Association of Clinical Pathologists. Irish Branch Meeting 27-28 March 1998

In the past conventional bacteriological techniques have misidentified Gram negative organisms such as Stenotrophomonas maltophilia as Burkholderia cepacia due to colonial and phenotypic similarities. Identification is extremely important as B. cepacia has severe consequences for CF patients which ultimately lead to death. Categorisation with B. cepacia has implications for infection control, psychosocial issues and patient management. The aim of this study was to provide a faster and more specific technique for the detection of Pseudomonas species, Pseudomonas aeruginosa, and B. cepacia. The techniques employed were split into conventional and molecular. Conventional methods involved using Pseudomonas Isolation Agar Medium, which supports the growth of Pseudomonas species and also B. cepacia. MAST cepacia agar was also used which is specific for the growth ofB. cepacia. However false positives may occur and can include Ralstonia spp., Alcaligenes spp., and S. maltophilia which maybe misidentified as B. cepacia. The molecular method employs sequence specific primers for Pseudomonas spp., P. aeruginosa and B. cepacia to amplify the bacterial DNA by; PCR. The DNA is then visualised using a 2% (w/v) agarose gel stained with ethidium bromide. Sputa samples from 66 adult CF patients were examined using both techniques. For Pseudomonas spp. conventional methods detected 41 positives compared to 47 strong and 6 weak positives by PCR. For P. aeruginosa, conventional methods detected 38 positives and PCR detected 40 strong and 7 weak positives. Conventional methods detected 20 positive B. cepacia patients compared to 25 strong and 12 weak positive by PCR. PCR provides rapid and specific detection of small number of these Gram negative organisms. Conventional methods can be slow and sometimes it can be difficult to grow the organism at it is extremely difficult to mimic the in vivo growth conditions. Presently the clinical significance of PCR weak positive/culture negative patients is being assessed. Bone marrow biopsies were divided and fixed in 10% formalin and Bouins solutions. Morphology and immunocytochemical reactivity with various antibodies using the Avidin Biotin technique for visualisation ofreactions was assessed on sections from each. Lymphoplasmacytoid morphology was clearly demonstrable in the Bouins fixed portion of one biopsy, whereas the only diagnosis possible in the formalin fixed portion was non-Hodgkin's lymphoma. Reactions with anti IgG, A and M heavy chain and IgK and L anti-immunoglobulin antibodies were far stronger in Bouins fixed samples whereas equivalent staining for both fixatives was obtained with anti CD15, muramidase, CD3, CD19, VIII and glycophorin A. …