LETTER

A 30-year-old man was referred to the Hospital of Basurto in October 2000 for evaluation of a 48-h attack of fever, shivering, unproductive cough, left chest pain, and diarrhea. The patient had a long history of chronic granulomatous disease (CGD) which had been diagnosed 11 years earlier, in 1989, with nocardial pneumonia (13). Pulmonary auscultation revealed crackles over the anterior plane of the right hemithorax and over the medial axillary line of the left hemithorax. A chest radiograph with an air bronchogram showed an area of consolidation with alveolar characteristics in the posterior segment of the right upper lobe. Initial treatment with levofloxacin and trimethoprim-sulfamethoxazole was started, with clinical and radiological improvement.

Considering the past medical history of the patient, various sputum specimens were sent to be examined for usual and opportunistic organisms. Right upper lobe bronchoalveolar lavage, bronchial aspiration, and telescopic catheter samples were obtained when bronchoscopy was done. Cultures of sputum and bronchial washing were positive for Nocardia spp. on Mueller-Hinton, blood, and chocolate agars, although no suggestive organisms were observed with Gram stain. The isolate was identified by standard biochemical tests for actinomycetes (1, 8).

Susceptibility testing by disk diffusion onto Mueller-Hinton agar (14) showed that the isolate was susceptible to broad-spectrum cephalosporins, amikacin, amoxicillin-clavulanic acid, and trimethoprim-sulfamethoxazole and was resistant to erythromycin and ofloxacin. With this susceptibility pattern, and according to the new taxonomic classifications (1, 15), the isolate was identified as Nocardia asteroides sensu stricto.

After revision of the microbiological history of the first isolate and by comparison of its susceptibility pattern with that of the present isolate, we noted that the first isolate had a susceptibility pattern different from the present isolate. The first isolate was resistant to broad-spectrum cephalosporins and susceptible to amikacin, imipenem, trimethoprim-sulfamethoxazole, tetracyclines, and amoxicillin-clavulanic acid. Because of this, the first isolate was reidentified as Nocardia farcinica and not N. asteroides as it had been identified before (14). The first isolate was recovered from our archive, and its antimicrobial susceptibility pattern was confirmed. The identification of both isolates was confirmed by PCR restriction fragment length polymorphism (RFLP) analysis. Primers Tb11 and Tb12 were used for the PCR amplification of a 439-bp fragment of the hsp-65 gene (12). Twenty microliters of PCR product was digested using 5 U of either MspI or HinfI for 60 min at 37°C, with 2.5 µl of the corresponding buffer. The digestion product was loaded onto a 3% MS-8 agarose gel, and the fragments were visualized using ethidium bromide staining and UV light (10). The type strains N. asteroides CECT 3051 and N. farcinica CECT 3053 were used as control strains. They were obtained from Colección Española de Cultivos Tipo.

PCR-RFLP analysis of the first isolates and the type strain CECT 3053 produced only one band of 440 bp by digestion with both enzymes, while the second isolate and the type strain CECT 3051 produced one band of 440 bp by digestion with HinfI and three bands of 180, 145, and 120 bp by digestion with MspI. By comparison of these patterns with those of published data (10), the first isolate was identified as N. farcinica and the second was identified as N. asteroides type VI (Fig. 1).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   RFLP band patterns from MspI and HinfI digests of PCR-amplified sequences of the hsp-65 gene. Lanes 1 to 4 show products of HinfI digestion (lane 1, CECT 3051; lane 2, CECT 3053; lane 3, second clinical isolate; lane 4, first clinical isolate), and lanes 5 to 8 show products of mspI digestion (lane 5, CECT 3051; lane 6, CECT 3053; lane 7, second clinical isolate; lane 8, first clinical isolate). Lanes M, molecular size marker VIII.

Patients with CGD suffer from repeated bacterial and fungal infections as a result of the inability of macrophages to eliminate catalase-positive bacteria. In these patients, there is a severe alteration in the activity of the reduced NADPH oxidase system (3, 4). The aerobic actinomycetes, including the family Nocardiaceae, are bacteria that belong to the order Actinomycetales, with complex and evolving taxonomy (6, 7). The standard biochemical and hydrolysis tests were not able to delineate the true heterogeneity of the N. asteroides taxon. Recently, by using different methods, including DNA homology studies, 11 species of the genus Nocardia were recognized and two specific subgroups of the N. asteroides complex have been accepted as distinct species, N. nova and N. farcinica (1, 16). Infection with N. farcinica, according to the described cases, seems to be more virulent and disseminated than infection with other species of the genus (3, 7, 9). The difference in the clinical courses of the two episodes described in the present report may be due to the early treatment of the second episode. Many reports of cases of recurrent infection by Nocardia have been published; all of them demonstrate reinfection by the same species in patients with CGD or with some other respiratory pathology (2, 5, 11). Recurrence of infection usually occurs a short time after the initial infection. To our knowledge, this is the first documented case of infection by different Nocardia species, in different pulmonary areas and with 10 years separating the first and second infections.

PCR-RFLP represents a rapid and sensitive method for the identification of clinical isolates of Nocardia spp. and can be readily implemented for routine clinical use.