Tuberculosis Imaging Program

该研究项目过去的主要活动集中在优化在新扫描仪上对恒河猴、NZW 兔和普通狨猴进行成像的方法和程序，同时在 Mtb 模型中进行各种化疗和基础免疫学实验。作为这些努力的一部分，我们优化了扫描仪质量控制系统、数据控制系统、麻醉方案、通气程序和屏气方法，以便为动物受试者和正在进行的实验尽可能安全地生成最佳图像。该过程仍在进行中，但针对中型（3 至 6 公斤）动物的标准方案已经建立，并已成功应用于 Mtb 感染的恒河猴和 NZW 兔。 由于我们拥有超过10年的数据和多篇出版物，使用CT Hounsfield单位（HU）密度范围和PET FDG摄取值作为结核病灶的描述性和定量特征，因此我们对以前的小型临床CT扫描仪和新型扫描仪（LFER）采集的图像的定量差异进行了系统研究。一项研究使用了 LFER 附带的常见 CATPHAN 模型，在两个系统上都成像。这种类型的体模通常用于扫描仪质量控制 (QC)，采用六种具有不同平均 HU 的标准材料，模拟活体受试者中发现的密度（HU 类似于骨骼、空气、肌肉、脂肪），并在具有多种能量设置（kVp 和 mAs）的两台扫描仪上成像。在绘制不同材料的线性衰减系数 (LAC) 和 HU 测量结果后，我们发现至少有 2 例模型不是采用美国电气制造商协会 (NEMA) 标准材料制成的。 2020年，我们的持照技术专家与经过认证的公司合作，设计了一种新的CT模体，适合用NIST和NEMA标准材料对猴胸部进行建模。新的线性斜率计算表明 Ceretom CT 和 Mediso 装置的 y 截距不同，为 155 HU 而不是 115。重新运行这些测试后，对于恒河猴和兔子，LFER 上可用的最佳技术仍然是 80 kVp 980 A 80 ms (65.6 As)。收集并分析使用该技术的扫描，特别关注识别病变的 HU 范围。 用于优化 PET 图像的标准化参数是给予受试者的与体重和习惯相关的探针剂量、允许停留的时间以及数据收集的持续时间。我们决定将停留时间保持在 1 小时，并研究数据收集时间和剂量。 2019 年，我们将恒河猴的最佳 FDG 剂量确定为 0.5 mCi/Kg，以最大程度地减少生成图像中的噪声和变异性。肉眼看来，不同剂量产生的图像相似，但详细分析表明，0.5 mCi 剂量获得了最佳定量结果。 2020 年，我们使用 0.2mCi/kg、0.5mCi/kg、1.0mCi/kg 和 2.0mCi/kg 对呼吸门控的幼稚狨猴、幼稚 NZW 兔和慢性感染 Mtb 的兔子应用相同的方案。我们发现对于兔子来说，1 mCi/kg 是最佳剂量。 2019 年 10 月，我们终于能够完全整合新的门控硬件，与为 LFER 扫描仪开发的新软件编程配合使用，以扫描狨猴。该系统有助于在动物呼吸周期的选定阶段创建伪影较低的 CT 数据集。初步测试表明，门控通过缓解胃中的空气并消除长时间机械屏气导致呼吸性酸中毒的可能性，有助于提高狨猴的稳定性和舒适度，不幸的是，其代价是辐射暴露。我们正在尝试确定可接受的最低扫描长度，以收集必要的数据，以最大限度地减少辐射剂量。由于狨猴体型小、表面积大，FDG 剂量优化结果仍为 2 mCi/kg。 为了确保我们收集的数据的质量，并使用我们的成像方式进行高质量和一致的疾病量化，我们建立并正在维护一个全面的质量控制体系。质量措施清单请参见2019年报告。此外，我们与 Mediso 科学家和工程师保持合作，维护系统，同时探索改进收集数据的方法和分析数据的方法。当我们与多个小组和研究模型合作时，保持实验数据的良好记录和组织非常重要。我们继续维护两个网络驱动器来存储每个 PI 的这些数据。这种设置使我们能够将所有重要数据集中在一个位置，供 PI 实验室中的每个人参考，并为修改敏感的原始成像数据提供单独的有限访问位置。 2019 年，我们在免疫抑制剂研究中对 PET 图像进行分析，成功检测到恒河猴肺部和淋巴结异常区域与疾病相关的 FDG 摄取（SUV > 2.5）。然而，在这些动物中，并非所有患病组织的 FDG 摄取量均升高。因此，我们应用了一种自动化方法，使用全肺技术来分离低密度和高密度范围。这种方法使我们能够密切、准确地监测疾病变化，即使单个病变在 CT 图像中很难分离。我们已经为该小组的出版物准备了实验室和分析方法。去年进行的其他抗结核药物相关实验包括 Herbert 博士进行的三项单剂量 PK 实验、一项使用 2 种药物和细胞色素 P450 抑制剂进行的长期稳态 PK 实验，以鉴定任何药物间相互作用，以及五项抗结核活性研究。我们在兔子身上继续进行了另外两个阶段的宿主定向治疗研究，并开发了一种对有限数量的患有真正肉芽肿的小鼠进行成像的方案。已经完成了三项大型基础免疫学研究，研究 SIV 合并感染或用细胞因子或外源性物质操纵宿主免疫系统。我们已经使用上述方法来分析这些实验的扫描数据，但也在寻找更具体的方法来测量微小的变化。 TBIP 已协助 VRC 提交动物协议文件。最后，为了便于VRC研究的启动，我们订购了一种特殊的Mtb菌株，制作了库存瓶，并进行了高精度的滴度测定，这样当他们的批准文件到位时，我们就不必等待他们做这3个月的工作。当大流行导致工作暂停时，我们正计划与 VRC 调查员进行第一次感染。 TBIP 团队在 2020 年春季完成了所有正在进行的已登记物种实验，没有丢失样本或动物。最后，我们订购了设备并审查和修改了我们的 SOP，以实施 SARs-CoV-2 研究。我们计划了模拟程序，以便对工作人员进行更新方法的培训，以便我们能够在研究人员准备好时快速实施研究。

The major activities of this research project in the past have centered around optimizing the methods and procedures for imaging rhesus macaques, NZW rabbits, and common marmosets on new scanners while conducting a variety of chemotherapy and basic immunology experiments in Mtb models. As a part of these efforts we have optimized scanner quality control systems, data control systems, anesthesia protocols, ventilation procedures and breath-hold methods to produce the best images as safely as possible for the animal subjects and the experiments being conducted. The process is still ongoing, but a standard protocol for medium sized (3 to 6 kg) animals was established and has been applied successfully in Mtb-infected rhesus and NZW rabbits. As we have more than 10 years of data and multiple publications using CT Hounsfield unit (HU)density ranges and PET FDG uptake values as descriptive and quantitative features for tuberculosis lesions, we have made systematic study of the quantitative differences in images collected on the previous small clinical CT scanner and the new scanner (LFER). One study used a common CATPHAN phantom supplied with the LFER that was imaged on both systems. This type of phantom, typically used for scanner quality control (QC), with six standard materials with varying mean HUs that mimic densities found in a live subject (HUs similar to bone, air, muscle, fat) was imaged on both scanners with several energy settings (kVp and mAs). After plotting the linear attenuation coefficient (LAC) and the HU measurement of the different materials, we discovered that the phantom was not made with National Electrical Manufacturers Association (NEMA) standard materials in at least 2 cases. In 2020, our licensed technologist worked with certified company to design a new CT phantom for appropriate for modeling the monkey chests with NIST and NEMA standard materials. The new linear slope calculations indicate the Ceretom CT and the Mediso unit have differing y intercepts of 155 HU rather than 115. After rerunning these tests, the optimal technique available on the LFER was still 80 kVp 980 A 80 ms (65.6 As) for the rhesus and the rabbit. Scans using this technique were collected and analyzed with specific attention to the HU ranges where lesions were identified. Parameters to standardize for optimization of the PET image are the probe dose administered to the subjects as related to body weight and habitus, the time it is allowed to dwell, and the duration of the data collection. We decided to hold dwell time to 1 h and investigated data collection time and dose. In 2019 we established the optimal FDG dose for the rhesus as 0.5 mCi/Kg to minimize both noise and variability in the resulting images. To the naked eye, the images resulting from various doses were similar, but a detailed analysis showed that the best quantitative results were obtained with the 0.5 mCi dose. In 2020, we applied the same protocol to respiratory-gated naive marmosets and both nave NZW rabbits and chronically infected ones with Mtb using 0.2mCi/kg, 0.5mCi/kg, 1.0mCi/kg and 2.0mCi/kg. We found that for the rabbit, 1 mCi/kg was optimal. In October 2019 we were finally able to fully incorporate new gating hardware to work with new software programing developed for the LFER scanner in order to scan the marmosets. This system is helping create a lower artifact CT dataset during a selected phase of the animals breathing cycle. Preliminary tests suggest gating has helped improve the marmosets stability and comfort by alleviating air in the stomach and eliminating the potential for respiratory acidosis from a prolonged mechanical breath hold, unfortunately the tradeoff is radiation exposure. We are trying to determine the lowest acceptable scan length to collect the necessary data to minimize radiation dose. The results of the FDG dose optimization for the marmoset, because of their small size and large surface area, was still 2 mCi/kg. To assure the quality of the data we are collecting and to conduct high quality and consistent disease quantification with our imaging modalities, we have established and are maintaining a comprehensive quality control system. Please see the 2019 report for the list of quality measures. In addition, we maintain a collaboration with the Mediso scientists and engineers to maintain the systems but to also explore ways to improve the data collected and ways to analyze it. As we work with multiple groups and research models, it is important to keep the experimental data well documented and organized. We continue to maintain two network drives to store these data per PI. This setup allows us to have all the important data in one spot for everyone in the PIs lab to reference and a separate limited access-location for the modification-sensitive original imaging data. In 2019, our analysis of PET images in an immune inhibitor study was successful in detecting disease related FDG uptake (SUV > 2.5) in abnormal regions in lungs and lymph nodes of rhesus macaques. However, in these animals, not all of the diseased tissue had an elevated FDG uptake. Therefore, we applied an automated method that segregates low- and high-density ranges using a whole lung technique. This approach allowed us to closely and accurately monitor disease changes even if the individual lesions were very difficult to separate in the CT images. We have prepared our laboratory and analysis methods for the groups publication. Other anti-TB drug related experiments undertaken in the last year include three single dose PK experiments with Dr Herbert, one very long steady-state PK with 2 drugs and a cytochrome P450 inhibitor to identify any drug-drug interactions as well as five anti-TB activity studies. We have continued two more phases of the host directed therapy studies in rabbits and developed a protocol for imaging a limited number of mice with true granulomas. There have been three large basic immunology studies looking at either co-infection with SIV or manipulation of the host immune system with cytokines or exogenous agents also completed. We have used the methods mentioned above to analyze the scan data from these experiments but are looking at more specific methods for measuring small changes as well. TBIP has assisted the VRC with their animal protocol documents for submission. Finally, in order to facilitate the start of VRC studies, we ordered, made stock vials, and titered a special strain of Mtb to high accuracy, so that when their approval documents were in place, we would not have to wait for them to do this 3 months of work. We were just planning the first infection with the VRC investigator when the pandemic caused the work to pause. The TBIP team completed all ongoing experiments with registered species during the spring of 2020 with no loss of samples or animals. Finally, we have ordered equipment and reviewed and modified our SOPs in order to implement SARs-CoV-2 studies. We have planned mock procedures in order to train staff in the updated methods so that we can rapidly implement the studies when the investigators are ready.