Detection of specific antibodies to an antigenic mannoprotein for diagnosis of Penicillium marneffei penicilliosis

Abstract

The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marneffei. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis.

FIG. 1

Western blot analysis of the purified recombinant Mp1p protein antigen with serum from a P. marneffei-inoculated guinea pig. Molecular size markers (in kilodaltons) are shown on the left.

FIG. 2

ELISA-based antibody test detects high levels of specific antibodies only in P. marneffei-inoculated guinea pigs. Serial dilutions of the guinea pig sera were made, and an ELISA was performed to determine the antibody levels in the animal sera. OD₄₀₅ values were obtained and plotted. Standard deviations for each serum group were obtained, with the exception of H. capsulatum, for which only one serum specimen was tested. The numbers of guinea pigs used for each group were as follows: preimmune, 3; P. marneffei inoculated, 3; C. albicans inoculated, 3; A. fumigatus inoculated, 3; H. capsulatum inoculated, 1; and B. dermatitidis inoculated, 2.

FIG. 3

Evaluation of sensitivity and specificity of P. marneffei antibody test of penicilliosis patients. To evaluate the sensitivity and specificity of the P. marneffei antibody test, serum specimens were obtained from 17 documented penicilliosis patients with HIV infection. In addition, serum specimens from two penicilliosis patients without HIV infection or other conditions of immunodeficiency were also included in the evaluation. The control serum specimens for the test came from 90 healthy blood donors, 55 tuberculosis patients, and 20 typhoid fever patients. All serum samples were diluted 1:100 before the ELISA test was performed. The test results at OD₄₀₅ were plotted. The cutoff line for positive diagnosis is drawn at a value that equals the sum of the mean value and 10 times the standard deviation of healthy blood donors.