What can be learned from genotyping of fungi?

Multiple genotyping studies have been carried out in order to clarify the epidemiology of fungal infections, more specifically to determine the sources, transmission routes, and colonization patterns of fungal isolates. In this review, the results obtained in genotyping investigations of Aspergillus isolates are summarized and discussed. Furthermore, we examine the epidemiologic studies of Candida albicans, Exophiala dermatitidis and Scedosporium apiospermum infections in patients with cystic fibrosis. Relative to Aspergillus fumigatus, colonization of the respiratory tract by multiple strains, and of deep organs by only a single strain were observed. On the other hand, the few studies which focused on other fungi isolated from patients with cystic fibrosis have suggested that colonization occurs primarily by a dominant genotype.

Rapid quantification of itraconazole-resistant Aspergillus fumigatus in air.

Solid-phase cytometry (SPC) was used to determine the total number and the number of itraconazole-resistant Aspergillus fumigatus cells in 60 air samples. Of the 570 A. fumigatus cells that were recovered, 10 (1.8%) were resistant. SPC proved more specific and rapid than culture and allowed high-troughput susceptibility testing.

Rapid and direct quantification of viable Candida species in whole blood by use of immunomagnetic separation and solid-phase cytometry.

Candida species are a common source of nosocomial bloodstream infections in critically ill patients. The sensitivity of the traditional diagnostic procedure based on blood culture is variable, and it usually takes 2 to 4 days before growth of Candida species is detected. We developed a 4-h method for the quantification of Candida species in blood, combining immunomagnetic separation (IMS) with solid-phase cytometry (SPC) using viability labeling. Additionally, Candida albicans cells could be identified in real time by using fluorescent in situ hybridization. By analysis of spiked blood samples, our method was shown to be sensitive and specific, with a low detection limit (1 cell/ml of blood). In a proof-of-concept study, we applied the IMS/SPC method to 16 clinical samples and compared it to traditional blood culture. Our method proved more sensitive than culture (seven samples were positive with IMS/SPC but negative with blood culture), and identification results were in agreement. The IMS/SPC data also suggest that mixed infections might occur frequently, as C. albicans and at least one other Candida species were found in five samples. Additionally, in two cases, high numbers of cells (175 to 480 cells/ml of blood) were associated with an endovascular source of infection.

Rapid detection and quantification of Aspergillus fumigatus in environmental air samples using solid-phase cytometry.

Aspergillus fumigatus is an ubiquitous fungus capable of causing severe infections such as aspergilloma, allergic bronchopulmonary aspergillosis, and invasive aspergillosis, especially in immunocompromised patients. Monitoring the number of Aspergillus fumigatus spores in the air is crucial for infection control. In the present study, a novel approach for the quantification of Aspergillus fumigatus, based on solid-phase cytometry (SPC) and immunofluorescent labeling, was developed. The sensitivity and specificity of the assay were confirmed by testing pure cultures. Paecilomyces variotii and Rhizopus stolonifer were codetected but could be excluded on the basis of morphology of the microcolonies. The SPC method has considerable advantages compared to the culture-based method, including its low detection limit (4 cells/m3), its speed (results are obtained within 24 h), and the straightforward microscopic identification of Aspergillus fumigatus. Additionally, comparison of results obtained with both methods demonstrated that they are equally accurate for the quantification of Aspergillus fumigatus in environmental air samples.

Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry.

This protocol describes the use of solid-phase cytometry for the enumeration of airborne bacteria and fungi. In contrast with conventional methods, accurate results can be obtained in real time, especially for air samples with low numbers of microorganisms. Air samples are collected by impaction on a water-soluble polymer that is subsequently dissolved. Part of the sample can be filtered over two membrane filters with different pore sizes. One filter is used to obtain a total count of all viable microorganisms, and a second filter is used to determine the number of airborne fungi. Microorganisms present on the filter are labeled with a viability substrate and subsequently detected and quantified using a solid-phase cytometer. The detected spots are microscopically validated using an epifluorescence microscope to discriminate between bacteria, fungi and fluorescent particles. The whole procedure takes 5 h to complete and results in the accurate quantification of airborne bacteria and fungi for samples with a low or high microbial load.

Microsatellite typing of Aspergillus fumigatus isolates recovered from deep organ samples of patients with invasive aspergillosis.

Microsatellite typing was used to analyze 41 Aspergillus fumigatus isolates from 9 patients with proven invasive aspergillosis hospitalized in 2 different centers. No strains were shared between patients. For 8 of 9 patients, a single genotype was found for the isolates recovered from all anatomic sites involved.

Enumeration of airborne bacteria and fungi using solid phase cytometry.

Conventional methods for the enumeration of airborne micro-organisms are inaccurate and time-consuming, hence the interest in novel approaches is increasing. In the present study, the use of solid phase cytometry (SPC) was evaluated for the enumeration of airborne micro-organisms. A 4 h SPC procedure based on viability staining was applied to samples from 50 locations and compared with an optimised culture-based method. Plate counts after air sampling were repeatable but strongly dependent on sampling volume. Samples with low or high microbial load were difficult to analyse using the culture-based method, unlike with SPC. Results show that SPC can be considered superior to the culture-based method because of its much higher dynamic range, its speed and its ability to enumerate not only culturable but all viable micro-organisms.