Improved protocols for isolation of Mycobacterium ulcerans from clinical samples.

Abstract

Background: The isolation and culture of Mycobacterium ulcerans (Mu) as a primary diagnostic modality for Buruli ulcer (BU) disease are limiting due to their low sensitivity and slow-growing nature. M. ulcerans cultures can also be overgrown with other bacteria and fungi. Culture, however, remains an important tool for the study of persisting viable M. ulcerans, drug susceptibility tests, and other molecular assays to improve management of the disease. The challenge of contamination with other fast-growing bacteria necessitates decontamination of clinical samples prior to culturing, but current methods may be too harsh, resulting in low yields of M. ulcerans. We aimed to evaluate a Tika-Kic decontamination process for M. ulcerans that uses supplements to stimulate M. ulcerans growth to improve recovery.

Methods: Swab and Fine Needle Aspirate (FNA) samples were collected from 21 individuals with confirmed BU at baseline (week 0) and weeks 2 and 4 after initiating antibiotic treatment. Samples were decontaminated with Tika-Kic decontamination medium and the modified Petroff (NaOH) methods then inoculated each into Mycobacterium Growth Indicator Tube (MGIT) or Löwenstein Jensen (LJ) medium. Time to growth detection and confirmation by qPCR as well as the proportion of positive cultures for all three methods and the proportion of positive cultures for all three time points were documented. Common contaminating bacteria were also isolated and identified.

Results: The proportion of M. ulcerans positive cultures obtained was higher for Tika-MGIT samples [14/43 (32%)] compared to Petroff-MGIT samples [10/43 (23%)] and Petroff-LJ samples [8/43 (19%)]. Baseline samples had a higher isolate proportion [17 (53%)] compared to samples collected after treatment initiation [9 (28%) for week 2 and 6 (19%) for week 4]. Contaminating bacteria isolated include Burkholderia cepacia, Pseudomonas aeruginosa, Pasteurella pneumotropica, Proteus mirabilis, Morganella morganii, Staphylococcus aureus and Enterococcus.

Conclusion: Our study shows an advantage for culturing Mycobacterium ulcerans from clinical samples using the Tika-Kic decontamination and growth medium. Further research is needed to refine sample processing to improve M. ulcerans recovery.