Limits...
Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring.

Sarfo FS, Le Chevalier F, Aka N, Phillips RO, Amoako Y, Boneca IG, Lenormand P, Dosso M, Wansbrough-Jones M, Veyron-Churlet R, Guenin-Macé L, Demangel C - PLoS Negl Trop Dis (2011)

Bottom Line: This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids.We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry.However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out.

View Article: PubMed Central - PubMed

Affiliation: Komfo Anokye Teaching Hospital, Kumasi, Ghana.

ABSTRACT

Background: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/principal finding: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone.

Conclusions/significance: Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.

Show MeSH

Related in: MedlinePlus

Comparison of the performances of the TLC-Fluo and HPLC approaches for mycolactone quantitative detection.A representative picture of the fluorescent signals and elution peaks obtained for mycolactone (10–500 ng) detection by TLC-Fluo (A) and HPLC (B) are shown, with the corresponding standard curves (C). Arbitrary units correspond to fluorescence intensity of the mycolactone band (TLC-Fluo) and area of the mycolactone elution peak (HPLC). Similar results were obtained in at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3139662&req=5

pntd-0001237-g001: Comparison of the performances of the TLC-Fluo and HPLC approaches for mycolactone quantitative detection.A representative picture of the fluorescent signals and elution peaks obtained for mycolactone (10–500 ng) detection by TLC-Fluo (A) and HPLC (B) are shown, with the corresponding standard curves (C). Arbitrary units correspond to fluorescence intensity of the mycolactone band (TLC-Fluo) and area of the mycolactone elution peak (HPLC). Similar results were obtained in at least three independent experiments.

Mentions: We first compared the TLC-Fluo and HPLC/MS/MS approaches for mycolactone detection in the 0–500 ng range, using purified mycolactone as a reference. Both methods were highly sensitive, yielding a detectable signal with only 10 ng mycolactone (Fig. 1A,B). However the fluorescent signal of mycolactone modified by coupling to 2-naphtalene boronic acid was not a linear function of its concentration, whereas the areas of mycolactone elution peaks in HPLC were proportional to mycolactone concentration in the 10–500 ng domain (Fig. 1C). To evaluate the efficacy of mycolactone extraction from biological samples, serum samples (1 ml) or mononuclear cell pellets (106 cells) were spiked with purified mycolactone. We then proceeded to solvent extraction as described in the Methods section, and estimated the proportion of recovered mycolactone using the above-described standard curves. The estimated yields of extraction with organic solvents were of 20% from cell pellets and 10% from serum samples (not shown). Several combinations of solvents were tried that did not improve the recovery of mycolactone.


Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring.

Sarfo FS, Le Chevalier F, Aka N, Phillips RO, Amoako Y, Boneca IG, Lenormand P, Dosso M, Wansbrough-Jones M, Veyron-Churlet R, Guenin-Macé L, Demangel C - PLoS Negl Trop Dis (2011)

Comparison of the performances of the TLC-Fluo and HPLC approaches for mycolactone quantitative detection.A representative picture of the fluorescent signals and elution peaks obtained for mycolactone (10–500 ng) detection by TLC-Fluo (A) and HPLC (B) are shown, with the corresponding standard curves (C). Arbitrary units correspond to fluorescence intensity of the mycolactone band (TLC-Fluo) and area of the mycolactone elution peak (HPLC). Similar results were obtained in at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139662&req=5

pntd-0001237-g001: Comparison of the performances of the TLC-Fluo and HPLC approaches for mycolactone quantitative detection.A representative picture of the fluorescent signals and elution peaks obtained for mycolactone (10–500 ng) detection by TLC-Fluo (A) and HPLC (B) are shown, with the corresponding standard curves (C). Arbitrary units correspond to fluorescence intensity of the mycolactone band (TLC-Fluo) and area of the mycolactone elution peak (HPLC). Similar results were obtained in at least three independent experiments.
Mentions: We first compared the TLC-Fluo and HPLC/MS/MS approaches for mycolactone detection in the 0–500 ng range, using purified mycolactone as a reference. Both methods were highly sensitive, yielding a detectable signal with only 10 ng mycolactone (Fig. 1A,B). However the fluorescent signal of mycolactone modified by coupling to 2-naphtalene boronic acid was not a linear function of its concentration, whereas the areas of mycolactone elution peaks in HPLC were proportional to mycolactone concentration in the 10–500 ng domain (Fig. 1C). To evaluate the efficacy of mycolactone extraction from biological samples, serum samples (1 ml) or mononuclear cell pellets (106 cells) were spiked with purified mycolactone. We then proceeded to solvent extraction as described in the Methods section, and estimated the proportion of recovered mycolactone using the above-described standard curves. The estimated yields of extraction with organic solvents were of 20% from cell pellets and 10% from serum samples (not shown). Several combinations of solvents were tried that did not improve the recovery of mycolactone.

Bottom Line: This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids.We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry.However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out.

View Article: PubMed Central - PubMed

Affiliation: Komfo Anokye Teaching Hospital, Kumasi, Ghana.

ABSTRACT

Background: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/principal finding: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone.

Conclusions/significance: Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.

Show MeSH
Related in: MedlinePlus