Fig 1.
Flow diagram of the selection of 460 clinical samples included in this study.
The culture-confirmed T. marneffei infection group included 79 patients (Table A in S1 File). The non-T. marneffei control group consisted of 381 participants, including 139 patients with other microbial infections (Table B in S1 File), 170 patients without evidence of infection, and 72 healthy individuals from endemic areas. The 309 patients with or without additional isolated pathogens were epidemiologically similar to the 79 patients with T. marneffei. The serum samples were obtained from the Clinical Pathology Unit, Maharaj Nakorn Chiang Mai Hospital (a tertiary hospital affiliated with Chiang Mai University), Chiang Mai, Thailand, and the Department of Clinical Laboratory, Affiliated Hospital of Youjiang Medical University for Nationalities, China.
Table 1.
Baseline clinical characteristics of study participants.
Table 2.
Antigen preparations were derived from the diverse fungal isolates.
Fig 2.
Schematic representation of the biotin-labeled 4D1-based sandwich ELISA for the detection of TMCYA.
(1) Capture mAb 4D1 is coated on the solid phase. (2) TMCYA antigen binds to the immobilized antibody via its Asn(Asparagine)-containing epitope. (3) Biotin-labeled 4D1 detection antibody binds to a second epitope on TMCYA, forming the sandwich complex. (4) Streptavidin-HRP associates with the biotin-labeled 4D1. (5) Addition of chromogenic substrate yields an HRP-catalyzed colored product for detection.
Fig 3.
Verification of biotin-labeled 4D1 antibody conjugation and its binding specificity for TMCYA.
(A) Coomassie Brilliant Blue-stained 10% SDS-PAGE of ten fungal cytoplasmic antigen preparations (10 μg/well), demonstrating successful protein extraction and clear band separation across a broad molecular weight range (11-180 kDa). (B) Western blot of the same antigen panel probed with biotin-labeled 4D1 followed by streptavidin-HRP detection. A positive signal was observed exclusively in the TMCYA lane (lane 2), producing a broad immunoreactive band spanning approximately 50-150 kDa. No reactivity was detected against cytoplasmic antigens derived from T. marneffei conidia or mold forms, or from other fungal species tested under the experimental conditions, supporting the high specificity of the 4D1 conjugate for TMCYA. Lane assignments are as follows: lane 1, molecular weight marker; lane 2, T. marneffei yeast; lane 3, T. marneffei conidia; lane 4, T. marneffei mold; lane 5, C. albicans; lane 6, P. kudriazevii; lane 7, Geotrichum sp.; lane 8, S. apiospermum; lane 9, Trichosporon sp.; lane 10, S. schenckii (yeast); lane 11, C. neoformans; lane 12, P. citrinum; lane 13, A. fumigatus.
Fig 4.
Standard curve of the biotin-labeled 4D1 sandwich ELISA for the detection of TMCYA.
A standard curve was generated using serial dilutions of pooled serum spiked with TMCYA at concentrations ranging from 0.781 to 1000 μg/mL. Each concentration was assayed in triplicate, and the results are presented as mean ± standard deviation (SD) from three independent experiments. The curve was fitted using a four-parameter logistic (4-PL) regression model and demonstrated excellent reproducibility. Within this concentration range, the coefficient of determination (R²) between the OD values and TMCYA concentrations was 0.997, indicating a highly reliable fit. The detection limit of the assay for T. marneffei antigen spiked in pooled serum was approximately 19.398 μg/mL.
Fig 5.
The specificity of the biotin-labeled 4D1 sandwich ELISA.
Serial concentrations (0.781-1000 μg/mL) of cytoplasmic antigens from T. marneffei (yeast, mold, and conidia) and 9 non-T. marneffei fungi were evaluated using the biotin-labeled 4D1 sandwich ELISA. Each concentration was assayed in duplicate across three independent experiments, and data are presented as mean ± SD. A clear dose-dependent increase in OD450 was observed exclusively for TMCYA, whereas T. marneffei mold and conidia as well as all other fungal antigens produced only minimal background signals without a detectable response curve. Two-way ANOVA revealed a highly significant interaction between fungal species and antigen concentration (P < 0.0001), demonstrating that only TMCYA is specifically recognized by the assay.
Fig 6.
Diagnostic performance of the biotin-labeled 4D1 sandwich ELISA for detecting TMCYA in serum.
(A) Comparison of OD450 values between culture-confirmed T. marneffei-positive patients and the control group. Each point represents an individual serum sample. The median OD450 in the culture-positive group was significantly higher than that of controls (Mann-Whitney test, P < 0.0001). A diagnostic cut-off value of 0.268 (dashed blue line) was determined based on ROC curve analysis. (B) Receiver operating characteristic (ROC) curve of the assay. The area under the curve (AUC) was 0.983 (95% CI: 0.972-0.994, P < 0.0001), indicating excellent diagnostic accuracy. The optimal cut-off point was identified at the maximal Youden index.
Table 3.
Diagnostic performance of the biotin-labeled 4D1 sandwich ELISA compared with culture (reference standard).
Table 4.
Diagnostic performance of the biotin-labeled 4D1 sandwich ELISA when testing serum samples from individuals with or without T. marneffei infection.