Question

Discuss, in detail, methods which may be employed to confirm mycotic infections other than culture and direct staining methodologies.

Answer 1

Diagnosis of fungal infection has relied primarily on methods such as direct microscopic examination of clinical samples, histopathology, and culture. The growth in the number of fungi that clinical mycologists must identify has forced investigators to develop and apply new methods for fungal identification that go beyond classical phenotypic methods. Culture, direct microscopy, and histopathology have been the foundation for diagnosis of fungal infection for many decades. Microscopy, histopathology, and use of fungal-specific stains play important roles in diagnosis.

Serologic tests for patient antibodies have been useful for non-culture-based diagnosis of fungal infection. Serology is of greatest value in diagnosis of endemic mycoses. Available technologies include immunodiffusion (ID), complement fixation (CF), and enzyme immunoassay (EIA). CF and immunodiffusion are the most common serologic tests for diagnosis of histoplasmosis. The ID test detects precipitating antibodies to Histoplasma H and M antigens. Serologic testing for histoplasmosis is most useful if an increase in CF titer is observed between acute and convalescent sera in acute histoplasmosis. Depending on the antigen used in the test, the qualitative ID test will determine the presence of coccidioidal IgM with a result that is similar to a tube precipitin test (IDTP) or coccidioidal IgG that detects antibody recognized by the CF test (IDCF). Detection of IgM is useful in diagnosis of acute primary coccidioidomycosis in which the sensitivity may be >80%. CF detects IgG antibodies. IgG antibodies are produced during the convalescent phase of disease or during chronic infection. CF is more sensitive than IDCF and provides quantitative results. A commercially available EIA can be used to detect IgM or IgG antibodies.

Molecular methods for fungal identification generally work best when pure cultures are available. However, because polymerase chain reaction (PCR) plays a role in many molecular identification methods, molecular identification can work in the absence of live cells if template nucleic acid is available in patient specimens, including fixed tissue. PCR is a central component for many molecular methods, either as the main diagnostic strategy or as one of the preliminary steps in the diagnostic assay. Consequently, diagnostic PCR encompasses a number of different approaches. The simplest consists of conventional PCR in which species-specific primers that have been designed based on existing sequence or data, are used to amplify fungal DNA from clinical specimens.

MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) is the most popular and fastest growing non-nucleic acid sequence based molecular diagnostic assay for fungi. The technique generates species-specific spectra that provide a unique signature characteristic of the species. MALDI-TOF instrumentation consists of an ion source that transfers sample molecules into a gas phase, a mass analyzer that resolves ions based on mass-to-charge ratio, and a component that detects the ions. Samples are mixed with a matrix of small acidic molecules that crystallizes the specimen and facilitates ionization because the matrix absorbs energy in the range of the laser used for sample excitation. The TOF component consists of a tube that the excited ions travel through, with the transit time (time-of-flight) of individual ions providing the method for identification. The generated spectra are screened against a library of reference spectra, which correspond to individual species.