Identification Of Fungal Organisms Research Articles

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

Diagnosis of deep cutaneous fungal infections: Correlation between skin tissue culture and histopathology

Deep cutaneous fungal infections (DCFIs) are responsible for significant morbidity and mortality, particularly in immunocompromised patients. Although a direct correlation between histopathologic examination and culture is expected, discordant findings may be seen, presenting a unique diagnostic and therapeutic challenge. We sought to determine the correlation between skin tissue cultures and histopathologic examination in patients with DCFI. This is a 10-year retrospective review (2003-2012) of patients with a diagnosis of DCFI seen at a single tertiary care institution. Tissue cultures and histopathologic findings were reviewed. In 8 of 33 cases, fungal elements were seen on routine histopathologic sections but skin cultures were negative. Three of 8 of the discordant cases had concurrent positive non-skin tissue cultures that correlated with the pathology interpretation, and 3 of 8 patients in the discordant group died of systemic fungal infection. This was a retrospective study design and a single tertiary care institution experience. The histopathologic interpretation of skin tissue specimens is critical for rapid and accurate diagnosis of DCFI. Despite the identification of fungal organisms on histopathologic assessment of skin tissue specimens, skin tissue culture may fail to show fungal growth. A diagnosis of a DCFI and initiation of appropriate treatment should always be considered in spite of discordant results.

Prevalence and impact of invasive fungal infections in intensive care

Invasive fungal infections (IFI) affect 1% of ICU patients and are increasing in incidence. IFIs are associated with a poor prognosis, which is further complicated by difficulties in identification of fungal organisms by traditional culture methods and the emergence of Candida species resistant to triazole therapy [1,2]. This study aimed to assess the prevalence of IFIs, the organisms responsible and outcomes of patients affected.

Molecular monitoring of fungal communities in air samples by denaturing high-performance liquid chromatography (D-HPLC)

To describe a new molecular technique for the assessment of fungal diversity in the air. Air samples were collected every week in a henhouse in France during a 15-week period. After air sampling, the collecting membrane was diluted, and the liquid was used for subsequent cultivation and molecular analysis: PCR-temperature temporal gradient electrophoresis (TTGE), which has already been used for the identification of fungal species in air samples and PCR-denaturing high-performance liquid chromatography (D-HPLC), a new technique for the analysis of complex microbial populations. D-HPLC profiles were reproducible from run-to-run, and several fungal organisms could be identified at the species level by sequencing. PCR-D-HPLC enabled the identification of fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The new technique allowed the detection of more fungal species than did the PCR-TTGE technique. However, some fungal species were detected only by PCR-TTGE, suggesting that PCR-D-HPLC and PCR-TTGE are complementary. PCR-D-HPLC represents a considerable saving in time over currently available procedures for detection and identification of fungal organisms in air. However, the fungal diversity detected by PCR-D-HPLC or by PCR-TTGE was lower than that revealed by culture.

Immunoreactivity of the fungal cell wall

The cell wall is the major fungal structure involved in the interaction with the host and most of the immunological effects observed with intact fungal cells have been reproduced with cell-wall components. As a result of the exposure to fungal antigens, most individuals develop both cellular and antibody responses intended to limit the invasiveness or to eradicate the fungus from the infected tissues. However, a number of fungi including Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Trichophyton spp. and Histoplasma capsulatum can also induce T- and B-suppressive activities. A wide diversity of immunodominant cell-wall antigens for both cell-mediated and humoral responses have been identified in the most important fungal pathogens, although considerable differences exist in the information available at the molecular level among the different mycoses. Cellular responses require macrophage and Th1 activation, whereas humoral responses comprise the activation of the complement system and the induction of antibodies. The ability of fungal cell-wall components to elicit cellular or humoral immune responses has been traditionally used in the serodiagnosis of mycoses, the identification of fungal organisms and the development of vaccines for the prevention of mycoses. In the future, the analysis of such molecules will provide critical information in understanding the nature of host- fungus interactions.

Fluorescence of fungi in superficial and deep fungal infections.

Fluorescence of many fungi is noted when H&E stained sections are examined under a fluorescent microscope. In theory, this phenomenon could aid in the diagnosis of cutaneous and disseminated fungal infections without the delay associated with special stains. Seventy-six cases of superficial and deep fungal infections and 3 cases of protothecosis were studied to determine the clinical usefulness of this technique. In most cases, fluorescence was noted, but was not intense. Fluorescence of fungi did not correlate with the age of the specimen. In most cases, organisms in H&E stained sections were more easily identified with routine light microscopy than with fluorescent microscopy. This report suggests that in H&E stained skin specimens, fluorescent microscopy is of little benefit in the identification of fungal organisms.

Immunoreactivity of the fungal cell wall

The cell wall is the major fungal structure involved in the interaction with the host and most of the immunological effects observed with intact fungal cells have been reproduced with cell-wall components. As a result of the exposure to fungal antigens, most individuals develop both cellular and antibody responses intended to limit the invasiveness or to eradicate the fungus from the infected tissues. However, a number of fungi including Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Trichophyton spp. and Histoplasma capsulatum can also induce T- and B-suppressive activities. A wide diversity of immunodominant cell-wall antigens for both cell-mediated and humoral responses have been identified in the most important fungal pathogens, although considerable differences exist in the information available at the molecular level among the different mycoses. Cellular responses require macrophage and Th1 activation, whereas humoral responses comprise the activation of the complement system and the induction of antibodies. The ability of fungal cell-wall components to elicit cellular or humoral immune responses has been traditionally used in the serodiagnosis of mycoses, the identification of fungal organisms and the development of vaccines for the prevention of mycoses. In the future, the analysis of such molecules will provide critical information in understanding the nature of host-fungus interactions.