Zeng Xianyu, Kong Fanrong, Halliday Catriona, Chen Sharon, Lau Anna, Playford Geoffrey, Sorrell Tania C
Centre for Infectious Diseases and Microbiology-Public Health (CIDM-PH), Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, Australia.
J Clin Microbiol. 2007 Sep;45(9):2872-80. doi: 10.1128/JCM.00687-07. Epub 2007 Jul 18.
We evaluated a combined panfungal PCR-reverse line blot (RLB) hybridization assay based on internal transcribed spacer 1 (ITS1) and ITS2 region polymorphisms to identify 159 Candida, Cryptococcus neoformans, and Aspergillus isolates (22 species). Its utility to identify fungal pathogens directly from 27 clinical specimens was also determined. ITS sequence analysis was performed to resolve discrepant identifications or where no RLB result was obtained. Species-specific ITS2- and ITS1-based probes correctly identified 155 of 159 isolates (98%) and 149 (93.7%) isolates, respectively. All strains were unambiguously differentiated with the exception of cross-reactivity between the Candida norvegensis probe and Candida haemulonii DNA product. Species identification of the pathogen was made for all 21 specimens (sensitivity of 100%) where species-specific probes were included in the RLB; however, there was no ITS2 probe-based hybridization signal for two specimens. Results were concordant with the culture results for 18 (85.7%) specimens. The assay was able to provide species identification in the absence of a culture result (two specimens) and to detect mixed infection (one specimen). The results indicate that the RLB assay is capable of reliably detecting yeasts and Aspergillus spp. in clinical specimens and that the incorporation of both ITS1- and ITS2-targeted probes is required for optimal sensitivity. The test has potential utility in the early diagnosis of invasive fungal infection, since "fungal" DNA was detected in all 27 specimens. Prior to incorporation of probes to detect other fungal species, ITS sequencing may be performed to achieve species identification.
我们评估了一种基于内转录间隔区1（ITS1）和ITS2区域多态性的联合泛真菌聚合酶链反应-反向线杂交（RLB）检测方法，以鉴定159株念珠菌、新型隐球菌和曲霉分离株（22个种）。还确定了该方法直接从27份临床标本中鉴定真菌病原体的实用性。进行ITS序列分析以解决不一致的鉴定结果或未获得RLB结果的情况。基于ITS2和ITS1的种特异性探针分别正确鉴定了159株分离株中的155株（98%）和149株（93.7%）。除挪威念珠菌探针与哈氏念珠菌DNA产物之间存在交叉反应外，所有菌株均能明确区分。在RLB中包含种特异性探针的所有21份标本中均进行了病原体的种鉴定（敏感性为100%）；然而，有两份标本没有基于ITS2探针的杂交信号。18份（85.7%）标本的结果与培养结果一致。该检测方法能够在没有培养结果的情况下（两份标本）提供种鉴定，并能检测混合感染（一份标本）。结果表明，RLB检测方法能够可靠地检测临床标本中的酵母和曲霉属，并且为了获得最佳敏感性需要同时使用针对ITS1和ITS2的探针。该检测方法在侵袭性真菌感染的早期诊断中具有潜在应用价值，因为在所有27份标本中均检测到了“真菌”DNA。在纳入检测其他真菌物种的探针之前，可进行ITS测序以实现种鉴定。
