02723nas a2200313 4500000000100000008004100001653003000042653002700072653001200099653001900111653001400130653001700144100001800161700001500179700001500194700001100209700001000220700001500230700001200245700001200257700001300269700001600282245011500298856009000413300001300503490000700516520187200523022001402395 2016 d10apolymerase chain reaction10aMycobacterium ulcerans10aLesions10aHistopathology10adiagnosis10aBuruli ulcer1 aYeboah-Manu D1 aPluschke G1 aBratschi M1 aBolz M1 aRuf M1 aUm Boock A1 aVogel M1 aBayi PF1 aSopho GE1 aJunghanss T00aSpatial distribution of Mycobacterium ulcerans in Buruli ulcer lesions: implications for laboratory diagnosis. uhttp://journals.plos.org/plosntds/article/asset?id=10.1371%2Fjournal.pntd.0004767.PDF ae00047670 v103 a

BACKGROUND: Current laboratory diagnosis of Buruli ulcer (BU) is based on microscopic detection of acid fast bacilli, quantitative real-time PCR (qPCR), histopathology or cultivation. Insertion sequence (IS) 2404 qPCR, the most sensitive method, is usually only available at reference laboratories. The only currently available point-of-care test, microscopic detection of acid fast bacilli (AFB), has limited sensitivity and specificity.

METHODOLOGY/ PRINCIPAL FINDINGS: Here we analyzed AFB positive tissue samples (n = 83) for the presence, distribution and amount of AFB. AFB were nearly exclusively present in the subcutis with large extracellular clusters being most frequently (67%) found in plaque lesions. In ulcerative lesions small clusters and dispersed AFB were more common. Beside this, 151 swab samples from 37 BU patients were analyzed by IS2404 qPCR and ZN staining in parallel. The amount of M. ulcerans DNA in extracts from swabs correlated well with the probability of finding AFB in direct smear microscopy, with 56.1% of the samples being positive in both methods and 43.9% being positive only in qPCR. By analyzing three swabs per patient instead of one, the probability to have at least one positive swab increased from 80.2% to 97.1% for qPCR and from 45% to 66.1% for AFB smear examination.

CONCLUSION / SIGNIFICANCE: Our data show that M. ulcerans bacteria are primarily located in the subcutis of BU lesions, making the retrieval of the deep subcutis mandatory for examination of tissue samples for AFB. When laboratory diagnosis is based on the recommended less invasive collection of swab samples, analysis of three swabs from different areas of ulcerative lesions instead of one increases the sensitivity of both qPCR and of smear microscopy substantially.

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