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

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

Melioidosis Research Programme Clinical samples Blood Respiratory secretion Urine Pus and fluid Spleenic pus from Melioidosis patient Key specimens to collect include blood, throat swabs, respiratory secretions including throat swab, sputum, tracheal suction, pleural effusion , urine, and when available, pus and swabs from wounds or lesions.

Ashdown et al. Pathology 1979a, Howard et al. Clinical Micro 2003, 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) Culture remains the diagnostic gold standard for meiloidosis although has low sensitivity and low negative predictive values. B. pseudomallei grows well on most routine laboratory media and can be isolated from sterile sites using standard techniques. Specimens from non-sterile sites can benefit from the use of selective media which help to promote the growth of B. pseudomallei while reducing the growth of other organisms. Higher B. pseudomallei counts in clinical samples correlate with higher mortality rates. 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 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. pseudomallei in blood, mortality rates reach 96% where <1 CFU/ml of B. pseudomallei has a relative mortality rate of 42% MORTALITY 100 50 10 <1 1-100 >100 Blood for culture is the most common specimen used in the diagnosis of melioidosis. The collection of blood cultures should occur prior to commencement of antimicrobial treatment. cfu/mL 1 B.pseudomallei cfu/mL Walsh et.al Clin Infect Dis 1995 Wuthiekanun et.al Clin Infect Dis 2006

Value of throat swab in diagnosis of melioidosis Throat swab:1011 melioidosis, 3524 healthy (4,625) Specificity 100% (no carrier) Sensitivity 79% (sputum) The specificity of TS culture for the diagnosis of melioidosis was 100%, and the overall sensitivity was 36% (79% for sputum-positive patients). The throat swab culture also identified an additional 6% of patients with pulmonary melioidosis for whom sputum cultures were negative. Cultures are often positive for patients with pulmonary or disseminated melioidosis and may be positive for a significant number of patients who are unable to expectorate or who are not bacteremic. The throat swab should be complementary to other methods of diagnosis. 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 Quantitative urine culture is a standard, inexpensive diagnostic test that is widely undertaken to provide laboratory evidence of urinary tract infection. On this graph you can see clearly that the mortality rose with the increasing number of B. pseudomallei. Patients with negative urine cultures had the lowest death rate (39%) and the overall in-hospital mortality rate was 45%. Mortality rates rose with increasing B. pseudomallei counts in urine, from 58% for those with positive spun pellets only to 71% for those with > or = 10(5) CFU/ml. This was independent of age, presence of bacteremia, known risk factors for melioidosis such as diabetes, and the prior administration of antibiotics. The presence of B. pseudomallei in urine during systemic infection is associated with a poor prognosis. The finding of asymptomatic bacteriuria (76%) has been considered to represent renal filtration of bacteria present in the blood, previously termed “spillover.” What do you think? Correlations between bacterial counts in blood and other samples were not found. <103 103 -104 >105 Limmathurotsakul et al. J Clin Micro., 2005

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

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 Culture has long been a gold standard diagnostic test for melioidosis and I don’t think any method can replaced culture particularly in developing countries where PCR isn’t commonly available. Isolation and identification of Burkholderia pseudomallei from any clinical specimens indicate melioidosis infection but it remains the pitfall in identification because of unfamiliar with organism. Contaminated samples need selective agar and also the broth that can enhance the growth of small number of organism in the sample. However in my opinion the combination of culture, PCR and serology will gave the best results for earliest diagnosis.

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 12