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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.

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Related in: MedlinePlus

Mycolactone presence is maintained in ulcer exudates during antibiotic therapy.Mean concentration of mycolactone in ulcer exudates harvested before (0 week), during (2 to 8 weeks of treatment), or after completion of the 8 week antibiotic treatment. Dashed horizontal line indicates detection threshold.
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pntd-0001237-g003: Mycolactone presence is maintained in ulcer exudates during antibiotic therapy.Mean concentration of mycolactone in ulcer exudates harvested before (0 week), during (2 to 8 weeks of treatment), or after completion of the 8 week antibiotic treatment. Dashed horizontal line indicates detection threshold.

Mentions: Exudate samples were split into two equivalent parts, which were analyzed in parallel for the presence of mycolactone by HPLC/MS/MS or TLC-Fluo. We could not conclude on the presence of mycolactone by TLC-Fluo, because of the co-migration of auto-fluorescent compounds (Fig. 2A). Using HPLC determination, elution peaks corresponding to mycolactone were observed in 3/6 newly diagnosed patients and all patients undergoing or completing their course of antibiotic treatment (13/13, 4/4 respectively) (Table 1). To confirm that they effectively contained mycolactone, elution peaks were collected in three patients, and analyzed by MS/MS. In all of them, the characteristic spectrum of mycolactone parent ion (m/z 765) and products was observed [14], demonstrating the presence and structural integrity of mycolactone in ulcer exudates (Fig. 2B). Notably, the presence of mycolactone in these samples persisted during and after completion of antibiotic therapy (Fig. 3).


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)

Mycolactone presence is maintained in ulcer exudates during antibiotic therapy.Mean concentration of mycolactone in ulcer exudates harvested before (0 week), during (2 to 8 weeks of treatment), or after completion of the 8 week antibiotic treatment. Dashed horizontal line indicates detection threshold.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001237-g003: Mycolactone presence is maintained in ulcer exudates during antibiotic therapy.Mean concentration of mycolactone in ulcer exudates harvested before (0 week), during (2 to 8 weeks of treatment), or after completion of the 8 week antibiotic treatment. Dashed horizontal line indicates detection threshold.
Mentions: Exudate samples were split into two equivalent parts, which were analyzed in parallel for the presence of mycolactone by HPLC/MS/MS or TLC-Fluo. We could not conclude on the presence of mycolactone by TLC-Fluo, because of the co-migration of auto-fluorescent compounds (Fig. 2A). Using HPLC determination, elution peaks corresponding to mycolactone were observed in 3/6 newly diagnosed patients and all patients undergoing or completing their course of antibiotic treatment (13/13, 4/4 respectively) (Table 1). To confirm that they effectively contained mycolactone, elution peaks were collected in three patients, and analyzed by MS/MS. In all of them, the characteristic spectrum of mycolactone parent ion (m/z 765) and products was observed [14], demonstrating the presence and structural integrity of mycolactone in ulcer exudates (Fig. 2B). Notably, the presence of mycolactone in these samples persisted during and after completion of antibiotic therapy (Fig. 3).

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