Specific Diagnostic Tests Research Articles

P058 Proteomic analysis-based discovery of a novel biomarker that differentiates intestinal Behçet’s disease from Crohn’s disease

Abstract Background Intestinal Behçet’s disease (BD) and Crohn’s disease (CD) present similar manifestations, but there are no specific pathognomonic clinical, laboratory, or histological diagnostic tests to differentiate intestinal BD from CD. We used a proteomic approach to discover a novel diagnostic biomarker specific to intestinal BD. Methods The colon mucosa tissue samples were obtained using colonoscopy-guided biopsy of the affected bowel from patients with intestinal BD or CD at the Inflammatory Bowel Disease Clinic of Severance Hospital, Seoul, Korea. Peptides from seven intestinal BD and seven CD patients were extracted and labeled using tandem mass tag (TMT) reagents. The labeled peptides were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The differentially expressed proteins were further validated using immunohistochemical (IHC) analysis with tissue samples and an ELISA test with serum samples from 20 intestinal BD and 20 CD patients. Results A total of 3,266 proteins were identified using TMT/LC-MS/MS-based proteomic quantification, including 39 candidate proteins differentially expressed between intestinal BD and CD. Beta-2 glycoprotein 1 (APOH) and maltase-glucoamylase (MGAM) showed significantly higher intensity in the IHC staining of the intestinal BD tissues than that of CD tissues. Furthermore, the serum MGAM level was significantly higher in patients with intestinal BD than in patients with CD. Conclusion Our proteomic analysis revealed that some proteins were differentially expressed in intestinal BD patients compared to CD patients. Differential MGAM expression in intestinal BD suggests its role as a potential novel diagnostic biomarker in the differentiation of intestinal BD from CD.

Proteomic analysis-based discovery of a novel biomarker that differentiates intestinal Beh\xe7et\u2019s disease from Crohn\u2019s disease

Intestinal Behçet’s disease (BD) and Crohn’s disease (CD) present similar manifestations, but there are no specific diagnostic tests to differentiate them. We used a proteomic approach to discover novel diagnostic biomarkers specific to intestinal BD. Colon mucosa tissue samples were obtained from patients with intestinal BD or CD using colonoscopy-guided biopsy of the affected bowel. Peptides from seven intestinal BD and seven CD patients were extracted and labeled using tandem mass tag (TMT) reagents. The labeled peptides were identified and quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The proteins were further validated using immunohistochemical (IHC) analysis with tissue samples and an ELISA test with serum samples from 20 intestinal BD and 20 CD patients. Using TMT/LC–MS/MS-based proteomic quantification, we identified 39 proteins differentially expressed between intestinal BD and CD. Beta-2 glycoprotein 1 (APOH) and maltase-glucoamylase (MGAM) showed higher intensity in the IHC staining of intestinal BD tissues than in CD tissues. The serum MGAM level was higher in intestinal BD patients. Proteomic analysis revealed that some proteins were differentially expressed in patients with intestinal BD compared with those with CD. Differential MGAM expression in intestinal BD suggests its role as a potential novel diagnostic biomarker.

One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei

This study highlights the development of a multiplex real-time loop-mediated isothermal amplification assay. The developed assay employed a dual-function oligonucleotide (DFO) which simultaneously monitors the emitted amplification signals and accelerates the amplification process. The DFO was a modification of loop primer (LP); the 5′-end and 3′-end of the LP was tagged with fluorophore and quencher, respectively. The DFO was quenched in its unbound state and fluoresces only when it anneals to the specific target during the amplification process. With the same working mechanism as LP, DFO allowed the detection of target genes in less than 1 h in a real time monitoring system. We demonstrated this detection platform with Burkholderia pseudomallei, the causative agent of melioidosis. An internal amplification control (IAC) was incorporated in the assay to rule out false negative result and to demonstrate that the assay was successfully developed in a multiplex system. The assay was 100% specific when it was evaluated against 96 B. pseudomallei clinical isolates and 48 other bacteria species. The detection limit (sensitivity) of the developed assay was 1 fg/μl of B. pseudomallei genomic DNA and 18.2 CFU/ml at the bacterial cell level. In spiked blood samples, the assay's detection limit was 14 CFU/ml. The assay's diagnostic evaluation showed 100% diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value. An integrated multiplex LAMP and real-time monitoring system was successfully developed, simplifying the workflow for the rapid and specific nucleic acid diagnostic test.

In Search of the Holy Grail: A Specific Diagnostic Test for Rheumatic Fever.

Current diagnosis of Acute Rheumatic Fever and Rheumatic Heart Disease (ARF/RHD) relies on a battery of clinical observations aided by technologically advanced diagnostic tools and non-specific laboratory tests. The laboratory-based assays fall into two categories: those that (1) detect “evidence of preceding streptococcal infections” (ASOT, anti-DNAse B, isolation of the Group A Streptococcus from a throat swab) and (2) those that detect an ongoing inflammatory process (ESR and CRP). These laboratory tests are positive during any streptococcal infection and are non-specific for the diagnosis of ARF/RHD. Over the last few decades, we have accumulated considerable knowledge about streptococcal biology and the immunopathological mechanisms that contribute to the development, progression and exacerbation of ARF/RHD. Although our knowledge is incomplete and many more years will be devoted to understanding the exact molecular and cellular mechanisms involved in the spectrum of clinical manifestations of ARF/RHD, in this commentary we contend that there is sufficient understanding of the disease process that using currently available technologies it is possible to identify pathogen associated peptides and develop a specific test for ARF/RHD. It is our view that with collaboration and sharing of well-characterised serial blood samples from patients with ARF/RHD from different regions, antibody array technology and/or T-cell tetramers could be used to identify streptococcal peptides specific to ARF/RHD. The availability of an appropriate animal model for this uniquely human disease can further facilitate the determination as to whether these peptides are pathognomonic. Identification of such peptides will also facilitate testing of potential anti-streptococcal vaccines for safety and avoid potential candidates that may pre-dispose potential vaccine recipients to adverse outcomes. Such peptides can also be readily incorporated into a universally affordable point of care device for both primary and tertiary care.

Peptide microarray-based analysis of antibody responses to SARS-CoV-2 identifies unique epitopes with potential for diagnostic test development.

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)‐2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross‐reactivity between SARS‐CoV‐2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n = 24 patient samples and n = 12 control samples were screened for antibodies against the entire SARS‐CoV‐2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, OC43, and 229E. While widespread cross‐reactivity was revealed across several immunodominant regions of S and N, IgG binding to several SARS‐CoV‐2‐derived peptides provided statistically significant discrimination between COVID‐19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID‐19‐specific diagnostic antibody tests.

Umbilical cord miRNAs to predict neonatal early onset sepsis.

ObjectiveTo determine if miRNA (miR) expression in umbilical cord blood and umbilical cord tissue differs between neonates with early onset sepsis (EOS) versus neonates without true infection.MethodsRetrospective case-control study design of human patients with EOS (n = 8), presumed sepsis (N = 12) and non-infected control patients (N = 21). Differential expression of >300 miRs was examined using the MIHS-3001ZE-miScript miRNA PCR Array Human miFinder 384HC. Expression levels of miRs were normalized using the global Ct mean of expressed miR and compared between groups. Data analysis was performed using GeneGlobe data analysis software. Ratios of over and under-expressed miRs were calculated and compared between groups using receiver operating characteristic (ROC) curves.ResultsBoth umbilical cord plasma and umbilical cord tissue revealed several miRs with differential expression with little overlap between the two specimen types. The most overexpressed miR in plasma of EOS patients was miR-211-5p and the most overexpressed in EOS cord tissue was miR-223-5p. ROC curves comparing the ratios of over and under-expressed miRs for EOS patients and controls resulted in an area under the curve of 0.787 for cord plasma (miR-211-5p/miR-142-3p) and 0.988 for umbilical cord tissue (miR-223-5p/miR-22-3p), indicating good discrimination.ConclusionsmiRs show differential expression in EOS versus non-infected controls and presumed sepsis. A ratio of over and under-expressed miRs can provide a potentially sensitive and specific diagnostic test for EOS.

The Efficiency of Manual Therapy and Sacroiliac and Lumbar Exercises in Patients with Sacroiliac Joint Dysfunction Syndrome

Manual therapy, exercise therapy, and the combination of these 2 are common treatments for sacroiliac joint dysfunction syndrome. The effects of these treatments have been discussed in several studies; the superiority of one over the other for patients with sacroiliac joint dysfunction syndrome is still the subject of discussion. This study aims to assess the effects of manual therapy for sacroiliac joints, sacroiliac joints home-based exercises, and home-based lumbar exercises. A comparative, prospective, single-blind, randomized, controlled trial. This trial was conducted at a single center at the Istanbul University, Istanbul Medical Faculty, Department of Physical Medicine and Rehabilitation. Within the scope of this study, 69 women diagnosed with sacroiliac joint dysfunction syndrome through specific sacroiliac joints clinical diagnostic tests were randomized into 3 groups. The first group was assigned manual therapy and a sacroiliac joints home-based exercise program (n = 23), the second group was assigned sacroiliac joints manual therapy and a home-based lumbar exercise program (n = 23), and the third group was assigned a home-based lumbar exercise program (n = 23). All patients who participated in the study were evaluated at the beginning of the study and on the twenty-eighth and ninetieth day. All 3 groups showed a significant decrease in the sacroiliac joints -related pain parameter, which is checked with the visual analogue scale (P < 0.05) after the treatment. The Gillet test, Vorlauf test, Posterior Shear test, Compression test, and irritation Point tests after the treatment yielded a significant (P < 0.05) negative trend in all groups. Short Form-36 health survey for screening form, Modified Oswestry Pain Questionnaire, and Douleur Neuropathique 4 questions patient interview questionnaire for the assessment of neuropathic pain forms revealed a significant (P < 0.05) improvement in patients' complaints after the treatment in all 3 groups. Significant improvement in patients with sacroiliac joint dysfunction syndrome in all 3 groups was identified after the treatment. The absence of a healthy control group is one of the important limitations of the study. Manual therapy is effective in the long term in sacroiliac joint dysfunction syndrome. Adding specific exercises for sacroiliac joints to the sacroiliac joints manipulation treatment further increases this effectiveness.

Consideration of Vector-Borne and Zoonotic Diseases during Differential Diagnosis.

Recognition and reporting of vector-borne and zoonotic disease (VBZD) cases is largely dependent upon the consideration of such diseases by healthcare practitioners during the initial diagnosis and ordering of specific confirmative diagnostic tests. This study was conducted to assess the general knowledge and understanding of VBZD transmission and clinical presentation. Healthcare practitioners were surveyed to determine the extent of training and educational experiences they received relative to VBZDs, and their likelihood to consider such diseases during differential diagnoses. In addition, an assessment of their knowledge of arthropod species that may transmit VBZD pathogens was conducted. Having postprofessional school training relevant to VBZDs significantly influenced diagnostic accuracy for such disease cases based on the presented clinical signs and symptoms. The prevalence of VBZDs in the United States likely is significantly underestimated. The authors suggest the enhancement of VBZD-focused education as an important initiative that would significantly improve timely diagnosis, treatment, and, ultimately, prevention of these diseases.

Kawasaki Disease and Systemic Juvenile Idiopathic Arthritis - Two Ends of the Same Spectrum.

Kawasaki disease (KD) and systemic juvenile idiopathic arthritis (sJIA) are two distinct systemic inflammatory diseases of childhood. Each diagnosis is based on criteria, but numerous clinical features are overlapping. As no specific diagnostic tests are available, differentiation between both disease entities can be challenging. Here, we describe the disease course of patients with co-diagnosis of both KD and sJIA (KD/sJIA). All our KD (n = 1765) and sJIA (n = 112) cases were critically reviewed for co-diagnosis of KD/sJIA. Eight KD/sJIA cases were identified and their clinical presentation, treatment regimens, coronary artery outcome and complications are herein described. Each KD/sJIA patient fulfilled diagnostic criteria for KD and for sJIA. Ongoing fever, rash and arthritis were present in each patient. The KD/sJIA patients had recalcitrant KD requiring multiple doses of intravenous immunoglobulin and steroids. Five patients had coronary artery dilatation at KD diagnosis, which resolved in all by 6 weeks. Pericardial effusion was present in 5 patients. One KD/sJIA patient developed macrophage activation syndrome. In conclusion, a small proportion (0.5%) of our KD patients evolved into sJIA, and 7% of our sJIA population presented initially as KD. KD/sJIA patients were characterized by a recalcitrant KD course and a high prevalence of coronary artery dilatation. Patients with co-diagnoses may provide a clue to potentially shared immunopathology in KD and sJIA, leading us to posit that both entities may be part of the same clinical spectrum.

Specific Tests and Inflammatory Biomarkers in the Evaluation of Brucellosis Disease

Brusellosis is the world's most common bacterial zoonotic infection and is still endemic in many developing countries. The clinical appearance of brucellosis is not specific, but the course and severity of the infection varies. In this study, it was aimed to determine the relationship between laboratory parameters and clinical response and organ involvement in patients with Brucellosis diagnosed with specific diagnostic tests. In the study, 100 patients without previous diagnosis of Brucellosis who have admitted to the Department of Infectious Diseases and Clinical Microbiology and had positive Brucella tube agglutination tests and whose clinic was compatible with Brucellosis were evaluated prospectively. Patients were invited to be checked in the 1st, 2nd and 6th weeks . Patients with hip pain and low back pain were evaluated with sacroiliac magnetic resonance imaging (MRI) and lumbar MRI for sacroiliitis and spondylodiscitis. Patients with liver and bone marrow involvements, sacroiliitis, spondylodiscitis and orchitis were recorded as the patients with organ involvement. After six weeks, the decline of the complaints was considered as a clinical response. In the 6th week of the treatment, it was observed that platelet distribution width (PDW) and mean platelet volume (MPV) levels were lower in patients with a clinical response compared to the patients with no clinical response which was statistically significant (p= 0.01, p= 0.02). Platelet and platelet lymphocyte ratio (PLR) level in patients with organ involvement in the 1st and 6th weeks of the treatment, were observed to be lower than the patients without organ involvement which was statistically significant (week 1: p= 0.001, p= 0.01; week 6: p= 0.03, p= 0.01). Among patients with organ involvement and non-organ involvement, the area under the curve was 66% in the ROC curve analysis for PLR at the onset of the treatment. When the cut-off value was taken as 128.8%, the sensitivity was 55%, and the specificity was 78%. Depending on the level of platelet at the beginning of the treatment, in the ROC curve analysis carried out among patients with organ involvement and non-organ involvement, the area under the curve was 73% and when the cut off value was taken as 256000, the sensitivity was 71%, and the specificity was 68%. In the 6th week of the treatment, the area under the curve was observed as 67% in the ROC curve analysis of the PDW level among patients from whom clinical responses were received and not received. When the cut-off value was taken as 10.75%, the sensitivity was 65%, and the specificity was 70%. In the ROC curve analysis of the MPV value for clinical response, the area under the curve was 66%, and when the cut-off value was taken as 9.95, the sensitivity was observed as 52%, and the specificity was 74%. As a result, in the evaluation of the clinical response, which is important in the termination of the treatment in patients with Brucellosis, the use of MPV and PDW values in the evaluation of organ involvement and platelet level and PLO in the follow up, are cheap, easily accessible biomarkers that can be used clinically.

Diagnostic Accuracy of High-Resolution CT in Early Diagnosis of COVID-19 Patients with Comparison to Reverse Transcriptase-PCR

Objective: To determine the accuracy of high-resolution CT (HRCT) in early diagnosis of COVID-19 in comparison to Reverse-Transcription Polymerase Chain Reaction (RT-PCR). Study Design and Setting: This was a retrospective study, conducted in Department of Radiology, PNS Shifa Hospital, Karachi from April 2020 to June 2020. Methodology: A total of 115 patients were included in the study who presented with symptoms. Patient’s data such as demographic details, clinical presentations, RT-PCR reports and HRCT reports were collected from the record of the hospital. Data was entered and analyzed using SPSS-25. Results: Out of 115 study participants, 98(85.2%) were male and 17(14.8%) were female with the average age of 50.53 (±18.64) years. Fever along with cough was the most common clinical presentation, presented by 91(79.1%) subjects. In comparison to RT-PCR, sensitivity of HRCT was 96.2%, its specificity standing at 60% with the overall accuracy of 93.19%. Moreover, mortality Rate of these symptomatic patients was 11.30% (13 out of 115). The average age of the expired patients was 67.07 (±11.45) years and the age of discharged patients was 48.31 (±17.92) years. The difference in age was statistically significant (p-value &lt;0.001). Conclusion: Highly specific and sensitive diagnostic tests for COVID-19 have not yet been developed. High resolution CT chest has high sensitivity for early diagnosis of COVID-19, hence HRCT should be incorporated as a primary tool in assessment criteria of hospitalized patients for early diagnosis of COVID-19.

NAFLD: a multi-faceted morbid spectrum with uncertain diagnosis and complicated management.

Non-alcoholic steatohepatitis (NASH) can be considered the pandemic of these times, because of its global diffusion alongside obesity, diabetes, and metabolic dysfunction. It is a disease that often poses many difficulties, since making an early diagnosis is often impossible since specific diagnostic tests and criteria are missing: so, it needs a high degree of suspicion. Most of the times the evolution to its more severe and terminal step, NASH cirrhosis, is unavoidable and so are the social pressure on health system and economic consequences it brings back. The aim of this article is to provide a review of the literature about both non-alcoholic fatty liver disease (NAFLD) and NASH, thus structuring a wide, comprehensive, 360-degree work with a focus on all major aspects of NAFLD, spanning from diagnosis, physiopathology and its repercussions on liver transplantation. Moreover, we also focus on patients related issues both in pre- and post-transplant management (when these patients are listed for liver transplant). NAFLD and NASH are a contemporary plague, and an exhaustive knowledge of the problem throughout all its aspects is necessary in order to lower economic weight that metabolic issues bring back and to have an open view to possible solutions to all management issues that NASH patients have and that are often prohibitive to a definitive cure (for example cardiovascular risk in patients otherwise eligible to liver transplantation). We aim to offer a complete view on the actual knowledge about NAFLD and NASH, by an extensive review of the literature.

Cost Savings From a Policy to Diagnose and Prevent Transmission of Adenoviral Conjunctivitis in Employees of a Large Academic Medical Center

Adenoviral conjunctivitis is highly contagious, can be associated with systemic infections, and can cause chronic visual impairment. It accounts for a large proportion of acute conjunctivitis. Outbreaks of epidemic keratoconjunctivitis (EKC) are costly in terms of productivity loss from work furloughs and spread to patients and have resulted in clinic and departmental closures. To examine the institutional cost savings of a policy to diagnose adenoviral conjunctivitis and triage and furlough medical center employees with this condition to prevent outbreaks. This quality improvement study assessed Johns Hopkins Medicine employees with red eye from November 1, 2011, through October 31, 2018, who were triaged at the occupational health clinic whose conditions were diagnosed using polymerase chain reaction (PCR) validated for adenoviral conjunctivitis. Only employees with positive PCR test results were furloughed, with furlough length tailored to subtype (a minimum of 2 weeks for EKC and 1 week otherwise). Total number of furloughs avoided and cost savings associated with reducing unnecessary furloughs. Of 2142 employees with red eye, 1520 (71.0%) underwent PCR testing; 130 (8.6%) had positive adenoviral PCR test results, of whom 41 (31.5%) had EKC. Furloughing 130 employees with positive PCR test results vs furloughing all 1520 employees clinically suspected of having adenoviral conjunctivitis represented an estimated annual savings of $442 073, or $3 094 511 during 7 years. The cost of performing PCR on employees suspected of having adenoviral conjunctivitis was 5.0% of the cost associated with furloughing all employees with red eye. No outbreaks occurred. In this quality improvement study, this policy, notable for development and use of PCR for adenoviral conjunctivitis on a large scale, resulted in substantial cost savings from fewer work furloughs compared with the number of employees who would have been furloughed based on clinical diagnosis. These results may provide impetus for policy adoption by other institutions and for development of a rapid, sensitive, and specific diagnostic test for adenoviral conjunctivitis.

Public awareness and knowledge of liver health and diseases in Singapore.

Background and AimDespite efforts in controlling and managing liver diseases, significant health issues remain. This study aims to evaluate the degree of public awareness and knowledge regarding liver health and diseases in Singapore.MethodsA cross‐sectional, self‐reported, web‐based questionnaire was administered to 500 adult individuals. Questionnaire items pertained to knowledge and awareness of overall liver health, liver diseases and their associated risk factors.ResultsSixty‐four percent of respondents were ≥35 years old and 54.0% were male. While majority agreed that regular screening was important for liver health (91.2%), only 65.4% attended health screening within recent 2 years. Hepatitis B had more awareness than hepatitis C among the respondents. About 70% agreed the consequences of viral hepatitis included liver cirrhosis, failure, and/or cancer. Yet, only 15% knew hepatitis C is not preventable by vaccination and more than half mistaken hepatitis B and C are transmissible via contaminated or raw seafood. Despite 75% being aware of non‐alcoholic fatty liver disease, many were not aware of the related risk factors and complications. Awareness of specific screening and diagnostic tests for liver health was poor as one‐fifth correctly identified the diagnostic tests for viral hepatitis. Preferences for doctor's consultation, TV, or newspapers (online) as information channels contrasted those currently used in the public health education efforts.ConclusionsThe levels of understanding of liver diseases, risk factors, and potential complications are suboptimal among the Singapore public. More public education efforts aligned with respondents' information‐seeking preferences could facilitate addressing misperceptions and increase knowledge about liver diseases.

Diagnosis and Management of Severe Asthma in Switzerland: Analysis of Survey Results Conducted with Specialists and General Practitioners

Background: Severe asthma commonly affects 5–10% of the asthmatic population and accounts for approximately 50% of the overall asthma costs. Objective: This analysis investigated how severe asthma is diagnosed, treated, and managed by specialists and general practitioners (GPs) in Switzerland. Methods: Two surveys, one each among specialists (N = 44) and GPs (N = 153), were conducted to understand their self-perception on diagnosis, treatment, and management of severe asthma. Results: Fifty-five percent of the specialists felt very confident and 43% confident in recognizing the symptoms of severe asthma and diagnosing severe asthma. In contrast, 9% of the GPs were very confident and 59% confident in diagnosing severe asthma. More specific diagnostic tests for severe asthma, like total and specific immunoglobulin E levels and measurement of the fraction of exhaled nitric oxide, were run by specialists (χ2 = 171.4; df = 15, p < 0.001). GPs and specialists were using different measurements to assess severe asthma (χ2 = 385.2; df = 13, p < 0.001) and their prescribing patterns differed significantly (χ2 = 189.8; df = 10, p < 0.001). GPs referred patients with severe asthma if the diagnosis was unclear (24%), if treatment failure occurred (26%), and if the patients were at high risk (41%). Conclusions: Oral corticosteroids (OCSs) are considered as background therapy for severe asthma by GPs and specialists. In order to reduce the OCS burden, there is a need to improve the awareness for other add-on therapies. A joint collaboration between GPs and specialists is the key to leverage therapeutic strategies together.

Artritis psoriásica

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy linked to the presence of cutaneous psoriasis and usually-negative rheumatoid factor (RF). The development of this disease depends on predisposing genetic factors, immunological determinants that perpetuate the local inflammatory cycle, and the participation of the Th1/Th17 pathways and those of the IL-9, IL-12/23 and IL-17 interleukins. Part of PsA genesis has been explained by association studies, but knowledge about the mechanisms that make it possible is currently incomplete. There are no specific diagnostic tests; diagnosis is based on the Classification Criteria for Psoriatic Arthritis (CASPAR) scheme, backed by imaging techniques. The last few years have seen great strides in immunosuppressive therapy for PsA, with the development of new treatments against different targets.

Urinary Extracellular Vesicles as Potential Biomarkers for Urologic Cancers: An Overview of Current Methods and Advances.

Simple SummaryThe diagnostic and monitoring techniques used for urologic cancers comprise a group of invasive methodologies that still lack sensitivity and specificity. Therefore, the search for a non-invasive alternative is of extreme importance. Urinary extracellular vesicles are an emerging source of biomarkers that have the potential to be used in cancer detection and management, in a minimally invasive way. However, the increasing interest, allied to the absence of standardization and consensus in strategies to isolate and characterize these vesicles, results in a vast list of candidate biomarkers that present no significant overlap. In this review, we show the variability in the methods implemented to obtain these vesicles and focus on microRNA and protein-derived urinary extracellular vesicles as candidate biomarkers for prostate, bladder and kidney cancers.Urologic cancers are a heterogeneous group of tumors, some of which have poor prognosis. This is partly due to the unavailability of specific and sensitive diagnostic techniques and monitoring tests, ideally non- or minimally invasive. Hence, liquid biopsies are promising tools that have been gaining significant attention over the last decade. Among the different classes of biomarkers that can be isolated from biofluids, urinary extracellular vesicles (uEVs) are a promising low-invasive source of biomarkers, with the potential to improve cancer diagnosis and disease management. Different techniques have been developed to isolate and characterize the cargo of these vesicles; however, no consensus has been reached, challenging the comparison among studies. This results in a vast number of studies portraying an extensive list of uEV-derived candidate biomarkers for urologic cancers, with the potential to improve clinical outcome; however, without significant validation. Herein, we review the current published research on miRNA and protein-derived uEV for prostate, bladder and kidney cancers, focusing on different uEV isolation methods, and its implications for biomarker studies.

Diagnostic Challenges in Pediatric Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune dysregulation that encompasses a broad range of underlying genetic diseases and infectious triggers. Monogenic conditions, autoimmune diseases, and infections can all drive the phenotype of HLH and associated immune hyperactivation with hypercytokinemia. A diagnosis of HLH usually requires a combination of clinical and laboratory findings; there is no single sensitive and specific diagnostic test, which often leads to “diagnostic dilemmas” and delays in treatment initiation. Ferritin levels, one of the most commonly used screening tests, were collected across a large tertiary care pediatric hospital to identify the positive predictive value for HLH. Herein, we present several cases that illustrate the clinical challenges of confirming an HLH diagnosis. Additionally, we report on the utility of establishing a formal multi-disciplinary group to aid the prompt diagnosis and treatment of patients presenting with HLH-like pathophysiologies.

Recent advances in the diagnosis and management of amyloid cardiomyopathy.

Amyloidosis is a disorder characterized by misfolded precursor proteins that form depositions of fibrillar aggregates with an abnormal cross-beta-sheet conformation, known as amyloid, in the extracellular space of several tissues. Although there are more than 30 known amyloidogenic proteins, both hereditary and non-hereditary, cardiac amyloidosis (CA) typically arises from either misfolded transthyretin (ATTR amyloidosis) or immunoglobulin light-chain aggregation (AL amyloidosis). Its prevalence is more common than previously thought, especially among patients with heart failure and preserved ejection fraction (HFpEF) and aortic stenosis. If there is a clinical suspicion of CA, focused echocardiography, laboratory screening for the presence of a monoclonal protein (serum and urinary electrophoresis with immunofixation and serum free light-chain ratio), and cardiac scintigraphy with 99mtechnetium-labeled bone-tracers are sensitive and specific initial diagnostic tests. In some cases, more advanced/invasive techniques are necessary and, in the last several years, treatment options for both AL CA and ATTR CA have rapidly expanded. It is important to note that the aims of therapy are different. Systemic AL amyloidosis requires treatment targeted against the abnormal plasma cell clone, whereas therapy for ATTR CA must be targeted to the production and stabilization of the TTR molecule. It is likely that a multistep treatment approach will be optimal for both AL CA and ATTR CA. Additionally, treatment of CA includes the management of restrictive cardiomyopathy with preserved or reduced ejection fraction in addition to treating the amyloid deposition. Future studies are necessary to define optimal management strategies for AL CA and ATTR CA and confirm cardiac response to therapy.

The occurrence and clinical importance of infectious stage of Echinocephalus (Nematoda: Gnathostomidae) larvae in selected Australian edible fish.

Cases of gnathostomiasis, an infection caused by consuming infected seafood, have been reported in Australia. However, doubt exists over the validity of these diagnoses as there are no reports of Gnathostoma spp. in Australian teleost fish. Also, the diagnoses in human cases were based on a serological test developed in Thailand. The specificity and sensitivity of this test in non-endemic areas are uncertain. Interestingly, parasites belonging to the genus Echinocephalus, which morphologically are very similar to Gnathostomum, are commonly found in Australian fish and shellfish and can potentially infect humans. The aim of this study was to determine the occurrence of these zoonotic nematodes within commercial fish and to characterise nematode larvae in order to provide insights into the specific identity of the potential causative agents of gnathostomiasis in Australia. Six edible fish species (n=163) were examined. Gnathostomid-type larvae were found only in Acanthopagrus australis and Rhabdosargus sarba. Detailed examination and sequence data suggested parasite larvae belonged to the genus Echinocephalus. Further investigation of the occurrence of zoonotic nematodes within marine environments and observation of their spatial and temporal patterns will help raise awareness of the significance of this food safety issue within global fishing industries and health sectors. The accurate identification of zoonotic nematodes is a key component of disease surveillance and control. This information can also be used to develop specific and sensitive diagnostic test.