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

Abstract

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.

Fig 1. Diagram of latex agglutination.

(A) Reaction between gp43-sensitized latex particles (SLPs) for detection of specific antibodies and (B) between the monoclonal antibody mAb17c-SLPs for detection of circulating gp43 antigen in the sample.

Fig 2. Appearance of typical latex agglutination test.

In positive reactions, the LA tests formed loose aggregates within 5 to 10 min (left). In negative reactions, the LA test remained as a milky suspension (right).

Fig 3. (A) Detection of anti-gp43 antibody in the sera of patients using gp43-SLPs and (B) gp43 antigen in the sera of patients using mAb17c-SLPs.

Samples were from paracoccidioidomycosis (PCM; n = 65), histoplasmosis (HIS; n = 18), aspergillosis (ASP; n = 18), candidiasis (CAN; n = 13), no fungal disease (NFD; n = 12), and healthy controls (NHS; n = 38). Agglutination score: (4+) 100%, (3+) 75%, (2+) 50%, (1+) 25%, and (N) negative.

Fig 4. (A) Detection of anti-gp43 antibody in the cerebrospinal fluid of PCM patients using gp43-SLPs and (B) gp43 antigen in the cerebrospinal fluid of PCM patients using mAb17c-SLPs.

Agglutination score: (4+) 100%, (3+) 75%, (2+) 50%, (1+) 25%, and (N) negative. Samples were from 14 PCM patients and 6 negative controls with no fungal disease (NFD).

Fig 5. (A) Detection of anti-gp43 antibody in the bronchoalveolar lavage fluid from PCM patients using gp43-SLPs and (B) gp43 antigen in the bronchoalveolar lavage fluid from PCM patients using mAb17c-SLPs.

Agglutination score: (4+) 100%, (3+) 75%, (2+) 50%, (1+) 25%, and (N) negative. Samples were from 13 PCM patients and 6 negative controls with no fungal disease (NFD).

Fig 6. Representative curves for serological follow-up.

Samples were from 10 PCM patients with acute (n = 5) or chronic (n = 5) forms of the disease before (T1, at the moment of disease diagnosis), during (T2 = 3 months), and after treatment (T3, between 18–24 months). (A, C) Anti-gp43 antibody detection using gp43-SLPs and (B, D) gp43 antigen detection using mAb17c-SLPs.

Fig 7. Receiver operating characteristics (ROCs) depicting assay sensitivity and specificity.

Samples were from 65 PCM patient sera and 99 control sera, including patients with histoplasmosis, aspergillosis, non-fungal diseases, and healthy subjects. The ROC is plotted between the true-positive rate (sensitivity) on the y-axis and the false-positive rate (100-specificity) on the x-axis. The area under the curve (AUC) represents the accuracy of the LA test, which was 0.962±0.0143 (95% CI 0.920–0.986, P<0.0001) for the gp43-SLPs, 0.929±0.0192 (95% CI 0.878–0.963, P<0.0001) for the mAb17c-SLPs, and 0.992±0.00769 (95% CI 0.964–1.000, P<0.0001) for the DID. The more the AUC is greater than 0.5, the better the test.