Molecular Study of Persistence ofNocardia asteroidesand Nocardia otitidiscaviarumStrains in Patients with Long-Term Nocardiosis

Nocardiaspp. are gram-positive, aerobic actinomycetes with rudimentary to extensively branched vegetative hyphae which fragment into bacteroid, rod-shaped to coccoid elements. No- cardiae have a worldwide distribution in soil, and at least six species are pathogenic for humans and animals. They may enter the body via inhalation of contaminated dust particles or via wounds contaminated with dust or soil (14). Nocardiosis is an indolent process, most frequently charac- terized by primary pulmonary lesions that may be typically subacute or chronic. The disease tends to be more acute in immunodepressedpatients.Remissionsandexacerbationslast- ing for days or weeks are characteristic. The disease may spread hematogenously to other organs and has a predilection for the central nervous system. In the skin, several types of lesions may be seen, including mycetoma. The mycetoma is a slowlyprogressiveprocessthatmaytakeseveralyearstoevolve (2). No report describes long-term persistent nocardiosis in whichseveralisolatesfromthesamepatientwereobtainedand geneticallycompared.Similarly,comparisonofrelapseisolates with original strains in recurrences ofNocardiainfection after repeated, prolonged, and apparently successful chemotherapy are generally not performed. However, a recurrent Nocardia pneumonia was diagnosed in a patient with chronic granulo- matous disease over a 2.5-year interval on the basis of plasmid analysis and antibiotic susceptibility data (11). Accurate methods for strain identification are essential for comparisonofclinicalisolates.Conventionalphenotypicmeth- ods based on serotyping (19), biotyping (22), and typing with a yeast killer system (17) have been applied to nocardiae. New molecular genotyping methods based on analysis of chromo- somal DNA provide more reproducible data and avoid depen- dence on potentially variable phenotypic traits (21). Plasmid profiling could be a reliable method (11), but only 25 and 50% of Nocardia asteroides and Nocardia farcinica isolates, respec- tively, carry plasmids (20). Moreover, plasmid acquisition or loss cannot be excluded. Although ribosomal DNA gene pro- filing (ribotyping) is of value in typing numerous bacterial pathogens (3), this technique did not allow the differentiation of reference strains from strains isolated during a limited out- break (9). Moreover, ribotyping is time-consuming and is not well suited to routine use. However, this method could allow species diagnosis (13). A PCR method based on the random amplification of polymorphic DNA (RAPD) fragments by us- ing a single primer with an arbitrary sequence has been de- scribed previously (25) and has been applied to the typing of various pathogenic microorganisms (24). In the present study, we evaluated the application of RAPD fingerprinting with 10- and 12-mer primers of arbitrary se- quence for comparison of N. asteroides and Nocardia otitidis- caviarum strains isolated from patients with long-term persis- tent nocardiosis.