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Burkholderia pseudomallei levels in specimens and use of selective media. Burkholderia pseudomallei. Vanaporn Wuthiekanun Senior Microbiologist. Culture remains the diagnostic gold standard for meiloidosis although it has low sensitivity - PowerPoint PPT Presentation
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Vanaporn Wuthiekanun Senior Microbiologist
Burkholderia pseudomallei levels in specimens and use of selective media
Burkholderia pseudomallei
• 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.
Introduction
Limmathurotsakul et.al PLOS One 2010
Wuthiekanun et al. Am J. Trop. Med. Hyg 2007
Spleenic pus from Melioidosis patient1. Blood
2. Respiratory secretion
3. Urine
4. Pus and fluid
Clinical samplesClinical samples
• 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)
•
Selective media
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
• 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
Summary of selective media
Peacock et al. Clinical Micro 2005, Glass et al. Am J. Trop. Med. Hyg 2009
Ceftazidime resistant strains due to deletion of PBP3
Ceftazidime-sensitive initial isolate
Ceftazidime-resistant secondary isolate
Etests Gram stain RTM3 Microscopy
Chantratita N et al. PNAS 2012
1000
10
100
1
B.pseudomallei cfu/mL
<1 1-100 >100
100
0
50
MORTALITY
cfu/mL
Bacteraemia and outcome
Walsh et.al Clin Infect Dis 1995Wuthiekanun et.al Clin Infect Dis 2006
• 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 rate after 24 hours.
• Quantitatively, if there are more than 100 colony forming units (CFU)/ml of B. pseudomallei in blood, mortality rates reach 96% where <1 CFU/ml of B. pseudomallei has a relative mortality rate of 42%
• 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 rate after 24 hours.
• Quantitatively, if there are more than 100 colony forming units (CFU)/ml of B. pseudomallei in blood, mortality rates reach 96% where <1 CFU/ml of B. pseudomallei has a relative mortality rate of 42%
• T hroat swab:1011 melioidosis, 3524 healthy (4,625)• Specificity 100% (no carrier)• Sensitivity 79% %%%%%%%)
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• 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
39
58 6171
020
4060
80
% m
orta
lity
rate
1 2 3 4
Count number of B. pseudomallei (CFU/ml) in urine
0 <103 >105103 -104
Limmathurotsakul et al. J Clin Micro., 2005
Quantitative number of B. pseudomallei in clinical specimens
Wuthiekanun et al. Am J. Trop. Med. Hyg 2007
• 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
• 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 ChantratitaDr Wirongrong ChierakulDr Direk Limmathurotsakul Dr Rapeephan RattanawongnaraProf Nicholas WhiteProf Nicholas Day
Menzies School of Health ReserachDr Bart J Currie
Dr. Allen Cheng
Sappasithiprasong Hospital
Prof Wipada Chaowagul
Dr Prapit Teparrukkul
Nitaya Teerawattanasuk
Medical staff and nurses
laboratory staff
Khon Kaen UniversityDr Surasukdi Wongratanacheewin
Dr Rasana Wongratanacheewin
University of Cambridge, UK
Prof Sharon Peacock