Cerebrospinal Fluid from Alzheimer's Disease Patients Contains Fungal Proteins and DNA.

The identification of biomarkers for Alzheimer's disease is important for patient management and to assess the effectiveness of clinical intervention. Cerebrospinal fluid (CSF) biomarkers constitute a powerful tool for diagnosis and monitoring disease progression. We have analyzed the presence of fungal proteins and DNA in CSF from AD patients. Our findings reveal that fungal proteins can be detected in CSF with different anti-fungal antibodies using a slot-blot assay. Additionally, amplification of fungal DNA by PCR followed by sequencing distinguished several fungal species. The possibility that these fungal macromolecules could represent AD biomarkers is discussed.

Immunodiagnosis of paracoccidioidomycosis due to Paracoccidioides brasiliensis using a latex test: detection of specific antibody anti-gp43 and specific antigen gp43.

BACKGROUND: Paracoccidioidomycosis (PCM) is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations. METHOD/PRINCIPLE FINDINGS: A latex immunoassay using sensitized latex particles (SLPs) with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43) was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL) from patients with PCM due to P. brasiliensis. The gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7-100.0), specificity (93.94%, 95% CI 87.3-97.7), and positive (91.4%) and negative (98.9%) predictive values. In addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3-99.6), specificity (88.89%, 95% CI 81.0-94.3), and positive (85.1%) and negative (97.8%) predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924) and mAb17c-SLPs (k = 0.850), which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. In addition, the latex test was useful for monitoring PCM patients receiving therapy. CONCLUSIONS/SIGNIFICANCE: The high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but mainly in remote locations with limited laboratory infrastructure and/or minimally trained community health workers.

Serology of paracoccidioidomycosis.

This review provides the background for understanding the role of a battery of diagnostic methods in paracoccidioidomycosis (PCM). This systemic mycosis is a disease endemic in many regions of Latin America, with sporadic cases also occurring throughout the world (mycosis of importation). Although excellent laboratory methods for diagnosis are available, there are deficiencies that must be met by continued research. Understanding the uses and limitations of a battery of laboratory methods is essential to diagnose PCM. Clinicians and laboratory directors must be familiar with the uses and limitations of a battery of serologic and mycological tests to accurately diagnose of PCM. Antibody and antigen detections are valuable adjuncts to histopathology and culture. More recently, the gp43 and gp70 antigen detection assay have improved the methodology of diagnosis of this mycosis, which improves reproducibility and facilitates monitoring antigen clearance during antifungal treatment. Furthermore, detection of antigen in cerebrospinal fluid and in bronchoalveolar lavage fluid increases the sensitivity for diagnosis of PCM in central nervous system and in pulmonary infections, respectively.

Diagnosis of neuroparacoccidioidomycosis by detection of circulating antigen and antibody in cerebrospinal fluid.

Neuroparacoccidioidomycosis (neuroPCM) is the central nervous system infection by the fungus Paracoccidioides brasiliensis. Its diagnosis is a difficult task that depends on neuroimaging techniques such as computed tomography and magnetic resonance imaging. However, the detection of circulating P. brasiliensis antigens in body fluids by inhibition enzyme-linked immunosorbent assay (inh-ELISA) has provided encouraging results. In this study, 14 cerebrospinal fluid (CSF) and 11 serum samples of patients with neuroPCM were analyzed by inh-ELISA for detection of circulating glycoprotein antigens of 43 kDa (gp43) and 70 kDa (gp70). Circulating gp43 and gp70 antigens were detected in all CSF samples from patients with neuroPCM at mean concentrations of 19.3 and 6.8 mug/ml, respectively. In addition, both gp43 and gp70 antigens were detected in 10 of 11 serum samples analyzed at mean concentrations of 4.6 and 4.0 mug/ml, respectively. By immunodiffusion test, CSF samples were determined to be negative in 13 of 14 samples. The detection of anti-gp43 and anti-gp70 antibodies by conventional ELISA showed positive results for all CSF samples, with titers ranging from 1:50 to 1:51,200. Therefore, the high sensitivity of the inh-ELISA technique in detecting gp43 and gp70 antigens in the CSF of neuroPCM patients strongly indicates that this assay can be considered as a powerful diagnostic tool. In addition, the finding of anti-gp43 and anti-gp70 antibodies in CSF samples by conventional ELISA also seems to be a promising diagnostic method for this special modality of PCM.

Detection of circulating gp43 antigen in serum, cerebrospinal fluid, and bronchoalveolar lavage fluid of patients with paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is an important systemic fungal disease, particularly among individuals living and working in rural areas of endemicity in Latin America, who, without antifungal therapy, may develop fatal acute or chronic infection. For such patients, the detection of antibody responses by immunodiffusion is of limited value due to false-negative results. In contrast, the detection of Paracoccidioides brasiliensis gp43 circulating antigen may represent a more practical approach to the rapid diagnosis of the disease. Accordingly, an inhibition enzyme-linked immunosorbent assay (inh-ELISA) was developed for the detection of a 43-kDa P. brasiliensis-specific epitope incorporating a species-specific murine monoclonal antibody. With sera from patients with acute and chronic forms of the disease (n = 81), the overall sensitivity of the test was found to be 95.1%, while specificity was found to be 97.5% compared to that with normal human sera from blood donors (n = 93) and sera from patients with other chronic fungal infections (histoplasmosis [n = 33] and cryptococcosis [n = 20]). The inh-ELISA detected circulating antigen in 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM according to the patient's clinical presentation. Cerebrospinal fluid from 14 patients with neuroparacoccidioidomycosis and 13 samples of bronchoalveolar lavage fluid from patients with pulmonary unifocal PCM were also tested for gp43 detection, with the test showing 100% sensitivity and specificity. This novel, highly specific inh-ELISA represents a significant addition to the existing tests for the diagnosis of PCM.