Tuberculosis Imaging Program

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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.