背景:那他珠单抗是复发性多发性硬化症(MS)的有效治疗方法。在治疗期间,个体发生进行性多灶性白质脑病(PML)的风险增加.到目前为止,PML型JC多瘤病毒(JCV)的相关库仍然难以捉摸。我们在这里测试了用那他珠单抗治疗的MS患者粪便中JCV-DNA的检测是否可能是PML风险评估的未来工具。
方法:粪便中存在JCV-DNA,尿液,研究了接受那他珠单抗治疗的MS患者的全血和已知的血清抗JCV抗体指数值(IV)。不同的DNA提取方法,比较了实时(RT)和液滴数字(dd)PCR技术。通过测序筛选JCV分离株的PML相关变体。
结果:筛选了30例用那他珠单抗治疗的MS患者。对于21名患者,血,凳子,和尿液样本可用。这些患者根据其血清抗JCV抗体IV进行分层(高(>1.5,n=12);中(1.5-0.9,n=2);低(<0.9,n=1);阴性(n=6))。在全血或粪便样品中未检测到JCV-DNA。四个尿液样本有可测量的JCV-DNA,RT-PCR检测范围为1.71×104-1.07×108国际单位(IU)/mL,对应于通过ddPCR测量的4.62×104-9.85×106拷贝/mL。所有JCV变体都是野生型的并且来源于具有高抗体IV的患者。
结论:粪便特异性DNA提取方法提供了最高质量的DNA,而ddPCR和RT-PCR的敏感性相当。我们的发现不支持评估粪便样本对MS患者的PML风险分层。需要进一步的研究来探索PML相关病毒变体出现的地方。
BACKGROUND: Natalizumab is an effective treatment for relapsing multiple sclerosis (MS). During therapy, individuals are at increased risk of developing progressive multifocal leukoencephalopathy (
PML). So far, the relevant reservoir for
PML-type JC polyomavirus (JCV) remains elusive. We here tested if the detection of JCV-DNA in stool of persons with MS treated with natalizumab could be a future tool for
PML risk assessment.
METHODS: The presence of JCV-DNA in stool, urine, and whole blood of MS patients treated with natalizumab and known serum anti-JCV antibodies index values (IV) was studied. Different DNA extraction methods, real-time (RT) and droplet digital (dd) PCR techniques were compared. JCV isolates were screened for
PML-associated variants by sequencing.
RESULTS: Thirty MS patients treated with natalizumab were screened. For 21 patients, blood, stool, and urine samples were available. These patients were stratified according to their serum anti-JCV antibody IV (high (>1.5, n = 12); medium (1.5-0.9, n = 2); low (<0.9, n = 1); negative (n = 6)). JCV-DNA could not be detected in the whole blood or stool samples. Four urine samples had measurable JCV-DNA, ranging from 1.71×104-1.07×108 international units (IU)/mL detected by RT-PCR, corresponding to 4.62×104-9.85×106 copies/mL measured by ddPCR. All JCV variants were wild-type and derived from patients with high antibody IV.
CONCLUSIONS: Stool-specific DNA extraction methods provided the highest quality of DNA, while the sensitivity of ddPCR and RT- PCR was comparable. Our findings do not support assessing stool samples for PML risk stratification in persons with MS. Further studies are needed to explore where
PML-associated viral variants arise.