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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 concentration in the serum of BU patients during antibiotic therapy.Mean concentration of mycolactone in serum samples collected 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-g005: Mycolactone concentration in the serum of BU patients during antibiotic therapy.Mean concentration of mycolactone in serum samples collected 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: Total lipids were extracted from 1 ml serum samples then analyzed by TLC-Fluo (Ghana samples) or HPLC (Ivory Coast samples) (Table 1). Again, the identification of mycolactone by TLC-Fluo was difficult, because of the co-migration of auto-fluorescent compounds (Fig. 4A). Using HPLC, mycolactone detection was also hampered by the co-elution of UV-absorbing contaminants (Fig. 4B). We could nevertheless identify mycolactone-like peaks in 3/5 newly diagnosed patients, 1/8 patients undergoing treatment, and 0/4 patients completing treatment. These peaks were collected in two patients and analyzed by MS/MS. In both of them, the characteristic spectrum of mycolactone parent and product ions could be observed (Fig. 4B). The concentration of circulating mycolactone was evaluated in positive samples by measuring the area of mycolactone elution peaks and subtracting the mean signal of two healthy controls (Fig. 5). Calculated values were in the 40–200 ng/ml range.


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 concentration in the serum of BU patients during antibiotic therapy.Mean concentration of mycolactone in serum samples collected 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-g005: Mycolactone concentration in the serum of BU patients during antibiotic therapy.Mean concentration of mycolactone in serum samples collected 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: Total lipids were extracted from 1 ml serum samples then analyzed by TLC-Fluo (Ghana samples) or HPLC (Ivory Coast samples) (Table 1). Again, the identification of mycolactone by TLC-Fluo was difficult, because of the co-migration of auto-fluorescent compounds (Fig. 4A). Using HPLC, mycolactone detection was also hampered by the co-elution of UV-absorbing contaminants (Fig. 4B). We could nevertheless identify mycolactone-like peaks in 3/5 newly diagnosed patients, 1/8 patients undergoing treatment, and 0/4 patients completing treatment. These peaks were collected in two patients and analyzed by MS/MS. In both of them, the characteristic spectrum of mycolactone parent and product ions could be observed (Fig. 4B). The concentration of circulating mycolactone was evaluated in positive samples by measuring the area of mycolactone elution peaks and subtracting the mean signal of two healthy controls (Fig. 5). Calculated values were in the 40–200 ng/ml range.

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