Application of QuantiFERON ELISA for Detection of Interferon-Gamma Autoantibodies in Adult-Onset Immunodeficiency Syndrome.

OBJECTIVE: Patients who develop interferon-gamma autoantibodies (IFN-ɤ autoAbs) in adult-onset immunodeficiency (AOID) syndrome are more likely to develop opportunistic and recurrent intracellular infections. The assay to detect IFN-ɤ autoAbs is essential for the diagnosis and therapeutic monitoring of AOID syndrome. Therefore, this study applied the QuantiFERON assay for the detection of IFN-ɤ autoAbs. METHODS: Serum from patients with AOID syndrome (n = 19) and serum from healthy patients (n = 20) was collected and applied using 2 neutralizing platforms of enzyme-linked immunosorbent assay (ELISA) kits (the BD ELISA and the QuantiFERON ELISA) for IFN-ɤ autoAbs detection. RESULTS: The pooled serum from patients with AOID syndrome showed >50% inhibition at 1:5000 dilution (positive), whereas the pooled serum from healthy patients showed <50% inhibition at 1:5000 dilution (negative) according to the neutralizing QuantiFERON ELISA. Each specimen showed the same result according to both the neutralizing BD ELISA and the neutralizing QuantiFERON ELISA. Moreover, the patient serum showed a variation in titer ranging from 1:5000 to >1:5,000,000 according to the neutralizing QuantiFERON ELISA. CONCLUSION: The QuantiFERON ELISA kit could be applied for the detection of IFN-ɤ autoAbs for the diagnosis and therapeutic monitoring of AOID syndrome.

A peptide ELISA to detect antibodies against Pythium insidiosum based on predicted antigenic determinants of exo-1,3-beta-glucanase.

Human pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Diagnosis of pythiosis relies on culture identification, serodiagnosis, and molecular-based assay. Preparation of a serodiagnostic test requires culture filtrate antigen (CFA) extracted from the live pathogen. A 74-kDa immunoreactive protein of P. insidiosum, is encoded by the exo-1,3-beta-glucanase gene (PinsEXO1). PinsEXO1 protein is recognized by sera from pythiosis patients but not by sera from uninfected patients; therefore, this protein could be used to detect anti-P. insidiosum antibodies. In this study we aimed to: identify, synthesize, and evaluate an antigenic determinant (epitope) of PinsEXO1 to be used to serodiagnose pythiosis based on peptide ELISA, and to compare the diagnostic performance of that test with the current CFA-based ELISA. Two antigenic determinants of PinsEXO1 (Peptide-A and -B) were predicted using the PREDITOP program. The sera from 34 pythiosis patients and 92 control subjects were evaluated. Peptide-A, Peptide-B, and CFA-based ELISAs all had a specificity of 100%. Peptide-B ELISA had a sensitivity of 91% and an accuracy of 98% and both Peptide-A and CFA-based ELISAs had a sensitivity of 100% and an accuracy of 100%. Peptide-A is a more efficient epitope than Peptide-B, and can be used as an alternative antigen to develop a serodiagnostic assay for pythiosis.

Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis.

Pythiosis is a life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. Morbidity and mortality rates of pythiosis are high. The treatment of choice for pythiosis is surgical debridement of infected tissue. Early and accurate diagnosis is critical for effective treatment. In-house serodiagnostic tests, including immunodiffusion (ID), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICT) and hemagglutination (HA) have been developed to detect antibodies against P. insidiosum in sera. This study compares the diagnostic performance of ID, ELISA, ICT, and HA, using sera from 37 pythiosis patients and 248 control subjects. ICT and ELISA showed optimal diagnostic performance (100% sensitivity, specificity, positive predictive value and negative predictive value). ICT was both rapid and user-friendly. ELISA results were readily quantitated. ID is relatively insensitive. HA was rapid, but diagnostic performance was poor. Understanding the advantages offered by each assay facilitates selection of an assay that is circumstance-appropriate. This will promote earlier diagnoses and improved outcomes for patients with pythiosis.

False-positive serum and bronchoalveolar lavage Aspergillus galactomannan assays caused by different antibiotics.

Our objective was to identify false-positive serum and bronchoalveolar lavage (BAL) fluid galactomannan (GM) tests caused by various antibiotics commonly used in general practice. Serum and BAL samples from patients who did not have the diagnostic criteria of invasive aspergillosis and received different antibiotics were prospectively analyzed for GM. Serum and BAL samples were also collected from patients who did not receive antibiotics. At the cut-off index of >or=0.5, false-positive serum results were found in patients who received amoxicillin-clavulanate, piperacillin-tazobactam, cefepime, and cefoperazone-sulbactam (26.7%, 58.3%, 14.3%, and 66.7%, respectively). Fungal colonization in BAL samples had a higher BAL GM than those without fungal colonization. In 71 patients who had a negative BAL culture for fungi, at the cut-off value of >or=1.0, false-positive BAL fluid results were found in patients who received amoxicillin-clavulanate (27.3%), piperacillin-tazobactam (50%), cefepime (16.7%), carbapenem (45.5%), and ceftriaxone (45.5%). False-positive serum and BAL GM assays were also detected in patients who did not receive any antibiotics. In summary, this study demonstrates the false-positive GM levels in serum and BAL caused by beta-lactam antibiotics that are commonly used in general practice. Physicians should be aware of this possible interference.

The 74-kilodalton immunodominant antigen of the pathogenic oomycete Pythium insidiosum is a putative exo-1,3-beta-glucanase.

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.