超灵敏CRISPR-TB快速血筛用于结核病诊断和治疗监测
Editor: He Sarina Yang, PhD, DABCC, Assistant Professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine
Authors: Zhen Huang, PhD, Postdoc fellow, Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine; Sylvia M LaCourse, MD, Assistant Professor, Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA; Bo Ning, PhD, Assistant professor, Tulane University School of Medicine; Tony Y Hu, PhD, Professor in Biochemistry and Molecular Biology, Biomedical Engineering, and Microbiology and Director, Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, remains a leading cause of global mortality, with an estimated 10 million TB cases and 1.2 million TB-related deaths annually. However, TB diagnosis is challenging in young children and individuals living with HIV due to difficulty obtaining respiratory samples and their frequent paucibacillary nature. This has led to a recognized need for more effective non-sputum-based TB diagnostics in these populations. A new and easy way to rapidly screen those susceptible to TB infections was recognized by WHO and other public health officials as the major technological hurdle needed to overcome the disease.
A multinational research team led by Prof. Tony Y. Hu (Weatherhead Presidential Chair in Biotechnology Innovation at Tulane University) has developed a rapid blood test to diagnose and monitor treatment. This study evaluated the diagnostic sensitivity and specificity of an ultrasensitive CRISPR-based fluorescence assay to detect Mtb cell-free DNA (Mtb-cfDNA) in blood samples from adults and children, including children with HIV. This assay demonstrated high diagnostic sensitivity and specificity (>90%) in a pooled adult and pediatric group, and diagnostic sensitivity remained high (85%) in children diagnosed with TB by non-microbiologic clinical findings.
"TB diagnosis remains challenging and is still widely dependent on sputum-based microbiologic methods with unsatisfied sensitivity and specificity, and spend weeks," said Prof. Hu, "what's more they don't apply to young children, HIV co-infected or extrapulmonary TB cases who usually have higher mortality due to untimely diagnosis".
Researchers newly developed a CRISPR-powered TB assay (CRISPR-TB) by ultrasensitive detection of Mtb-cfDNA in the blood for rapidly diagnosing TB, predicting TB progression, and monitoring TB treatment responses. This technology employs CRISPR/Cas12a, a widely used gene editor, to amplify nucleic acid detection signals for ultrasensitive tracking of trace circulating Mtb-cfDNA that is a highly degraded circulating short DNA fragment secreted by apoptotic Mtb or Mtb-infected cells, can reflect systemically infection, support real-time analysis, and keep homeostasis with host pathological states.
Current TB assays often demonstrate reduced performance with children, HIV-positive TB patients, or those with TB infections in non-lung tissues, and these patients can require tissue biopsies for diagnosis. CRISPR-TB, however, detected non-HIV-infected adults (96.4%) and children (83.3%) as well as HIV-infected children (100%) with comparable high sensitivity by using a small volume of blood sample in 2 hours.
“We are particularly excited that the blood Mtb-cfDNA level of HIV-infected children began to decline within 1 month of treatment, and most of the children's blood Mtb-cfDNA was cleared after treatment, which means that CRISPR-TB has the potential to monitor treatment and will give physicians the ability to better treat worldwide TB infections" Prof. Hu said.
The researchers also adapted this assay to a lateral flow platform that allows running Mtb-cfDNA test in 30 min without any equipment and readout with naked-eye, which will benefit millions of people living in resource-limited areas with high TB burden.
“This assay may be a game-changer for TB diagnoses that not only provides accurate diagnosis results but also has the potential to predict disease progression and monitor treatment", Prof. Hu said, “This will help the clinic to rapidly intervene in treatment and reduce the risk of death, especially for children living with HIV. I hope it can be pushed forward quickly to reach these children as early as possible”.
The study, “CRISPR detection of circulating cell-free M. tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study,” was published in Lancet Microbe6.
结核病（TB）仍然是全球死亡的主要原因之一，每年有约1000万新发病例和120万TB相关死亡病例1。快速准确的诊断是实现“终结全球TB流行”战略的先决条件，但TB的诊断仍具挑战2。广泛应用的传统诊断技术依赖痰液样本，对肺外结核、儿童和HIV共感染患者诊断性能较差。亟需灵敏、特异、简便和快速的非痰样本检测技术，以改善TB诊断并评估TB治疗。
灵敏检测循环中痕量的结核分枝杆菌游离DNA（Mtb-cfDNA）有望成为一种通用的TB诊断方法，而且具有评估TB负担和监控治疗的巨大潜力。但受限于传统核酸检测方法较低的检测灵敏度，先前基于Mtb-cfDNA诊断TB的尝试均不理想，诊断灵敏度低且不稳定3,4。基于CRISPR增强的新型核酸检测技术有望解决该问题5。由Tony Y. Hu教授领导的多国联合科研团队采用超灵敏CRISPR荧光检测系统分析血液中的Mtb-cfDNA水平（CRISPR-TB），用于结核病诊断和治疗监测。这项研究发表在最新一期的Lancet Microbe杂志6。研究表明，CRISPR-TB诊断可以：
灵敏且特异性地诊断TB：CRISPR-TB对成人和儿童TB，诊断灵敏度高（94.1%；95% CI：80.3-99.3%）特异性强（94.9%；95% CI：82.7-99.4%），且成功诊断所有受试肺外TB病例（6/6）。此外，对于最具临床诊断挑战性的（受HIV感染处于严重免疫抑制状态并表现出TB症状）的幼儿病例，CRISPR-TB成功诊断了所有的确诊TB（13/13）和大多数的未确诊TB病例（80%；52/65）。
表明易感人群中与TB相关的死亡风险增加：基线CRISPR-TB阳性的HIV感染儿童的6月死亡风险是基线阴性幼儿的2.4倍（p=0.073），对于未接受TB治疗的儿童，CRISPR-TB阳性幼儿的死亡风险进一步增加至3.9倍（p=0.015）。
允许快速监测抗TB治疗效果：抗TB治疗导致患者Mtb-cfDNA水平显著下降，研究完成时，大多数具有纵向血清的HIV感染儿童显示Mtb-cfDNA清除。其中28.6%的HIV感染儿童Mtb-cfDNA水平在开始治疗后的一个月内即显著降低，并在50（IQR：24-77）天时显示Mtb-cfDNA清除。
促进潜在的早期诊断：CRISPR-TB提前43.5(IQR: 24.5-83)天诊断出83%(10/12)的受HIV感染的新发TB幼儿，表明早期诊断的潜力。
可转化POCT方法：整合免疫层析平台，开发了纸基CRISPR-TB便携检测技术，该方法无需复杂的仪器设备，可在30分钟内完成检测，适宜于在高结核病负担但资源有限地区的推广使用。
上述结果表明，CRISPR-TB不仅可以提供准确的诊断，还可以预测疾病进展和监测治疗，这将有助于临床快速干预治疗和降低死亡风险，尤其是HIV感染儿童的死亡风险。
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Huang Z, LaCourse SM, Kay AW, Stern J, Escudero JN, Youngquist BM, Zheng W, Vambe D, Dlamini M, Mtetwa G, Cranmer LM, Njuguna I,Wamalwa DC, Maleche-Obimbo E, Catanzaro DG, Lyon CJ, John-Stewart G, DiNardo A, Mandalakas AN, Ning B, Tony Y Hu, CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study. Lancet Microbe 2022. DOI: https//doi.org/10.1016/S2666-5247(22)00087-8.