Evaluating And Improving Microbiological Methods For The Diagnosis of Buruli Ulcer Disease

ABSTRACT 

Background Challenges associated with early diagnosis of Buruli ulcer disease (BUD), an infection caused by Mycobacterium ulcerans (M. ulcerans) is a major setback in public health and disease control. Lack of simple, convenient, rapid and sensitive diagnostic procedures, readily available to rural endemic communities has hampered control efforts. Improving the sensitivity of simple diagnostic methods such as microscopy for AFB detection constitutes a crucial effort in this direction. M. ulcerans isolation by culture though slow, provides isolates critical to performing important investigations to provide information on drug susceptibility profiles of M. ulcerans isolates and molecular epidemiology of the disease. Techniques aimed at improving the sensitivities of the two diagnostic methods will facilitate early disease diagnosis and subsequently improve disease control. Objective This study assessed procedures aimed at improving the sensitivities of methods of AFB detection by microscopy and the isolation of M. ulcerans in culture. Methodology The performances of eight smear preparation protocols were assessed for the effective detection of acid fast bacilli (AFB). Swabbed samples from ulcer lesions of Buruli ulcer (BU) patients in two BU endemic districts of the Eastern region of Ghana were used. Smear preparation was based on physical and chemical modifications, of the conventional methods for Zeihl-Neelsen (ZN) staining protocols, for the detection of acid fast bacilli (AFB) from BU cases. xvii Additionally, two dilutions of 5 selected chemical agents were investigated for their potential use as decontamination agents in M. ulcerans in culture. The activities of the chemical agents were preliminarily assessed against clinical isolates of potential skin contaminants. Effective chemical agents were simultaneously evaluated for (i) their decontamination activity against contaminants in BU samples and (ii) their effect on the growth of M. ulcerans in culture. Broth dilution methods were applied in these investigations. M. ulcerans from the study isolates were tested on rifampicin and streptomycin by the agar proportion method described by Canetti to assess the drug susceptibility. Results A total number of 135 clinically diagnosed BUD patients were recruited for the study. The age of the patients ranged between 1 to 92 years, with a mean age of 39 years. Swabs were taken form 123 cases whiles fine needle aspirates (FNAs) was taken from the 12 remaining BUD cases. Out of 123 swabs taken, PCR detected 111 positive cases, followed by microscopy with 62 cases and then culture with 52 cases. The observed differences between the three methods was statistically significant (p