Molecular identification of Burkholderia spp. directly from sputum samples of South American cystic fibrosis patients reveals genetic heterogeneity

CASTAÑEDA N.C, PIVETTA O. AND CENTRÓN D.

Buenos Aires, Argentina

Congreso; 6thWorld Congress of the World Society for Pediatric Infectious Diseases (WSPID); 2009

World Society for Pediatric Infectious Diseases

  Cystic fibrosis (CF) is the most common lethal inherited disorder affecting Caucasians, with an incidence of approximately one in 3500. Respiratory tract infection associated to failure (90%) is the major cause of morbidity and mortality in CF patients. The most common pathogen in respiratory secretions of CF patients are Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and members of the Burkholderia cepacia complex (BCC). The latest is an emergent opportunist with a high level of antibiotic resistance, a high rate of transmission among CF patients and requirement of molecular tools for accurate diagnosis. On the other hand, mucoid strains of Pseudomonas aeruginosa are frequently isolated from chronic infection with accelerated decline in lung function. Multiplex PCR (m-PCR) reactions were performed on sputum sample of one hundred and twenty four patients (55 male, and 69 female) attending our laboratory from December 2005 to April 2009 from Brazil. paraguay By using different primer pairs targeting specific genomic sequences of P. aeruginosa, MRSA (meticillin-resistance-Staphyloccocus aureus) and BCC, we developped m-PCR with high values of sensibility and specificity. RFLP-PCR of the RecA gene revealed not to have enough discrimination since sequence analysis evidenced novel BCC species as well as Burkholderia species that are rarely encountered from human specimens. PCR and sequencing 348 bp of the recA gene identified non cultivable, indeterminated and new species of  Burkholderia spp.. The majority of patients were infected with Burkholderia cenocepacia (formerly genomovar III), B. multivorans, and B. contaminans. In addition, this technique led us to detect  brother-to-sister transmission as well as persistence and/or replacement of Burkholderia spp. species. The early molecular detection of low numbers of P. aeruginosa in the CF lungs by PCR  have been very successful to efficient control, prevention of cross-infection and early aggressive antibiotic therapy of P. aeruginosa colonization in our patients. Since MRSA can spread easily from patient to patient via personnel, hospitals over the world are confronted with the problem to control MRSA. Consequently, there is a need to develop rapid and simple screening or diagnostic tests for detection and/or identification of MRSA to reduce its dissemination and improve the diagnosis and treatment of infected patients. Given the diversity of these CF isolates, the rapid diagnostic based in molecular approaches from respiratory samples appears to be the optimum choice for identification of atypical Burkholderia spp, P. aeruginosa and MRSA in CF patients from differents geographical region of latin America.