Marathon-Induced Cardiac Strain as Model for the Evaluation of Diagnostic microRNAs for Acute Myocardial Infarction

Background: The current gold standard biomarker for myocardial infarction (MI), cardiac troponin (cTn), is recognized for its high sensitivity and organ specificity; however, it lacks diagnostic specificity. Numerous studies have introduced circulating microRNAs as potential biomarkers for MI. This study investigates the MI-specificity of these serum microRNAs by investigating myocardial stress/injury due to strenuous exercise. Methods: MicroRNA biomarkers were retrieved by comprehensive review of 109 publications on diagnostic serum microRNAs for MI. MicroRNA levels were first measured by next-generation sequencing in pooled sera from runners (n = 46) before and after conducting a full competitive marathon. Hereafter, reverse transcription quantitative real-time PCR (qPCR) of 10 selected serum microRNAs in 210 marathon runners was performed (>10,000 qPCR measurements). Results: 27 potential diagnostic microRNA for MI were retrieved by the literature review. Eight microRNAs (miR-1-3p, miR-21-5p, miR-26a-5p, miR-122-5p, miR-133a-3p, miR-142-5p, miR-191-5p, miR-486-3p) showed positive correlations with cTnT in marathon runners, whereas two miRNAs (miR-134-5p and miR-499a-5p) showed no correlations. Upregulation of miR-133a-3p (p = 0.03) and miR-142-5p (p = 0.01) went along with elevated cTnT after marathon. Conclusion: Some MI-associated microRNAs (e.g., miR-133a-3p and miR-142-5p) have similar kinetics under strenuous exercise and MI as compared to cTnT, which suggests that their diagnostic specificity could be limited. In contrast, several MI-associated microRNAs (miR-26a-5p, miR-134-5p, miR-191-5p) showed different release behavior; hence, combining cTnT with these microRNAs within a multi-marker strategy may add diagnostic accuracy in MI.

Validity of Chemiluminescent Immunoassay Serology Test for Anti-Sars Cov-2 Antibodies IgM and IgG 1

In December 2019, an outbreak of acute pneumonia occurred in Wuhan, China. The disease was transmitted betweenhumans through droplets (coughing or sneezing) of infected patients, causing this outbreak to spread rapidly in variouscountries in the world, including Indonesia. On February 11, 2020, WHO announced the pneumonia was caused byCoronavirus Disease 2019 (COVID-19), which was caused by a new type of Coronavirus, the SARS-CoV-2. A rapid andaccurate diagnosis is critical for the control of the COVID-19 outbreak. The widely used test is a serology-based test thatdetects the presence of SARS-CoV-2 IgM/IgG antibodies in the patient's body. One of the methods used for this test isChemiluminescent Immunoassay (CLIA). This study aimed to determine the reliability of CLIA. The study was conductedfrom August to September 2020. The number of samples was 63 patients' serum. Polymerase chain reaction examination atHusada Utama Hospital, Surabaya, revealed that 21 patients were confirmed positive for COVID-19 with positive PCRresults, and 42 patients were healthy with negative COVID-19 results. The results showed that IgM had a diagnosticsensitivity of 85.7%, diagnostic specificity of 92.8%, a positive predictive value of 85.7%, a negative predictive value of 92.8%,and accuracy of 90.4%. In comparison, IgG had a diagnostic sensitivity of 90.4%, diagnostic specificity of 90.4%, a positivepredictive value of 82.6%, a negative predictive value of 90.5%, and accuracy of 90.4%. In conclusion, IgG has a highersensitivity than IgM, while IgM had higher specificity, positive predictive value, and negative predictive value than IgG.However, the positive, negative predictive value and efficiency values were the same for IgM and IgG.

Combination of shear wave elastography and BI-RADS in identification of solid breast masses

Background To explore the value of quantitative shear wave elastography (SWE) plus the Breast Imaging Reporting and Data System (BI-RADS) in the identification of solid breast masses. Methods A total of 108 patients with 120 solid breast masses admitted to our hospital from January 2019 to January 2020 were enrolled in this study. The pathological examination served as the gold standard for definitive diagnosis. Both SWE and BI-RADS grading were performed. Results Out of the 120 solid breast masses in 108 patients, 75 benign and 45 malignant masses were pathologically confirmed. The size, shape, margin, internal echo, microcalcification, lateral acoustic shadow, and posterior acoustic enhancement of benign and malignant masses were significantly different (all P < 0.05). The E mean, E max, SD, and E ratio of benign and malignant masses were significantly different (all P < 0.05). The E min was similar between benign and malignant masses (P > 0.05). The percentage of Adler grade II-III of the benign masses was lower than that of the malignant masses (P < 0.05). BI-RADS plus SWE yielded higher diagnostic specificity and positive predictive value than either BI-RADS or SWE; BI-RADS plus SWE yielded the highest diagnostic accuracy among the three methods (all P < 0.05). Conclusion SWE plus routine ultrasonography BI-RADS has a higher value in differentiating benign from malignant breast masses than color doppler or SWE alone, which should be further promoted in clinical practice.

Development and validation of a novel counter-immunoelectrophoresis assay for the detection of antibodies against extractable nuclear antigens

<p>The identification of autoantibodies is a primary diagnostic marker for the diagnosis of some autoimmune diseases. The sera of patients with connective tissue diseases commonly contain autoantibodies that target nuclear antigens. As such these antibodies are called antinuclear antibodies (ANAs). Furthermore, the clinical identification of ANAs in patient sera to specific nuclear antigens is a primary tool for the diagnosis of connective tissue diseases. For this purpose specific extractable nuclear antigens (ENAs) are used. The most commonly employed laboratory technique for the detection of ENA antibodies is the enzyme linked immunoabsorbant assay (ELISA). ELISA is a simple technique that can be automated and provides high diagnostic sensitivity for the detection of ENA antibodies comparatively to a counter immunoelectrophoresis (CIE) assay. However, compared to CIE ELISA lacks diagnostic specificity. Therefore there is a demand for an assay with high diagnostic specificity as a secondary diagnostic test for the detection of ENA antibodies. The central aim of this project was to develop and validate a novel CIE assay that used fluorescently labelled ENAs to detect ENA antibodies in patient serum. Development of the assay began with comparative testing of fluorescently labelled and unlabelled antigens to confirm that the immunochemical properties of the antigen remained intact. The CIE assay conditions were optimised for the detection of four ANAs SSA, SSB, RNP/Sm, and Sm using their corresponding ENAs. Assay conditions optimised were flurophore type, gel composition, buffer composition, antigen concentration, the running time, and analytical specificity. Evaluation of 281 clinical sera samples known to have previously tested positive test by ANA indirect immunofluorescence were performed using the optimised CIE assay and ELISA. The inter-rater agreement between the CIE assay and ELISA results was determined. Clinical details were used to retrospectively classify the clinical sera samples into clinical categories and case groups. This data was used for the diagnostic comparison of the CIE assay and ELISA results. There was strong inter-rater agreement between the SSA CIE assay and ELISA results. The diagnostic specificity and positive likelihood ratio values of the SSA CIE assay were superior to those of the ELISA, while diagnostic sensitivity and the negative likelihood ratio values were approximately the same. There was strong inter-rater agreement between the RNP/Sm CIE assay and ELISA. However, it was observed that the statistical measures of assay accuracy (diagnostic specificity and sensitivity, positive likelihood ratio, and negative likelihood ratio) for the RNP/Sm ELISA were superior to those of CIE assay. The diagnostic specificity of the SSB and Sm CIE assays were higher than that of their respective ELISAs. This was also true for the positive likelihood ratio values of the SSB and Sm CIE assays when compared to their respect ELISAs. The SSA CIE assay results suggest that this assay maybe a suitable replacement for ELISA as a diagnostic tool for the identification of anti-SSA antibodies and Sjogren’s syndrome. The SSB and Sm CIE assay results suggest that these assays would be suitable as secondary confirmatory diagnostic assays for the identification of their respective ENA antibodies. However, further performance evaluation of the assays within a routine diagnostic laboratory is required.</p>

Development and validation of a novel counter-immunoelectrophoresis assay for the detection of antibodies against extractable nuclear antigens

<p>The identification of autoantibodies is a primary diagnostic marker for the diagnosis of some autoimmune diseases. The sera of patients with connective tissue diseases commonly contain autoantibodies that target nuclear antigens. As such these antibodies are called antinuclear antibodies (ANAs). Furthermore, the clinical identification of ANAs in patient sera to specific nuclear antigens is a primary tool for the diagnosis of connective tissue diseases. For this purpose specific extractable nuclear antigens (ENAs) are used. The most commonly employed laboratory technique for the detection of ENA antibodies is the enzyme linked immunoabsorbant assay (ELISA). ELISA is a simple technique that can be automated and provides high diagnostic sensitivity for the detection of ENA antibodies comparatively to a counter immunoelectrophoresis (CIE) assay. However, compared to CIE ELISA lacks diagnostic specificity. Therefore there is a demand for an assay with high diagnostic specificity as a secondary diagnostic test for the detection of ENA antibodies. The central aim of this project was to develop and validate a novel CIE assay that used fluorescently labelled ENAs to detect ENA antibodies in patient serum. Development of the assay began with comparative testing of fluorescently labelled and unlabelled antigens to confirm that the immunochemical properties of the antigen remained intact. The CIE assay conditions were optimised for the detection of four ANAs SSA, SSB, RNP/Sm, and Sm using their corresponding ENAs. Assay conditions optimised were flurophore type, gel composition, buffer composition, antigen concentration, the running time, and analytical specificity. Evaluation of 281 clinical sera samples known to have previously tested positive test by ANA indirect immunofluorescence were performed using the optimised CIE assay and ELISA. The inter-rater agreement between the CIE assay and ELISA results was determined. Clinical details were used to retrospectively classify the clinical sera samples into clinical categories and case groups. This data was used for the diagnostic comparison of the CIE assay and ELISA results. There was strong inter-rater agreement between the SSA CIE assay and ELISA results. The diagnostic specificity and positive likelihood ratio values of the SSA CIE assay were superior to those of the ELISA, while diagnostic sensitivity and the negative likelihood ratio values were approximately the same. There was strong inter-rater agreement between the RNP/Sm CIE assay and ELISA. However, it was observed that the statistical measures of assay accuracy (diagnostic specificity and sensitivity, positive likelihood ratio, and negative likelihood ratio) for the RNP/Sm ELISA were superior to those of CIE assay. The diagnostic specificity of the SSB and Sm CIE assays were higher than that of their respective ELISAs. This was also true for the positive likelihood ratio values of the SSB and Sm CIE assays when compared to their respect ELISAs. The SSA CIE assay results suggest that this assay maybe a suitable replacement for ELISA as a diagnostic tool for the identification of anti-SSA antibodies and Sjogren’s syndrome. The SSB and Sm CIE assay results suggest that these assays would be suitable as secondary confirmatory diagnostic assays for the identification of their respective ENA antibodies. However, further performance evaluation of the assays within a routine diagnostic laboratory is required.</p>

Immunoprecipitation-targeted proteomics assays facilitate rational development of SARS-CoV-2 serological diagnostics

Current design of serological tests employs conservative immunoassay approaches and is often focused on convenience, speed of manufacturing, and affordability. Limitations of such serological tests include semi-quantitative measurements, lack of standardization, potential cross-reactivity, and inability to distinguish between antibody subclasses. As a result of cross-reactivity, diagnostic specificity of serological antibody tests may not be sufficiently high to enable screening of the general asymptomatic populations for the acquired immunity against low-prevalence infectious diseases, such as COVID-19. Likewise, lack of a single standard for assay calibration limits inter-laboratory and international standardization of serological tests. In this study, we hypothesize that combination of immunoaffinity enrichments with targeted mass spectrometry measurements would enable rational design of serology diagnostics of infectious diseases, such as COVID-19. The same instrumental platform allows for sensitive and specific measurements of viral protein antigens, as wells as anti-viral antibodies circulating in human serum. Our proof-of-concept immunoprecipitation - parallel reaction monitoring (IP-PRM) assays quantified NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum. In addition, a multiplex IP-selected reaction monitoring (IP-SRM) assay facilitated differential quantification of anti-SARS-CoV-2 antibody isotypes and subclasses in patient sera. Simultaneous evaluation of numerous antigen-antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic specificity and sensitivity. Anti-RBD IgG1, IgG3, IgM and IgA1 subclasses, but not IgG2, IgG4 and IgA2, were found elevated in COVID-19-positive sera. Synthetic heavy isotope-labeled peptide internal standards as calibrators revealed elevated anti-RBD IgG1 in positive (510-6700 ng/mL; 0.02-0.22% of total serum IgG1) versus negative sera (60 [interquartile range 41-81] ng/mL). Likewise, anti-RBD IgM was elevated in positive (190-510 ng/mL; 0.06-0.16% of total serum IgM) versus negative sera (76 [31-108] ng/mL). Further validation of immunoprecipitation-targeted proteomics assays as a platform for serological assays will facilitate standardization and improvement of the existing serological tests, enable rational design of novel tests, and offer tools for comprehensive investigation of antibody isotype and subclass cooperation in immunity response.

Comparison of Molecular and Parasitological Methods for Diagnosis of Human Trichostrongylosis

Human trichostrongyliasis is a zoonotic disease that is prevalent among rural populations in some countries. This study was performed to evaluate various parasitological methods and polymerase chain reaction (PCR) for the diagnosis of human trichostrongyliasis. A total of 206 fresh stool samples were collected from residents of endemic villages of Northern Iran. All samples were examined using conventional parasitological methods, including wet mount, formalin ethyl acetate concentration (FEAC), agar plate culture (APC), Harada–Mori culture (HMC), and Willis, along with the PCR technique. Among the total of 206 individuals examined, 72 people (35%) were found infected with Trichostrongylus species using combined parasitological methods. By considering the combined results of parasitological methods as the diagnostic gold standard, the Willis technique had a sensitivity of 91.7% compared with 52.8% for the APC, 40.3% for the HMC, 37.5% for FEAC, and 5.6% for the wet mount technique. The diagnostic specificity of all the parasitological methods was 100%. Furthermore, the PCR method detected Trichostrongylus spp. DNA in 79 fecal samples (38.3%) with a sensitivity of 97.2% and a specificity of 93.3%. According to the current findings, the Willis method was more sensitive than are the other parasitological methods in the diagnosis of human trichostrongyliasis. However, the PCR assay was more sensitive and more reliable in the detection of human trichostrongyliasis in comparison with the parasitological methods.

A-74 COVID-19 and Post-Acute Sequelae of SARS-CoV-2 Infection (PASC): Acute and Longitudinal Analysis of Anosmia and Ageusia with Delayed Sudden-Onset Neuropsychiatric Symptoms

Objective Patients with COVID-19 and PASC may exhibit chemosensory dysfunction associated with acute neuroinflammation from immune system overactivation (Uzunova, Pallanti, & Hollander, 2021). Neuropsychiatric disturbances in patients with no history of anxiety or depression have also been reported. These central nervous system manifestations of COVID-19 may be sequelae of trans-olfactory and infralimbic tract penetration (Speth et al., 2020). Methods Our case involved a 52-year-old, right-handed, American Indian female, who at three months post neuropsychological evaluation, was diagnosed with laboratory confirmed COVID-19 with onset of complete anosmia and ageusia. Two months later, a sudden-onset of panic and depression occurred with no precipitating event. All symptoms were documented daily until return of function. Results Pre-COVID-19 neuropsychological testing revealed findings consistent with ophthalmologic/vestibular migraine and ruled out dementia, and formal anxiety and depressive disorders. Post-COVID-19 neuropsychological analysis and follow-up revealed that anosmia and ageusia had largely resolved after 8-months, and that the delayed sudden-onset panic and depression also resolved within that same time period. Conclusions A paucity of data exists concerning COVID-19 and PASC anosmia and ageusia, and sudden-onset neuropsychiatric symptoms. Our case is unique since neuropsychological testing preceded the COVID-19 infection, which provided a baseline of functioning (e.g., Pocket Smell Test: 3/3 baseline and 0/3 acute COVID-19) and pre-morbid diagnostic specificity. The present case findings align with Cappali and Gatti (2021) whereby 91% of patients reported olfactory recovery, with 53% total recovery after 8-months. No other known reports simultaneously documented detailed recovery of anosmia, ageusia and delayed sudden-onset panic and depression, and COVID-19 antibody laboratory testing.

Serum Adsorption Study to Validate the Specificity of a Rapid Test to Detect Strongyloides stercoralis Infection

A lateral flow rapid test for strongyloidiasis will greatly facilitate the control and elimination of the disease. Previously SsRapid™ prototype rapid test showed high diagnostic specificity to detect Strongyloides infection, determined using non-Strongyloides sera negative by IgG-ELISAs. Since high specificity is crucial before a test is used for public health control activities, further validation of its specificity is needed. Also, it needs to be ascertained whether non-Strongyloides sera positive by IgG-ELISAs and SsRapid are truly positive for Strongyloides or are cases of cross-reactivity. We performed 84 rapid tests (two types of dipsticks and cassettes) using 34 serum samples. They were divided into four groups based on Strongyloides infection and coinfection with other parasites and the availability of recombinant proteins and rapid tests for the latter. Sera was adsorbed using polystyrene microspheres beads separately coated with four recombinant parasite proteins. The small sample size is a limitation of this study; however, the overall results showed that the sera adsorption procedure was successful, and the SsRapid test is specific.

Diagnostic accuracy of rapid point-of-care tests for diagnosis of current SARS-CoV-2 infections in children: A systematic review and meta-analysis

Objective: To systematically assess the diagnostic accuracy of rapid point-of-care tests for diagnosis of current SARS-CoV-2 infections in children under real-life conditions. Study design: Multiple bibliographic databases including MEDLINE and Embase, clinical trial registries and further information sources were systematically searched for literature (last bibliographic search: May 7, 2021). Diagnostic cross-sectional or cohort studies that included paediatric study participants and evaluated rapid point-of care tests for diagnosing current SARS-CoV-2 infections against RT-PCR as the reference standard were eligible for inclusion. QUADAS-2 was used to assess the risk of bias and the applicability of the included studies. Bivariate meta-analyses with random effects were performed. Variability was assessed by subgroup analyses. Results: We included 17 studies with a total of 6355 paediatric study participants. All included studies compared antigen tests against RT-PCR. Only one study was at low risk of bias. The pooled overall diagnostic sensitivity and specificity in paediatric populations was 64.2% (95% CI: 57.4%-70.5%) and 99.1% (95% CI: 98.2%-99.5%), respectively. In symptomatic children, the pooled diagnostic sensitivity was 71.8% (95% CI: 63.6%-78.8%) and the pooled diagnostic specificity was 98.7% (95% CI: 96.6%-99.5%). The pooled diagnostic sensitivity in asymptomatic children was 56.2% (95% CI: 47.6%-64.4%) and the pooled diagnostic specificity was 98.6% (95% CI: 97.3%-99.3%). Conclusions: Performance of current antigen tests under real-life conditions varies broadly. Policymakers should especially be aware of the low diagnostic sensitivity of current antigen tests. Results should be interpreted with caution since risk of bias was predominantly judged as unclear due to poor reporting. Study Registration: CRD42021236313 (PROSPERO).