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)
Figure 3. Analytical specificity of Taqman PCR with CF sputum
CF sputum specimens
101
Culture positive
Culture negative
4
97
PCR positive
PCR negative
PCR positive
PCR negative
(concordant)
(discordant)
(discordant)
(concordant)
4
0
1
96
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.
Figure 4. Analytical indices of Taqman BCC assay with CF sputum
AIMS
Parameter
Percent
•Develop a real-time PCR specific for BCC detection.
Sensitivity
100
•Test the newly-developed assay on both culture isolates & sputum samples.
Specificity
99.0
•Adapt a DNA extraction method suitable for PCR of BCC in sputa.
•Investigate the impact of removing extracellular DNA prior to DNA extraction.
Positive Predictive Value
(PPV)
Negative Predictive Value
(NPV)
RESULTS:
80
100
Figure 1. Illustration of method. A) Protocol for culture isolates. B) Protocol for sputum
samples. C) Protocol for sputum samples with inclusion of PMA.
Freshly
cultured
bacterial
colonies
CF
sputum
2.5μl
100mM
PMA
30 mins in
dark
500μl
C.
B.
A.
White
light
2x 60
secs
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)
1 colony in
100μl water,
heated at
95°C for 10
minutes.
Relative DNA detected
1/CT (530nm)
Figure 5. Impact of PMA on the amount of DNA detected in one CF
patient's sputa following treatment for an exacerbation.
0.06
0.05
0.04
without PMA
0.03
with PMA
0.02
0.01
0
07/05/2008
19/05/2008
Dates of sputum samples
DISCUSSION
Roche
LightCycler 2.0
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.
Roche MagNApure
extraction system
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.
Figure 2. Specificity of Taqman PCR with cultured isolates
Isolates
27
This technique can be completed within the working day, fulfilling the requirement for a rapid detection
system for BCC in CF patients.
BCC-positive strains
Mixture of non-BCC strains
9
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.
18
Additional acknowledgement to Andy Tuck for facilitating this project and to the British
Society of Microbial Technology who provided some funding.
PCR-positive
9
PCR-negative
0
PCR-positive
1
• 1 isolate from 8 S.
maltophilia isolates
PCR-negative
17
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.
© Collette Allen 2008