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. 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<0.001). Out of 12 FNA samples collected, PCR detected 10 positive cases, followed by microscopy with 6 cases, whiles laboratory diagnosis from culture detected 5 cases. The difference between the three methods for the detection of M. ulcerans in FNA samples was also statistically significant (p<0.001). Eight smear preparation protocols were investigated. Among the protocols, Ziehl-Neelsen (Z-N) stained smears from samples processed with 5% phenol in 4% ammonium sulphate (5%/4% phenol ammonium sulphate) and concentrated by gravitational sedimentation had the best outcome at a score rating of 686, whilst the least effective protocol was 3.5% sodium hypochlorite concentrated by gravitational sedimentation at a score rating of 360. Four (4) out of five (5) chemical agents at 2 dilutions each, tested on potential skin contaminants had activities against all. Povidone iodine at 0.5% and 1%, 2%CPC/4%NaCl, 1% virkon and 10% oxalic acid inhibited the growth of all microbes tested. Forty percent (40%) benzalkonium chloride , 1% CPC/2%NaCl, 5% oxalic acid could not inhibit the growth of two of the microbes. The least effective was twenty percent (20%) benzalkonium chloride (BC), inhibiting the growth of 6 out of 9 microbes tested. Of the 35 BU samples tested in duplicates against the selected chemical agents, 1% virkon and 1% povidone iodine (PI) recorded the highest activity of 99%.Whilst the least activity was recorded by 20% BC at 86%. Outcomes for assessing the usefulness of the selected chemical agents as decontamination agents in M. ulcerans culture indicated that, 2 % CPC / 4% NaCl and 0.5% virkon were the most effective at 52.8 %, and 51.4%, M. ulcerans recovery rates respectively. M. ulcerans recoveries were arithmetically higher than that of the conventional method (5% oxalic acid) at a yield of 44%. The observed differences were however not statistically significant (2 % CPC / 4% NaCl, p=0.7608 and 0.5% virkon, p=0.4070). The highest contamination rate of 29 % was exhibited by 5% oxalic acid and 1% CPC/2% NaCl of 21% in culture. Six (6) M. ulcerans isolates showed resistance to rifampicin, whilst 1 was resistant to streptomycin in-vitro. Resistance to both drugs was not observed among any of the isolates. Conclusions and recommendations It conclusion, BU specimens processed with 5% phenol /4% ammonium sulphate and concentration by gravitational sedimentation will improved AFB detection. In the peripheral centres where equipments like centrifuge may not be readily available, this sedimentation procedure can be used with adequate training. 2% CPC / 4% NaCl and 0.5% virkon are effective decontamination agents for the isolation M. ulcerans. Potential resistance to drugs (rifampicin and streptomycin), could also be a concern in the study area. In view of this, regular susceptibility testing of isolates and surveillance of susceptibility results (as is being done for Mycobacterium tuberculosis), is recommended.