Outbreaks of infectious disease often result from exposure to a common source of the etiologic agent. Generally, the etiologic agent causing an outbreak of infection is derived from a single cell whose progeny are genetically identical or closely related to the source organism. In epidemiological terms, the organisms involved in the outbreak are clonally related; that is, they have a common origin. Clonally related organisms are members of the same species that share virulence factors, biochemical traits, and genomic characteristics. However, there is sufficient diversity at the species level that organisms isolated from different sources at different times and in different geographical regions may be differentiated or classified into subtypes or strains.

Principles and Applications of Methods for DNA-Based Typing of Microbial Organisms

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies.

The Ins and Outs of DNA Fingerprinting the Infectious Fungi

We sought the reservoir of Fusarium species in a hospital with cases of known fusarial infections. Cultures of samples from patients and the environment were performed and evaluated for relatedness by use of molecular methods. Fusarium species was recovered from 162 (57%) of 283 water system samples. Of 92 sink drains tested, 72 (88%) yielded Fusarium solani; 12 (16%) of 71 sink faucet aerators and 2 (8%) of 26 shower heads yielded Fusarium oxysporum. Fusarium solani was isolated from the hospital water tank. Aerosolization of Fusarium species was documented after running the showers. Molecular biotyping revealed multiple distinct genotypes among the isolates from the environment and patients. Eight of 20 patients with F. solani infections had isolates with a molecular match with either an environmental isolate (n = 2) or another patient isolate (n = 6). The time interval between the 2 matched patient-environment isolates pairs was 5 and 11 months, and 2, 4, and 5.5 years for the 3 patient-patient isolate pairs. The water distribution system of a hospital was identified as a reservoir of Fusarium species.

Fusariosis Associated with Pathogenic Fusarium Species Colonization of a Hospital Water System: A New Paradigm for the Epidemiology of Opportunistic Mold Infections

Nosocomial Fungal Infections: Epidemiology, Infection Control, and Prevention

Molecular characterization of Candida albicans isolates is essential for understanding the epidemiology of nosocomial infections caused by this yeast. Here, we investigated the potential value of multilocus sequence typing (MLST) for characterizing epidemiologically related or unrelated C. albicans strains of various clinical origins. Accordingly, we sequenced the internal regions (loci) of six selected housekeeping genes of 40 C. albicans clinical isolates and 2 reference strains. In all, 68 polymorphic nucleotide sites were identified, of which 65 were found to be heterozygous in at least one isolate. Ten to 24 different genotypes were observed at the different loci, resulting, when combined, in 39 unique genotype combinations or diploid sequence types (DSTs). When MLST was applied to 26 epidemiologically unrelated isolates and the 2 reference strains, it allowed the identification of 27 independent DSTs, thus demonstrating a discriminatory power of 99.7. Using multidimensional scaling together with the minimum spanning tree method to analyze interstrain relationships, we identified six groups of genetically related isolates on the basis of bootstrap values of greater than 900. Application of MLST to 14 epidemiologically related isolates showed that those recovered from patients in the same hospital ward during the same 3 months had specific DSTs, although 73% of these isolates were genetically very close. This suggests that MLST can trace minute variations in the sequences of related isolates. Overall, MLST proved to be a highly discriminatory and stable method for unambiguous characterization of C. albicans.

Usefulness of Multilocus Sequence Typing for Characterization of Clinical Isolates of Candida albicans

Burn patients are particularly exposed to deep-seated nosocomial infections caused by Candida species. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. We report the use of random amplified polymorphic DNA to type isolates of C. albicans in the Hopital Cochin burn unit. This molecular typing method, which is based on PCR with arbitrary short primers, was evaluated on a panel of 32 C. albicans strains isolated from various anatomical sites of unrelated patients, and the strains showed 22 different patterns. Random amplified polymorphic DNA was then used in the epidemiological surveillance of the patients in the burn unit over a 9-month period. Seven patterns were identified among 84 isolates from 18 patients. One pattern (pattern A) corresponding to isolates from 7 of the 18 patients (68% of isolates) predominated throughout the 9-month study, while some strains with other profiles were isolated only once. Some profiles appeared to show a particular geographic pattern within the unit, suggesting transmission from room to room. These results underline the importance of fungal surveillance in such patients and the need to inform nursing staff of measures to prevent the spread of Candida spp. from patient to patient.

M Schlotterer

Papers

Use of random amplified polymorphic DNA as a typing method for Candida albicans in epidemiological surveillance of a burn unit.