Division of Infectious Diseases, Department of Medicine, University Health Network/Mount Sinai Hospital, Toronto, ON, Canada.
BMC Infect Dis. 2013 Jul 16;13:324. doi: 10.1186/1471-2334-13-324.
C. difficile (CD) real-time polymerase chain reaction (PCR) for toxin B gene (tcdB) is more sensitive, and reduces turnaround time when compared to toxin immunoassay. We noted typical amplification curves with high tcdB cycle thresholds (Ct) and low endpoints (Ept) that are labeled negative by the Xpert(®) C. difficile assay (Cepheid) and undertook this study to determine their significance.
We defined an indeterminate CD assay result as detection of a typical PCR amplification curve with an Ept >10 that was interpreted as negative by the Xpert(®) assay. Samples with indeterminate Xpert(®) result were collected for 5 months and retested by Xpert(®), cultured for toxigenic CD, and isolates subjected to PCR ribotyping, detection of toxin genes and multilocus variable-number tandem repeat analysis (MLVA) typing. Chart reviews were completed to assess if patients met the Society of Healthcare Epidemiology of America and the Infectious Diseases Society of America CD infection (CDI) clinical case definition. Illness severity was compared with tcdB Ct and culture results.
During the 5-month study period, 48/3620 (1%) of specimens were indeterminate and 387/3620 (11%) were positive. Of the 48 patients with indeterminate results, 39 (81%) met the clinical case definition of CDI, and 7 of these (18%) met criteria for severe CDI. Toxigenic stool cultures were positive for 86% (6/7) of patients with severe CDI, 19% (6/32) of patients with non-severe CDI, and 44% (4/9) of patients who did not meet the clinical case definition of CDI (p = 0.002). Lower tcdB Ct and higher Ept were associated with greater likelihood of toxigenic culture positivity (p = 0.03) and more severe symptoms (p = 0.06). Indeterminate results were not associated with a particular technologist or instrument module, or CD strain type.
A subset of specimens (1%) using the Xpert(®) C. difficile assay have typical amplification curves and are interpreted as negative. At least one-third of these results are associated with positive CD culture. The mechanism of these indeterminate results is not technique-related, equipment-related, or due to particular CD strains. Clinicians should be aware that even PCR testing has the potential to miss CDI cases and further highlights the importance of clinical context when interpreting results.
与毒素免疫检测相比，艰难梭菌实时聚合酶链反应（PCR）检测毒素 B 基因（tcdB）更为敏感，且能缩短检测周转时间。我们发现，Xpert®艰难梭菌检测（Cepheid）标记为阴性的高 tcdB 循环阈值（Ct）和低终点（Ept）的典型扩增曲线很多，为此我们进行了这项研究以确定其意义。
我们将 Xpert®检测结果不确定的艰难梭菌检测定义为检测到典型的 PCR 扩增曲线，Ept >10，而 Xpert®检测将其解释为阴性。收集了 5 个月的 Xpert®检测结果不确定的样本，再次进行 Xpert®检测、培养产毒艰难梭菌，并对分离株进行 PCR 核糖体分型、毒素基因检测和多位点可变数串联重复分析（MLVA）分型。完成图表回顾以评估患者是否符合美国医疗保健流行病学学会和美国传染病学会艰难梭菌感染（CDI）临床病例定义。比较疾病严重程度与 tcdB Ct 和培养结果。
在 5 个月的研究期间，3620 份标本中有 48/3620（1%）的检测结果不确定，3620 份标本中有 387/3620（11%）为阳性。48 例检测结果不确定的患者中，39 例（81%）符合 CDI 的临床病例定义，其中 7 例（18%）符合严重 CDI 的标准。产毒粪便培养阳性率为 7 例严重 CDI 患者中的 86%（6/7）、32 例非严重 CDI 患者中的 19%（6/32）和 9 例不符合 CDI 临床病例定义的患者中的 44%（4/9）（p=0.002）。较低的 tcdB Ct 和较高的 Ept 与产毒培养阳性的可能性更大（p=0.03）和更严重的症状相关（p=0.06）。不确定的结果与特定的技术人员或仪器模块或艰难梭菌菌株类型无关。
使用 Xpert®艰难梭菌检测的部分标本（1%）有典型的扩增曲线，被解释为阴性。这些结果中至少有三分之一与阳性 CD 培养结果相关。这些不确定结果的机制不是技术相关、设备相关或特定 CD 菌株所致。临床医生应注意，即使是 PCR 检测也有可能漏诊 CDI 病例，这进一步强调了在解释结果时临床背景的重要性。
