Non-culture methods being developed and evaluated for mycotic
infections include polymerase chain reaction (PCR),
galactomannan (GM) antigenemia, Western blot (WB) to detect
antibodies, and detection of the fungal metabolites
D-arabinitol and
(1,3)-beta-D-glucan. Sample preparation for PCR from blood specimens depends on fractionation of peripheral blood, its pre-incubation in blood culture broth, or a total
DNA method, which does not rely on fractionation, or pre-incubation. Targets for PCR of fungi in the 18S or ITS2 subunits of the ribosomal RNA genes facilitated the design of Aspergillus and Candida genus and species probes. Amplicons were identified using PCR-
enzyme linked
immunosorbent assay (ELISA) or reverse line-blot formats. A pilot study indicated that PCR tests on blood specimens were positive at least once in patients with confirmed invasive
aspergillosis (IA). When serum-PCR and serum-GM tests were compared in IA patients, antigenemia was more often positive. PCR detected Aspergillus
DNA in bronchoalveolar lavage specimens from patients at risk even when cultures were negative.
D-Arabinitol can be detected as a marker of
candidiasis with gas chromatography-mass spectrometry or
enzyme dependent-fluorometry. Each method can differentiate the microbial D- and host L-enantiomers.
(1,3)-beta-D-Glucan is produced by most genera of pathogenic fungi and can be detected in plasma by the 'G-test'. In patients with
febrile neutropenia the efficacy of
azole therapy correlated with plasma
(1,3)-beta-D-glucan concentrations of > or = 10 pg ml(-1). The diagnosis of early acute
pulmonary histoplasmosis can be improved by a WB test utilizing deglycosylated M
antigen, a 94-kDa
glycoprotein. The identity of M
antigen as a
catalase was deduced from the sequence of the cloned gene. PCR identification of Histoplasma capsulatum cultures was accomplished with primer pairs selected from H and M
antigen gene sequences.