1. Development and evaluation of a rapid same-day direct real time PCR assay for viable Burkholderia cepacia complex cells from cystic fibrosis patient sputa Allen, C.*, Marsh, P.* Rogers, G. # * HPA South-East, Southampton General Hospital, Southampton. # King ’ s College, London. Cystic fibrosis (CF) patients face a lifetime of multiple respiratory infections which contribute to morbidity and mortality. Among the most problematic infectious agents are microorganisms belonging to the Burkholderia cepacia complex (BCC) which comprises of at least 9 morphologically similar, genetically distinct species, formally known as genomovars. All members of the BCC have been isolated from CF patients and result in a range of clinical conditions, from asymptomatic carriage to an acceleration of pulmonary decline and the often fatal ‘cepacia syndrome’. (1) Accurate and fast detection of BCC is essential in care of CF patients in order to implement infection control procedures and facilitate timely therapeutic interventions. There is an alarming rate of BCC misidentification when using standard phenotypic tests, as well as a lengthy culture and identification process. (2) This necessitates an urgent need for a rapid accurate assay to discriminate BCC in CF sputa. A direct DNA extraction and real-time PCR protocol for specific, sensitive detection of BCC in CF sputa was developed. To minimise the impact of residual DNA in CF sputa affecting the clinical utility of this assay, this method incorporated the selective extraction of DNA from viable/intact cells. Removal of extracellular DNA was conducted using the high affinity photoreactive DNA binding dye propidium monoazide (PMA; Biotium, CA). (3) The method described can be completed in one working day, instead of the 5 required for culture and identification. The purpose of this investigation was to develop a real-time PCR technique for the rapid detection of BCC from sputum. AIMS Adapt a DNA extraction method suitable for PCR of BCC in sputa. Develop a real-time PCR specific for BCC detection. Test the newly-developed assay on both culture isolates & sputum samples. Investigate the impact of removing extracellular DNA prior to DNA extraction. RESULTS: Figure 1. Illustration of method. A) Protocol for culture isolates. B) Protocol for sputum samples. C) Protocol for sputum samples with inclusion of PMA. DISCUSSION The sensitivity and specificity data for this assay are very promising. Interestingly, with the CF sputum specimen that was found to be negative for BCC culture but positive via the newly designed PCR assay (see figure 3), a look-back at previous results showed that this patient had been recently infected with BCC. This demonstrates that either the PCR assay is more sensitive than culture, or that incorrect identification of organisms cultured from the patient sample occurred. The impact of removing extracellular DNA is demonstrated in figure 5. Without PMA, a similar detection level is observed in both samples, suggesting similar quantities of BCC DNA. Following the use of PMA a reduction is seen in the level of DNA detected within each sample and between the 2 samples. PMA binds to DNA in its free state or in damaged cells, preventing amplification by PCR. Only DNA subsequently extracted from intact cells will be detected by PCR. The clinical utility of a PCR result following the use of PMA may be greater, i.e. more useful, as it might provide an indication as to whether an infection is resolving. This technique can be completed within the working day, fulfilling the requirement for a rapid detection system for BCC in CF patients. Figure 3. Analytical specificity of Taqman PCR with CF sputum ParameterPercent Sensitivity100 Specificity99.0 Positive Predictive Value (PPV) 80 Negative Predictive Value (NPV) 100 Figure 2. Specificity of Taqman PCR with cultured isolates PCR-positive 9 PCR-negative 0 PCR-positive 1 1 isolate from 8 S. maltophilia isolates PCR-negative 17 Figure 4. Analytical indices of Taqman BCC assay with CF sputum CF sputum 2.5 μ l 100mM PMA 500 μ l 30 mins in dark White light 2x 60 secs 1 colony in 100μl water, heated at 95°C for 10 minutes. A. B. C. Roche LightCycler 2.0 Roche MagNApure extraction system Freshly cultured bacterial colonies External lysis: 100μl sample + 130μl lysis buffer + 20μl proteinase K. 65°C for 10 minutes, 95°C for 10 minutes. (Roche MagNApure Extraction Kit III) References: 1)Mahenthiralingam E, Urban T and Goldberg J. The multifarious, multireplicon Burkholderia cepacia complex. Nature Reviews Microbiology 2005;3:144-156. 2)McMenamin J, Zaccone T, Coenye T et al. Misidentification of Burkholderia cepacia in US Cystic Fibrosis treatment centres: An analysis of 1,051 sputum isolates. Chest 2000;117:1661-1665. 3)Nocker A, Sossa-Fernandez P, Burr M and Camper A. Use of propidium monoazide for live/dead distinction in microbial ecology. Applied and Environmental Microbiology 2007;73:5111-5117. Acknowledgements: The authors wish to acknowledge the help and support of staff within HPA Southampton, especially those with the molecular diagnostics unit & those dealing with CF sputum samples. Additional acknowledgement to Andy Tuck for facilitating this project and to the British Society of Microbial Technology who provided some funding. Isolates 27 BCC-positive strains 9 Mixture of non-BCC strains 18 CF sputum specimens 101 Culture positive 4 Culture negative 97 PCR positive (concordant) 4 PCR negative (discordant) 0 PCR positive (discordant) 1 PCR negative (concordant) 96 © Collette Allen 2008