Burkholderia pseudomallei levels in
specimens and use of selective media
Burkholderia pseudomallei
Vanaporn Wuthiekanun
Senior Microbiologist
Introduction
• Culture remains the diagnostic gold standard for
meiloidosis although it has low sensitivity
• B. pseudomallei grows well on routine laboratory media,
while selective media help with non-sterile specimens
• Higher B. pseudomallei counts in clinical samples
correlate with higher mortality rates.
Limmathurotsakul et.al PLOS One 2010
Wuthiekanun et al. Am J. Trop. Med. Hyg 2007
Clinical samples
1. Blood
2. Respiratory secretion
3. Urine
4. Pus and fluid
Spleenic pus from
Melioidosis patient
Selective media
• Specimens from non-sterile sites can benefit from the
use of selective media
• A number of selective media have been developed to
facilitate growth of B. pseudomallei from sites with
mixed flora.
• Ashdown agar (ASH)
• B. pseudomallei selective agar (BPSA)
• B. cepacia selective media (BCSA)
• Pseudomonas cepacia agar (PCA)
Ashdown et al. Pathology 1979a, Howard et al. Clinical Micro 2003,
Peacock et al. Clinical Micro 2005, Glass et al. Am J. Trop. Med. Hyg 2009
Summary of selective media
• BCSA is an equivalent selective agar to ASH
• The most sensitive medium for the growth of
B. pseudomallei was ASH
• PCA is a commercial selective agar that may
be suitable for B. pseudomallei and B. mallei
Peacock et al. Clinical Micro 2005, Glass et al. Am J. Trop. Med. Hyg 2009
Ceftazidime resistant strains due to
deletion of PBP3
Etests
Gram stain
RTM3 Microscopy
Ceftazidime-sensitive initial isolate
Ceftazidime-resistant secondary isolate
Chantratita N et al. PNAS 2012
Bacteraemia and outcome
1000
• If a blood culture becomes
positive within 24 h, the mortality
rate has been found to be at 73.7%
compared with a 40.9% mortality
100 after 24 hours.
rate
• Quantitatively, if there are more
than
100 colony forming units
10
(CFU)/ml of B. pseudomallei in
blood, mortality rates reach 96%
where <1 CFU/ml of B.
pseudomallei
has a relative
1
mortality rate of 42%
B.pseudomallei cfu/mL
100
MORTALITY
50
<1
1-100
cfu/mL
0
>100
Walsh et.al Clin Infect Dis 1995
Wuthiekanun et.al Clin Infect Dis 2006
• Throat swab:1011 melioidosis, 3524 healthy (4,625)
• Specificity 100% (no carrier)
• Sensitivity 79% (sputum)
Value of throat swab in diagnosis of melioidosis
Wuthiekanun et al. J Clin Microbiol 2001
The role and significance of quantitative urine
cultures in the diagnosis of melioidosis
80
Count number of B. pseudomallei (CFU/ml) in urine
71
61
40
39
0
20
% mortality rate
60
58
1
0
3
2
<10
1033 -104
5
4
>10
Limmathurotsakul et al. J Clin Micro., 2005
• Mortality rose with the
increasing urine bacterial
quantity
• Only 24% of patients with
positive urine cultures had
urinary symptoms
• The presence of B.
pseudomallei in urine is
associated with a poorer
prognosis
Quantitative number of B. pseudomallei in
clinical specimens
• Data of positive samples:
• total 376/730
• blood 203/414 (49%)
• urine 56/268 (21%)
• respiratory secretions
94/120 (78%)
• pus 23/28 (82%)
• Blood samples had the
lowest count from <0.1 to
>100 CFU/ml
Wuthiekanun et al. Am J. Trop. Med. Hyg 2007
• Pus and respiratory
secretions had the highest
median count
Summary
• Pitfalls in identification can include technical
unfamiliarity with the disease and organism
• Specimens from non-sterile sites can benefit from
the use of selective media
• Routine use of culture, in addition with PCR and
serological test, may lead to earlier diagnosis of
melioidosis
Collaborators and Contributors
MORU,
Faculty of Tropical Medicine,
Mahidol University
Dr Narisara Chantratita
Dr Wirongrong Chierakul
Dr Direk Limmathurotsakul
Dr Rapeephan Rattanawongnara
Prof Nicholas White
Prof Nicholas Day
University of Cambridge, UK
Prof Sharon Peacock
Sappasithiprasong Hospital
Prof Wipada Chaowagul
Dr Prapit Teparrukkul
Khon Kaen University
Nitaya Teerawattanasuk
Dr Surasukdi Wongratanacheewin
Medical staff and nurses
Dr Rasana Wongratanacheewin
laboratory staff
Menzies School of Health Reserach
Dr Bart J Currie
Dr. Allen Cheng