Zaman Kamran, Rudramurthy Shivaprakash Mandya, Das Ashim, Panda Naresh, Honnavar Prasanna, Kaur Harsimran, Chakrabarti Arunaloke
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India.
Department of Histopathology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India.
J Med Microbiol. 2017 Aug;66(8):1124-1129. doi: 10.1099/jmm.0.000560. Epub 2017 Aug 9.
We aimed to evaluate a PCR-based technique for the diagnosis of mucormycosis and the identification of fungi from fresh tissue specimens in patients with rhino-orbito-cerebral-mucormycosis (ROCM).
Fifty cases of ROCM were included in the study. Conventional identification was performed using microscopy and culture. Molecular diagnosis was performed by amplifying the ribosomal DNA using pan-fungal ITS primers and semi-nested Mucorales-specific primers of the 18S region. The amplified products were sequenced to identify the agents. The utility of PCR-RFLP of the 18S region of rDNA was evaluated to identify the Mucorales.
The ROCM cases were diagnosed by the demonstration of aseptate ribbon-like hyphae in biopsy specimens collected from the patients. Isolation was possible in 24 (48 %) samples. The ITS2 PCR confirmed mucormycosis in 27 cases (54 %; CI 59.4-68.2). By comparison, Mucorales-specific PCR was able to amplify DNA and the sequence enabled the identification of Mucorales speciesin all the patients. PCR-RFLP of the 18S region of rDNA could only identify the agent to genus level.
The molecular technique was able to identify Mucorales species in 26 (42 %) cases that were negative by culture. Mucorales-specific semi-nested PCR targeting the 18S region is a better technique than ITS2 PCR for diagnosis. PCR-RFLP of the 18S region helps in identification to genus level.
我们旨在评估一种基于聚合酶链反应（PCR）的技术，用于诊断毛霉病，并从鼻眶脑毛霉病（ROCM）患者的新鲜组织标本中鉴定真菌。
本研究纳入了50例ROCM患者。采用显微镜检查和培养进行传统鉴定。通过使用泛真菌内部转录间隔区（ITS）引物和18S区域的半巢式毛霉目特异性引物扩增核糖体DNA进行分子诊断。对扩增产物进行测序以鉴定病原体。评估核糖体DNA 18S区域的PCR-限制性片段长度多态性（PCR-RFLP）用于鉴定毛霉目的效用。
通过在患者采集的活检标本中发现无隔带状菌丝确诊ROCM病例。24份（48%）样本能够分离出真菌。ITS2 PCR在27例（54%；可信区间59.4 - 68.2）中确诊为毛霉病。相比之下，毛霉目特异性PCR能够扩增DNA，并且测序能够在所有患者中鉴定毛霉目菌种。核糖体DNA 18S区域的PCR-RFLP仅能将病原体鉴定到属水平。
分子技术能够在26例（42%）培养阴性的病例中鉴定出毛霉目菌种。针对18S区域的毛霉目特异性半巢式PCR比ITS2 PCR是更好的诊断技术。18S区域的PCR-RFLP有助于鉴定到属水平。
