Tunsjø H S, Kalyanasundaram S, Worren M M, Leegaard T M, Moen A E F
Department of Health Sciences, Oslo and Akershus University College, Oslo, Norway.
Department of Microbiology and Infection Control, Akershus University Hospital, Lørenskog, Norway.
Eur J Clin Microbiol Infect Dis. 2017 Jan;36(1):65-74. doi: 10.1007/s10096-016-2771-0. Epub 2016 Sep 9.
Rapid nucleic acid amplification tests for methicillin-resistant Staphylococcus aureus (MRSA) diagnostics commonly target the mec resistance gene, genes specific for S. aureus, and the integration site for the SCCmec resistance cassette, orfX. Due to poor specificity when these target genes are used individually, additional culture is required to verify positive results. The combination of these targets is useful, but the optimal algorithm may depend on the presence of the genetic markers in S. aureus isolates, as well as the prevalence of MRSA in a population. The aim of the present study was to identify a rapid, low-cost, and functional screening algorithm in order to reduce the response time for MRSA diagnostics. An in-house orfX-SCCmec polymerase chain reaction (PCR) assay was established and evaluated. The results were compared with an existing mec/nuc PCR assay and traditional culture. Methicillin-sensitive S. aureus (MSSA) that tested false-positive in the orfX-SCCmec PCR assay were further investigated with full genome sequencing using the Ion PGM™ System to verify results and causality. Based on these data, a two-step screening algorithm with initial mec/nuc PCR followed by orfX-SCCmec PCR on positive samples was suggested and tested on 1443 patient samples. 22.5 % of MSSA isolates tested false-positive with the orfX-SCCmec PCR. Full genome sequencing of these isolates identified genetic variation in the attB region of S. aureus, including empty cassette variants and non-mec SCC. The suggested two-step MRSA screening algorithm allowed us to report MRSA results for 95.6 % of all samples and 99 % of MRSA-negative samples after one day.
用于耐甲氧西林金黄色葡萄球菌（MRSA）诊断的快速核酸扩增试验通常靶向mec耐药基因、金黄色葡萄球菌特异性基因、SCCmec耐药盒的整合位点orfX。由于单独使用这些靶基因时特异性较差，需要额外培养以验证阳性结果。这些靶标的组合是有用的，但最佳算法可能取决于金黄色葡萄球菌分离株中遗传标记的存在情况以及人群中MRSA的流行率。本研究的目的是确定一种快速、低成本且有效的筛查算法，以缩短MRSA诊断的响应时间。建立并评估了一种内部orfX-SCCmec聚合酶链反应（PCR）检测方法。将结果与现有的mec/nuc PCR检测方法和传统培养方法进行比较。对在orfX-SCCmec PCR检测中呈假阳性的甲氧西林敏感金黄色葡萄球菌（MSSA），使用Ion PGM™系统通过全基因组测序进一步研究，以验证结果和因果关系。基于这些数据，提出了一种两步筛查算法，即首先进行mec/nuc PCR，然后对阳性样本进行orfX-SCCmec PCR，并在1443份患者样本上进行了测试。22.5%的MSSA分离株在orfX-SCCmec PCR检测中呈假阳性。对这些分离株的全基因组测序确定了金黄色葡萄球菌attB区域的基因变异，包括空盒变体和非mec SCC。建议的两步MRSA筛查算法使我们能够在一天后报告95.6%的所有样本和99%的MRSA阴性样本的MRSA结果。
