Name: analysis of 18fdg pet/ct imaging as a tool for studying mycobacterium tuberculosis infection and treatment in non-human primates
Uploaded: 2017-09-05T15:00:00
Description: Watch this Scientific Journal Video about Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates at JoVE.com

The authors wish to acknowledge Mark Rodgers for outlining the infection procedures and L. Eoin Carney and Brian Lopresti for guidance in establishing these imaging procedures. Funding for this work has been provided by The Bill and Melinda Gates Foundation (J.L.F., P.L.L.), National Institutes of Health, National Institutes of Allergy and Infectious Diseases R01 AI111871 (P.L.L.), National Heart Lung and Blood Institute R01 HL106804 (J.L.F.), R01 HL110811.

All methods outlined in this work have been approved by the University of Pittsburgh Institutional Animal Care and Use Committee. All procedures followed institutional biosafety and radiation safety requirements. CT scanning requires donning lead apron and throat cover. Biosafety Level 3 (BSL3) garb and procedures for working with non-human primates must be followed according to institutional guidelines. All scanning was performed in a BSL3 facility.
1. Animal Infection Procedure
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	<li>Barry, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+latent+tuberculosis:+rethinking+the+biology+and+intervention+strategies.">The spectrum of latent tuberculosis: rethinking the biology and intervention strategies.</a> Nat Rev Microbiol. 7 (12), 845-855 (2009).</li><li>Lin, P. L., Flynn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+latent+tuberculosis:+a+moving+target.">Understanding latent tuberculosis: a moving target.</a> J Immunol. 185 (1), 15-22 (2010).</li><li>Pawlowski, A., Jansson, M., Skold, M., Rottenberg, M. E., Kallenius, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis+and+HIV+co-infection.">Tuberculosis and HIV co-infection.</a> PLoS Pathog. 8 (2), e1002464 (2012).</li><li>Keane, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-blocking+agents+and+tuberculosis:+new+drugs+illuminate+an+old+topic.">TNF-blocking agents and tuberculosis: new drugs illuminate an old topic.</a> Rheumatology (Oxford). 44 (6), 714-720 (2005).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+CT+Identifies+Reactivation+Risk+in+Cynomolgus+Macaques+with+Latent+M.+tuberculosis.">PET CT Identifies Reactivation Risk in Cynomolgus Macaques with Latent M. tuberculosis.</a> PLoS Pathog. 12 (7), e1005739 (2016).</li><li>Flynn, J. L., Klein, E., Dick, T., Leong, V. D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A color atlas of comparative pulmonary tuberculosis histopathology. , 83-106 (2011).</li><li>Signore, A., Mather, S. J., Piaggio, G., Malviya, G., Dierckx, R. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Changes+by+(18)Fluorodeoxyglucose+positron+emission+tomography+coregistered+with+computed+tomography+predict+outcome+after+Mycobacterium+tuberculosis+infection+in+cynomolgus+macaques.">Early Changes by (18)Fluorodeoxyglucose positron emission tomography coregistered with computed tomography predict outcome after Mycobacterium tuberculosis infection in cynomolgus macaques.</a> Infect Immun. 82 (6), 2400-2404 (2014).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiologic+Responses+in+Cynomolgus+Macaques+for+Assessing+Tuberculosis+Chemotherapy+Regimens.">Radiologic Responses in Cynomolgus Macaques for Assessing Tuberculosis Chemotherapy Regimens.</a> Antimicrob Agents Chemother. 57 (9), 4237-4244 (2013).</li><li>Diedrich, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+latent+tuberculosis+in+cynomolgus+macaques+infected+with+SIV+is+associated+with+early+peripheral+T+cell+depletion+and+not+virus+load.">Reactivation of latent tuberculosis in cynomolgus macaques infected with SIV is associated with early peripheral T cell depletion and not virus load.</a> PLoS One. 5 (3), e9611 (2010).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multistage+vaccine+H56+boosts+the+effects+of+BCG+to+protect+cynomolgus+macaques+against+active+tuberculosis+and+reactivation+of+latent+Mycobacterium+tuberculosis+infection.">The multistage vaccine H56 boosts the effects of BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection.</a> J Clin Invest. 122 (1), 303-314 (2012).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+cell+depletion+exacerbates+acute+Mycobacterium+tuberculosis+while+reactivation+of+latent+infection+is+dependent+on+severity+of+tissue+depletion+in+cynomolgus+macaques.">CD4 T cell depletion exacerbates acute Mycobacterium tuberculosis while reactivation of latent infection is dependent on severity of tissue depletion in cynomolgus macaques.</a> AIDS Res Hum Retroviruses. 28 (12), 1693-1702 (2012).</li><li>Mattila, J. T., Diedrich, C. R., Lin, P. L., Phuah, J., Flynn, J. 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J., Zumla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tuberculosis. , 243-258 (2015).</li><li>Kita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+therapeutic+and+prophylactic+vaccine+against+Tuberculosis+using+monkey+and+transgenic+mice+models.">Development of therapeutic and prophylactic vaccine against Tuberculosis using monkey and transgenic mice models.</a> Hum Vaccin. 7, 108-114 (2011).</li><li>Langermans, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+effect+of+bacillus+Calmette-Guerin+(BCG)+vaccination+on+Mycobacterium+tuberculosis+infection+in+highly+related+macaque+species:+implications+for+primate+models+in+tuberculosis+vaccine+research.">Divergent effect of bacillus Calmette-Guerin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: implications for primate models in tuberculosis vaccine research.</a> Proc Natl Acad Sci U S A. 98 (20), 11497-11502 (2001).</li><li>Okada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+prophylactic+and+therapeutic+vaccine+against+tuberculosis.">Novel prophylactic and therapeutic vaccine against tuberculosis.</a> Vaccine. 27 (25-26), 3267-3270 (2009).</li><li>Reed, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+tuberculosis+vaccine,+Mtb72F/AS02A,+evidence+of+protection+in+cynomolgus+monkeys.">Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys.</a> Proc Natl Acad Sci U S A. 106 (7), 2301-2306 (2009).</li><li>Capuano, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Mycobacterium+tuberculosis+infection+of+cynomolgus+macaques+closely+resembles+the+various+manifestations+of+human+M.+tuberculosis+infection.">Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection.</a> Infect Immun. 71 (10), 5831-5844 (2003).</li><li>Srinivas, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recovery+coefficient+method+for+partial+volume+correction+of+PET+images.">A recovery coefficient method for partial volume correction of PET images.</a> Ann Nucl Med. 23 (4), 341-348 (2009).</li><li>Kumar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+modern+imaging+techniques+for+diagnosis+of+infection+in+the+era+of+18F-fluorodeoxyglucose+positron+emission+tomography.">Role of modern imaging techniques for diagnosis of infection in the era of 18F-fluorodeoxyglucose positron emission tomography.</a> Clin Microbiol Rev. 21 (1), 209-224 (2008).</li><li>Martin, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitally+Barcoding+Mycobacterium+tuberculosis+Reveals+In+Vivo+Infection+Dynamics+in+the+Macaque+Model+of+Tuberculosis.">Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis.</a> MBio. 8 (3), (2017).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metronidazole+prevents+reactivation+of+latent+Mycobacterium+tuberculosis+infection+in+macaques.">Metronidazole prevents reactivation of latent Mycobacterium tuberculosis infection in macaques.</a> Proc Natl Acad Sci U S A. 109 (35), 14188-14193 (2012).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+neutralization+results+in+disseminated+disease+in+acute+and+latent+Mycobacterium+tuberculosis+infection+with+normal+granuloma+structure+in+a+cynomolgus+macaque+model.">Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model.</a> Arthritis Rheum. 62 (2), 340-350 (2010).</li><li>Phuah, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+B+Cell+Depletion+on+Early+Mycobacterium+tuberculosis+Infection+in+Cynomolgus+Macaques.">Effects of B Cell Depletion on Early Mycobacterium tuberculosis Infection in Cynomolgus Macaques.</a> Infect Immun. 84 (5), 1301-1311 (2016).</li><li>Martinez, V., Castilla-Lievre, M. 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Data acquired from PET/CT can be used as surrogate measurements for many aspects of M. tuberculosis infection that would be unobservable without such technology. PET/CT is much more sensitive than X-ray technology, which is often used in macaques studies. PET/CT provides structural, spatial and functional information. The analyses described above have many practical applications such as monitoring disease progression, assessing effectiveness of drug treatment, and providing risk factors for reactivation<sup clas...

Mycobacterium tuberculosis has plagued humans for millennia and causes more mortality than any other single infectious agent in the world today. In 2015, there were 10.5 million reported new cases of tuberculosis (TB) globally1 with the majority of cases emanating from India, Indonesia, China, Nigeria, Pakistan, and South Africa. Estimates place the global death toll from TB at 1.4 million people during that same time period. This value is nearly 25% lower than the death rate 100 years ago. Although drug sensitive TB is treatable, the regimen is lengthy requiring multiple medications and compliance is a concern. The emergence of multi drug-resistant (MDR) strains accounted for ~580,000 of the new TB cases in 2015. The successful treatment rate of patients with MDR strains of M. tuberculosis is only estimated to be around 50%. Even more alarming is the emergence of extensively resistant (XDR) strains of M. tuberculosis, which are resistant to nearly every available drug. Thus, new techniques are needed within the TB research field that enhance the ability to diagnose TB, increase the immunological understanding of the disease process, and allow for the screening of novel treatments and prevention strategies including antibiotic regimens and vaccine efficacy studies.
M. tuberculosis is an aerobic acid-fast bacillus that is physically characterized by its very complex outer cell wall and slow growth kinetics. Infection generally occurs through inhalation of individual bacteria contained in aerosolized droplets that are expelled from a symptomatic, infected individual while coughing, sneezing, or singing. Of the exposed individuals that develop infection, only 5 - 10% of people develop active clinical TB. The remaining 90% have a varying spectrum of asymptomatic infections that ranges from subclinical infection to no disease at all, all of which is classified clinically as latent TB infection (LTBI)2,3. Of the population that has this asymptomatic infection, approximately 10% will develop active TB by reactivation of the contained infection in their lifetime. The risk of reactivation dramatically increases if a person with asymptomatic infection contracts HIV or undergoes treatment with an immunosuppressive drug, such as TNF inhibitors4,5,6. Active TB disease also presents as a spectrum, with most people having pulmonary TB, which affects the lungs and thoracic lymph nodes. However, M. tuberculosis can infect any organ, so that the infection can also present in extrapulmonary sites of involvement.
The pathologic hallmark of M. tuberculosis infection is an organized spherical structure of host cells, called the granuloma. Macrophages, T cells and B cells are major components of the granuloma, with variable numbers of neutrophils7. The center of the granuloma is often necrotic. Thus, granulomas function as an immune microenvironment to kill or contain the bacilli, preventing spread to other parts of the lungs. However, M. tuberculosis can subvert killing by the granuloma, and persist within these structures for decades. Consistent and regular monitoring for the development of active TB disease after new infection or reactivation of LTBI is impractical, scientifically challenging and time consuming. Techniques that study these processes longitudinally, in humans and human-like animal models, are extremely useful to the scientific community in furthering the understanding of the many complexities of M. tuberculosis infection and disease.
PET/CT is an extremely useful imaging technique that has been employed to study a vast range of disease states in humans and animal models8. PET is a functional technique that uses positron-emitting radioactive compounds as a reporter. These radioisotopes are typically functionalized to a metabolic compound, such as glucose, or to a targeting group that is designed to bind to a receptor of interest. Since the radiation emitted from PET isotopes is powerful enough to penetrate tissue, very low concentrations can be used which allows for study below saturation levels in receptor-targeting compounds and at a low enough concentration to have no impact on metabolic processes when using agents such as 2-deoxy-2-(18F)Fluoro-D-glucose (FDG). CT is a three-dimensional x-ray imaging technique that uses varying levels of x-ray attenuation to identify physical characteristics of organs within the body9. When paired with PET, CT is used as a map to determine specific locations and structures that show uptake of a PET radiotracer. PET/CT is a powerful tool for in vivo imaging of both humans and animal models infected with M. tuberculosis infection that has led to many important insights into pathogenesis, response to drug treatment, disease spectrum, etc6,10,11,12. This work describes specific PET/CT analytical methods to study TB in non-human primate models longitudinally using parameters such as granuloma size, FDG uptake in individual lesions, whole lung and lymph node FDG avidity, and detection of extrapulmonary disease6,10,11,12.
This manuscript describes methodologies of imaging analysis in non-human primates (NHPs), specifically cynomolgus macaques, which are used to longitudinally evaluate disease progression and drug treatment following infection with M. tuberculosis. NHPs are a valuable animal model because when inoculated with a low dose of M. tuberculosis Erdman strain, animals show a variety of disease outcomes with ~50% developing active TB and the remaining animals having asymptomatic infection (i.e. controlling the infection, LTBI), providing the closest model to the clinical disease spectrum seen in humans3,13,14,15,16. Reactivation of LTBI in macaques is triggered by the same agents that cause reactivation in humans, examples of which include human immunodeficiency virus (HIV, using simian immunodeficiency virus (SIV) as the macaque version of HIV), CD4 depletion or tumor necrosis factor (TNF) neutralization13,16. In addition, macaques present with pathology that is extremely similar to that seen in humans, including the organized granulomas that form in lungs or other organs17. Thus, this model has provided important insights into the basic host-pathogen interactions in M. tuberculosis infection, as well as valuable knowledge about drug regimens and vaccines for tuberculosis14,18,19,20,21.
PET/CT imaging provides the ability to follow the appearance, distribution, and progression of individual granulomas. This work has primarily used FDG as a probe, which, as a glucose analogue, incorporates into metabolically active host cells, such as macrophages, neutrophils, and lymphocytes8, all of which are in granulomas. Thus, FDG is a proxy for host inflammation. The analysis procedures detailed herein uses OsiriX, a widely used DICOM viewer available for purchase and use. The image analysis methods described track the shape, size, and metabolic activity (via FDG uptake) of individual granulomas over time and uses imaging as a map for identifying specific lesions upon animal necropsy. Additionally, a separate method has been developed that quantifies the summation of FDG uptake in the lung above a specific threshold (SUV &#8805; 2.3) and uses this value to evaluate differences between control and experimental groups across studies ranging from vaccine trials to co-infection models. These data support that this overall measure of FDG uptake in lungs is correlated with bacterial burden, thus providing information about the disease status. Similar analyses can be performed on the FDG uptake of thoracic lymph nodes to study progression of disease as well. The following protocol describes the experimental process from animal infection through image analysis.

<table>
	<tbody>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>23061</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td>4866</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Cetacaine</td>
			<td>Patterson Vet Generics</td>
			<td>07-892-6862</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>07-800-9721</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>7H11 agar</td>
			<td>BD</td>
			<td>283810</td>
			<td>BD Biosciences</td>
		</tr>
		<tr>
			<td>IV catheter</td>
			<td>Surflash</td>
			<td>07-806-7659</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>18F-FDG</td>
			<td>Zevacor</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Jorgensen Labs Inc</td>
			<td>07-887-0284</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Artificial tears</td>
			<td>Patterson Vet Generics</td>
			<td>07-888-1663</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>07-806-3204</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Neurologica Ceretom CT</td>
			<td>Samsung Neurologica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Siemens Focus 220 microPET</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inveon Research Software</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>OsiriX</td>
			<td>Pixmeo</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of 18fdg pet/ct imaging as a tool for studying mycobacterium tuberculosis infection and treatment in non-human primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Identification and Analysis of Individual Lesions
Individual granulomas can be visualized for number, size, and FDG uptake qualitatively to understand the general scope of the infection process (Figure 1). Using these images, counting granulomas over time is a quantitative measure of disease spread. Figure 2 depicts individual granuloma counts over time in a gr...

Watch this Scientific Journal Video about Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates at JoVE.com

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

The overall goal of this positron emission tomography computer tomography or PET/CT image analysis procedure, is to study the disease process and potential treatment methods of Mycobacterium tuberculosis infections in non-human primates. This method can help answer key questions in the study of tuberculosis, including infection location over time, the reactivation from latency and the outcome of specific treatment regiments. The main advantage of this technique is that Mycobacterium tuberculosis infection and treatment can be tracked in real time without the need for periodic animal necropsies.Begin by exporting the co-registered PET/CT images to the appropriate software and opening the images from the software database in the axel orientation. Click on the CT scan and change the window level, window width to CT pulmonary. The lung tissue will become dark and the anatomical features will appear lighter with the airways in black and the vasculature in white.Focusing on one lobe at a time, scroll through the fused PET/CT image to identify the lesions. Small lesions can be distinguished from the vessels by hovering the cursor over the structure in question and scrolling up and down a slice or two. If the structure remains under the cursor, it is a lesion.To measure a lesion of interest, remove the PET signal, so that only the CT is visible and select the length tool. Scroll until the slice that contains the largest portion of lesion is visible and draw a long line across the longest length of the lesion. The data included in this region of interest will represent the length in millimeters of the diameter of the lesion.In the window level, window width and color lookup table menu, select set window level, window width manually in the window level, window width drop down menu and enter zero for from, and 20 for to, in the dialog box. Select the oval tool from the mouse button function tool drop down menu and scroll over the lesion to assess the hardest portion of the tissue. Then draw an oval around the lesion to obtain descriptive statistics for all of the specific threshold levels within the region in voxels.To measure the total lung FDG avidity, click anywhere on the CT scan. Under the region of interest drop down menu, select grown region 2D 3D segmentation and set the lower threshold to 1024 and the upper threshold to 200. When the thresholds have been set, click anywhere inside the lung to highlight the entire slice in green.In the segmentation parameters dialog box, click compute to expand the grown region from one slide to the entire lung volume. Next, click on the small icon to the left of the name of the CT scan and drag the icon to the PET scan. Then, select copy regions of interest to overlay the lungs onto the PET scan.To fill in the high density areas of the lung that appear as gaps on the PET scan. Under the region of interest drop down menu, select brush regions of interest and closing. In the pop up window, slide the arrow to three so that the top of the box reads, structuring element radius 3 and check apply to all regions of interest with the same name.Close any remaining gaps manually as necessary. When the entire lung is visible on the PET scan, delete all of the pixels outside of the lung, open the region of interest menu and select, set pixel values to, click the outside region of interest checkbox and set all of the pixels outside of the region of interest to zero. To isolate the hot"pathology, select set pixel values to again and click the inside regions of interest checkbox, clicking the end box, so that all of the values between zero and 2.3, are set to zero.To make sure that only the disease pathology is measured, under the region of interest menu, select delete all regions of interest in this series and grow region 2D 3D segmentation. Change the lower threshold to 2.3 and the upper threshold to 100 and scroll through the entire PET window, clicking on any disease pathology and compute. After repeating for every area of heart disease, open the regions of interest menu and select save regions of interest, to save the whole lung region of interest.Then export the raw data values into a spreadsheet. To determine the FDG uptake in the hot"lymph nodes, highlight the PET/CT fusion image, open the color lookup table drop down box in the toolbar and select the UCLA lookup table setting. To draw a manual region of interest around the lymph node of interest, click the drop down menu on the right side of the mouse button function in the main toolbar and select closed polygon.Click on the edge of the lymph node to establish the first point of the region of interest, continuing to click and trace the tissue segment until the entire lymph node is almost enclosed. Then double click to close the region of interest and record the specific threshold values in a separate spreadsheet. To normalize the disease pathology FDG data values, click on the co-registered PET and CT image and scroll through the image, until the slice containing the meeting point of the main bronchial tube is reached.Use the oval tool to draw regions of interest of approximately the same size on the back muscle and the muscle posterior and lateral to the spinal column, to obtain background specific threshold values. Track the icon to the left of the co-registered PET/CT scan to the PET window and select, copy regions of interest. The regions of interest should appear in the PET scan window.Under the main menu, select the mode check box and make sure that max intensity projection is selected in the dropdown menu. Then confirm that the thick slab sliding scale is set to 10 and record the mean specific threshold values of the two muscle regions of interest in a new spreadsheet. Individual granulomas can be visualized for number, size and FDG uptake qualitatively, to understand the general scope of the infection process.Counting granulomas from the images over time, allows a quantitative measure of the disease spread. For example, of the 10 animals in this representative experiment, three developed active disease and six developed latent infection. From six weeks after infection and thereafter, animals that would later develop active disease, demonstrated statistically higher numbers of granulomas than the animals that would develop latent infection.In all of the active infection animals, an increase in FDG uptake was measured in every granuloma from three to six weeks post infection. While the animals that went on to latency, exhibited a variation in FDG uptake. With some lesions increasing, decreasing or demonstrating the same uptake over the same period.While the FDG uptake in the mediastinal lymph nodes is similar between active and latent animals at three weeks. The lymph nodes from active animals exhibit a significantly higher uptake at six, eight and 12 weeks. Indeed, animals with a higher total lung FDG avidity are overall more likely to reactivate with over 90%of the animals demonstrating a more than 1 x 10 to the three lung FDG avidity or with at least one scan visible extrapulmonary site of infection reactivating after tumor necrosis vector neutralization in this experiment.Once mastered, this technique can be completed in one to three hours per animal, depending on disease severity, if it is properly performed. While attempting this procedure, it is important to pay attention to the details and always save your regions of interest. Following this procedure, other assessments such as, determining the immunologic and bacteria logic parameters can be performed to answer additional questions about the relationship of the size and SUV of granulomas to infection outcome.After its development, this technique paved the way for researchers in the field of tuberculosis to evaluate specific treatments and vaccines in a variety of non-human primate species. After watching this video, you should have a good understanding of how to qualitatively and quantitatively evaluate PET/CT scans from M tuberculosis infected non-human primates. Don't forget, that working with Mycobacterium tuberculosis can be extremely hazardous and precautions such as, wearing the appropriate bio-safety level 3 PPE and following approved protocols should always be taken while performing imaging on infected non-human primates.

Research

JoVE Journal

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...