell tumor of testis. Carney syndrome is a rare complex multisystemic genetic disorder, including multiple and recurrent cardiac myxomas. We strongly suggest that reporting familial Carney syndrome is still critical in the 21st century to augment the awareness of this situation among clinicians and pathologists.

MASLD and aspartame: are new studies in the horizon?

Fatty liver disease has been on the rise in the past few decades, and there is no hope that it will stop. The terminology change that has been recently proposed may not be sufficient to advocate for a reduction of steatogenic foods and a change in lifestyle. A course change may be supported by the recent labeling of aspartame sweetener as a possible carcinogenic compound by the International Association for Research on Cancer (IARC), an agency of the World Health Organization (WHO). Aspartame sweeteners and other edulcorating molecular compounds besides colorings may trigger liver cancer other than fatty liver disease, despite limited data supporting it. An essential bias in human cohort studies is indeed the exclusion of all confounding factors, which may be barely impossible for human studies. In this perspective, we suggest that the activation of the NOD-like receptor-enclosing protein 3 (NLRP3) inflammasome and the stimulation of the tumor suppression gene TP53 may be critical in the progression from fatty liver to liver inflammation and liver cancer. Aspartame reduces a transcriptional coactivator, precisely the peroxisomal proliferator-initiated receptor-γ (gamma) coactivator 1-α (alpha) (or PGC1α). This coactivator upregulates mitochondrial bioformation, oxidative phosphorylation, respiratory capacity, and fatty acid ß-oxidation. Aspartame acts in this way, probably through the activation of TP53. These events have been accountable for the variations in the lipid outline in serum and total lipid storage as well as for the impairment of gluconeogenesis in the liver, as supported by the downregulation of the gluconeogenic enzymes in experimental animals, and may be relevant in humans as well.

Analysis of risk factors for community-acquired Clostridioides difficile diarrhea in children: a case-control study in Chenzhou, China.

Background: Most previous studies on Clostridium difficile infection (CDI) mainly focused on adults with underlying diseases or critical illnesses. However, the number of CDI cases in children has also significantly increased, especially the growth of community-acquired CDI, which has attracted attention. This study was conducted to examine the toxin gene characteristics and the risk factors associated with community-acquired CDI (CA-CDI) in children with diarrhea. Methods: Children with diarrhea before admission or within 48 hours of hospitalization were included in the study. Stool samples were collected from children with community-acquired diarrhea who were treated at the Children's Hospital of the First People's Hospital of Chenzhou, China from June of 2021 to June of 2022. Fluorescence real-time polymerase chain reaction was utilized to detect Clostridioides difficile (CD) toxins A (tcdA) and B (tcdB) genes as well as binary toxin gene A (cdtA) and B (cdtB) in the specimens cultured for CD. Each child with CA-CDI was matched with four control children of the same sex, age, and place of residence. Necessary clinical data were extracted from the hospital's electronic medical record system. Then, a multivariate conditional logistic regression analysis was applied to identify potential risk factors for CA-CDI. Results: Sixteen (8.3%) of the 193 stool specimens who tested positive for CD were selected for the case group, and their matching 64 control patients were in the study cohort. The breakdown of the CD genotypes of the 16 positive cases were follows: 14 (tcdA+ and tcdB+) (7.25%) and 2 (tcdA+ and tcdB-) (1.04%). The cdtA and cdtB binary toxin genes were negative in all. The results of multivariate conditional logistic regression analysis identified antibiotic use within the previous month [odds ratio (OR) =5.13; 95% confidence interval (CI): 1.65-15.91] and non-breastfeeding (OR =4.89; 95% CI: 1.11-21.53) as independent risk factors for CDI in pediatric patients experiencing community-acquired diarrhea. Conclusions: Children who had been treated with antibiotics and not breastfed were more susceptible to CDI. Therefore, in order to prevent and to control the spread of CD infection, being prudent to the aforementioned high-risk factors is strongly advocated in clinical practice.

Stillbirth Investigations: An Iconographic and Concise Diagnostic Workup in Perinatal Pathology.

Introduction Stillbirth is a dramatic event for the parents, health care team, and anyone close to the expectant parents. Multidisciplinary team (MDT) meetings are essential to improve communication in health care. We review the most frequent findings discussed at MDT meetings. Methods A PubMed search was conducted through December 2021 since the inception (1965) using clinical queries with the key terms "stillbirth" AND "investigation" AND "pathology" AND "human." The search strategy included reviews, meta-analyses, randomized controlled trials, clinical trials, and observational studies. This systematic review is based on, but not limited to, the search results. It is the experience of more than 30 years of pediatrics, obstetrics, and pathology staff. Results Two hundred and six articles were screened and complemented through the perusal of congressional activities and personal communications. Pathological findings following perinatal death can be divided into macroscopic, histologic, and placental findings. The placenta is crucial in fetal medicine and is key in determining the cause of stillbirth in a substantial number of events. Perinatal lung disease is essential to evaluate the response of newborns to extrauterine life and address newborns' outcomes appropriately. Conclusions Stillbirth remains one of the less explored areas of medicine, and we can determine the cause in a limited number of cases. Nevertheless, placental pathology is critical in the etiology discovery pathway. Accurate investigations and discussion of photography-supported findings are vital in promoting communication at MDT meetings.

Congenital segmental dilatation of the intestine: an in-depth review.

OBJECTIVE: Congenital segmental dilatation of the intestine (CSDI) is a rare gastrointestinal condition. We conducted a scoping review through MEDLINE and Google Scholar, collecting data from 1959 through August 2020 to better understand this peculiar disease. METHODS: The clinical and pathological features of 150 patients were reviewed. RESULTS: The mean age was 25.9 days, and 61.3% of patients were male. An antenatal diagnosis was made in 15.3% of patients. Predominant symptoms included abdominal distension (83.9%) and vomiting (61.3%). Pallor and anemia were associated with ileal CSDI. The most common sites of the lesion were the ileum (56%) and colon (27.3%). Associated anomalies occurred in 57.3% of the patients, of which the most common included other abnormalities of the digestive system (69.8%), abdominal wall (19.8%), and cardiovascular system (11.6%). Resection and anastomosis was performed in 83.3% of patients. Postoperative complications occurred in 10%. Normal ganglion cells were commonly found (97.3%), while muscle layer hypertrophy and atrophy were found in 14.7% and 13.3% of the patients, respectively. Abnormal interstitial cells of Cajal were identified in four patients. Death occurred in 12.7% of patients. Demise was significantly associated with the duodenal location of CSDI (Mantel-Cox test, p = 0.002). CONCLUSION: CSDI remains poorly understood, and mortality is associated chiefly with its duodenal location. Further research is needed, and biorepositories should be promptly set up to study this disease in the future better.

What is currently known about the topic? CSDI is a sporadic condition, which can represent a challenge for neonatologists.What new information is contained in this article? Pallor and anemia are found explicitly in ileal CSD, and the duodenal location of CSDI is associated with the highest mortality rate.

LipidR - a Commentary on a Lipidomics Software Platform Useful for Potential Liquid Biopsies Data Analysis.

LipidR is a recent software platform that uses R. This commentary briefly reviews the data published in the literature using this new tool for data mining and analyzing lipidomics datasets. This software is maintained by the R Foundation specifically for Statistical Computing. The R language is widely used among statisticians and data miners for developing statistical software and data analysis. LipidR is a novel open-source R package with enormous functionalities. LipidR filled three significant gaps in lipidomics. First, LipidR has the potential to mine public lipidomics datasets more efficiently and effectively. Second, the deconstructing function of the lipid name is unique to LipidR. Third, the adjustment for confounding factors, which is required for complex clinical lipidomic profiles, is implemented in LipidR. We expect LipidR will be crucial in analyzing lipidomic profiles of liquid biopsies in the near future.

COVID-19 vaccination-related autoimmune hepatitis-a perspective.

Autoimmune hepatitis (AIH) is the inflammation of the liver with clear-cut interface hepatitis and piecemeal necrosis located at the boundary between portal areas and periportal hepatocytes, and characterized by autoimmunity to hepatocytes with an increase in the antinuclear antibody. After the disastrous SARS-CoV-2 pandemic flagellated several countries, several vaccines have been commercialized and have become a ground for social responsibility. The mRNA vaccines, issued by Pfizer-BioNTech (BNT162b2) and Moderna (mRNA-1273), do not use prebuilt viruses to supply the antigen in the subject's body and are not perfect but have been useful in tackling the pandemic. Nevertheless, both myocarditis and AIH have been reported as side effects of the vaccination programs in addition to thromboembolic events. Here, we explore this topic and give a data-based perspective, gathering a comparison between the titin protein of the sarcomere and myocarditis. The isolation of a Drosophila gene using the serum from a patient with autoimmune scleroderma recognized an epitope on chromosomes (condensed mitotic form) in both human cultured cells and early Drosophila embryos. It revealed that this gene encodes a Drosophila homolog of the vertebrate titin (D-Titin). Moreover, anti-titin antibodies have been found in a subset of patients with myasthenia gravis, a neuromuscular junction disease that is mostly associated with autoimmune antibodies, such as the anti-acetylcholine receptor antibody. The co-existence of myasthenia gravis and autoimmune hepatitis is rare, and a cohort of patients with myasthenia gravis anti-titin antibodies seems to be highly relevant. In consideration of these data and the number of patients who may not be symptomatic, we postulated that autoimmune phenomena may not be exceedingly rare, following the administration of mRNA technology-based vaccines, and a balance between pros and cons in administrating boosters is critical.

Intestinal Lesions Due to Rhodococcus equi in a Patient With Advanced Retroviral Disease and Pulmonary Infection: A Case of Colonic Malakoplakia.

In humans, Rhodococcus equi (R. equi) is a zoonotic infection usually involving immunocompromised subjects, only rarely affecting immunocompetent subjects. Herein, we describe an R. equi infection in a 50-year-old Russian man with acquired immune deficiency syndrome (AIDS) who presented with pulmonary cavitary lesions and clinical manifestation of colonic malakoplakia. A colonoscopy examination showed ulceration and mucosal erosion, and the histological findings confirmed the colonic malakoplakia. The patient recovered from pulmonary and gastrointestinal disease after four weeks of antibiotic treatment with intravenous ciprofloxacin and oral azithromycin and also underwent subsequent long-term oral antibiotic treatment to achieve clinical and immune restoration after antiretroviral therapy. Infectious disease pathology subspecialties should always consider R. equi chronic infection as a cause of malakoplakia in patients with AIDS. As only a few cases of colonic malakoplakia associated with R. equi are reported in the literature, these cases are important to describe, especially for clinical and treatment management.

A Serological Analysis of the Humoral Immune Responses of Anti-RBD IgG, Anti-S1 IgG, and Anti-S2 IgG Levels Correlated to Anti-N IgG Positivity and Negativity in Sicilian Healthcare Workers (HCWs) with Third Doses of the mRNA-Based SARS-CoV-2 Vaccine: A Retrospective Cohort Study.

BACKGROUND: With SARS-CoV-2 antibody tests on the market, healthcare providers must be confident that they can use the results to provide actionable information to understand the characteristics and dynamics of the humoral response and antibodies (abs) in SARS-CoV-2-vaccinated patients. In this way, the study of the antibody responses of healthcare workers (HCWs), a population that is immunocompetent, adherent to vaccination, and continuously exposed to different virus variants, can help us understand immune protection and determine vaccine design goals. METHODS: We retrospectively evaluated antibody responses via multiplex assays in a sample of 538 asymptomatic HCWs with a documented complete vaccination cycle of 3 doses of mRNA vaccination and no previous history of infection. Our sample was composed of 49.44% males and 50.56% females, with an age ranging from 21 to 71 years, and a mean age of 46.73 years. All of the HCWs' sera were collected from April to July 2022 at the Sant'Elia Hospital of Caltanissetta to investigate the immunologic responses against anti-RBD, anti-S1, anti-S2, and anti-N IgG abs. RESULTS: A significant difference in age between HCWs who were positive and negative for anti-N IgG was observed. For anti-S2 IgG, a significant difference between HCWs who were negative and positive compared to anti-N IgG was observed only for positive HCWs, with values including 10 (U/mL)-100 (U/mL); meanwhile, for anti-RBD IgG and anti-S1 IgG levels, there was only a significant difference observed for positive HCWs with diluted titers. For the negative values of anti-N IgG, among the titer dilution levels of anti-RBD, anti-S1, and anti-S2 IgG, the anti-S2 IgG levels were significantly lower than the anti-RBD and anti-S1 levels; in addition, the anti-S1 IgG levels were significantly lower than the anti-RBD IgG levels. For the anti-N IgG positive levels, only the anti-S2 IgG levels were significantly lower than the anti-RBD IgG and anti-S1 IgG levels. Finally, a logistic regression analysis showed that age and anti-S2 IgG were negative and positive predictors of anti-N IgG levels, respectively. The analysis between the vaccine type and mixed mRNA combination showed higher levels of antibodies in mixed vaccinated HCWs. This finding disappeared in the anti-N positive group. CONCLUSIONS: Most anti-N positive HCWs showed antibodies against the S2 domain and were young subjects. Therefore, the authors suggest that including the anti-SARS-CoV-2-S2 in antibody profiles can serve as a complementary testing approach to qRT-PCR for the early identification of asymptomatic infections in order to reduce the impact of potential new SARS-CoV-2 variants. Our serological investigation on the type of mRNA vaccine and mixed mRNA vaccines shows that future investigations on the serological responses in vaccinated asymptomatic patients exposed to previous infection or reinfection are warranted for updated vaccine boosters.

Computer-assisted diagnostics.

Healthcare is at the edge of a profound renovation or collapse due to the rapid inflow of machine learning protocols and procedures able to optimize several processes. Clinical trials are key for the progress of science and the correct interpretation of data. Rickard et al., in this journal, report that data on misidentification rates in medical trials are scarce. In five trials involving more than 800 blood or histology specimens examined, data clarification forms (DCFs) were issued for 21% of instances, and 67% were related to sample identification. The authors suggest that a suitable number of de- recognized data points is critical. Moreover, a formalized process involving the specimen accession employed in routine care is key to mitigate recognition errors and their potential profound impact on clinical research and outcome. We fully agree with the authors and their report is highly relevant today that we face transformation in healthcare. We suggest that 3D barcoding may mitigate several issues on misidentification.