SOBA: Development and testing of a soluble oligomer binding assay for detection of amyloidogenic toxic oligomers.

The formation of toxic Amyloid ß-peptide (Aß) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aß oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aß oligomers produce strong SOBA signals, while monomeric and ß-sheet protofibrillar Aß do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aß oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

Development and preliminary evaluation toward a new tuberculosis treatment monitoring tool: the PATHFAST TB LAM Ag assay.

The PATHFAST TB LAM Ag assay is based on a chemiluminescent enzyme immunoassay to quantify lipoarabinomannan (LAM) in sputum within 1 h, and was developed as an alternative to conventional culture methods for monitoring tuberculosis (TB) treatment. This study aimed to evaluate the analytical performance and initial clinical feasibility of using five Mycobacterium tuberculosis variants, 178 non-tuberculous mycobacteria (NTM), 34 upper respiratory and oral cavity microorganisms, 100 sputum specimens from untreated patients, and potential interfering substances, including 27 drugs. The results reveled a single-site repeatability coefficient of variation (CV) of 5.2%-7.0%, and a multi-site reproducibility CV of 7.1%-8.4%. The limit of blank, limit of detection, and limit of quantification were 3.03 pg/mL, 6.67 pg/mL, and 7.44 pg/mL, respectively. Linearity was observed over the analytical measurement range (10.0 pg/mL-50,000 pg/mL), and no hook effect was observed. The assay tended to cross-react with slow-growing NTMs, but not with common upper respiratory and oral cavity microorganisms, except Nocardia asteroides, Nocardia farcinica, and Tsukamurella paurometabola. No interference was observed in the presence of mucin, blood, or major anti-TB, anti-HIV, and anti-pneumonia drugs. Regarding clinical performance, the assay had a sensitivity of 88.8% (95% CI: 80.0%-94.0%) and specificity of 100.0% (95% CI: 83.9%-100.0%) using mycobacterial culture as the reference standard, and a correlation (Spearman's r = -0.770) was observed between LAM concentration and time to detection of culture. These findings show, for the first time, that the PATHFAST TB LAM Ag assay has potential value for monitoring TB treatment.

Diagnosis of Coccidioidomycosis with the Second-Generation Miravista IgG and IgM Enzyme Immunoassay and the Role of Adding Miravista Coccidioides Antigen Detection to Immunodiagnostic Assays.

In the present study, we validate and compare the second-generation Miravista Coccidioides IgG and IgM enzyme immunoassays (EIA) (MiraVista Diagnostics [MVD] Ab EIA) to Meridian Diagnostics Coccidioides IgG and IgM EIA (Meridian Ab EIA), immunodiffusion (ID) and complement fixation (CF). We also evaluated whether the addition of Coccidioides antigen testing to anti-Coccidioides antibody testing increased the sensitivity for the diagnosis of currently active coccidioidomycosis. We retrospectively studied 555 patients evaluated at Valleywise Health Medical Center between January 2013 and May 2017 for whom coccidioidomycosis was suspected and samples were submitted to MVD for testing. Specimens were tested for antigen in the MVD antigen enzyme immunoassay (MVD Ag EIA) and for IgG and IgM antibodies with MVD and Meridian Diagnostics EIAs. ID and CF were obtained from medical records. Sensitivity and specificity were 83.0% and 91.1% or MVD Ab EIA, 69.3% and 99.7% for Meridian Ab EIA, 85.4% and 100% for ID and 65.5% and 100% for CF. Combined MVD antigen and antibody detection by EIA and ID resulted in increased sensitivity in disseminated and pulmonary disease (MVD Ag/MVD Ab: 100%, 88.3%; MVD Ag/Meridian Ab: 98.2%, 78.6%; and MVD Ag/ID: 100%, 91.7%). The detection of antibodies by MVD EIA was more sensitive than Meridian EIA or CF but similar to ID. This study supports the use of antigen testing in immunocompromised patients and those with suspected disseminated disease. Furthermore, the addition of antigen detection by EIA to antibody detection resulted in higher sensitivity of all serological tests.

The most common methods for the diagnosis of moderate or severe coccidioidomycosis rely on the detection of antibodies or antigens. Here we present the validation of a new Miravista Coccidioides antibody detection test combined with antigen detection and compare it to other immunodiagnostics.

Impact of the transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for the measurement of plasma aldosterone concentration (PAC) on the diagnosis of primary aldosteronism (PA) via retrospective analyses in Okinawa, Japan.

In Japan, the traditional method for measuring plasma aldosterone concentration (PAC) was radioimmunoassay (RIA), which had several challenges, including poor traceability of certified reference materials and reduced detection sensitivity at low concentrations. To overcome these issues, a chemiluminescent enzyme immunoassay (CLEIA) for PAC measurement was introduced in April 2021 and the Japan Endocrine Society published new guidelines for primary aldosteronism (PA). This study aimed to evaluate the impact of the transition from RIA to CLEIA for PAC measurement on PA diagnosis. Data from 190 patients admitted to the Second Department of Internal Medicine, University of the Ryukyus Hospital, between April 2012 and March 2021 were analyzed. Patients who were diagnosed with PA underwent adrenal venous sampling. The PAC measured by RIA (PAC(RIA)) was converted to the estimated PAC measured by CLEIA (ePAC(CLEIA)) using a conversion formula. The present study evaluated the discordance rates in diagnoses based on screening (SC), captopril challenge test (CCT), saline infusion test (SIT), and diagnosis of PA between results judged by PAC(RIA) according to the previous guidelines and those judged by ePAC(CLEIA) according to the new guidelines. The results revealed discordant diagnosis rates of 6.4% for SC and 10.1% for CCT, with no discordance for SIT. The discordant diagnosis rate for PA was 3.7%. Our study reveals the challenges in establishing appropriate diagnostic criteria for PA using PAC(CLEIA) and highlights the demand for further research on provisionally positive categories.

Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

Performance of the clarus Aspergillus galactomannan enzyme immunoassay prototype for the diagnosis of invasive pulmonary aspergillosis in serum.

BACKGROUND: Serum galactomannan (GM) testing is essential for diagnosing invasive aspergillosis (IA), particularly in immunocompromised individuals. The global lack of on-site GM testing capacities necessitates cost-effective alternatives, such as .the clarus Aspergillus GM enzyme immunoassay prototype (clarus AGM prototype). METHODS: This single-centre, cross-sectional study compared the diagnostic performance of the clarus AGM prototype (IMMY, Norman, Oklahoma) with the serological gold standard (=Platelia AGM assay; Bio-Rad, Marnes-la-Cocquette, France). IA was classified according to modified 2020 EORTC/MSG consensus and 2024 FUNDICU criteria. In total, 300 prospectively (May-Dec 2023) and retrospectively (2012-2015) collected samples were included. RESULTS: Among 300 samples from 232 patients, 49 (16%) were classified as proven (n = 1) or probable IA (n = 48). In non-IA cases (n = 250), one patient was classified as possible IA. With the manufacturer recommended cut-off of ≥0.2, sensitivity and specificity of the clarus AGM prototype were 27% (13/49; 95% confidence interval [CI]: 15%-41%) and 99% (248/250; 95% CI: 97%-100%), respectively, while sensitivity and specificity were 78% and 79% when using the optimised Youden's cut-off of 0.0045 ODI. ROC curve analysis demonstrated an area under the curve (AUC) of 0.829 (95% CI: 0.760-0.898) for the clarus AGM prototype in distinguishing between proven/probable IA and non-IA. The AUC for the Platelia AGM was 0.951 (95% CI: 0.909-994). Spearman's correlation analysis showed a weak correlation between the two assays (0.382; p < .001). CONCLUSIONS: The weak correlation between the clarus AGM prototype and Platelia AGM highlights the need for further investigation into the clinical performance of the clarus AGM prototype, giving the different antigen epitopes addressed.

Comparative Analysis of the Clarus Aspergillus Galactomannan Enzyme Immunoassay Prototype for the Diagnosis of Invasive Pulmonary Aspergillosis in Bronchoalveolar Lavage Fluid.

BACKGROUND: Galactomannan (GM) testing using Platelia Aspergillus enzyme immunoassay (Platelia AGM) from bronchoalveolar lavage fluid (BALF) aids in early diagnosis of invasive pulmonary aspergillosis (IPA). Globally, only a minority of laboratories have the capability to perform on-site GM testing, necessitating accessible and affordable alternatives. Hence, we conducted a comparative evaluation of the new clarus Aspergillus GM enzyme immunoassay prototype (clarus AGM prototype) with Platelia AGM using BALF samples. METHODS: This is a single-center, prospective, cross-sectional study, where Platelia AGM testing was routinely performed followed by clarus AGM prototype testing in those with true positive or true negative AGM test results according to the 2020 EORTC/MSG and the 2024 FUNDICU consensus definitions. Descriptive statistics, ROC curve analysis, and Spearman's correlation analysis were used to evaluate analytical performance of the clarus AGM prototype assay. RESULTS: This study enrolled 259 adult patients, of which 53 (20%) were classified as probable IPA, while 206 did not fulfill IPA-criteria. Spearman's correlation analysis revealed a strong correlation between the two assays (rho = 0.727, p < 0.001). The clarus AGM prototype had a sensitivity of 96% (51/53) and a specificity of 74% (153/206) for differentiating probable versus no IPA when using the manufacturer recommended cut-off. ROC curve analysis showed an AUC of 0.936 (95% CI 0.901-0.971) for the clarus AGM prototype, while the Platelia AGM yielded an AUC of 0.918 (95% CI 0.876-0.959). CONCLUSIONS: Clarus AGM prototype demonstrated a strong correlation and promising test performance, comparable to Platelia AGM, rendering it a viable alternative in patients at risk of IPA.

Is the Medium Still the Message? Culture-Independent Diagnosis of Gastrointestinal Infections.

Infectious diarrhea is caused by a variety of pathogens, including viruses, bacteria, and parasitic organisms. Though the causative agent of diarrhea has historically been evaluated via stool cultures, recently, culture-independent diagnostic tests (CIDT) have been developed and utilized with increasing frequency. Current practice guidelines recommend their use as adjuncts to stool cultures for diagnosing acute and chronic diarrhea. The three principal CIDT are microscopy, enzyme-based immunoassays (EIAs), and molecular based polymerase chain reaction (PCR). This review explores the common causes of infectious diarrhea, the basics of stool culture, the diagnostic utility of these three culture-independent modalities, and the strengths and weaknesses of all currently available clinical techniques. It also outlines considerations for specific populations including returning travelers and those with inflammatory bowel disease.

Fully automated chemiluminescence enzyme immunoassays showing high correlation with immunoprecipitation mass spectrometry assays for ß-amyloid (1-40) and (1-42) in plasma samples.

Blood based ß-amyloid (Aß) assays that can predict amyloid positivity in the brain are in high demand. Current studies that utilize immunoprecipitation mass spectrometry assay (IP-MS), which has high specificity for measuring analytes, have revealed that precise plasma Aß assays have the potential to detect amyloid positivity in the brain. In this study, we developed plasma Aß40 and Aß42 immunoassays using a fully automated immunoassay platform that is used in routine clinical practice. Our assays showed high sensitivity (limit of quantification: 2.46 pg/mL [Aß40] and 0.16 pg/mL [Aß42]) and high reproducibility within-run (coefficients of variation [CVs]: <3.7% [Aß40] and <2.0% [Aß42]) and within-laboratory (CVs: <4.6% [Aß40] and <5.3% [Aß42]). The interference from plasma components was less than 10%, and the cross-reactivity with various lengths of Aß peptides was less than 0.5%. In addition, we found a significant correlation between the IP-MS method and our immunoassay (correlation coefficients of Pearson's r: 0.91 [Aß40] and 0.82 [Aß42]). Our new method to quantify plasma Aß40 and Aß42 provides clinicians and patients with a way to continuously monitor disease progression.

Performance evaluation of antibody tests for detecting infant respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is a major cause of respiratory tract disease in young children and throughout life. Infant infection is also associated with later respiratory morbidity including asthma. With a prospective birth cohort study of RSV and asthma, we evaluated the performance of an RSV antibody enzyme-linked immunoassay (EIA) for detecting prior infant RSV infection. Infant RSV infection was determined by biweekly respiratory illness surveillance plus RSV polymerase chain reaction (PCR) testing in their first RSV season and serum RSV antibodies after the season at approximately 1 year of age. RSV antibodies were detected by RSV A and B lysate EIA. Antibody and PCR results on 1707 children included 327 RSV PCR positive (PCR+) and 1380 not RSV+. Of 327 PCR+ children, 314 (96%) were lysate EIA positive and 583 out of 1380 (42%) children not PCR+ were positive. We compared the lysate EIA to RSV F, group A G (Ga), and group B G (Gb) protein antibody EIAs in a subset of 226 sera, 118 PCR+ children (97 group A and 21 group B) and 108 not PCR+. In this subset, 117 out of 118 (99%) RSV PCR+ children were positive by both the F and lysate EIAs and 103 out of 118 (87%) were positive by the Ga and/or Gb EIAs. Comparison of the two G EIAs indicated the infecting group correctly in 100 out of 118 (86%) and incorrectly in 1 out of 118 (1%). The lysate and F EIAs are sensitive for detecting infant infection and the two G EIAs can indicate the group of an earlier primary infection.

Evaluation of a chemiluminescent enzyme immunoassay-based high-throughput SARS-CoV-2 antigen assay for the diagnosis of COVID-19: The VITROS® SARS-CoV-2 Antigen Test.

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.

Screening for parvovirus B19 antigen through chemiluminescent enzyme immunoassay is equivalent to B19 nucleic acid amplification test-based screening of pooled plasma.

BACKGROUND: Human parvovirus B19 (B19) is a pathogen that threatens the quality of plasma products. Therefore, health authorities have mandated measures against B19 contamination of plasma pools. The US FDA has recommended a B19 genome level of 104 IU/ml or lower in pooled plasma lots. Therefore, the B19 nucleic acid amplification test (B19-NAT) has been introduced in many plasma fractionators. However, in the Japanese Red Cross, which is the only approved blood collector in Japan, the B19 antigen test has been introduced for screening donated blood in Japan. Therefore, to clarify whether the antigen test is robust enough to screen blood samples according to the FDA recommendation, we evaluated B19 genome levels in each pooled plasma lot from 2003 to 2020. STUDY DESIGN AND METHODS: Data of 5576 pooled plasma lots from factories A and B, which were derived from plasma bags and passed the B19 antigen-based tests, receptor-mediated hemagglutination assay (B19-RHA), or chemiluminescent enzyme immunoassay (B19-CLEIA), during 2003 to 2020, were evaluated. The amount of B19 genome in each lot was determined using quantitative or semiquantitative B19-NAT. RESULTS: The B19 genome levels in pooled plasma lots screened using B19-RHA did not meet the FDA recommendation, whereas the lots derived from B19-CLEIA fulfilled the FDA recommendation, even during the B19 epidemic in Japan. DISCUSSION: The results suggest that the B19-CLEIA donor screening for plasma pools is also useful in light of the US FDA recommendation.

Estimation of the window period of human T-cell leukemia virus type 1 and 2 tests by a lookback study of seroconverters among Japanese voluntary blood donors.

BACKGROUND: Japan is endemic for human T-cell leukemia virus type 1 (HTLV-1), and the horizontal transmission of HTLV-1 is often reported. However, the window period (WP) for serologic or molecular screening is unclear. STUDY DESIGN AND METHODS: Results for anti-HTLV-1 screening and confirmatory tests obtained from 648 591 repeated blood donors in the Kyushu district, one of the most endemic areas of HTLV-1 in the world, were evaluated. A lookback study was conducted for seroconverters. RESULTS: During 2012 to 2019, 436 seroconverters (155 men, 281women) were identified with use of a screening chemiluminescence enzyme-immunoassay (CLEIA) and multiple confirmatory tests. Because the period between the latest seronegative donation and seroconversion was highly variable (2.1-276.7 months), 19 cases that seroconverted within 6 months were subjected to the analysis. The WP of the particle agglutination assay and CLEIA was estimated to be 2.2 ± 0.6 and 2.6 ± 1.7 months, respectively. The WP of the indirect immunofluorescence assay was 4.8 ± 6.5 months. Although the WP of western blotting was estimated to be 6.3 ± 8.7 months, four cases were still indeterminate through the study period. Chemiluminescence and line immunoassays, the current screening and confirmatory tests used in the Japanese blood program, showed the shortest WP of 2.2 ± 0.6 months. The WP of real-time polymerase chain reaction for HTLV-1 was estimated to be 4.1 ± 7.8 months. CONCLUSIONS: The WP in commercially available testing systems for HTLV-1/2 was determined for natural infection among repeated blood donors. Considering the HTLV-1 WP will help increase transfusion safety and facilitate the accurate diagnosis of HTLV-1 infection.

Clostridioides difficile laboratory diagnostic techniques: a comparative approach of rapid and molecular methods.

Clostridioides difficile infection is a public health problem because of it is easily spread; with harmful consequences, it is essential to reduce hospital costs and prevent its dissemination by having a precise diagnosis. The gold standard for its diagnosis is polymerase chain reaction (PCR); however, the technique is not available for all laboratories due to the high cost. New approaches using non-molecular tests to detect C. difficile and toxin A/B production has been proposed to improve cost benefits. The objective of this study is to compare molecular methods (PCR) and rapid methods (immunochromatographic test and enzymatic immunoassay). A series of tests comprising these diagnostic techniques was performed with 50 patients with a clinical diagnosis for Clostridioides difficile on GeneXpert® devices test; a calculation of the sensitivity was executed, followed by a comparison of the efficiency of all techniques. Greater sensitivity was observed in the PCR-based methods (BD MAX™ and BioFire FilmArray®) and the GDH-based assays (RIDASCREEN® and Alere Techlab®). The proposed algorithm represents minor monetary disadvantages but a significant temporal optimization of 10%. Future studies concerning both positive and negative results could be advantageous because of the possibility of calculating more method concordance indexes, such as the specificity and Kappa index, in addition to being able to indicate a monetary profit if the proposed algorithm was applied due to the nonproceeding PCR cases.

Serotonin-release assay-positive but platelet factor 4-dependent enzyme-immunoassay negative: HIT or not HIT?

IgG-specific and polyspecific PF4-dependent enzyme-immunoassays (EIAs) have exceptionally high sensitivity (≥99%) for diagnosis of heparin-induced thrombocytopenia (HIT), a drug reaction caused by platelet-activating antibodies detectable by serotonin-release assay (SRA). The IgG-specific EIAs are recommended for screening, as their high sensitivity is accompanied by relatively high specificity vis-à-vis polyspecific EIAs. We investigated the frequency of SRA-positive/EIA-negative (SRA+/EIA-) HIT, prompted by referral to our reference HIT laboratory of serial blood samples from a patient ("index case") with false-negative IgG-specific EIAs. Despite initial clinical suspicion for HIT, repeat negative IgG-specific EIAs prompted heparin resumption, which triggered recurrent thrombocytopenia and near-fatal cardiac arrest, indicating likely post-heparin HIT-associated anaphylactoid reaction. Further investigations revealed a strong-positive SRA, whether performed with heparin alone, PF4 alone, or PF4/heparin, with inhibition by Fc receptor-blocking monoclonal antibody (indicating IgG-mediated platelet activation); however, five different IgG-specific immunoassays yielded primarily negative (or weak-positive) results. To investigate the frequency of SRA+/EIA- HIT, we reviewed the laboratory and clinical features of patients with this serological profile during a 6-year period in which our reference laboratory investigated for HIT using both SRA and IgG-specific EIA. Although ~0.2% of 8546 patients had an SRA+/EIA- profile, further review of 15 such cases indicated clerical/laboratory misclassification or false-positive SRA in all, with no SRA+/EIA- HIT case identified. We conclude that while SRA+/EIA- HIT is possible-as shown by our index case-this clinical picture is exceptionally uncommon. Moreover, the requirement for a positive EIA is a useful quality control maneuver that reduces risk of reporting a false-positive SRA result.

Cross-reactivity of a Histoplasma capsulatum antigen enzyme immunoassay in urine specimens from persons with emergomycosis in South Africa.

Histoplasma antigen detection in urine is a rapid diagnostic method for disseminated histoplasmosis, although cross-reactivity has been reported in specimens from patients with other thermally dimorphic fungal infections. We tested urine specimens, from persons with suspected invasive fungal infections, using a commercial monoclonal antibody Histoplasma enzyme immunoassay (EIA) at a South African national mycology reference laboratory from August 2014 through December 2018. Corresponding fungal culture and histopathology results were obtained from an electronic laboratory information system. In some cases, cultured fungal isolates were sent with the urine specimen for species-level identification by phenotypic and molecular methods. Cross-reactivity was confirmed using culture filtrates of several fungal pathogens. Of 212 referred cases, 41 (19%) were excluded since they had no recorded clinical history (n = 1), alternative diagnoses were confirmed (n = 2), or no fungal culture or histopathology results (n = 38). Eighty-seven of 212 (41%) had laboratory evidence of an invasive fungal disease, while 84 (40%) did not. Of the 87 cases, 37 (43%) were culture-confirmed mycoses: emergomycosis (n = 18), histoplasmosis (n = 8), sporotrichosis (n = 6), cryptococcosis (n = 2), talaromycosis (n = 1), and other fungi isolated (n = 2). The sensitivity and specificity of the EIA were calculated for two groups: culture-confirmed (n = 37) and histology-confirmed invasive fungal disease (n = 50). The sensitivity and specificity of the EIA for diagnosis of histoplasmosis compared to culture were 88% (7/8, 95%CI 47-100%) and 72% (21/29, 95%CI 53-87%), respectively, and for diagnosis of emergomycosis/histoplasmosis compared to histology was 83% (29/35, 95%CI 66-93%) and 93% (14/15, 95%CI 68-100%), respectively. Cross-reactions occurred in urine specimens of patients with Emergomyces africanus infection and in culture filtrates of E. africanus, T. marneffei and Blastomyces species. A commercial Histoplasma EIA had satisfactory accuracy for diagnosis of culture-confirmed histoplasmosis, but cross-reacted in urine specimens from patients with invasive disease caused by the closely-related pathogen, E. africanus and in culture filtrates of E. africanus and other related fungi. LAY SUMMARY: Emergomyces africanus and Histoplasma capsulatum are fungi that cause a multi-system disease among HIV-seropositive persons with a low CD4 cell count. Handling live cultures of these fungi to confirm a diagnosis requires specialized laboratory equipment and infrastructure which is infrequently accessible in low-resource settings. The features of the two diseases (i.e., disseminated histoplasmosis and emergomycosis) may be indistinguishable when infected tissue is prepared, stained, and examined under a microscope. Enzyme immunoassays (EIA) have been developed as rapid diagnostic tools for the detection of a cell wall component of H. capsulatum in urine specimens, although cross-reactions have been reported in specimens from patients with other fungal infections. We evaluated the accuracy of a commercial Histoplasma EIA to diagnose histoplasmosis and to assess cross-reactions in urine specimens from persons with emergomycosis and in cultures of E. africanus and related fungi. We report a sensitivity and specificity of 88% (95%CI 47-100%) and 72% (95%CI 53-87%) for diagnosis of histoplasmosis compared to culture and 83% (95%CI 66-93%) and 93% (95%CI 68-100%) for diagnosis of either histoplasmosis/emergomycosis compared to a diagnosis made by microscopic examination of infected tissue. The assay cross-reacted in urine specimens from patients with emergomycosis and in culture filtrates of related fungi. Although the EIA cross-reacted with other related fungi, this test can decrease the time to diagnosis and facilitate early treatment of emergomycosis and histoplasmosis in South Africa.

Diagnostic Modality of Clostridioides difficile Infection Predicts Treatment Response and Outcomes in Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) patients are at increased risk of developing Clostridioides difficile infection (CDI). Common methods to diagnose CDI involve a combination of tests including the toxin A/B enzyme immunoassay (Toxin) and toxin gene polymerase chain reaction assay (PCR). Disease outcomes in Toxin+ versus Toxin-PCR+ IBD patients remain unclear. AIMS: This study aimed to examine the response to antibiotics and risk of IBD therapy escalation in Toxin+ versus Toxin-PCR+ patients. METHODS: IBD patients at an academic center with CDI diagnosis based on Toxin+ or Toxin-PCR+ from 2012 to 2017 were identified. Comparisons of response to antibiotics within 30 days and escalation of IBD therapy within 90 days of CDI diagnosis between these two groups were analyzed by Chi-square analysis. Multivariable regression analysis examined factors associated with antibiotic response. RESULTS: Among 92 patients included, 61% had Crohn's disease and 39% had ulcerative colitis. 70% tested Toxin-PCR+. 60% received vancomycin or fidaxomicin to treat CDI. 82% of Toxin+ patients responded to antibiotics compared to 25% of Toxin-PCR+ patients (p < 0.001). 21% of Toxin+ patients required IBD therapy escalation compared to 63% of Toxin-PCR+ patients (p < 0.001). When adjusted for the types of antibiotics used, IBD subtypes, and immunosuppression status, positivity to Toxin (OR 14.85, CI 4.62-47.72) was the most significant predictor of response to antibiotics. CONCLUSIONS: Toxin+ compared to Toxin-PCR+ IBD patients had a significantly higher rate of response to antibiotics and lower chances of requiring IBD therapy escalation. Future outcome studies involving CDI in IBD patients should be stratified by modality of diagnosis.

Comparison of the analytical and clinical performances of two different routine testing protocols for antinuclear antibody screening.

BACKGROUND: The diagnosis of systemic autoimmune rheumatic diseases (SARD) is based on the detection of serum antinuclear antibodies (ANA) for which indirect immunofluorescence (IIF) is the golden standard. New solid-phase immunoassays have been developed to be used alone or in combination with the detection of extractable antinuclear antibodies (ENA) to improve SARD diagnosis. The purpose of this study was to compare the clinical performances of different ANA screening methods alone or in combination with ENA screening methods for SARD diagnosis. METHODS: A total of 323 patients were screened for ANA by IIF, EliA™ CTD Screen, and ELISA methods. Agreements were calculated between the methods. Then, EliA™ CTD Screen positive samples were screened for ENA by line immunoassay (LIA) and fluorescence enzyme immunoassay (FEIA). RESULTS: The diagnostic accuracy of EliA™ CTD Screen (79% sensitivity and 91% specificity) was better than that of ELISA or IIF. The combination of EliA™ CTD plus IIF had the highest sensitivity (93%). ENA determination revealed that Ro52 and Ro60 were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36% of samples positive for Ro52, and 41% for Ro60. CONCLUSIONS: EliA™ CTD Screen has a better diagnostic performance when compared to IIF and ELISA. The combined use of EliA™ CTD Screen and IIF clearly improves the rate and accuracy of SARD diagnosis. The use of EliA™ CTD Screen as first-line screening technique allows the detection of antibodies, which could not be detected by IIF alone.

Technical note: Validation of an in-house bovine serum enzyme immunoassay for progesterone measurement.

Measuring circulating progesterone (P4) of dairy cows is a key component of many research studies dealing with basic and applied reproduction physiology. The gold standard in dairy cows for the measurement of P4 in serum is radioimmunoassay (RIA), but it generates radioactive waste and requires licensed facilities. The purpose of this study was to develop and validate an in-house competitive enzyme immunoassay (EIA) to measure the P4 concentration in serum of dairy cattle. The secondary objective was to validate a commercial EIA. In the present study, a competitive EIA was developed using commercially available antibodies and conjugates. Ninety-six well microtiter plates were coated with the secondary antibody and incubated overnight. Following a washing step, the wells were blocked using the primary antibody. Serum samples were prepared by first extracting P4 using petroleum ether, then diluted in working conjugate solution. Samples were pipetted into the coated and blocked plates, then the matching HRP conjugate label (P4-3-HRP, East Coast Bio, North Berwick, ME) was added. The plates were incubated for 2 h, then washed. The substrate solution was added, and the plate was incubated up to 1 h at room temperature in the dark until a blue color had developed. A stop solution was added, and the optical density measured on a microplate reader was set at 450 nm. The binding proportion was calculated by a visible spectrum absorbance reader, and the amount of P4 was calculated using a log-logit regression line. The commercial EIA was executed as suggested by the manufacturer. The validation of the in-house EIA was done by calculating the inter- and intraassay coefficients of variation (CV) and evaluating the parallelism of diluted samples. The results from the in-house and commercial EIA were also compared with the ones from the RIA graphically (scatterplots and Bland-Altman plots) and statistically, using the Spearman correlation coefficient (r) and the Cohen's kappa statistics using a threshold of 1.0 ng/mL (κ). For the in-house EIA, the intraassay CV were all <10%, but the interassay for samples with small and large P4 concentration had CV of 12.5 and 11.0%, respectively. The correlations between the results from the EIA and the RIA were strong (in-house: r = 0.90; commercial: r = 0.83). At small concentrations (<1.0 ng/mL), however, the correlation with the gold standard was weak (in-house: r = 0.27; commercial: r = 0.14). This was likely due to the lack of accuracy at small concentrations, also shown by the absence of parallelism in samples ≤0.4 ng/mL. In conclusion, results from both the in-house and commercial EIA strongly correlated with the gold standard, but less so at smaller concentrations. The in-house EIA offers good accuracy to measure P4 in samples with a concentration >0.4 ng/mL, and a perfect agreement with RIA using a threshold of 1.0 ng/mL.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.