Real-Time Reagent-Free Noninvasive Diagnosis of Tuberculosis

描述（由申请人提供）：尽管结核病（TB）给人类造成了损失，但这种疾病仍然难以诊断，因此难以控制。世界上三分之一的人口感染了结核分枝杆菌。每年有900多万人患上活动性结核病，170万人死于该病[WHO 2010]。目前广泛使用的诊断方法在识别活动性结核病患者方面表现不佳，大约50%的患者痰液抗酸杆菌（AFB）涂片呈阴性[Steingart 2006]。未确诊病例在不知不觉中继续传播疾病，威胁公共卫生。在美国，诊断的不确定性以及缺乏敏感、特异和快速的检测，导致住院费用显著增加，因为疾病控制和预防中心（CDC）指南建议在单人床、负压室中隔离结核病疑似病例，直到连续三次每日痰样本经直接涂片处理并被认为AFB阴性[CDC 2005]。由于后勤问题，这些患者在被清除之前平均要隔离5天。由于这些患者中绝大多数没有患病，住院层面的结核病控制成本主要由结核病疑似患者的住院隔离天数决定[Scott 1994]，并且最终往往由医院本身承担。使这种感染控制方法更加复杂的是，AFB痰涂片阴性并不能明确排除结核传播的风险[Behr 1999]。当未确诊病例继续传播疾病时，社会成本急剧增加[Miller 2010]。当前结核病诊断金标准的局限性重新激发了人们对从结核病嫌疑人中识别结核分枝杆菌的快速、准确、具有成本效益、非侵入性和用户友好的方法的兴趣。对患者咳嗽气雾剂进行结核分枝杆菌分析有可能满足所有这些要求。利弗莫尔仪器独特的单粒子气溶胶质谱（SPAMS）技术可能是临床真实环境中结核病诊断的答案。垃圾邮件系统以前曾被能源部用于检测炭疽杆菌。在这个应用中，我们描述了一个基于SPAMS的气溶胶分析系统，作为检测咳嗽和呼出气体中的结核分枝杆菌的潜在工具。我们现有的系统版本操作简单，坚固，包含很少的活动部件，而且重要的是，不含试剂，因此每次测试的边际成本非常低。SPAMS的工作原理是，在单个气溶胶粒子向双极性飞行时间质谱仪源区中心移动时，用激光跟踪它们。到达后，它们被一个单一的脉冲高功率激光解吸和电离，产生的离子由质谱仪测量。实时分析质量特征可以识别粒子。在许多微生物的情况下，它们可以被识别到物种水平。一个SPAMS系统每秒可以评估几十到几百个粒子，在90秒内收集1000个质谱，每个质谱都是一个单独的微生物。在这个应用程序中，我们描述了评价的SPAMS检测结核分枝杆菌从结核嫌疑人的咳嗽气溶胶。以前曾在临床环境中在气溶胶阶段检测到结核分枝杆菌，但这种检测是使用聚合酶链反应（PCR）对数小时内收集的样本进行分析的劳动密集型检测[Chen 2005]。我们将证明，结核病的实时诊断是一个可行的概念，通过快速和准确地检测结核分枝杆菌使用这一最先进的技术。这是一个具有许多潜在应用的变革性概念，包括医院急诊室或结核病控制诊所的快速分诊

DESCRIPTION (provided by applicant): Despite the human cost of tuberculosis (TB), the disease remains difficult to diagnose and thus to control. One-third of the world's population is infected with M. tuberculosis. Every year, more than 9 million people develop active TB and 1.7 million people die from the disease [WHO 2010]. Current widely used diagnostics perform poorly in identifying active TB in patients with roughly 50% of cases having sputa that are smear negative for acid fast bacilli (AFB) [Steingart 2006]. Undiagnosed cases unwittingly continue to transmit disease, threatening public health. In the US, the uncertainty in diagnosis, and the absence of sensitive, specific, and rapid tests, results in significant increases in hospitalizatio costs because Centers for Disease Control and Prevention (CDC) guidelines recommend that suspected cases of TB be isolated in single bed, negative-pressure rooms until three consecutive daily sputum samples have been processed by direct smear and deemed negative for AFB [CDC 2005]. Due to logistics issues, these patients remain in isolation an average of five days before being cleared. Because the vast majority of these patients do not have the disease, TB control cost at the inpatient level is dominated by these inpatient isolation days for TB suspects [Scott 1994] and is often ultimately borne by the hospital itself. Compounding this infection control approach is the fact that negative sputum smears for AFB do not definitively exclude risk of TB transmission [Behr 1999]. Societal costs increase dramatically when undiagnosed cases continue to transmit disease [Miller 2010]. The limitations of the current gold standards for TB diagnosis have stimulated renewed interest in rapid, accurate, cost-effective, non-invasive, and user-friendly methods of identifying M. tuberculosis from TB suspects. Patient cough aerosol analysis for M. tuberculosis has the potential to address all these requirements. Livermore Instruments' unique Single Particle Aerosol Mass Spectrometry (SPAMS) technology may be the answer to TB diagnosis in clinical real-world settings. SPAMS has been previously deployed by the Department of Energy for the detection of B. anthracis. In this application, we describe an aerosol analysis system based on SPAMS as a potential tool for detection of M. tuberculosis in coughed and exhaled breath. The existing versions of our system are simple to operate, sturdy, contain few moving parts, and, importantly, are reagent-free so that the marginal cost per test is extremely low. SPAMS functions by drawing in individual aerosol particles and tracking them with lasers as they proceed towards the center of the source region of a dual-polarity time-of-flight mass spectrometer. Upon their arrival, they are desorbed and ionized by a single, pulsed high power laser and the resulting ions are measured by the mass spectrometer. Real-time analysis of the mass signature allows the identification of the particles. In the case of many microorganisms, they can be identified to the species level. A SPAMS system can evaluate dozens to hundreds of particles per second, collecting 1000 mass spectra, each of an individual microorganism, in 90 seconds. In this application, we describe the evaluation of SPAMS for the detection of M. tuberculosis from coughed aerosol of TB suspects. M. tuberculosis has been detected in the aerosol phase in clinical settings previously but such detection was performed labor-intensively using the polymerase chain reaction (PCR) to analyze samples collected over a matter of hours [Chen 2005]. We will prove that the diagnosis of TB in real- time is a viable concept by rapidly and accurately detecting M. tuberculosis using this state-of-the-art technology. This is a transformative concept with many potential applications, including rapid triage in hospitals at the emergency room or at TB control clinics to identify patients in need of treatment and to prevent nosocomial transmission, as well as for rapid mass screening in non-clinical environments such as in the field in high incidence settings. Finally, another key advantage of this technology that distinguishes it from other breath analysis systems under development is the ability to train SPAMS for the detection of other respiratory pathogens as well. This makes SPAMS ideal as a diagnostic in TB patients in whom parenchymal damage and bronchiectasis often result in concomitant infection/colonization with other non-TB pathogens. The ability to diagnose other respiratory diseases further broadens SPAMS' potential use in a variety of clinical and non-clinical settings. Livermore Instruments Inc. is a San Francisco Bay Area startup company dedicated to bringing aerosol analysis to nontraditional fields. PUBLIC HEALTH RELEVANCE: Despite advances in medical technology, TB remains stubbornly difficult to diagnose even by experienced pulmonologists. On the one hand, missed diagnoses remain ill while continuing to spread the disease, possibly to patients and physicians in a hospital. On the other hand, patients falsely suspected of the disease are confined to negative pressure rooms until TB can be ruled out. Those uninfected with TB were, in fact, ill but were isolated for days with reduced physician contact. This technology will mitigate both of those problems. In addition, this research will provide a method for the noninvasive and rapid screening of large populations for TB. An example of this would be the use of a SPAMS instrument to detect TB in populations at border crossings and customs entry checkpoints. More than 50% of all domestic cases of TB were contracted outside of the United States, making their detection abroad or at points of entry critical to the mitigation of the problem. While SPAMS instruments appear complex, they are, in fact, highly robust, with the first generation of experimental instruments ever created still in service. While this study will include the use of disposable masks and tubing, future protocol development will seek to eliminate these. If successful, a SPAMS also removes significant logistical burdens for both institution and patient, including the collection, handling and testing of sputum by trained professionals and the delay in diagnosis resulting there from. Therefore, although SPAMS systems appear expensive, because SPAMS systems can streamline patient screening, last for decades and consume only electricity, they are, in fact, inexpensive on a per test basis. Also, in the future SPAMS systems may be tasked to diagnose multiple diseases simultaneously, with a similar savings in logistical burden per disease but at no increase in cost for the instrumentation. SPAMS systems have already been successfully fielded under extremely austere conditions including deserts, mountain tops, aboard ships at sea and to remote islands. We envision a future where a single SPAMS system in a single van can be driven on an anal circuit where it will evaluate the entire village population of villages along its route at a cost of roughly $0.50 per test, gradually removing TB as a health problem in that region.