Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity.

BACKGROUND: The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. METHODOLOGY/PRINCIPAL FINDINGS: The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. CONCLUSIONS/SIGNIFICANCE: The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

Serological diagnosis of strongyloidiasis: An evaluation of three commercial assays.

BACKGROUND: Strongyloidiasis is caused by a neglected nematode, manifesting as chronic intestinal infection with potentially severe manifestations. The disease is an emerging problem in non-endemic countries affecting travelers and migrants. Diagnosis of strongyloidiasis is hampered by the lack of standardization and absence of a gold standard. Since adequate direct methods to detect the motile larvae in stool samples are not widely available, other techniques such as serology have been developed. METHODS: We evaluated three commercial ELISA kits (DRG Instruments, IVD Research, and Bordier Affinity Products) to detect IgG antibodies against Strongyloides stercoralis assays utilizing serum samples from travelers with microscopically confirmed strongyloidiasis (n = 50) and other imported helminthic infections (n = 159) as well as healthy controls (n = 50). RESULTS: The DRG, IVD, and Bordier assays showed sensitivities of 58.0%, 64.0%, and 56.0%, respectively. Specificity values were 96.0%, 96.0%, and 92.0% in healthy controls, and 67.3%, 62.9%, and 76.7% in cases with other helminth infections, respectively. Cross-reactions were mostly observed in cases with other nematodes (37.5%, 42.5%, and 20.0%, respectively), but also in trematode (33.3%, 38.1%, and 19.0%, respectively) and in cestode infections (25.0%, 30.0%, and 32.5%, respectively). CONCLUSION: The study demonstrates the diagnostic limitations of serological assays to detect or exclude cases of strongyloidiasis in returning travelers, who frequently present with recent or acute infections.

Development of a diagnostic model for biliary atresia based on MMP7 and serological tests using machine learning.

OBJECTIVE: To develop a machine learning diagnostic model based on MMP7 and other serological testing indicators for early and efficient diagnosis of biliary atresia (BA). METHODS: A retrospective analysis was conducted on patient information from those hospitalized for pathological jaundice at Beijing Children's Hospital between January 1, 2019, and December 31, 2023. Patients with serum MMP7, liver stiffness measurements, and other routine serological tests were included in the study. Six machine learning models were constructed, including logistic regression (LR), random forest (RF), decision tree (DET), support vector machine classifier (SVC), neural network (MLP), and extreme gradient boosting (XGBoost), to diagnose BA. The area under the receiver operating characteristic curve was used to evaluate the diagnostic efficacy of the various models. RESULTS: A total of 98 patients were included in the study, comprising 64 BA patients and 34 patients with other cholestatic liver diseases. Among the six machine learning models, the XGBoost algorithm model and RF algorithm model achieved the best predictive performance, with an AUROC of nearly 100% in both the training and validation sets. In the training set, these two algorithm models achieved an accuracy, precision, recall, F1 score, and AUROC of 1. Through model interpretation analysis, serum MMP7 levels, serum GGT levels, and acholic stools were identified as the most important indicators for diagnosing BA. The nomogram constructed based on the XGBoost algorithm model also demonstrated convenient and efficient diagnostic efficacy. CONCLUSION: Machine learning models, especially the XGBoost algorithm and RF algorithm models, constructed based on preoperative serum MMP7 and serological tests can diagnose BA more efficiently and accurately. The most important influencing factors for diagnosis are serum MMP7, serum GGT, and acholic stools.

Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study.

BACKGROUND: Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future "screen and treat" strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. METHODS: During active screening, venous blood samples from 1095 individuals from Côte d'Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. RESULTS: One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1-99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5-88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7-84.2%); DCN HAT RDT 78.2% (95% CI: 75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n = 399) and 93.0-100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. CONCLUSIONS: Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based "screen and treat" strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. TRIAL REGISTRATION: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

A Case of Intravenous IVIg Interfering with Serological Test Results of HBV.

BACKGROUND: Since Imbach [1] first reported the use of high-dose intravenous immunoglobulin (IVIg) in the treatment of idiopathic thrombocytopenic purpura (ITP) in children, indications for IVIg therapy have been increaseing. At present, IVIg infusion has become an important means of clinical treatment. The phenomenon of anti-HBs and anti-HBc elevation caused by IVIg infusion in patients has been reported in journals, but similar reports in journals related to laboratory diagnosis are rare. METHODS: We reported a case of a patient with immune thrombocytopenia (ITP) which interfered with hepatitis B virus (HBV) serological detection after receiving intravenous IVIg. We used chemiluminescence immunoassay to detect serological markers of HBV. IU/mL was used to represent the detection data of HBsAg and HBsAb and cutoff value was used to represent the detection HBeAg, HBeAb, and HbcAb. RESULTS: The serological markers of HBV were all negative before IVIg infusion. One week after IVIG infusion, the item was tested again, and the results of HBsAb, HBeAb, and HBcAb were positive. As the time increased after infusion, HBsAb, HBeAb, and HBcAb in the patient gradually decreased. CONCLUSIONS: After IVIg infusion, the sudden positive change of HBsAb, HBeAb, and HbcAb in the patient's body was not caused by HBV infection, but caused by the infusion of foreign antibody. This case study shows that physicians should be particularly careful when interpreting results in patients treated with intravenous IVIg involving viral hepatitis B.

Evaluation of chimeric recombinant antigens for the serodiagnosis of Trypanosoma cruzi in dogs: a promising tool for Chagas disease surveillance.

BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.

Comparative Evaluation of Select Serological Assays for Zika Virus Using Blinded Reference Panels.

In response to the 2015 Zika virus (ZIKV) epidemic that occurred in Brazil, numerous commercial serological assays have been developed for clinical and research applications. Diagnosis of recent infection in pregnant women remains challenging. Having standardized, comparative studies of ZIKV tests is important for implementing optimal diagnostic testing and disease surveillance. This is especially important for serology tests used to detect ZIKV infection given that antibodies against ZIKV can cross-react with other arboviruses in the same virus family, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). We looked at the sensitivity and specificity of tests detecting ZIKV antibodies (IgM, IgG) from multiple manufacturers using panels of samples previously collected with known exposure to ZIKV and other arboviruses. We found that performance of the IgM tests was highly variable, with only one test (Inbios 2.0 IgM capture ELISA) having both high sensitivity and specificity. All IgG tests showed good sensitivity; however, specificity was highly variable, with some assays giving false-positive results on samples infected by another flavivirus. Overall, the results confirmed that accurate ZIKV antibody testing is challenging, especially in specimens from regions endemic for multiple other flaviviruses, and highlight the importance of available and suitable reference samples to evaluate ZIKV diagnostics.

A modified quality control protocol for infectious disease serology based on the Westgard rules.

When traditional statistical quality control protocols, represented by the Westgard protocol were applied to infectious disease serology, the rejection limits were questioned because of the high rejection probability. We first define the probability of false rejection (Pfr) and error detection (Ped) for infectious disease serology. QC data in 6 months were collected and the Pfr of each rule in the Westgard protocol and Rilibak protocol was evaluated. Then, as improvements, we chose different rules for negative and positive QC data to constitute an asymmetric protocol, furthermore, while reagent lot changes, the mean value of QC protocol is reset with the first 15 QC results of new lot reagent. QC materials and Standard Reference Materials were tested synchronously in the next 6 months, to verify whether the Pfr and Ped of the asymmetric protocol could meet the requirement. Protocol 1 exhibited the higher level of rejection rate among the two protocols, especially after reagent lot changes; Pfr below the lower control limit (LCL) was 1.39-21.78 times higher than the upper control limit (UCL); false rejections were more likely to occur in negative QC data, with Pfr-total of 27-65%. The asymmetric protocol can significantly reduce the proportion of analytes with Pfr by over 20%. Systematic error due to reagent lot changes and random error due to routine QC data variation were considered potential factors for excessive Pfr. Asymmetric QC protocol that can reduce Pfr by different control limits for negative and positive QC data.

Development of a Routine Serological Test Index Panel for the Surveillance of Gastric Cancer Risk in a High-Risk Population.

OBJECTIVE: This study aims to develop a predictive model for the detection of gastric cancer risk utilizing non-invasive parameters and to assess the model's effectiveness in risk stratification for gastric cancer (GC). METHODS: A case-control study was conducted among inpatients with various gastric diseases. These individuals were categorized into two groups: the gastric cancer group (138 cases) and the chronic non-atrophic gastritis (CNAG) group (319 cases). We employed a comprehensive panel of hematological, biochemical, and coagulation parameters derived from routine blood tests. Random Forest and Logistic regression analysis was used for feature selection and model building. Statistical analyses were performed using R version 4.2.3. RESULTS: Logistic regression analysis was employed to establish risk prediction models for GC, incorporating variables such as D-dimer, carcinoembryonic antigen (CEA), carbohydrate antigen 724 (CA724), and hemoglobin (HGB). A visual nomogram was generated as the final prediction model. The area under the receiver operating characteristic curve (AUC) for the training and test sets were 0.8093 [95% confidence interval (CI), 0.7541-0.8644], and 0.8076 [95% CI 0.7237-0.8915], respectively. Furthermore, we have developed an HTML file, featuring the Logistic equation, which enables real-time assessment of GC risk scores. CONCLUSION: The performance of this predictive model demonstrates its adequacy, making it a valuable and cost-effective noninvasive tool for identifying early gastric cancer (EGC) in patients. Consequently, this model may facilitate the implementation of targeted preventive and intervention strategies in clinical practice.

Acceptability and feasibility of tests for infection, serological testing, and photography to define need for interventions against trachoma.

BACKGROUND: Trachoma causes blindness due to repeated conjunctival infection by Chlamydia trachomatis (Ct). Transmission intensity is estimated, for programmatic decision-making, by prevalence of the clinical sign trachomatous inflammation-follicular (TF) in children aged 1-9 years. Research into complementary indicators to field-graded TF includes work on conjunctival photography, tests for ocular Ct infection, and serology. The perceived acceptability and feasibility of these indicators among a variety of stakeholders is unknown. METHODOLOGY: Focus group discussions (FGDs) with community members and in-depth interviews (IDIs) with public health practitioners in Tanzania were conducted. FGDs explored themes including participants' experience with, and thoughts about, different diagnostic approaches. The framework method for content analysis was used. IDIs yielded lists of perceived strengths of, and barriers to, implementation for programmatic use of each indicator. These were used to form an online quantitative survey on complementary indicators distributed to global stakeholders via meetings, mailing lists, and social media posts. RESULTS: Sixteen FGDs and 11 IDIs were conducted in October-November 2022. In general, all proposed sample methods were deemed acceptable by community members. Common themes included not wanting undue discomfort and a preference for tests perceived as accurate. Health workers noted the importance of community education for some sample types. The online survey was conducted in April-May 2023 with 98 starting the questionnaire and 81 completing it. Regarding barriers to implementing diagnostics, the highest agreement items related to feasibility, rather than acceptability. No evidence of significant differences was found in responses pertaining to community acceptability based on participant characteristics. CONCLUSIONS: All of the indicators included were generally deemed acceptable by all stakeholders in Tanzania, although community education around the benefits and risks of different sample types, as well as addressing issues around feasibility, will be key to successful, sustainable integration of these indicators into trachoma programs.

Improved serodiagnosis of Trypanosoma vivax infections in cattle reveals high infection rates in the livestock regions of Argentina.

Bovine trypanosomosis, caused by Trypanosoma vivax, currently affects cattle and has a significant economic impact in sub-Saharan Africa and South America. The development of new diagnostic antigens is essential to improve and refine existing methods. Our study evaluated the efficacy of two recombinant antigens in detecting specific antibodies in cattle. These antigens are derivatives of an invariant surface glycoprotein (ISG) from T. vivax. A fraction of a previously described antigen (TvY486_0045500), designated TvISGAf, from an African strain was evaluated, and a new ISG antigen from an American isolate, TvISGAm, was identified. The two antigens were expressed as fusion proteins in Escherichia coli: TvISGAf was fused to the MBP-His-tag, and TvISGAm was obtained as a His-tag fused protein. An ELISA evaluation was conducted using these antigens on 149 positive and 63 negative bovine samples. The diagnostic performance was enhanced by the use of a combination of both antigens (referred to as TvISG-based ELISA), achieving a sensitivity of 89.6% and specificity of 93.8%. Following the validation of the TvISG-based ELISA, the seroprevalence of T. vivax infection in 892 field samples from cattle in the central region of Argentina was determined. The mean seroprevalence of T. vivax was 53%, with variation ranging from 21% to 69% among the six departments studied. These results support the use of the TvISG ELISA as a valuable serological tool for the detection and monitoring of T. vivax infection in cattle. Furthermore, we report for the first time the seroprevalence of T. vivax in Argentina, which highlights the widespread endemic nature of the disease in the region. In order to effectively manage the increasing spread of T. vivax in the vast livestock production areas of South America, it is essential to implement consistent surveillance programs and to adopt preventive strategies.

Use of a diagnostic Puumala virus real-time RT-PCR in an orthohantavirus endemic region in the Netherlands.

Laboratory diagnosis of orthohantavirus infection is primarily based on serology. However, for a confirmed serological diagnosis, evaluation of a follow-up serum sample is essential, which is time consuming and causes delay. Real-time reverse transcription polymerase chain reaction (RT-PCR) tests, if positive, provide an immediate and definitive diagnosis, and accurately identify the causative agent, where the discriminative nature of serology is suboptimal. We re-evaluated sera from orthohantavirus-suspected clinical cases in the Dutch regions of Twente and Achterhoek from July 2014 to April 2016 for the presence of Puumala orthohantavirus (PUUV), Tula orthohantavirus (TULV), and Seoul orthohantavirus (SEOV) RNA. PUUV RNA was detected in 11% of the total number (n = 85) of sera tested, in 50% of sera positive for anti-PUUV/TULV IgM (n = 16), and in 1.4% of sera negative or indeterminate for anti-PUUV/TULV IgM (n = 69). No evidence was found for the presence of TULV or SEOV viral RNA. Based on these findings, we propose two algorithms to implement real-time RT-PCR testing in routine orthohantavirus diagnostics, which optimally provide clinicians with early confirmed diagnoses and could prevent possible further invasive testing and treatment. IMPORTANCE: The addition of a real-time reverse transcription polymerase chain reaction test to routine orthohantavirus diagnostics may better aid clinical decision making than the use of standard serology tests alone. Awareness by clinicians and clinical microbiologists of this advantage may ultimately lead to a reduction in over-hospitalization and unnecessary invasive diagnostic procedures.

Reexploring cytomegalovirus serology in allogeneic hematopoietic cell transplantation.

PURPOSE OF REVIEW: Discuss the recent evidence on cytomegalovirus (CMV) serology in allogeneic hematopoeic cell transplant (HCT) recipients. RECENT FINDINGS: Whereas the role CMV-specific cellular mediated immunity has recently emerged as an important factor of CMV DNAemia posttransplant, the value of CMV serology has remained unchanged through decades, associated with donor selection and posttransplant prophylactic and monitoring strategies. In this review, we describe and discuss the emerging reports on the association between the magnitude of pretransplant CMV immunoglobulin G (IgG) titer and the posttransplant incidence of CMV DNAemia, as CMV IgG titer could become an additional tool in CMV risk assessment in the future. SUMMARY: Pretransplant recipient CMV serology may have significant implications in posttransplant CMV reactivation in allogeneic HCT recipients.

A Paper-Based Multiplexed Serological Test to Monitor Immunity against SARS-COV-2 Using Machine Learning.

The rapid spread of SARS-CoV-2 caused the COVID-19 pandemic and accelerated vaccine development to prevent the spread of the virus and control the disease. Given the sustained high infectivity and evolution of SARS-CoV-2, there is an ongoing interest in developing COVID-19 serology tests to monitor population-level immunity. To address this critical need, we designed a paper-based multiplexed vertical flow assay (xVFA) using five structural proteins of SARS-CoV-2, detecting IgG and IgM antibodies to monitor changes in COVID-19 immunity levels. Our platform not only tracked longitudinal immunity levels but also categorized COVID-19 immunity into three groups: protected, unprotected, and infected, based on the levels of IgG and IgM antibodies. We operated two xVFAs in parallel to detect IgG and IgM antibodies using a total of 40 µL of human serum sample in <20 min per test. After the assay, images of the paper-based sensor panel were captured using a mobile phone-based custom-designed optical reader and then processed by a neural network-based serodiagnostic algorithm. The serodiagnostic algorithm was trained with 120 measurements/tests and 30 serum samples from 7 randomly selected individuals and was blindly tested with 31 serum samples from 8 different individuals, collected before vaccination as well as after vaccination or infection, achieving an accuracy of 89.5%. The competitive performance of the xVFA, along with its portability, cost-effectiveness, and rapid operation, makes it a promising computational point-of-care (POC) serology test for monitoring COVID-19 immunity, aiding in timely decisions on the administration of booster vaccines and general public health policies to protect vulnerable populations.

Exploratory Research for New Diagnostic Markers of Mucormycosis.

Mucormycosis is a fungal infectious disease caused by Rhizopus oryzae and other members of the order Mucorales, and it is known as one of the most lethal fungal infections. Early diagnosis of mucormycosis improves prognosis because of limited effective treatments and the rapid progression of the disease. On the other hand, the lack of characteristic clinical findings in mucormycosis and the challenge of early definitive diagnosis make early treatment difficult. Our goal was to establish a serodiagnostic method to detect Rhizopus specific antigen (RSA), and we have developed a diagnostic kit by Enzyme-linked immuno-sorbent assay (ELISA) using a monoclonal antibody against this antigen. RSA increased over time in the serum and alveolar lavage fluid of R. oryzae-infected mice. RSA was also detected in serum and alveolar fluid, even at an early stage (Day 1), when the tissue invasion of R. oryzae mycelium was not histopathologically detectable in the lungs of R. oryzae-infected mice. Further evaluation is needed to determine the feasibility of using this assay in clinical practice.

Reference Material Production and Milk Protein Concentration as Elements to Improve Bluetongue Serological Diagnosis in Bulk Tank Milk.

The serological surveillance of bluetongue in bulk tank milk is an efficient and cost-effective method for the early detection of bluetongue virus incursions in unvaccinated free areas of the disease. In addition, the availability of standardized and reliable reagents and refined diagnostic procedures with high sensitivity and specificity are essential for surveillance purposes. However, no available reference materials for bluetongue virus serological surveillance in bulk tank milk exist. This study shows the production and characterization of reference material for the implementation of a commercially available bluetongue milk ELISA test in official laboratories, as well as the evaluation of a procedure to increase the sensitivity in samples with low levels of antibodies. This procedure, based on milk protein concentration, allowed us to notably increase the ELISA test's analytical sensitivity, which is useful for milk samples from farms with low within-herd prevalence or pools of bulk tank milk samples. The standardized milk reference material produced here, together with the evaluated procedure to improve analytical sensitivity, could be applied as tools to ensure an accurate diagnosis by official laboratories in bluetongue unvaccinated free areas.