Evaluating and Improving Microbiological Methods for the Diagnosis of Buruli Ulcer Disease
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Date
2013-03
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University of Ghana
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.
Description
Thesis (PhD) - University of Ghana, 2013