Vanaporn Wuthiekanun Senior Microbiologist

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

2. 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

3. Melioidosis Research Programme Clinical samples • Blood • Respiratory secretion • Urine • Pus and fluid Spleenic pus from Melioidosis patient

4. 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.Pathology1979a, Howard et al. Clinical Micro 2003, Peacock et al. Clinical Micro 2005, Glass et al.Am J. Trop. Med. Hyg 2009

5. 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

6. 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

7. Bacteraemia and outcome 100 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 rate after 24 hours. • Quantitatively, if there are more than 100 colony forming units (CFU)/ml of B. pseudomalleiin blood, mortality rates reach 96% where <1 CFU/ml of B. pseudomallei has a relative mortality rate of 42% MORTALITY 50 100 10 0 <1 1-100 >100 cfu/mL 1 B.pseudomallei cfu/mL Walsh et.al Clin Infect Dis 1995 Wuthiekanun et.al Clin Infect Dis 2006

8. 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

9. 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 0 <103 103 -104 >105 Limmathurotsakul et al. J Clin Micro., 2005

10. 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 • Pus and respiratory secretions had the highest median count Wuthiekanun et al.Am J. Trop. Med. Hyg 2007

11. 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

12. Collaborators and Contributors MORU, Faculty of Tropical Medicine, Mahidol University University of Cambridge, UK Prof Sharon Peacock Dr Narisara Chantratita Dr Wirongrong Chierakul Dr Direk Limmathurotsakul Dr Rapeephan Rattanawongnara Prof Nicholas White Prof Nicholas Day Sappasithiprasong Hospital Prof Wipada Chaowagul Dr Prapit Teparrukkul Nitaya Teerawattanasuk Medical staff and nurses laboratory staff Khon Kaen University Dr Surasukdi Wongratanacheewin Dr Rasana Wongratanacheewin Menzies School of Health Reserach Dr Bart J Currie Dr. Allen Cheng