POLYMERASE CHAIN REACTION (PCR)AND RIBOSOMAL RNA GENE SEQUENCING IN THE DETECTION AND IDENTIFICATION OF NON-CULTURABLE NOVEL AETIOLOGICAL AGENTS IN PATIENTS WITH ACUTE LEUKAEMIA.
D.MIDDLETON, BP.MCILHATTON AND MD.CURRAN. (Northern Ireland Histocompatibility and Immunogenetics Laboratory, City Hospital, Belfast)

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In the past decade there has been a significant increase in the number of reports of infections in patients with haematological malignancies. Complications which arise from such infections are frequent and potentially life-threatening to the patient. For most febrile episodes observed in granulocytopenic patients, the specific organism is difficult to predict at the onset of fever, thus focused antimicrobial chemotherapy may be delayed until the aetiological agent is identified and antimicrobial susceptibility data obtained. It has been reported that only 20% of febrile patients have had a microbiologically documented infection with septicaemia. It is therefore important to have the ability to detect these aetiological agents of infection which are difficult to isolate using routine conventional techniques or which maybe are missed and termed "culture-negative". The traditional basis for the identification of pathogenic and commensal organisms has been their isolation or propagation in the laboratory using conventional techniques.

Here we present a molecular based PCR approach, targeting the 16S and 18S ribosomal RNA genes, capable of detecting and identifying non-culturable novel pathogens in the "culture-negative" blood culture bottles from immunocompromised patients (leukaemia) undergoing a febrile episode. This approach has led to the identification of bacterial DNA in the blood of patients deemed "culture-negative" corresponding to Lactococcus lactis in 86% of patients, while fungal DNA identified as Saccharomyces cerevisiae was found in 69% of patients. The clinical significance of these results are currently under investigation. These new approaches should lead to a better detection and identification of the non-culturable organisms associated with the infection. Ultimately this should lead to a better understanding of these organisms and to more effective treatment.