Bregenzer Andrea, Ottiger Cornelia, Krismer Cornelia, Sager Karin, Fux Christoph A
Department of Infectious Diseases and Hospital Hygiene, Cantonal Hospital Aarau, Switzerland.
Institute for Laboratory Medicine, Cantonal Hospital Aarau, Switzerland.
Swiss Med Wkly. 2021 Dec 23;151:w30089. doi: 10.4414/smw.2021.w30089. eCollection 2021 Dec 20.
For patients with difficult venous access after long-term intravenous drug use, rapid point-of-care hepatitis C virus (HCV) RNA quantification in capillary whole blood with the Xpert® HCV Viral Load Fingerstick (VL FS) test (60 minutes) is a convenient and reliable method for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection. However, an expensive GeneXpert® system must be available on site. In decentralised settings with a low case-load, dried blood spot (DBS) testing might be an alternative.
Between December 2019 and January 2021, patients with an indication for HCV RNA quantification and informed consent provided 100 µl capillary whole blood each for on-site Xpert® HCV VL FS testing (reference) and DBS testing in the laboratory. For the latter, 100 µl blood, collected with an EDTA Minivette®, were transferred to a Whatman® 903 filter card. After drying for at least 1 hour, the DBS sample was packed into a sealable plastic bag with desiccant and sent to the central laboratory of our hospital, where it was stored at -20°C. For HCV RNA extraction, the whole DBS was cut out with an 18-mm puncher and transferred into 1.3 ml guanidinium thiocyanate-containing buffer (provided by Cepheid®). After mixing and incubating at room temperature for 2-3 hours, 1 ml supernatant was analysed with the Xpert® HCV VL test (105 minutes) (filter paper absorbs 0.3 ml).
Of 109 paired samples from 67 patients, 38 (34.9%) were positive with the Xpert® HCV VL FS test. Sensitivity and specificity of DBS testing were 89.5% (34/38; 95% confidence interval [CI] 75.9-95.8%) and 97.2% (69/71; 95% CI 90.3-99.2%), respectively. The six (5.5%) discordant results (four false negative, two false positive) all were observed in samples with HCV RNA detectable below the limit of quantification after 2-8 weeks of pan-genotypic direct-acting antiviral treatment or 5 weeks after acute hepatitis C in a patient clearing HCV spontaneously. Quantifiable results (n = 30; 16 genotype 1, 7 genotype 3, 4 genotype 4, 1 genotype 1a and 3a, 2 unknown; HCV RNA range: 2.74-6.66 log IU/ml) correlated well (R2 = 0.981). On average, uncorrected DBS test results were 1.30 ± 0.14 log IU/ml lower than Xpert® HCV VL FS test results (~42 μl instead of the expected 1000 μl plasma used). Storage of DBS samples at room temperature for 7 days before freezing reduced HCV RNA by 0.29 ± 0.12 log IU/ml.
HCV RNA can reliably be quantified with the Xpert® HCV VL test in capillary dried blood spot samples. Thus, access to capillary HCV RNA quantification for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection can be extended to decentralised settings with a low case load.
对于长期静脉注射吸毒后静脉通路建立困难的患者，使用Xpert® HCV病毒载量指尖检测法（VL FS）（60分钟）在毛细血管全血中进行即时快速丙型肝炎病毒（HCV）RNA定量，是诊断慢性HCV感染、监测治疗及检测再感染的一种便捷可靠的方法。然而，现场必须配备昂贵的GeneXpert®系统。在病例数较少的分散环境中，干血斑（DBS）检测可能是一种替代方法。
2019年12月至2021年1月期间，有HCV RNA定量指征且签署知情同意书的患者每人提供100 μl毛细血管全血用于现场Xpert® HCV VL FS检测（参考方法）以及在实验室进行DBS检测。对于后者，用EDTA微量采血管采集的100 μl血液被转移至一张Whatman® 903滤纸上。干燥至少1小时后，将DBS样本装入带有干燥剂的可密封塑料袋中，送至我院中心实验室，在-20°C下保存。进行HCV RNA提取时，用直径18 mm的打孔器将整个DBS取出，转移至1.3 ml含硫氰酸胍的缓冲液中（由Cepheid®提供）。混合并在室温下孵育2 - 3小时后，取1 ml上清液用Xpert® HCV VL检测法（105分钟）进行分析（滤纸吸收0.3 ml）。
在来自67例患者的109对样本中，38例（34.9%）Xpert® HCV VL FS检测呈阳性。DBS检测的敏感性和特异性分别为89.5%（34/38；95%置信区间[CI] 75.9 - 95.8%）和97.2%（69/71；95% CI 90.3 - 99.2%）。6例（5.5%）不一致结果（4例假阴性，2例假阳性）均出现在泛基因型直接抗病毒治疗2 - 8周后或1例自发清除HCV的急性丙型肝炎患者发病5周后HCV RNA低于定量下限但仍可检测到的样本中。可定量结果（n = 30；16例基因1型，7例基因3型，4例基因4型，1例基因1a和3a型，2例未知型；HCV RNA范围：2.74 - 6.66 log IU/ml）相关性良好（R2 = 0.981）。平均而言，未经校正的DBS检测结果比Xpert® HCV VL FS检测结果低1.30 ± 0.14 log IU/ml（约42 μl而非预期的1000 μl血浆）。DBS样本在冷冻前于室温下保存7天，HCV RNA降低了0.29 ± 0.12 log IU/ml。
使用Xpert® HCV VL检测法可在毛细血管干血斑样本中可靠地定量HCV RNA。因此，用于诊断慢性HCV感染、监测治疗及检测再感染的毛细血管HCV RNA定量检测可扩展至病例数较少的分散环境。
