Association of Clinical Pathologists (Irish Branch) Spring Meeting 1997

Although various clinical signs and symptoms, as well as laboratory markers (e.g. pyrexia, raised white cell count, increase in C-reactive protein and other inflammatory markers) may aid in the diagnosis of infection, bacteriological culture is still the single most important laboratory investigation that is carried out. However, in a variety ofinfections, conventional bacteriological techniques do not detect the presence of any aetiological agent of infection. For example, in approximately 4-24% of cases of endocarditis, the blood culture investigation is negative and hence no microbiological causative agent(s) is identified. Present detection rates in blood culture of inununocompromised febrile patients with haematological malignancies is approximately 40%, thereby leaving the remaining 60% ofblood culture with no detectable aetiological agent of infection. Likewise, of 195 patients with aseptic acute meningitis (i.e. raised white cell count with no bacteriological indications in CSF), a virological agent was only detected in 18 (9.2%). Hence in 90.8% of these cases, the causative organism was not identified by conventional microbiological techniques. Overall, this causes difficulties in the clinical management of such patients presenting with culture-negative infections. The traditional basis for the identification of pathogenic bacteria has been their isolation or propagation in the laboratory. Biochemical, morphological and serological tests usually require growth of the organism. Reliance on these parameters may have significantly limited the awareness of true bacterial diversity and is impractical in many situations. The rapidly expanding use the polymerase chain reaction (PCR) coupled with the use of 16S rRNA sequences for phylogenetic, evolutionary and diagnostic studies offers an opportunity for alternative approaches. Such an approach has led for example to the identification of previously uncharacterised pathogens, in bacterial angioma-tosis in leukaemia. Hence, the study of such newly characterised pathogens with broad-range 16S rRNA PCR oligonucleotide primers necessitates sequence determination, so that specific PCR protocols may be developed for individual pathogens that may be targeted in such circumstances. Various different PCR broad-range 16S rRNA specific primer pairs have been screened with a wide variety of Gram-positive and Gram-negative flora. Identification of each bacterial strain was confirmed prior to PCR by the use of the appropriate API identification test strip (Biomerieux, France). Each 16S rRNA amplicon was further characterised by generating a database of SSCP profiles. To date, 41 culture-negative cerebrospinal fluid (CSF) specimens from patients with suspected meningitis have demonstrated bacterial presence in 7 (7/41; 17%) and are being currently sequenced in order to establish the bacterial identity …