Leigheb Giorgio, Zavattaro Elisa, Molicotti Paola, Cannas Sara, Zanetti Stephania, Clemente Claudio, Johnson Roch C, Sopoh Ghislain E, Dossou Ange D, Colombo Enrico
Dermatology Unit, Department of Translational Medicine, University of Piemonte Orientale Amedeo Avogadro, Novara, Italy.
Int J Dermatol. 2014 Feb;53(2):213-20. doi: 10.1111/ijd.12249. Epub 2013 Dec 10.
Buruli ulcer (BU) is an infected cutaneous lesion, the etiological agent of which is Mycobacterium ulcerans. Diagnosis is confirmed by the identification of acid-fast bacilli and culture. In clinically suspicious forms with negative bacteriological or Ziehl-Neelsen (ZN) findings, molecular tests are used. This study compared the concordance of nested polymerase chain reaction (PCR) (targeting IS2404) and PCR (targeting IS2606) in different clinical situations.
A total of 57 samples were sourced from 39 BU patients. Control samples (n = 43) were obtained from non-BU ulcers in 38 patients. Samples were divided into two pieces and submitted to, respectively, histological examination and ZN staining, and PCR. Subsamples submitted to PCR were divided and submitted to nested PCR IS2404 and PCR IS2606, respectively.
Of the 57 BU biopsies, positive results were obtained by nested PCR in 18 (31.6%) and by IS2606 PCR in 37 (64.9%) cases. Sequencing of the positive samples confirmed the specificity of amplicons in all nested PCR samples and in 26 of 37 (70.2%) samples positive to IS2606. Hence, nested PCR was more specific (100% vs. 93%) and less sensitive (32% vs. 46%) than IS2606 PCR. In the BU samples, nested PCR was negative in 15 instances, and IS2606 PCR was negative in 11 instances in which ZN histology had been positive (false negatives). Both PCRs were positive in six ZN-negative smears.
We considered 57 samples from 39 BU patients in various clinical stages and at different times after the beginning of therapy. These provided positive results in 18 cases with IS2404 nested PCR and in 37 cases with PCR IS2606; only 26 of the latter remained positive subsequent to sequencing. Hence, even if IS2404 PCR is considered more specific, in subjects who appear to fail to respond to therapy, it is advisable to also carry out IS2606 PCR. A possible interpretation of the discordance between the two techniques due to unavoidable technical errors as well as to different sensitivity of the two tests at M. ulcerans DNA low concentration (i.e. in recent infection and in well-treated cases) is discussed.
布鲁里溃疡（BU）是一种感染性皮肤病变，其病原体为溃疡分枝杆菌。通过鉴定抗酸杆菌和培养来确诊。对于细菌学或齐-尼（ZN）染色结果为阴性的临床可疑病例，则采用分子检测。本研究比较了巢式聚合酶链反应（PCR）（靶向IS2404）和PCR（靶向IS2606）在不同临床情况下的一致性。
共从39例布鲁里溃疡患者中获取了57份样本。对照样本（n = 43）取自38例患者的非布鲁里溃疡。样本分成两份，分别进行组织学检查、ZN染色和PCR检测。用于PCR检测的子样本再进一步分开，分别进行靶向IS2404的巢式PCR和靶向IS2606的PCR检测。
在57份布鲁里溃疡活检样本中，巢式PCR检测出18份（31.6%）呈阳性，IS2606 PCR检测出37份（64.9%）呈阳性。对阳性样本进行测序，证实所有巢式PCR样本以及37份IS2606阳性样本中的26份（70.2%）扩增子具有特异性。因此，巢式PCR比IS2606 PCR更具特异性（100%对93%），但敏感性更低（32%对46%）。在布鲁里溃疡样本中，有15例巢式PCR结果为阴性，11例ZN组织学检查呈阳性的样本中IS2606 PCR结果为阴性（假阴性）。在6份ZN染色阴性涂片样本中，两种PCR检测均为阳性。
我们研究了39例处于不同临床阶段且在治疗开始后不同时间的布鲁里溃疡患者的57份样本。其中，靶向IS2404的巢式PCR检测出18例阳性，靶向IS2606的PCR检测出37例阳性；后者经测序后仅有26例仍为阳性。因此，即使IS2404 PCR被认为更具特异性，但对于治疗效果不佳的患者，建议同时进行IS2606 PCR检测。本文讨论了由于不可避免的技术误差以及两种检测方法在溃疡分枝杆菌DNA低浓度情况下（即近期感染和治疗良好的病例）敏感性不同，导致两种技术结果不一致的一种可能解释。
