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Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

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Page 1: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

Vanaporn Wuthiekanun Senior Microbiologist

Burkholderia pseudomallei levels in specimens and use of selective media

Burkholderia pseudomallei

Page 2: 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

Page 3: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

Spleenic pus from Melioidosis patient1. Blood

2. Respiratory secretion

3. Urine

4. Pus and fluid

Clinical samplesClinical samples

Page 4: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

• 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

Page 5: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

• 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

Page 6: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

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

Page 7: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

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%

Page 8: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

• 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

Page 9: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

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

Page 10: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

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

Page 11: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

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

Page 12: Vanaporn Wuthiekanun Senior Microbiologist Burkholderia pseudomallei levels in specimens and use of selective media Burkholderia pseudomallei

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