Histoplasma antigens as novel players for the development of new enzyme immunoassays for the serodiagnosis of histoplasmosis: A comparative study of their analytical performance.

Definitive diagnosis of histoplasmosis relies on culture and/or cytology/histopathology; however, these procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has low sensitivity in immunocompromised individuals and uses histoplasmin (HMN), a crude antigenic extract, as reagent. Novel protein antigen candidates have been recently identified and produced by DNA-recombinant techniques to obtain standardized and specific reagents for diagnosing histoplasmosis. To compare the analytical performance of novel enzyme-linked immunosorbent assays (ELISAs) for antibodies testing for diagnosing histoplasmosis using different Histoplasma capsulatum antigens as reagents. The H. capsulatum 100 kDa protein (Hcp100), the M antigen and its immunoreactive fragment F1 were produced by DNA-recombinant techniques. Galactomannan was purified from both the yeast and mycelial cell walls (yGM and mGM, respectively). The analytical performance of the ELISA tests for the serological detection of antibodies against these antigens was evaluated and compared with those obtained using HMN as reagent. Antibodies detection by the Hcp100 ELISA demonstrated 90.0% sensitivity and 92.0% specificity, versus 43.3% sensitivity and 95.0% specificity of the M ELISA, 33.3% sensitivity and 84.0% specificity of the F1 ELISA, 96.7% sensitivity and 94.0% specificity of the yGM ELISA, 83.3% sensitivity and 88.0% specificity of the mGM ELISA, and 70.0% sensitivity and 86.0% specificity for the HMN ELISA. In summary, Hcp100 is proposed as the most promising candidate for the serodiagnosis of histoplasmosis. The primary immunoreactive element in HMN proved to be GM rather than the M antigen. Nevertheless, a higher incidence of cross-reactions was noted with GM compared to M.

Hcp100 is a promising serodiagnostic candidate for histoplasmosis, boasting high sensitivity and specificity. Notably, GM, rather than M antigen, emerged as the primary immunoreactive element in HMN, despite a higher incidence of cross-reactions with GM compared to M.

Effect of very long-term storage and multiple freeze and thaw cycles on 11-dehydro-thromboxane-B2 and 8-iso-prostaglandin F2α, levels in human urine samples by validated enzyme immunoassays.

Biological samples are often frozen and stored for years and/or thawed multiple times, thus assessing their stability on long-term storage and repeated freeze-thaw cycles is crucial. The study aims were to assess:-the long-term stability of two major enzymatic and non-enzymatic metabolites of arachidonic acid, i.e. urinary 11-dehydro-thromboxane-(Tx) B2, 8-iso-prostaglandin (PG)F2α, and creatinine in frozen urine samples;-the effect of multiple freeze-thaw cycles. Seven-hundred and three urine samples measured in previously-published studies, stored at -40 °C, and measured for a second time for 11-dehydro-TxB2 (n = 677) and/or 8-iso-PGF2α (n = 114) and/or creatinine (n = 610) were stable over 10 years and the 2 measurements were highly correlated (all rho = 0.99, P < 0.0001). Urine samples underwent 10 sequential freeze-thaw cycles, with and without the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (10 mM); urinary 11-dehydro-TxB2 and creatinine were stable across all cycles (11-dehydro-TxB2: 100.4 ± 21%; creatinine: 101 ± 7% of baseline at cycle ten; n = 17), while 8-iso-PGF2α significantly increased by cycle 6 (151 ± 22% of baseline at cycle ten, n = 17, P < 0.05) together with hydrogen peroxide only in the absence of antioxidant. Arachidonic acid metabolites and creatinine appear stable in human urines stored at -40 °C over 10 years. Multiple freeze-thaw cycles increase urinary 8-iso-PGF2α in urine samples without antioxidants. These data are relevant for studies using urine samples stored over long-term and/or undergoing multiple freezing-thawing.

A measles IgM rapid diagnostic test to address challenges with national measles surveillance and response in Malaysia.

INTRODUCTION: A lateral flow rapid diagnostic test (RDT) enables detection of measles specific immunoglobulin M (IgM) antibody in serum, capillary blood, and oral fluid with accuracy consistent with enzyme immunoassay (EIA). The objectives of the study were: 1) to assess measles RDT inter-reader agreement between two clinic staff; 2) to assess the sensitivity and specificity of the measles RDT relative to standard surveillance testing in a low transmission setting; 3) to evaluate the knowledge, attitudes, and practices of staff in clinics using the RDT; and 4) to assess the impact of RDT testing on the measles public health response in Malaysia. MATERIALS AND METHODS: The clinic-based prospective evaluation included all suspected measles cases captured by routine measles surveillance at 34 purposely selected clinics in 15 health districts in Malaysia between September 2019 and June 2020, following day-long regional trainings on RDT use. Following informed consent, four specimens were collected from each suspected case, including those routinely collected for standard surveillance [serum for EIA and throat swabs for quantitative reverse transcriptase polymerase chain reaction (RT-qPCR)] together with capillary blood and oral fluid tested with RDTs during the study. RDT impact was evaluated by comparing the rapidity of measles public health response between the pre-RDT implementation (December 2018 to August 2019) and RDT implementation periods (September 2019 to June 2020). To assess knowledge, attitudes, and practices of RDT use, staff involved in the public health management of measles at the selected sites were surveyed. RESULTS: Among the 436 suspect cases, agreement of direct visual readings of measles RDT devices between two health clinic staff was 99% for capillary blood (k = 0.94) and 97% for oral fluid (k = 0.90) specimens. Of the total, 45 (10%) were positive by measles IgM EIA (n = 44, including five also positive by RT-qPCR) or RT-qPCR only (n = 1), and 38 were positive by RDT (using either capillary blood or oral fluid). Using measles IgM EIA or RT-qPCR as reference, RDT sensitivity using capillary blood was 43% (95% CI: 30%-58%) and specificity was 98% (95% CI: 96%-99%); using oral fluid, sensitivity (26%, 95% CI: 15%-40%) and specificity (97%, 95% CI: 94%-98%) were lower. Nine months after training, RDT knowledge was high among staff involved with the public health management of measles (average quiz score of 80%) and was highest among those who received formal training (88%), followed by those trained during supervisory visits (83%). During the RDT implementation period, the number of days from case confirmation until initiation of public response decreased by about 5 days. CONCLUSION: The measles IgM RDT shows >95% inter-reader agreement, high retention of RDT knowledge, and a more rapid public health response. However, despite ≥95% RDT specificity using capillary blood or oral fluid, RDT sensitivity was <45%. Higher-powered studies using highly specific IgM assays and systematic RT-qPCR for case confirmation are needed to establish the role of RDT in measles elimination settings.

UHPLC-MS/MS for plasma lamotrigine analysis and comparison with a homogenous enzyme immunoassay.

Aims: To develop and validate a UHPLC-MS/MS method for lamotrigine (LTG) analysis in human plasma and evaluate its agreement with a homogenous enzyme immunoassay (HEIA). Materials & methods: The UHPLC-MS/MS method was developed and validated according to the USFDA/EMA guidelines. A Bland-Altman plot was used to evaluate the agreement between UHPLC-MS/MS and HEIA. Results: Samples were pretreated with one-step protein precipitation and separated in 2.6 min. The intra- and inter-day bias and imprecisions were -15.8 to 15.0% and less than 11.17%, respectively. The recovery and matrix factor were 98.30 to 111.97%. The mean overestimation of UHPLC-MS/MS compared with HEIA was 21.57%. Conclusion: A rapid, sensitive and robust UHPLC-MS/MS method for plasma LTG analysis was developed and validated and was a 21.57% overestimation compared with HEIA.

Colorimetric and chemiluminescent enzyme immunoassays based on the alkaline phosphatase-tagged single-chain variable fragment fusion tracer for detecting zearalenone in agro-products.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin and seriously threatens food safety, which requires rapid and sensitive detection methods for monitoring ZEN in agro-products. Herein, an alkaline phosphatase-tagged single-chain variable fragment fusion protein (ALP-scFv) was used as a bifunctional tracer to develop a colorimetric enzyme immunoassay (CEIA) and a chemiluminescent enzyme immunoassay (CLEIA) for ZEN. In addition, the interactions between scFv and ZEN were exploited by computer-assisted simulation, and four key amino acid sites were preliminarily identified. After optimization, the CEIA and CLEIA exhibited a limit of detection of 0.02 and 0.006 ng/mL, respectively. Furthermore, both methods showed favorable accuracy in recovery experiments and good selectivity in cross reactions. Moreover, the detection results of the actual samples from both methods correlated well with those from high-performance liquid chromatography. Overall, the ALP-scFv fusion tracer-based CEIA and CLEIA are demonstrated as reliable tools for ZEN detection in food.

Advances in single molecule arrays (SIMOA) for ultra-sensitive detection of biomolecules.

In the contemporary era of scientific and medical advancements, the accurate and ultra-sensitive detection of proteins, nucleic acids and metabolites plays a pivotal role in disease diagnosis and treatment monitoring. Single-molecule detection technologies play a great role in achieving this goal. In recent years, digital detection methods based on single molecule arrays (SIMOA) have brought groundbreaking contributions to the field of single-molecule detection. By confining the target molecules to femtoliter-sized containers, the SIMOA technology achieves detection sensitivity of attomolar. This review delves into the historical evolution and fundamentals of SIMOA technology, summarizes various approaches to optimize its performance, and describes the applications of SIMOA for the ultrasensitive detection of biomarkers for diseases such as cancer, COVID-19, and neurological disorders, as well as in DNA detection. Currently, some SIMOA technologies have been realized for high-throughput and multiplexed detection. It is believed that SIMOA technology will play a significant role in medical monitoring and disease prevention in the future.

Detection of Histoplasma capsulatum and Blastomyces dermatitidis antigens in serum using a single quantitative enzyme immunoassay.

Histoplasma and Blastomyces antigen detection assays are commonly used diagnostic tools. However, a high level of cross-reactivity between these antigens prevents definitive pathogen identification by these assays alone. Retrospective analysis of 3,529 patients with Histoplasma and Blastomyces antigen testing performed on the same serum sample yielded an overall percent agreement of 99.3% (3,506 of 3,529; kappa: 0.859) between the two assays, suggesting that use of a single assay to detect both antigens may be an alternative diagnostic approach. We assessed performance of the Gotham BioTech Blastomyces antigen (GBA) enzyme immunoassay (EIA) (Portland, Maine) for detection of Blastomyces and Histoplasma antigens in serum. Comparison to the MiraVista Diagnostics Blastomyces (MVB) EIA showed 100% positive (24 of 24), negative (57 of 57), and overall (81 of 81) percent agreement. Additionally, 171 sera were used to compare the GBA EIA to the MiraVista Diagnostics Histoplasma (MVH) EIA, which showed 91.3% (63 of 69), 98% (100 of 102), and 95.3% (163 of 171) positive, negative, and overall percent agreement, respectively. Among eight patients with discordant GBA/MVH EIA results, seven had additional fungal testing performed, and results suggested that the MVH and GBA results were inaccurate for two and five samples, respectively. Overall, this study suggests that the GBA EIA has a high level of agreement with both of the commonly used, individual Blastomyces and Histoplasma antigen EIAs. By taking advantage of the high level of cross-reactivity between Blastomyces and Histoplasma antigen EIAs, utilization of a single antigen detection assay for these fungi provides an opportunity to optimize test utilization and decrease patient cost while maintaining a high level of diagnostic accuracy.

Redeveloping antigen detection kits for the diagnosis of rat hepatitis E virus.

The emergence of Rocahepevirus ratti [species HEV ratti (r HEV)] as a causative agent of hepatitis E in humans presents a new potential threat to global public health. The R. ratti genotype 1 (r-1 HEV) variant only shares 50%-60% genomic identity with Paslahepevirus balayani [species HEV balayani (b HEV)] variants, which are the main causes of hepatitis E infection in humans. Here, we report antigen diagnoses for r-1 HEV and b HEV using an enzymatic immunoassay (EIA) method. We detected recombinant virus-like particles protein (HEV 239) of r HEV and b HEV using a collection of hepatitis E virus (HEV)-specific monoclonal antibodies. Two optimal candidates, the capture antibody P#1-H4 and the detection antibodies C145 (P#1-H4*/C145#) and C158 (P#1-H4*/C158#), were selected to detect antigen in infected rat samples and r-1 HEV- or b HEV-infected human clinical samples. The two candidates showed similar diagnostic efficacy to the Wantai HEV antigen kit in b HEV-infected clinical samples. Genomic divergence resulted in low diagnostic efficacy of the Wantai HEV antigen kit (0%, 0 of 10) for detecting r-1 HEV infection. Compared with the P#1-H4*/C145# candidate (80%, 8 of 10), the P#1-H4*/C158# candidate had excellent diagnostic efficacy in r-1 HEV-infected clinical samples (100%, 10 of 10). The two candidates bind to a discrete antigenic site that is highly conserved across r HEV and b HEV. P#1-H4*/C145# and P#1-H4*/C158# are efficacious candidate antibody combinations for rat HEV antigen detection.

Infecciones por Clostridioides difficile en pediatría. La visión del laboratorio a partir del diagnóstico microbiológico / Clostridioides difficile infections in pediatrics. The view of the laboratory based on microbiological diagnosis

La infección por Clostridioides difficile (ICD) es la principal responsable de diarreas nosocomiales en adultos. En los últimos años se registró un aumento en la incidencia de la ICD en la población adulta que, en cambio, no fue bien caracterizado en pediatría. El objetivo de este trabajo es analizar los datos resultantes del diagnóstico microbiológico de ICD en el Hospital de Pediatría "Prof. Dr. Juan P. Garrahan". Materiales y métodos: se realizó un estudio retrospectivo observacional descriptivo que abarcó desde el 01/01/2018 hasta el 31/12/2021. El diagnóstico se realizó mediante enzimoinmunoensayo para glutamato deshidrogenasa (GDH) y toxinas en materia fecal (MF). Cuando sólo se detectó GDH, se realizó un cultivo toxigénico (CT) de la MF para la detección de toxinas in vitro. Se registraron: edad, sexo y procedencia de los pacientes y recurrencias de las ICD. Se efectuaron estudios de sensibilidad de 387 cepas de C. difficile a metronidazol (MTZ) y vancomicina (VAN). Resultados: en 6632 muestras (1764 pacientes) se registraron 649 estudios positivos (9,8%) (139 pacientes), la mayoría correspondieron a pacientes internados en áreas no críticas. Edad promedio: 7 años (7 ± 4,7). Sexo: 55% masculino. Recurrencias: 62 (45%). Positivos detectados mediante CT: 43%. Sensibilidad antibiótica: 100% a MTZ y 99,7% a VAN. Conclusión: Nuestra población presenta un bajo porcentaje de positividad. Se destaca el rendimiento del CT que permitió el diagnóstico de más de un tercio de los casos. MTZ y VANCO tuvieron excelente actividad in vitro frente a C. difficile (AU)

Clostridioides difficile infection (CDI) is the main cause of nosocomial diarrhea in adults. In recent years there has been an increase in the incidence of CDI in the adult population; however, CDI has not been well characterized in pediatrics. The aim of this study was to analyze the data resulting from the microbiological diagnosis of CDI at Hospital de Pediatría Prof. Dr. Juan P. Garrahan. Materials and methods: a retrospective, observational and descriptive study was conducted from 01/01/2018 to 12/31/2021. Diagnosis was made using enzyme immunoassay for glutamate dehydrogenase (GDH) and toxins in stools. When only GDH was detected, toxigenic culture (TC) of stools was performed for in vitro toxin detection. The age, sex and origin of patients and CDI recurrences were recorded. Sensitivity studies of 387 strains of C. difficile to metronidazole (MTZ) and vancomycin (VAN) were performed. Results: In 6,632 samples (1,764 patients), 649 positive results (9.8%) were recorded (139 patients), most of which corresponded to patients hospitalized in noncritical areas. Mean age: 7 years (7 ± 4.7). Sex: 55% male. Recurrences: 62 (45%). TC-positive results: 43%. Antibiotic sensitivity: 100% to MTZ and 99.7% to VAN. Conclusion: A low percentage of positivity was found in our population. The performance of TC was outstanding, allowing for the diagnosis of more than one third of the cases. MTZ and VANCO had excellent in vitro activity against C. difficile (AU)

Evaluation of an enzyme immunoassay technique on detecting urinary Histoplasma capsulatum antigen in the diagnosis of disseminated histoplasmosis in Argentina.

INTRODUCTION: Histoplasmosis is a systemic mycosis of universal distribution, highly endemic in the Americas. It is caused by a dimorphic fungus Histoplasma capsulatum var. capsulatum. It affects both immunocompetent and immunocompromised individuals where progressive and disseminated forms are observed. A very important risk factor is HIV infection/AIDS, with a mortality rate of 20-40% in Latin America. The diagnosis of this mycosis is made by conventional and molecular methods or by antigen and antibody detection. METHODS: In this retrospective, longitudinal and analytical study, carried out over a period of 2 years, the sensitivity (S) and specificity (E) of a commercial kit for the detection of Histoplasma antigen by EIA technique (HC-Ag) was evaluated in 50 patients with AIDSassociated histoplasmosis. In addition, its performance was compared with that of other diagnostic techniques routinely used in our laboratory. RESULTS: HC-Ag had a S of 94%, E 96%, positive likelihood coefficient (CVP): 20.68 and negative likelihood coefficient (CVN): 0.06. The delay time of the results was 4 days, similar to that of antibody detection and n-PCR and much less than that of blood cultures. The combination of methods improved S to 100%; with similar values in E. CONCLUSION: The HC-Ag method demonstrated its usefulness in the diagnosis of progressive disseminated histoplasmosis and the combination of methods is a good option to increase sensitivity and decrease the time to reach the diagnosis of certainty. This allows improving the strategy in the management of the disease and decreasing its case-fatality rate.

Introducción: La histoplasmosis es una micosis sistémica de distribución universal, altamente endémica en las Américas. Es causada por un hongo dimórfico: Histoplasma capsulatum var. capsulatum. Afecta tanto a inmunocompetentes como a inmunocomprometidos, se observan formas progresivas y diseminadas. Un factor de riesgo muy importante es la infección por HIV/sida, con una tasa de mortalidad del 20-40% en América Latina. El diagnóstico de esta micosis se realiza por métodos convencionales y moleculares o por detección de antígenos y anticuerpos. Métodos: En este estudio retrospectivo, longitudinal y analítico, realizado en un periodo de 2 años, se evaluó la sensibilidad (S) y especificidad (E) de un kit comercial para la detección de antígeno de Histoplasma por técnica de EIA (HC-Ag) en 50 pacientes con histoplasmosis asociada a sida. Además, se comparó su rendimiento con el de otras técnicas diagnósticas utilizadas habitualmente en nuestro laboratorio. Resultados: HC-Ag tuvo una S del 94%, E del 96%, coeficiente de verosimilitud positiva (CVP) de 20.68 y coeficiente de verosimilitud negativa (CVN) de 0.06. El tiempo de demora de los resultados fue de 4 días, similar al de la detección de anticuerpos y n-PCR y mucho menor que el de los hemocultivos. La combinación de métodos mejoró la S a 100%; con valores similares en E. Conclusión: El método HC-Ag demostró su utilidad en el diagnóstico de histoplasmosis diseminada progresiva y la combinación de métodos es una buena opción para aumentar la sensibilidad y disminuir el tiempo para llegar al diagnóstico de certeza. Esto permite mejorar la estrategia en el manejo de la enfermedad y reducir su tasa de letalidad.

Diagnostic Utility of Galactomannan Enzyme Immunoassay in Invasive Aspergillosis in Pediatric patients with Hematological Malignancy.

OBJECTIVE: This study aims to determine the diagnostic utility of galactomannan enzyme immunoassay (GM EIA) in invasive aspergillosis (IA) in children with hematological malignancy (high risk population) in terms of sensitivity, specificity, negative predictive value (NPV) and positive predictive values (PPV) at various cut offs while validating the revised EORTC/MSG 2019 criteria in order to obtain the best cut-off. MATERIAL AND METHODS: For 100 pediatric patients, serum and respiratory samples were collected. Clinical, mycological workup (potassium-hydroxide mount, fungal culture) and GM EIA was done to classify proven, probable, and possible IA as per EORTC-MSG guidelines,2019. Sensitivity, specificity, PPV and NPV were calculated of GM indices at cut-off 0.5, 0.7 and 1, and validated with revised EORTC -MSG, 2019. RESULTS: Of 100 patients enrolled, 75 were diagnosed with ALL, 14 with AML, two with Hodgkin's, three had non-Hodgkin lymphoma, and six had undifferentiated leukemia. With routine mycological findings, 51 were classified as probable IA, 11 as possible IA, and 38 as no IA. Aspergillus flavus was the most prevalent on culture (56.9%, 29/51) followed by A. fumigatus (29%, 15/51) A. niger (7.8%, 4/51), A. terreus (3.9%, 2/51) and A. nidulans (2%, 1/51). GM EIA demonstrated sensitivity 82.3%, specificity 97.4%, PPV 98.1%, and NPV 77.1% at cut-off 0.67 when comparing probable/possible IA v/s no IA groups. The GM EIA had the best sensitivity (82.4%), specificity (81.8%), PPV (95.5%), and NPV (50%) at cut off 0.78 when the probable IA group was compared to the possible IA. Seven patients succumbed of whom 5 had GMI ≥ 2. CONCLUSION: This study deduces the optimal cut-off for serum GM EIA to be 0.67 obtained by ROC analysis when comparing possible and probable IA versus no IA and reinforces the definition of probable category of EORTC-MSG criteria, 2019. At 0.5 ODI the sensitivity (87.1%) and NPV (80.5%) are high, thus making it the most suitable cut-off for detecting true positive and ruling out IA respectively, in pediatric patients with hematological malignancy. GM EIA when performed adjunctive to clinico-radiological findings can prove to be screening, diagnostic and prognostic test for IA in pediatric hematological malignancy patients.

New chemiluminescent enzyme immunoassay for quantitative measurement of Mac-2 binding protein glycosylation isomer in chronic liver disease.

BACKGROUND: This study aimed to evaluate the quantitative measurement of Mac-2 binding protein glycosylation isomer (M2BPGi) levels using the new chemiluminescent enzyme immunoassay. METHODS: The data of a total of 347 patients with hepatitis C virus (HCV) infection and 150 health volunteers from 13 locations in Japan were evaluated. The quantitative system for measuring M2BPGi-Qt levels was based on a new chemiluminescent enzyme immunoassay. We evaluated the reproducibility and quantitation range in quantitative M2BPGi-Qt measurement. We also investigated the confidence ratio of M2BPGi-Qt levels measured by the new quantitative system to M2BPGi levels measured by the current semi-quantitative system for validating the clinical utility of the new method. RESULTS: The reproducibility of M2BPGi-Qt in HCV samples with negative, positive 1+, and positive 2+ was 0.77 ± 0.02 AU/mL, 2.25 ± 0.03 AU/mL, and 6.55 ± 0.21 AU/mL, respectively, and the corresponding coefficient of variation (CV)s were 2.1%, 1.3%, and 3.2%, respectively. The range of quantification assessment resulted that all CVs showed less than 5% in investigated range. Sample stability testing found that the mean percentage difference between the pre- and post-storage values of 6 samples ranged between 96.2 and 103.9%. The correlation coefficient between M2BPGi and M2BPGi-Qt in patients with HCV and the healthy volunteers was 0.986 and 0.991, respectively. M2BPGi-Qt could be quantitatively assessed in a patient with over 20 C.O.I. CONCLUSION: Compared with qualitative methods, the M2BPGi quantitative measurement system could provide a numerical value unaffected by interpretation bias, and measurements are more precise at high M2BPGi levels.

Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers.

The introduction of spacers in coating steroid protein complexes and/or enzyme conjugates or immunogens is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We investigated the impact of different homobifunctional spacers, ranging in atomic length from 3 to 10, on the sensitivity and specificity of prednisolone (PSL) enzyme immunoassays. In this study, four homo-bifunctional spacers, namely, carbohydrazide (CH), adipic acid dihydrazide (ADH), ethylene diamine (EDA), and urea (U), were incorporated between PSL and horseradish peroxidase (HRP) for preparing the enzyme conjugate with an aim to improve the sensitivity of the assay without compromising assay specificity. The assays were developed using these enzymes conjugated with antibodies raised against the PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of a bridge in the enzyme conjugate was 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL, and 0.018 ng/mL with ADH, CH, EDA, and urea as a spacer, respectively. Among the four combinations, the PSL-21-HS-BSA-antibody with PSL-21-HS-U-HRP-enzyme conjugate gave better sensitivity and less cross-reaction. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32%-102.50%. The intra and inter-assay CV% was< 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r2 = 0.98). The present study suggests that the nature of the spacer is related to assay sensitivity and not the spacer length.

An empirical comparison of several commercial enzyme immunoassays for the non-invasive assessment of adrenocortical and gonadal function in mountain gorillas.

Wildlife researchers seeking to non-invasively examine endocrine function in their study species are presented with a dense and technical 'garden of forking paths' to navigate between collecting a biological sample and obtaining a final measurement. In particular, the choice of which enzyme immunoassay (EIA) to use with collected fecal samples, out of the many options offered by different manufacturers and research laboratories, may be one of the most consequential for final results. However, guidance for making this decision is still emerging. With this gap in mind, we performed a head-to-head comparison of results obtained from four different EIAs for fecal glucocorticoid metabolites (FGCMs), and three different EIAs for fecal androgen metabolites (FAMs), applied to the same set of fecal samples collected from the mountain gorillas (Gorilla beringei beringei) monitored by the Dian Fossey Gorilla Fund in Volcanoes National Park, Rwanda. We provide a) an analytical validation of the different EIAs via tests of parallelism and linearity; b) an estimate of inter-assay correlation between EIA kits designed for the same metabolites; and c) a test of the kits' ecological validity, in which we examine how well each captures endocrine changes following events that theory predicts should result in elevated FGCM and/or FAM concentrations. Our results show that kits differ to some degree in their performance; at the same time, nearly all assays exhibited at least moderate evidence of validity and covariance with others for the same analyte. Our findings, which differ somewhat from similar comparisons performed in other species, demonstrate the need to directly assess assay performance in a species- and context-specific manner as part of efforts to develop the burgeoning discipline of wildlife endocrinology.

Clostridioides difficile PCR Tcdb Cycle Threshold predicts toxin EIA positivity but not severity of infection.

BACKGROUND: Diagnosis of Clostridioides difficile Infection (CDI) entails compatible clinical presentation and laboratory findings. We evaluated real-time polymerase chain reaction (qPCR) cycle threshold (CT) as a predictor for disease severity and TcdB enzyme immunoassay (EIA) results. METHODS: Inpatients or emergency department patients who tested positive for tcdB gene by PCR were evaluated. Patients' stools underwent testing for GDH and TcdA/B by EIA. Medical health records were reviewed for demographic, clinical presentation, laboratory, treatment and outcome data. Severity of CDI was calculated using various severity score indexes. RESULTS: The median CT of cases was 32.05 ± 5.45. The optimal cut-off for predicting toxin EIA positivity and severe CDI based on chart review was 32.6 and 29.8, respectively, with the area under the receiver operator characteristics curve (AUC) of 0.74 and 0.60 respectively. CONCLUSION: CT value was an acceptable predictor for EIA toxin but less so for clinical severity. Our study potentially supports a diagnostic algorithm including CT value to reduce the number of EIA toxin assays performed.

Doubtful Clinical Value of Subtyping Anti-U1-RNP Antibodies Regarding the RNP-70 kDa Antigen in Sera of Patients with Systemic Lupus Erythematosus.

The detection of antinuclear antibodies is central to the diagnosis and prognosis of systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS) and mixed connective tissue disease (MCTD). Anti-U1-RNP and anti-RNP70 antibodies were assayed in the sera of patients with SLE (n = 114), pSS (n = 54) and MCTD (n = 12). In the SLE group, 34/114 (30%) were anti-U1-RNP positive, and 21/114 (18%) were both anti-RNP70 positive and anti-U1-RNP positive. In the MCTD group, 10/12 (83%) were anti-U1-RNP positive, and 9/12 (75%) were anti-RNP70 positive. Only one individual with pSS was antibody positive (for both anti-U1-RNP and anti-RNP70). All anti-RNP70-positive samples were also anti-U1-RNP positive. Anti-U1-RNP-positive subjects with SLE were younger (p < 0.0001); showed lower concentrations of complement protein 3 (p = 0.03); had lower eosinophil (p = 0.0005), lymphocyte (p = 0.006) and monocyte (p = 0.03) counts; and had accrued less organ damage (p = 0.006) than the anti-U1-RNP-negative SLE patients. However, we observed no significant clinical or laboratory parameter differences between the anti-U1-RNP-positive individuals with/without anti-RNP70 in the SLE group. In conclusion, anti-RNP70 antibodies are not exclusive to MCTD but are rarely detected in pSS and healthy individuals. In SLE, anti-U1-RNP antibodies are associated with a clinical phenotype that resembles MCTD, with hematologic involvement and less damage accrual. Based on our results, the clinical value of subtyping anti-RNP70 in anti-U1-RNP-positive sera appears to be of limited value.

A Chemiluminescence Enzyme Immunoassay Based on Biotinylated Nanobody and Streptavidin Amplification for Diazinon Sensitive Quantification.

The advantages of genetic modification and preferable physicochemical qualities make nanobody (Nb) easy to develop a sensitive and stable immunosensor platform. Herein, an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) based on biotinylated Nb was established for the quantification of diazinon (DAZ). The anti-DAZ Nb, named Nb-EQ1, with good sensitivity and specificity, was obtained from an immunized library via a phage display technique, where the molecular docking results indicated that the hydrogen bond and hydrophobic interactions between DAZ and complementarity-determining region 3 and framework region 2 in Nb-EQ1 played a critical role in the Nb-DAZ affinity processes. Subsequently, the Nb-EQ1 was further biotinylated to generate a bi-functional Nb-biotin, and then an ic-CLEIA was developed for DAZ determination via signal amplification of the biotin-streptavidin platform. The results showed that the proposed method based on Nb-biotin had a high specificity and sensitivity to DAZ, with a relative broader linear range of 0.12-25.96 ng/mL. After being 2-folds dilution of the vegetable samples matrix, the average recoveries were 85.7-113.9% with a coefficient of variation of 4.2-19.2%. Moreover, the results for the analysis of real samples by the developed ic-CLEIA correlated well with that obtained by reference method GC-MS (R2 ≥ 0.97). In summary, the ic-CLEIA based on biotinylated Nb-EQ1 and streptavidin recognition demonstrated itself to be a convenient tool for the quantification of DAZ in vegetables.

Non-invasive assessment of fecal glucocorticoid and androgen metabolites in the pygmy hippopotamus (Choeropsis liberiensis).

The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species' reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3ß,11ß,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance.

Investigation of anti-PF4 versus anti-PF4/heparin reactivity using fluid-phase enzyme immunoassay for 4 anti-PF4 disorders: classic heparin-induced thrombocytopenia (HIT), autoimmune HIT, vaccine-induced immune thrombotic thrombocytopenia, and spontaneous HIT.

BACKGROUND: Four platelet-activating anti-platelet factor 4 (PF4) disorders have been recognized: classic heparin-induced thrombocytopenia (cHIT), autoimmune heparin-induced thrombocytopenia (aHIT), spontaneous heparin-induced thrombocytopenia (SpHIT), and vaccine-induced immune thrombotic thrombocytopenia (VITT). All test immunoglobulin G (IgG) positive using solid-phase enzyme immunoassay (solid-EIA) against PF4/heparin (PF4/H) and/or PF4 alone. Fluid-phase EIA (fluid-EIA) should better discriminate between anti-PF4 and anti-PF4/H antibodies since conformationally altered PF4 bound to solid phase is avoided. OBJECTIVES: To compare anti-PF4 vs anti-PF4/H antibody profiles for anti-PF4 disorders using solid- and fluid-EIA. METHODS: We developed a novel fluid-EIA to measure anti-PF4 vs anti-PF4/H antibodies. RESULTS: Using fluid-EIA, 27 of 27 (100%) cHIT sera tested IgG positive with PF4/H, but only 4 of 27 (14.8%) tested positive against PF4 alone; all 27 exhibited heparin-enhanced binding. In contrast, 17 of 17 (100%) VITT sera tested IgG positive against PF4 alone, with markedly reduced binding against PF4/H; this distinct VITT antibody profile was not evident using solid-EIA. All 15 aHIT sera and all 11 SpHIT sera tested IgG positive against PF4 alone, with variable reactivity in PF4/H-EIA (heparin-enhanced binding in 14 of 15 and 10 of 11 aHIT and SpHIT sera, respectively). Remarkably, 1 SpHIT patient with a VITT-mimicking fluid-EIA profile (PF4 >> PF4/H) also clinically resembled patients with VITT (postviral cerebral vein/sinus thrombosis), with anti-PF4 reactivity correlating inversely with platelet count recovery; moreover, the single aHIT patient with a VITT-mimicking fluid-EIA profile also developed postviral cerebral vein/sinus thrombosis. CONCLUSION: cHIT and VITT sera showed opposite fluid-EIA profiles (cHIT: PF4/H >> PF4, with most testing negative against PF4 alone; VITT: PF4 >> PF4/H, with most testing negative against PF4/H). In contrast, all aHIT and SpHIT sera reacted against PF4 alone but with variable (usually enhanced) reactivity against PF4/H. VITT-mimicking clinical/serologic profiles occurred in only a minority of patients with SpHIT and aHIT.

Comparison of measles IgG enzyme immunoassays (EIA) versus plaque reduction neutralization test (PRNT) for measuring measles serostatus: a systematic review of head-to-head analyses of measles IgG EIA and PRNT.

BACKGROUND: As countries move towards or achieve measles elimination status, serosurveillance is an important public health tool. However, a major challenge of serosurveillance is finding a feasible, accurate, cost-effective, and high throughput assay to measure measles antibody concentrations and estimate susceptibility in a population. We conducted a systematic review to assess, characterize, and - to the extent possible - quantify the performance of measles IgG enzyme-linked assays (EIAs) compared to the gold standard, plaque reduction neutralization tests (PRNT). METHODS: We followed the PRISMA statement for a systematic literature search and methods for conducting and reporting systematic reviews and meta-analyses recommended by the Cochrane Screening and Diagnostic Tests Methods Group. We identified studies through PubMed and Embase electronic databases and included serologic studies detecting measles virus IgG antibodies among participants of any age from the same source population that reported an index (any EIA or multiple bead-based assays, MBA) and reference test (PRNT) using sera, whole blood, or plasma. Measures of diagnostic accuracy with 95% confidence intervals (CI) were abstracted for each study result, where reported. RESULTS: We identified 550 unique publications and identified 36 eligible studies for analysis. We classified studies as high, medium, or low quality; results from high quality studies are reported. Because most high quality studies used the Siemens Enzygnost EIA kit, we generate individual and pooled diagnostic accuracy estimates for this assay separately. Median sensitivity of the Enzygnost EIA was 92.1% [IQR = 82.3, 95.7]; median specificity was 96.9 [93.0, 100.0]. Pooled sensitivity and specificity from studies using the Enzygnost kit were 91.6 (95%CI: 80.7,96.6) and 96.0 (95%CI: 90.9,98.3), respectively. The sensitivity of all other EIA kits across high quality studies ranged from 0% to 98.9% with median (IQR) = 90.6 [86.6, 95.2]; specificity ranged from 58.8% to 100.0% with median (IQR) = 100.0 [88.7, 100.0]. CONCLUSIONS: Evidence on the diagnostic accuracy of currently available measles IgG EIAs is variable, insufficient, and may not be fit for purpose for serosurveillance goals. Additional studies evaluating the diagnostic accuracy of measles EIAs, including MBAs, should be conducted among diverse populations and settings (e.g., vaccination status, elimination/endemic status, age groups).