Rabbits infected by aerosol with M. tuberculosis strain HN878 develop slowly progressing chronic disease.
Thirty female rabbits (3.0 to 3.4 kg) were infected with M. tuberculosis strain HN878 (a recent clinical isolate of the Beijing clade virulent in rabbits [29, 35]) by nose-only aerosol exposure in groups of 12 animals; 5 animals each were sacrificed at 0, 2, 4, 6, 10, and 18 weeks. Aerosol infection delivered an average of 2.2 ± 0.13 (mean ± standard error of the mean [SEM]) log CFU/g lung at week 0 (2 h postinfection [p.i.]). By 2 weeks p.i., although lesions were invisible by gross inspection of the lung parenchyma, the number of bacteria had risen to 4.4 ± 0.13 log CFU/g (). By 4 to 5 weeks p.i., visible granulomas had formed, with diameters ranging from 1 mm to 3 mm, and the bacterial load reached its maximum at 5.2 ± 0.19 log CFU/g, for approximately 6.5 log CFU/lung. By 6 weeks p.i. the bacterial load had dropped to 4.0 ± 0.15 log CFU/g. At subsequent time points, the bacterial burden was stable at 4.1 ± 0.30 (8 to 10 weeks) and 3.9 ± 0.20 log CFU/g (17 to 18 weeks). At the later time points of this experiment, the rabbits weighed about 4 kg and the infection had generated an average of 58 ± 16 (mean ± SD) granulomas per animal, demonstrating that M. tuberculosis HN878 was able to establish a chronic infection in the rabbits.
Fig 1 NZW rabbits infected with M. tuberculosis strain HN878 experience a transient replicative phase, followed by chronic disease with lesions slowly increasing in density. (A) Time course of M. tuberculosis HN878 infection in the NZW rabbit lung compiled (more ...)
During the inflammatory stage at peak bacterial load (weeks 4 to 5), HN878 lesions were typically solid cellular granulomas widely distributed in single focal lesions or in multifocal clusters () often located adjacent to the pleural surface. The small (1- to 3-mm) lesions typically consisted of central histocytes and neutrophils, with a thin layer of lymphocytes making up the periphery, but lacked central necrosis, which is often observed by 5 weeks in M. bovis
-infected rabbits (9
). By 8 to 10 weeks p.i., most granulomas were surrounded by a thicker lymphocytic cuff, and within this cuff were more centrally located epithelioid macrophages mixed with rare multinucleated giant cells and a necrotizing center with eosinophilic staining material, typically with punctate nuclear debris (). Cavities were occasionally observed among the necrotizing lesions, and these often still contained an interior partially filled with necrotic material. Cavities were identified either during CT scans as lesions with centers approaching −1,000 HU (the density of air in Hounsfeld units) or during dissection as lesions that contained air. The cavity structure included an extensive central necrotic zone surrounded by a layer of intact macrophages, lymphocytes, and fibroblasts contained within an outer fibrotic wall. The chronic stage of progressive pulmonary infection with M. tuberculosis
HN878 was characterized by the development of heterogeneous lesions within the same animal, similar to those described in the human lung: solid granulomas that were exclusively cellular, tuberculomas containing a caseous necrotic center, and fibrotic lesions undergoing liquefaction.
PET-CT imaging reveals that M. tuberculosis HN878 induced granulomatous inflammation peaks in the rabbit lung at 4 to 5 weeks p.i.
Six age- and weight-matched rabbits with negative preinfection scans were serially scanned after infection with HN878. These animals were imaged every 2 to 3 weeks until 10 weeks p.i. and then once every 4 to 5 weeks until 20 weeks p.i. using a standardized imaging protocol where the feeding status, dose (mBq/kg), tracer uptake period, and calibration of the scanner were carefully controlled (39
). Three animals were scanned and necropsied at 4 weeks, and three more animals were scanned and necropsied at 9 weeks in order to match the PET-CT observations with histopathological observations and obtain data on bacterial burden in lesions.
Preliminary PET-CT studies had indicated that uninfected, commercially bred NZW rabbits lacked significant [18F]FDG uptake above background in the lung and thoracic lymph nodes. Scanning at 2 weeks p.i. produced CT and [18F]FDG PET images similar to baseline scans. Slight increases in lung density as measured by CT and diffuse uptake of [18F]FDG in the lung was detectable beginning at 3 weeks p.i. (). shows the progression of disease by CT and PET over 20 weeks of infection in one representative animal from this group. Each panel of three images depicts (from left to right) the CT, fused PET-CT, and PET images from 3 merged axial slices (3.75 mm of lung) carefully matched in absolute position throughout the infection. Although the lung appeared superficially unaffected in the 3-week scans, the background mean SUV of the lung increased to ~2 at 50 min after [18F]FDG injection from a baseline of 1 preinfection. By 4 weeks, a pronounced granulomatous inflammation was present with many small, apparently homogeneous lesions. PET-CT revealed a surprising variability in the FDG avidity of lesions that appear highly similar by CT. The fate of individual lesions was likewise highly variable. For example, two adjacent lesions with similar volumes and densities also had a similar SUVmax at 4 weeks (SUVmax = 23 and 22) and at 6 weeks (SUVmax = 24 and 21), yet the more FDG-avid lesion maintained FDG avidity and eventually formed a cavity that continued to increase in size from 9 to 20 weeks, while the slightly less avid lesion had almost completely resolved by 9 weeks (SUVmax = 10) before coalescing with the growing cavity around 14 weeks. Across all of these animals, the formation and fate of individual lesions appeared to be largely independent of each other, and the factors that controlled the outcome of individual lesions were not clear.
Fig 2 FDG PET-CT scanning of M. tuberculosis-induced lung disease reveals an early inflammatory phase of disease that partially resolves, followed by slow progression. A group of 6 rabbits was infected and monitored by scanning 6 to 8 times over 20 weeks of (more ...)
By 9 weeks, many of the original lesions had resolved, and those that remained appeared to be stable or to slightly increase in volume over the remaining 2 months. Notably, we also observed that many of the transient lesions formed during the inflammatory phase of the infection showed quantitative decreases in the [18F]FDG PET uptake prior to a slow disappearance from the CT scans, indicating a decrease in metabolic activity prior to the loss of volume. After this time, both lesion volume and lesion SUV remained reasonably constant or increased until at least 20 weeks p.i. This experiment suggests the existence in the rabbit of a transient stage of early inflammation where small, mostly cellular lesions form and regress in the rabbit lung while other apparently indistinguishable lesions progress, cavitate, and develop a suppurative, semiliquid core similar to that shown in . The mediastinal and hilar lymph nodes occasionally showed [18F]FDG PET activity, but few bacteria (<3 log/g) were recovered (data not shown).
CT analysis reveals that persisting lesions increase in density during progression to chronic infection.
Radiodensity in the lung parenchyma is often an indication of active pathology, so we examined changes in lung CT density during infection. The total three-dimensional lung volume of the 6 rabbits was extracted from serial CT images, the volumes of tissue at specific densities (expressed in Hounsfield units [HU]) were calculated, and these volumes were grouped into three categories: high density (HD) (175 to −224 HU), medium density (MD) (−225 to −624 HU), and low density (LD) (−625 to −1,000 HU). In the naïve uninfected rabbit lung, the HD tissue, including arterial blood vessels and bronchi, makes up less than 2% of the volume of the lung, and the MD tissue, including thinner blood vessels, bronchioles, and lymphatic lung tissue, makes up about 15% of the volume, with the remaining LD volume being mostly parenchyma and air. shows the progression of density changes in the animal represented in . While no granulomatous nodules were visible on the CT at 3 weeks, a clear increase in the volume of HD and MD tissue in the whole lung was observed. This abnormal lung volume continued to increase and had more than doubled by 4 weeks p.i., when visible lesions could be detected. Although the percentage of affected lung decreased by 6 weeks, as did the number of lesions observed on the CT scans (), the percentage of affected lung did not return to baseline levels and in fact began to increase at later time points as the rabbits developed expanding cavitary disease (compare at weeks 14 and 20 and with the evolution of MD and HD lung volume in ). This pattern was common among the 6 rabbits that were followed for the full 20 weeks () and the increase in HD and MD lung volume as a function of time PI was significant in 4 of 5 time points compared to naïve lung (P < 0.001).
The images were also examined to identify individual CT-dense nodules and other CT abnormalities located in the lung parenchyma. In order to determine the density of a lesion, an ROI was drawn using the brush tool in the axial slice with the greatest area of the lesion, and the mean HU ± SD were recorded for the ROI. As the time postinfection increased, the mean density of individual lesions increased, as shown in (P < 0.0001, ANOVA and posttest for linear trend). Each symbol on the graph represents a single lesion (>20/time point), and each time point included lesion measurements from at least five of six rabbits. Once the rabbits reached 10 to 11 weeks p.i., the increasing mean HU of the lesions began to level off, and the difference in mean density at 11, 14, and 20 weeks p.i. was not significant. This observation was not simply a by-product of disappearance of the less dense lesions in the animals over time, as tracking of individual lesions showed the same density changes in successive scans. Lesions that were in the process of disappearing from the CT scan often decreased in density (as expressed in HU) prior to disappearing. This analysis revealed an overall hardening of persisting lesions over time.
Comparison of bacteriologic response of 1 and 8 weeks of treatment with INH or RIF.
To assess the effects of chemotherapy on the radiologic features of this chronic disease, a group of 24 rabbits were infected with M. tuberculosis HN878 and treated with either INH or RIF. Daily doses of either INH or RIF were selected based on pharmacokinetic parameters determined in uninfected rabbits () and human exposure observed at doses in clinical use. Since the tolerability in rabbits of these two drugs for 2 months was unknown, the lower range of the calculated dose (30 mg/kg/day INH or 24 mg/kg/day RIF) was selected. The infected rabbits (8 to 9 weeks p.i.) were arranged randomly into six groups of 4 rabbits each. One group was euthanized after scanning at ~9 weeks; two groups were scanned, given 1 week of orally administered treatment, scanned again, and euthanized. The final three groups received 2 months of daily therapy with INH, RIF, or vehicle only and were then scanned a final time and euthanized.
Pharmacokinetic parameters of INH and RIF in uninfected rabbits and human equivalent doses based on clinical exposurea
After treatment, two types of lung samples, the entire right middle lobe and individual small granulomas from other areas of the lungs, were assessed for bacterial load. We included additional control data from animals in the disease progression experiment whose sacrifice times aligned with those of the treatment experiment. Animals treated for 1 week showed only a small, nonsignificant change in the mean CFU in the whole right middle lobe () and in individual lesions () compared with the control animals. After 2 months of single drug treatment, even by gross pathology it was apparent that the number and size of lesions in the lung were reduced. Although lesions were less obvious after 2 months of treatment, fibrotic scars on the pleural surface of the lung were still evident, and these as well as caseous lesions were collected for CFU assessment. Both the INH and RIF treatment groups had a significant reduction in CFU in the whole right middle lobe (−1.9 log [P < 0.001] and −1.4 log [P < 0.01], respectively) () and in individual lesions (INH, −3.6 log/g, and RIF, −2.7 log/g, both P < 0.001) () compared with the vehicle control animals.
Fig 3 Comparison of M. tuberculosis bacterial burden after 1 week or 8 weeks of INH or RIF treatment. (A) Mean bacterial burden (log CFU/g) (error bars show SD) of the right middle lobe of rabbits treated with either INH (30 mg/kg) or RIF (24 mg/kg) for 1 or (more ...) Pathology score changes in response to 1 or 8 weeks of treatment with INH or RIF.
The gross findings at necropsy were tabulated into a pathology score (PS) that allowed comparisons between groups of animals exposed to different treatments or times of infection. It enumerated the number and size of pathological lesions in the lung and associated lymph nodes within the thorax and extrapulmonary organs (6
). Additional factors, such as the presence of focal parietal pleural adhesions, thickening of the parenchyma, and cavitation, were also included. The PS was a composite number per rabbit; the data for the groups of rabbits given chemotherapy are summarized in . Uninfected rabbits housed in same facility routinely had a PS of 0. The scores for untreated animals 9 to 10 weeks p.i. ranged from 16 to 35 (mean ± SD, 25 ± 6.9). Animals sacrificed 2 months later after having been administered only vehicle control had a similar PS of 20 ± 7.3 (). Animals treated for 1 week had lower but not significantly different mean scores: 15 ± 7 and 16 ± 8 for INH and RIF, respectively. Animals treated with INH for 8 weeks had a significantly lower mean PS (7.6 ± 5.2; CI, 1.61 to 23.59; P
< 0.05 in Bonferroni's multiple comparison test for difference) than the vehicle controls. Treatment with RIF did not achieve a high level of statistical significance using this scoring system despite the highly significant reduction in bacterial burden. While this scoring system has proved to be useful in macaque studies, where the animals typically have more varied lesion types in more organs and drug treatment is begun with the onset of disease symptoms (6
), the usefulness of this score in rabbits, where treatment is initiated in asymptomatic animals, is less clear.
Histopathological and morphometric changes in response to treatment with INH or RIF.
For each animal, at least one cross-sectional slice of each lung lobe (other than the right middle lobe, which was used for CFU enumeration) was prepared by paraffin embedding and sectioning, and hematoxylin-and-eosin (H&E)- and Ziehl-Nielsen-stained sections were prepared. The H&E sections were scanned, and the resulting files were examined using ImageScope version 10 (Aperio Technologies). The percentage lesion area for each section was calculated by morphometric image analysis by using Matlab, as described previously (20
). shows a composite of representative low-magnification images of rabbit lungs. Well-organized solid and necrotizing granulomas existed ( to ) at 9 to 10 weeks p.i. Most granulomas were surrounded by a lymphocytic cuff, and within this cuff were more centrally located epithelioid macrophages mixed with occasional plasma cells and multinucleated giant cells; in addition, necrotizing granulomas contained eosinophilic staining material (best seen in , , And ). There was no apparent difference in the sections collected at 9 to 10 weeks ( and ), which contained 3.4% ± 2.3% (mean ± SD) lesion tissue by area, and those collected after 1 week of treatment (INH, and ; RIF, and ), which contained 2.1% ± 1.6% and 1.9% ± 1.5% lesion areas, respectively. These sections contained frequent small (1- to 2-mm) lesions located adjacent to the blood vessels ( and ) and the bronchioles ( and ) along with regions of bronchiolar thickening (A and E), as well as lesions on the pleural surface (B, D, and H). Lesions near the vessels along with bronchiolar thickening were visible by microscopic examination but were more difficult to measure with CT scanning, although [18
F]FDG activity colocalizing with vessels was often detected in the lung regions ( to ). In the animals treated with INH ( and ) and RIF ( and ) for 2 months, these small lesions were infrequently observed compared with the vehicle control animals ( and ) and the majority of lesions remaining after treatment were either small aggregates of histocytes (), occasional necrotizing granulomas, or a cavity showing some mineralization (). These measurements represent a significant response to treatment (P
= 0.015) with either INH (lesion area [mean ± SD], 0.3% ± 0.2%) or RIF (mean lesion area, 0.5% with the cavity and 0.3% ± 0.2% [SEM] without the cavity) compared to the vehicle control (lesion area [mean ± SD], 6.6% ± 5.7%) (). Both mean lesion number and mean lesion size calculated by the morphometry analysis were reduced in the 8-week drug treatment groups, but only mean lesion area reached significance (data not shown). Cavities were observed in both untreated animals (; 9, 14 and 20 weeks) and in the RIF-treated group (; also, see ).
Fig 4 Histopathologic changes in response to 1 or 8 weeks of INH or RIF treatment. (A and B) Granulomas 1 to 3 mm in diameter (both necrotizing and cellular) were observed in animals euthanized just prior to experimental treatment initiation at 9 to 10 weeks (more ...)
Fig 5 INH or RIF treatment of infected rabbits results in a slow resolution of both FDG PET and CT findings. (A) Groups of 4 rabbits were treated orally with 30 mg/kg INH daily for 8 weeks; images from a representative rabbit are shown. The top panel in each (more ...) PET-CT changes in lesion volume and [18F]FDG avidity quantified the efficacy of 2 months of treatment with INH and RIF in vivo.
Studies of human lungs using serial imaging by CT show that there is a relatively slow response to treatment (25
), so it was hypothesized that rabbits receiving only 1 week of treatment would show little or no change in lesion structure (assessed by CT) but greater changes in metabolic activity (assessed by [18
F]FDG PET uptake). The rabbit groups receiving 2 months of treatment were expected to show changes in both imaging modalities compared to control animals. The [18
F]FDG PET-CT images clearly documented changes in lesion volume and number after 4 and 8 weeks of treatment with INH or RIF (). Examples of this response can be seen in , which shows two CT-dense lesions and a larger consolidation that shrank as treatment was administered (4 and 8 weeks). The middle lesion of the three indicated with arrows had an initial volume of 130 mm3
, which was reduced to 49 and 14 mm3
after 4 and 8 weeks of therapy, respectively. The lesions showed reduction in [18
F]FDG PET activity with treatment as well (e.g., the SUVmax
of the lesion mentioned above was initially 8.6 and then dropped to the local background in the subsequent two scans). In images from a representative animal treated with RIF (), one lesion cavitated and became denser, according to the mean HU. This lesion initially had a volume of 59 mm3
and an SUVmax
of 14. After 4 weeks of RIF administration, the lesion was reduced slightly in volume to 48 mm3
with an SUVmax
of 6.7, but after 8 weeks the activity of the central cavity had increased (SUVmax
, 11.6), perhaps due to an influx of neutrophils into the caseum or an increase in metabolic activity of the epithelioid macrophages surrounding the necrotic center (for the histological appearance of this lesion at necropsy, see ). Another lesion in this axial slice was also reduced in size and activity with therapy but increased in density. Three-dimensional images of the same rabbit's lungs were reconstructed to show that the metabolically active lesions decreased in size with treatment (), with the exception of the cavity that developed at 4 weeks. Representative images from one of the rabbits treated orally with 2 ml raspberry syrup daily for 8 weeks (see Fig. S1 in the supplemental material) contained lesions with unchanging or increasing [18
F]FDG PET activity.
CT volume and SUVmax
changes in individual lesions were measured in animals from the control and 2-month treatment groups, and all volumes were plotted along with SUVmax
over time for each animal (). In the control animal whose data are shown in , these lesions were generally stable, although some lesions increased in size while maintaining a constant SUVmax
(maroon spheres). Other lesions increased slightly in SUVmax
while decreasing in size (dark blue spheres), and still others disappeared entirely (green spheres). Each lesion had a slightly different pattern of change, illustrating the dynamic and highly local nature of a TB lesion even in the chronic stage of disease (). This pattern was observed in all control animals. The lesions in the animals treated with INH became both smaller and less metabolically active, with three lesions being reduced below the detection limit after 8 weeks of treatment (); the lesions in the animals treated with RIF followed a similar pattern, although none of these lesions disappeared completely in the treatment period (). In order to assess the quantitative changes in these lesions across all animals, CT lesion volume and mean FDG uptake were multiplied to calculate the glycolytic index at each time point (3
) (). Treatment with either INH or RIF significantly reduced the lesional glycolytic index (P
= 0.015 and P
= 0.035, respectively; repeated-measures ANOVA).
Fig 6 Comparison of changes in individual lesion volume and [18F]FDG PET avidity in rabbits undergoing INH or RIF chemotherapy. The lesions from a representative animal in the vehicle control (A), the INH treatment (B), and the RIF treatment (C) groups were (more ...) Assessing quantitative measures of drug efficacy from PET-CT images.
Using manual analysis tools, the total volume of pulmonary lesions in each animal was measured from the series of CT scans collected during treatment; these totals included all abnormal findings in the lungs, including granulomas, consolidations, and obvious thickening of the airways or vessels (see Fig. S2 in the supplemental material). A repeated-measures ANOVA was used to assess the change in volume over time. The total lesion volume of the animals given vehicle was not significantly reduced from 8 to 16 weeks p.i., as expected (see Fig. S2A). Although the total volume of lesions was reduced in 6 of 8 animals receiving INH treatment (see Fig. S2B), the change for the group as a whole was not significant. This is despite reduction of the bacterial burden and reduction in the volume of lesions calculated from histological morphometry. One possible explanation for this observation was an increase in lung consolidations in one animal completing treatment (two consolidations each with a volume of at least 700 mm3 but relatively low SUVmaxs (5 to 5.7) formed late in treatment, and this tissue had low bacterial numbers (1.9 to 2.2 log10 CFU/g lung) that were not INH resistant. A second animal in the 8-week INH group experienced weight loss and symptoms consistent with INH-induced hepatic encephalopathy and had to be sacrificed prior to the final scan. The change in lesion volume in RIF-treated animals was significant for the repeated-measures ANOVA after 4 weeks (P < 0.01; CI, 111.2 to 593.1) and 8 weeks (P < 0.01; CI, 136 to 618) of therapy but not after only 1 week of treatment (see Fig. S2C).
We also analyzed three independent lesion features with respect to time receiving treatment and drug: volume, density, and corrected SUVmax (). The CT volumes of individual lesions in each rabbit scan were determined by manually tracking lesions in sequential scans during therapy. There were nonsignificant reductions in CT volume as early as 1 week posttreatment (). These became statistically significant after 4 weeks and 8 weeks of treatment, but the development of consolidations in one animal confounded the analysis at 8 weeks (). We also investigated if treatment was associated with a change in density of the lesions due to reduced bacterial burden resulting in more fibrosis or calcification. One rabbit treated with RIF ( and in ) did have lesions that increased in density as treatment continued, but the majority of animals had lesions that decreased in density during treatment. Although the changes were not significant at 1 week (), the reduction in lesion density was highly significant and correlated with treatment response after 4 and 8 weeks of treatment. Finally we looked at changes in [18F]FDG PET avidity for individual lesions as measured by SUVmax. After 1 week of therapy, SUVmax corrected values were significantly decreased in both the INH and RIF therapy groups () (for INH, P = 0.003; CI, 0.9456 to 4.035; for RIF, P = 0.007; CI, 0.862 to 4.596). FGD uptake of most lesions decreased with increasing treatment time for both INH (P < 0.0001 by one-way ANOVA and P < 0.0001 for linear trend, negative slope) and RIF (P < 0.0001 by one-way ANOVA and P < 0.0001 for linear trend, negative slope) as shown in 7E. In animals not receiving treatment, the changes in [18F]FDG PET avidity and lesion volume over time were not significant, but lesion density (P = 0.02) increased over time and had a positive linear slope.
Fig 7 Assessment of lesion volume, lesion density, and lesion SUVmax as biomarkers for response to treatment. Individual small nodules from serial scans for each rabbit in the drug treatment groups were analyzed by either a paired t test (2 scans) or a repeated-measures (more ...)
To see if there was a direct relationship between SUVmax and bacterial burden in individual lesions, we isolated lesions at necropsy and plated them for assessment of bacterial burden. The number of lesions available for this analysis was limited, as many lesions, especially in the treated animals, were no longer visible by CT and showed only background SUV. The data for small (1- to 4-mm-diameter) nodules presented in Fig. S3 in the supplemental material are suggestive but not conclusive that a positive linear relationship exists between SUV and bacterial burden in untreated animals.