Name: spatial quantification of drugs in pulmonary tuberculosis lesions by laser capture microdissection liquid chromatography mass spectrometry (lcm-lc/ms)
Uploaded: 2018-04-18T14:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS at JoVE.c

We thank Paul O&#39;Brien, Marizel Mina and Isabella Freedman for animal experiments, Jacquie Gonzalez and Danielle Weiner from NIH/NIAID for help with gamma irradiation of rabbit tissues prior to laser capture microdissection and Jansy Sarathy for manuscript thoughts and advice. This work was supported by funding from the Bill and Melinda Gates Foundation (OPP1174780) and NIH shared instrumentation grant 1S10OD018072. We thank Eliseo A. Eugenin for providing access to the Leica LMD 6500 microscope and sharing expertise and advice. The purchase, and ongoing support of, the LMD 6500 was funded by The National Institute of Mental Health grant, MH096625, the National Institute of Neurological Disorders and Stroke, NS105584, PHRI funding (to E.A.E) and GSK contributions (to E.A.E).

All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with approval from the Institutional Animal Care and Use Committee of the NIAID (NIH), Bethesda, MD.
1. Animal Experiments and Tissue Collection
This section of the protocol describes animal procedures and sample collection under Biosafety Level 3 (BSL3) conditions. Detailed protocols of the Mycobacterium tuberculosis aerosol infection procedure and drug administration protocols in rabbits have been described previously11,12.
<ol>
	<li>Infect New Zealand White rabbits (male and female at 4 - 5 months old) with M. tuberculosis HN878 using a nose-only aerosol system, as previously described11.</li>
	<li>Administer the chosen drugs (Ethambutol in the example presented here) via the preferred route and euthanize the animals at 2, 6, and 24 h following administration. First, anesthetize the rabbit by intramuscular injection of Ketamine at 35 mg/kg and Xylazine at 5 mg/kg. Wait for ten minutes and confirm proper anesthetization by pinching the tail and gently touching the eye. If there is no reaction, euthanize by intravenous administration of pentobarbital and phenytoin (see Table of Materials) at 1 mL/4.5 kg in 2 mL sterile saline. 
	NOTE: These timepoints are optimal to cover the pharmacokinetic profile for Ethambutol and may require adjusting/optimizing for other study drugs.</li>
	<li>Using forceps, scissors, and/or scalpel, remove the lungs from the chest cavity, resect lung biopsies containing large necrotic granulomas embedded in surrounding uninvolved lung tissue (as described previously3). Necrotic granulomas appear beige in color and typically protrude slightly from the surrounding red/pink-colored lung. To facilitate easy cryosectioning, ensure that biopsies are no larger than 2 x 1.5 x 1.5 cm.</li>
	<li>Using forceps, place the biopsy onto a pre-labeled cryomold tray with the desired cutting surface in direct contact with the base of the tray. After freezing, this will provide a flat surface from which cryosections will be cut.</li>
	<li>Freeze the biopsy in liquid nitrogen vapor. Fill a styrofoam container to a depth of 2 inches with liquid nitrogen and place a metal wire tube rack. The rack should protrude above the surface of the liquid nitrogen providing a flat surface on which the tissue trays are placed. Place the lid back on the styrofoam container and leave tissues for 10 minutes to fully freeze.</li>
	<li>Remove the tissue trays, quickly wrap in aluminium film and place in individually labeled resealable plastic bags and seal. Transfer to -80 &#176;C freezer for storage. 
	NOTE: Steps 1.1 - 1.6 are performed in BSL3 conditions (including all animal work and handling of infected organs and tissues). Gamma irradiate the lung biopsies at 3 Megarads to enable handling outside of BSL3 containment. Laser capture microdissection on unsterilized tissue may be performed within the BLS3 facility if approved safety protocols are in place. However, the remainder of this protocol describes downstream processing in a BSL-2 facility.</li>
</ol>
2. Tissue Sectioning
<ol>
	<li>Set the cryostat to the desired cutting temperature. Transfer the gamma-irradiated lung biopsy from -80 &#176;C storage to the cryostat and leave for 30 minutes to equilibrate the tissue temperature. Note: -20 to -22 &#176;C is optimal for TB lesion biopsies.</li>
	<li>Using tweezers, fix the biopsy to the cryostat chuck using a small amount of optimal cutting temperature adhesive (OCT) to adhere the base of the tissue to the chuck. Orient the tissue so that the flat surface (that was in contact with the base of the cryomold) is the exposed surface for cutting. Ensure the OCT does not contaminate the tissue surface, as this may interfere with the subsequent mass spectrometry analysis.</li>
	<li>Cut three tissue sections at 25 &#181;m thickness and mount onto PET membrane slides. Gently touch the membrane to the tissue section and remove. If too much pressure is applied, the thin membrane may tear.
	<ol>
		<li>Avoid excessive handling of the slides prior to mounting as this will result in the PET membrane becoming charged and poor adhesion of the tissue sections. Ensure that the membrane slide is kept at room temperature to enable thaw-mounting and successful adhesion of the tissue to the membrane.</li>
	</ol>
	</li>
	<li>Remove the slide from the cryostat and allow to air-dry for 3 minutes. If LCM-LC/MS/MS will not be performed immediately, seal the slide in a small airtight sealable bag and transfer to -80 &#176;C storage until required for dissection.</li>
	<li>Cut an adjacent section at 10 - 12 &#181;m and thaw-mount on a standard glass slide for Hematoxylin and Eosin (H&#38;E) staining and reference. Additional sections can be cut at this time for other desired histochemistry stains (such as Ziehl-Niellsen for visualizing Mycobacterium tuberculosis (MTB)).</li>
</ol>
3. Microdissection
<ol>
	<li>Remove sealed bag containing the slide from -80 &#176;C storage and allow to reach room temperature for 5 minutes. 
	NOTE: If the cold slide is immediately exposed to the laboratory atmosphere, the tissue will become coated with condensation, and the spatial integrity of the drug may be compromised.</li>
	<li>Turn on the microscope and laser (laser requires 5 - 10 minutes warm up before cutting can commence). Load flat-cap 0.20 mL PCR tubes into the holder.</li>
	<li>Remove slide from the bag and take an optical image of the tissue section on the PET slide using a flatbed scanner.</li>
	<li>Place the slide into the slide holder (tissue side facing down) and assign separate collection tubes to specific granuloma regions of interest using the microscope software. Typically, these will be &#39;uninvolved lung,&#39;&#160;&#39;cellular granuloma,&#39; and &#39;caseum&#39; (necrotic center), but may vary depending upon the specific pathology of the granuloma/biopsy.</li>
	<li>Focus on the tissue using the 5X microscope objective. This magnification should provide a good overview of the tissue containing both cellular and necrotic granuloma areas. In the software select the tube designated &#39;caseum&#39; to move it into position under the tissue.</li>
	<li>Enter the desired dissection parameters. Typical settings for a 25 &#181;m thick lung section are laser power 30, speed 15, and aperture 35 (arbitrary units). However, these will differ depending upon the microscope used and potential declining power due to the age of the laser.</li>
	<li>Select the &#39;free-draw&#39; tool and, using either a mouse or touchscreen pen, outline the desired region for dissection. The surface area of the region will be displayed in the software. Keep selected regions under 500,000 &#181;m2 (0.5 mm2) to facilitate easier dissection. Repeat the dissection until 3 million &#181;m2 (3 mm2) have been collected in total in the tube cap.
	<ol>
		<li>On occasion, the dissected region may remain stuck to the surrounding membrane (for example due to static attraction) and not fall into the collection cap. Remove these regions from the cumulative surface area total by selecting and removing manually within the software.</li>
	</ol>
	</li>
	<li>Select the cap for &#39;cellular lesion&#39; and collect 3 million &#181;m2 of tissue using the same process as outlined in step 3.7.</li>
	<li>Select the cap for &#39;uninvolved lung&#39; and collect 3 million &#181;m2 of tissue using the same process as outlined in step 3.7. Note that uninvolved lung tissue contains many bronchioles and alveolar spaces. Pay careful attention to exclude these from the defined tissue regions for dissection.</li>
	<li>Remove the cap holder and carefully unclip, seal and label each tube. Protect the dissected tissues from surrounding air disturbances (such as from air flow disruption from an opening door). Analyze the dissected tissues immediately, or store at -80 &#176;C and thaw prior to processing and LCMS analysis.</li>
</ol>
4. Extraction and LCMS Analysis
<ol>
	<li>Prepare extraction solution of 1:1 acetonitrile/methanol containing Ethambutol d-10 internal standard. When selecting an internal standard, use a stable labeled form of the analyte drug (such as deuterium-labeled EMB used in this demonstration) with sufficient mass shift to avoid isotope cross talk between the analyte drug and standard (usually a minimum of 4 daltons). 
	NOTE: Creating standards in homogenate of each respective tissue type is difficult because there is very limited control tissue with which to create homogenate standards. As an alternative to making standards from a spiked homogenate sample, a standard can be created by adding blank tissue and test compound together and extracting. A volume of control tissue in a homogenate that matches the target volume of the study sample tissue sections is combined directly with an amount of the test compound that would be present at a given concentration.</li>
	<li>Calculate the targeted tissue volume based on the surface area and thickness of the tissue section and determine the necessary dilution factor for the homogenate using the volume of homogenate that will be added to the standard and QC samples. Calculations are illustrated below for a 3 million &#181;m2 (3 mm2) target dissected area with a 25 &#181;m thickness and 2 &#181;L volume of homogenate. 
	<img alt="Equation 1" src="/files/ftp_upload/57402/57402eq1.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/57402/57402eq2.jpg" /> 
	<img alt="Equation 3" src="/files/ftp_upload/57402/57402eq3.jpg" /> 
	<img alt="Equation 4" src="/files/ftp_upload/57402/57402eq4.jpg" /></li>
	<li>Assuming a tissue density of 1 g/mL, prepare the homogenate stock by weighing 50 mg of control tissue and adding PBS buffer to dilute (using the 26.67 homogenate dilution factor calculated in step 4.2, the diluent is 1.283 mL). Homogenize by bead beating lung tissue and PBS buffer for 5 minutes at 1750 rpm on a bead homogenizer. 
	<img alt="Equation 5" src="/files/ftp_upload/57402/57402eq5.jpg" /> 
	<img alt="Equation 6" src="/files/ftp_upload/57402/57402eq6.jpg" /></li>
	<li>Dilute 1 mg/mL drug stocks concentration in 1:1 acetonitrile/water to create standard curve spiking solutions. Determine spiking standard concentrations based on the spike volume and the target tissue volume. The illustrated example is for a 100 ng/mL standard using a 10 &#181;L spike volume. 
	<img alt="Equation 7" src="/files/ftp_upload/57402/57402eq7.jpg" /> 
	<img alt="Equation 8" src="/files/ftp_upload/57402/57402eq8.jpg" /></li>
	<li>Remove the tubes containing the microdissected tissues from -80 &#176;C storage and allow to reach room temperature for 5 minutes.</li>
	<li>Add 10 &#181;L of 1:1 acetonitrile/water solution and 2 &#181;L of PBS buffer to the tubes containing microdissected tissue.</li>
	<li>For standard curve and quality control tubes, add 10 &#181;L of spiking solution to 2 &#181;L of control lung homogenate.</li>
	<li>Add 50 &#181;L of extraction solution to each tube.</li>
	<li>Vortex each tube for 5 minutes, sonicate for 5 minutes and centrifuge at 5000 RPM for 5 minutes to form a pellet of film and tissue in each tube.</li>
	<li>Transfer 50 &#181;L of supernatant to a 96-well deep-well plate and dilute with an additional 50 &#181;L of deionized water in each well.</li>
	<li>Perform LC/MS/MS analysis using optimized instrument parameters for Ethambutol and Ethambutol-d10 internal standard (as previously described in detail12).</li>
	<li>Use a dilution factor to correct for the exact amount of tissue dissected for each sample. 
	<img alt="Equation 9" src="/files/ftp_upload/57402/57402eq9.jpg" /></li>
</ol>
5. Method Validation
<ol>
	<li>Create a homogenate in control lung tissue by combining 1 part lung, 2 parts PBS, and 3 - 4 steel beads. Beat lung tissue and PBS buffer for 5 minutes at 1750 rpm using a bead homogenizer.</li>
	<li>Spike the homogenate by adding 10 &#181;L of 1 mg/mL Ethambutol DMSO stock into 990 &#181;L homogenate to create a final concentration of 10,000 ng/mL (10 mg/mL) and vortex for 1 minute.</li>
	<li>Create a frozen homogenate block by pouring the homogenate into a cryomold and rapidly freezing on dry ice for 5 minutes.</li>
	<li>Prepare 25 &#181;m thick sections from the homogenate block as described in steps 2.1 - 2.5.</li>
	<li>Dissect the target tissue area as specified in steps 3.2 - 3.10.</li>
	<li>Add 10 &#181;L 1:1 acetonitrile/water and 2 &#181;L of PBS buffer to the tubes containing microdissected tissue.</li>
	<li>Add 50 &#181;L of extraction solution to each tube. Follow steps 4.9 - 4.12 to create a standard curve and determine the drug concentration in the tissue homogenate block.</li>
	<li>Calculate extraction efficiency using the formula below: 
	<img alt="Equation 10" src="/files/ftp_upload/57402/57402eq10.jpg" /></li>
</ol>

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The authors have nothing to disclose.

Spatially-resolved quantification of drugs within pulmonary TB lesions is required to determine whether drug exposure reaches sterilizing concentrations to bacterial populations residing within the different lesion compartments. The LCM-LC/MS method described here enables absolute quantification of anti-TB drugs within all lesion compartments, including the bacteria-rich caseum, using only 1 - 3 tissue sections in total. Traditional tissue homogenization and LC/MS approaches for drug quantification in tissue often lack the spatial specificity to resolve specific lesion compartments and even when it is possible, there is significant potential to cross contaminate cellular and necrotic lesion areas during the manual extraction process.
The LCM-LC/MS approach has several key advantages to mass spectrometry-based imaging technologies. Primary among these are the fully quantitative capabilities of the method and the additional sensitivity of the LC/MS analysis due to resolving drug from interfering/suppressing endogenous species via chromatographic separation. However, MALDI-MSI provides more detailed spatial information regarding drug distribution. As both MALDI-MSI and LCM-LC/MS/MS require the same sample preparation steps to generate frozen tissue sections, the two techniques can be performed in tandem on the same tissue biopsy providing full quantification alongside highly-detailed imaging of drug distribution. An example of the requirement for higher analytical sensitivity is shown in Figure 3. Only very low levels of EMB signal were detected in the central necrotic granuloma region of the MALDI MS image (shown in Figure 3B), suggesting caseum penetration of the drug is poor. However, due to the superior limit of detection of LCM-LC/MS the drug was clearly demonstrated to reach sterilizing concentrations within that compartment and the drug concentration present was predominantly undetectable by MALDI (Figure 3A).
Tissue staining protocols are typically used prior to LCM for proteomic applications to enable easy identification of tissue areas and the cell populations located within. Routine histochemical stains such as H&#38;E are not compatible with drug quantification, as the many solvent washing steps involved would delocalize the drug from the tissue section. Adjacent cut sections can be stained with H&#38;E and used as a guide for microdissection. However, this is not usually necessary, as the lesion compartments can be optically resolved under the standard LCM brightfield microscope (Figure 2).
Optimization of the sectioning and microdissection steps is crucial for successful lesion quantification. During development of the method, we evaluated two different membrane materials, polyethylene terephthalate (PET) and polyethylene naphthalate (PEN), as substrate for mounting the tissue sections for microdissection. PEN membranes suffered from increased static charging in comparison to PET membranes and approximately 30% of all dissected tissue areas were lost due to static attraction between the dissected tissue region and the membrane. For this reason, PET membranes were chosen for future dissections due to the significantly reduced static attraction observed (only 5% of regions dissected from PET membranes were lost during the LCM process).
The stability of drugs within tissue sections is an important consideration when designing an LCM-LC/MS study. During the sectioning and dissection process, the tissue sections are exposed to lab temperature and light for a period of at least 1 hour. Other issues to consider include the extraction efficiency of the drug from the tissue sections, as there is no homogenization step involved (extraction is only performed by vortex mixing and sonication). In our experience, the extraction does not suffer from the lack of tissue homogenization due to the high extraction solvent to tissue ratio used and the thinness of the tissue sections dissected. Our observations are in agreement with a previously-described online LCM-LC/MS method developed to quantify propranolol in microdissected tissue sections of brain and liver, where complete extraction of drug from tissue was observed from 20 &#181;m and 40 &#181;m thick sections10. We validated the efficiency of drug extraction and the drug stability during the LCM process by directly comparing lung tissue concentrations (from the same rabbit and lung lobe) analyzed by LCM-LC/MS with concentrations determined by standard tissue homogenization and LC/MS (an example is shown in Table 1). This comparison allows us to determine whether any change of signal is occurring between the two methodologies.
The density of dissected tissue regions is also an important consideration when quantifying drug levels based upon the tissue surface area or volume. This is of particular concern for lung and lesions biopsies, in which the uninvolved lung tissue areas have an overall lower density than cellular and caseum (less tissue covering the same relative surface area) due to the presence of multiple open bronchioles and alveolar spaces. The effects of this difference in density can be mitigated by carefully drawing around the open spaces to avoid including them within the cumulative surface area of collected tissue (as shown in Figure 2).
An additional limitation is that the presented LCM-LC/MS method only quantifies total drug concentration within the isolated tissue (in common with all tissue homogenization, solvent extraction, and LC/MS quantification approaches) and does not resolve protein-bound drug concentrations from unbound fractions. Microdialysis is an alternative approach for quantifying unbound drug within tissue and enables accurate quantification of free drug concentrations reaching extracellular populations of bacteria13. However, the technique would be best applied as a complementary approach, as it lacks the spatial specificity of LCM-LC/MS and only quantifies soluble drug levels within tissue extracellular fluid, not the intracellular content.
We have combined, for the first time, LCM and LC/MS to spatially quantify antibiotics at the site of tuberculosis infection. The methodology is tremendously powerful since it can be applied to any small molecule drug used in any disease state. Indeed, we have recently quantified an antifungal drug candidate in a mouse model of abdominal candidiasis14. Differential drug partitioning into heterogeneous tumor compartments (including necrotic cores) is a primary concern in the treatment of cancer, and a critical area of research in cancer drug discovery15. LCM-LC/MS is ideally suited to approach these questions. Furthermore, LCM-LC/MS can be used for biomarker discovery to quantify metabolic, lipidomic and proteomic changes occurring in tissue regions and cell populations during disease pathogenesis.

The ability to spatially resolve and quantify drug levels is a crucial requirement for determining whether anti-tuberculosis drugs reach bacterial subpopulations within pulmonary lesions at sterilizing concentrations1. Of particular importance is determining drug penetration into the necrotic core of the lesion (called caseum), which typically contains the highest number of bacilli and may be poorly accessible to drugs due to the absence of vascularization.
Traditional methods to assess lesion penetration, which involve homogenization of excised pulmonary lesions followed by solvent extraction and liquid chromatography mass spectrometry (LC/MS) analysis, are highly sensitive and selective for the drugs of interest. However, these methods offer poor spatial information, limited to the size of the original homogenized tissue. Mass spectrometry-based imaging approaches, such as matrix-assisted laser desorption ionization (MALDI)2,3, desorption electrospray ionization (DESI)4 or liquid-enhanced surface extraction5,6 offer highly spatially-resolved imaging capabilities, but direct quantification can be extremely challenging or impossible due to heterogeneous ion suppression effects and differing extraction efficiencies of analyte from the various cell or tissue types7. Additionally, most direct tissue MS imaging approaches are inherently less sensitive than LC/MS due to the lack of chromatographic separation of endogenous species competing for ionization and the lower solvent extraction efficiency of the drug from tissue.
Laser capture microdissection (LCM) combined with LC/MS analysis has been routinely applied to isolate and characterize distinct tissue regions for proteomic studies8,9 and recently utilized for drug quantification in dosed animal tissue10. Here we present an optimized protocol applying LCM combined with LC/MS (LCM-LC/MS) analysis to quantify anti-TB drugs within distinct granuloma compartments. In the laser capture microdissection process, a UV laser is focused through the microscope objective onto the tissue section, which cuts and isolates the desired tissue area by following a path defined by the user. For gravity-assisted LCM (the technique used for this research), the tissue section is mounted onto a thin polymer membrane slide (PET or PEN) and the tissue is captured in a collection tube cap sited below the slide. The drugs are extracted from the excised tissue and quantified using standard LC/MS approaches. The amount of tissue required to be collected is ultimately determined from the expected concentration of the drug present in the tissue and the sensitivity of the LC/MS method. For most analyses of drugs dosed at therapeutic levels and analyzed using a routine triple quadrupole mass spectrometer, 3 million &#181;m2 (3 mm2) of tissue surface area is sufficient.
This protocol describes the powerful combination of spatial profiling and full quantification offered by LCM-LC/MS, providing absolute drug concentrations within all compartments of TB granulomas. The technique may also be applied to determining drug concentrations in many different diseased tissues providing vital drug discovery and development information.

<table><tbody><tr><td>New Zealand White rabbits</td><td>Covance</td><td>N/A</td><td></td></tr><tr><td>HN878 Mycobacterium tuberculosis</td><td>BEI Resources</td><td>NR-13647</td><td></td></tr><tr><td>Ketathesia (Ketamine) 100 mg/mL C3N</td><td>Henry Schein Animal Health</td><td>56344</td><td></td></tr><tr><td>Anased (Xylazine) 100 mg/mL</td><td>Henry Schein Animal Health</td><td>33198</td><td></td></tr><tr><td>Euthasol (pentobarbital sodium and phenytoin sodium) Solution</td><td>Virbac</td><td>710101</td><td></td></tr><tr><td>Acetonitrile (LC-MS grade)</td><td>Fisher</td><td>A955-212</td><td></td></tr><tr><td>Methanol (LC-MS grade)</td><td>Fisher</td><td>A456-212</td><td></td></tr><tr><td>Formic Acid (LC-MS grade)</td><td>Fisher</td><td>A117-50</td><td></td></tr><tr><td>Water (LC-MS grade)</td><td>Fisher</td><td>W6212</td><td></td></tr><tr><td>0.2 mL flat-cap PCR tubes</td><td>Corning</td><td>07-200-392</td><td></td></tr><tr><td>Steel frames, PET-membrane</td><td>Leica</td><td>11505151</td><td></td></tr><tr><td>Premium Frosted Microscope Slides</td><td>Fisher</td><td>12-544-2</td><td></td></tr><tr><td>96 Deep well plate 2.0ML PP RB</td><td>Fisher</td><td>NC0363259</td><td></td></tr><tr><td>Zorbax SB-C8 column (4.6 by 50 mm; particle size, 3.5 &amp;mu;m)</td><td>Agilent</td><td>820631-001D</td><td></td></tr><tr><td>&quot;Zipper&amp;rdquo; Seal Sample Bags</td><td>Fisher</td><td>01-816-1B</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CM1850 cryostat</td><td>Leica</td><td>Discontinued</td><td>Leica CM1860 is the current model</td></tr><tr><td>Laser Microdissection System 6500</td><td>Leica</td><td>Discontinued</td><td>Leica LMD 6 is the current model</td></tr><tr><td>Agilent 1260 Infinity II HPLC</td><td>Agilent</td><td></td><td></td></tr><tr><td>API 4000 QTRAP Mass Spectrometer</td><td>Sciex</td><td></td><td></td></tr></tbody></table>

spatial quantification of drugs in pulmonary tuberculosis lesions by laser capture microdissection liquid chromatography mass spectrometry (lcm-lc/ms)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

An overview of the LCM-LC/MS approach is shown in Figure 1. After sterilizing the tissue by gamma-irradiation, all subsequent steps (from tissue sectioning onwards) take place outside of BSL3 conditions. Figure 2 shows the lesion biopsy sections before and after tissue isolation by LCM. Necrotic and cellular areas of TB lesions can be easily identified and isolated by visual inspection of optical images alone (without the requirement to refer to histologically-stained adjacent tissue sections). The dissection process produces a clean cut with minimal disturbance to the surrounding tissue, and dissecting 3 million &#181;m2 (3 mm2) of each region of interest from the lesions takes approximately 1 hour in total.
The extraction efficiency and stability of the LCM-LC/MS method was assessed using Ethambutol (EMB)-spiked lung homogenate (Table 1). Complete extraction of the drug was observed, and no drug stability issues were detected through the dissection and extraction process. The LCM-LC/MS extraction and quantification protocol was further validated by comparing to established LC/MS quantification methods applied to the same tissues. Due to the inherent heterogeneity of necrotic pulmonary TB lesions and the poor spatial specificity offered by standard tissue homogenization, we validated the LCM-LC/MS method by directly comparing drug concentrations in uninvolved lung analyzed by both analytical techniques (due to its relatively high tissue homogeneity). Table 2 shows the drug concentrations in uninvolved lung from biopsies taken from three Ethambutol-dosed rabbits as evaluated by: 1) homogenizing 25 &#181;m thick tissue sections and analyzing by standard LC/MS, and 2) LCM-LC/MS analysis of uninvolved lung areas taken from an adjacent 25 &#181;m thick tissue section. The data shows that two approaches produce consistent quantification data, demonstrating the suitability for routine spatial quantification.
We have applied LCM-LC/MS to spatially-quantify many existing and novel anti-TB drugs within pulmonary lesions. Figure 3A shows example data from MTB-infected rabbits dosed steady-state with Ethambutol. LCM-LC/MS enabled full quantification of the drug resolved within caseum, cellular lesion, and uninvolved lung tissue areas. EMB was observed to penetrate well into the lesion and reach sterilizing concentrations within the necrotic caseum. The corresponding MALDI-MS image of EMB distribution acquired from an adjacent tissue section is shown in Figure 3B. The qualitative MALDI-MS images correlates well with the quantitative LCM-LC/MS data with lower drug concentrations detected in the necrotic caseum in comparison to the cellular rim. LCM-LC/MS data was validated by direct comparison to tissue homogenates analyzed by standard LC/MS.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57402/57402fig1.jpg" /> 
Figure 1: Schematic of the LCM-LC/MS process. Rabbit lung biopsies containing necrotic lesion along with surrounding uninvolved lung are collected and frozen. Cryosections are cut onto thin PET membranes and areas of uninvolved lung, cellular, and caseous lesion are dissected and isolated for quantification by LC/MS. Adapted from Zimmerman et al.12 <a href="https://cloudflare.jove.com/files/ftp_upload/57402/57402fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57402/57402fig2.jpg" /> 
Figure 2: Lesion (A,B) and uninvolved lung (C,D) as they appear when viewed using the microscope before (A,C) and after (B,D) microdissection. Caseous lesion areas appear darker and &#39;cracked&#39; in the optical image scan (A, green outline). Cellular lesion is lighter in color and more solid in structure (A, red outline). Uninvolved lung should be sampled at least 5 mm away from the lesion border and appears red/pink in color (C, cyan outline). Scale bars (black, blue, and purple) = 400 &#181;m. Note that only solid areas of uninvolved lung should be selected to avoid including alveolar spaces and bronchioles in the total surface area of tissue collected (as shown in in D). <a href="https://cloudflare.jove.com/files/ftp_upload/57402/57402fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57402/57402fig3.jpg" /> 
Figure 3: Example LCM-LC/MS dataset from lesion biopsies taken from two rabbits dosed with 100 mg/kg EMB for 7 days. (A) Favorable penetration of the drug into all lung and lesion compartments was observed. Drug concentrations quantified by LCM-LC/MS (hollow bars)/MS were in strong agreement with those quantified from homogenized dissected lesions by standard LC/MS (solid bars, mean &#177; standard deviation, n = 3 - 8). The minimum concentrations required to kill 99% of extracellular replicating bacilli (MBC99) and 99% of intracellular bacilli in macrophages (iMBC99) are indicated. (B) Top panel: MALDI-MS image showing the distribution of EMB [M+H]+ ion (m/z 205.193) within an adjacent tissue section. Note that the MALDI-MS image suggests poor penetration of EMB into the caseum due to the present drug concentrations being below the lower limit of detection (LOD) of the technique. However, the spatial specificity and superior LOD of LCM-LC/MS show the drug is reaching sterilizing concentrations within all lesion compartments including the caseum. Lower panel: Hematoxylin &#38; Eosin stained tissue section directly adjacent to the section used for MALDI MSI. Cellular (C) and necrotic granulomas (NG) were present in the tissue. The caseum core is outlined in white. Adapted from Zimmerman et al.12 <a href="https://cloudflare.jove.com/files/ftp_upload/57402/57402fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dissected&#160; area (&#181;m2)</td>
			<td>Measured EMB conc. (ng/g) (n = 3)</td>
			<td>EMB recovery (%) (n = 3)</td>
		</tr>
		<tr>
			<td>3 million</td>
			<td>10633 (&#177;404)</td>
			<td>106 (&#177;4)</td>
		</tr>
		<tr>
			<td>5 million</td>
			<td>10057 (&#177;1132)</td>
			<td>101 (&#177;11)</td>
		</tr>
		<tr>
			<td>10 million</td>
			<td>10563 (&#177;1128)</td>
			<td>105 (&#177;11)</td>
		</tr>
	</tbody>
</table>
Table 1: Extraction efficiency of the LC-LC/MS method for quantifying EMB in lung. Complete recovery of EMB was observed in the EMB spiked lung homogenate dissected tissues for all evaluated tissue volumes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rabbit ID</td>
			<td>LC/MS EMB (ng/g)</td>
			<td>LCM-LC/MS EMB (ng/g)</td>
			<td>&#160;Difference (%)</td>
		</tr>
		<tr>
			<td>899</td>
			<td>2910</td>
			<td>3320</td>
			<td>14</td>
		</tr>
		<tr>
			<td>904</td>
			<td>2010</td>
			<td>1870</td>
			<td>-7</td>
		</tr>
		<tr>
			<td>911</td>
			<td>2150</td>
			<td>2370</td>
			<td>9</td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of EMB quantification in uninvolved lung biopsies from 3 dosed rabbits by LC/MS and LCM-LC/MS. Equivalent drug concentrations were detected by both methods and no loss of signal due to degradation or extraction during the LCM-LC/MS process was observed.

Watch this Scientific Journal Video about Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS at JoVE.c

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

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9.0K Views.  Rutgers, The State University of New Jersey. Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Video: Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

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) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

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

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

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

Immunology and Infection

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Biochemistry

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 common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

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

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis 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

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 understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

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

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

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 ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

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.

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.

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

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Chemical Cartography Approaches to Study Trypanosomatid Infection

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. Human-infective trypanosomatid parasites include Trypanosoma cruzi, the causative agent of Chagas disease; Trypanosoma brucei, the&#160;causative agent of sleeping sickness; and Leishmania species, causative agents of leishmaniasis. Jointly, they affect 20 million people across the globe. These parasites show specific tropism: heart, esophagus, colon for T. cruzi, adipose tissue, pancreas, skin, circulatory system and central nervous system for T. brucei, skin for dermotropic Leishmania strains, and liver, spleen, and bone marrow for viscerotropic Leishmania strains. A spatial perspective is therefore essential to understand trypanosomatid disease pathogenesis. Chemical cartography generates 3D visualizations of small molecule abundance generated via liquid chromatography-mass spectrometry, in comparison to microbiological and immunological parameters. This protocol demonstrates how chemical cartography can be applied to study pathogenic processes during trypanosomatid infection, beginning from systematic tissue sampling and metabolite extraction, followed by liquid chromatography-tandem mass spectrometry data acquisition, and concluding with the generation of 3D maps of metabolite distribution. This method can be used for multiple research questions, such as nutrient requirements for tissue colonization by T. cruzi, T. brucei, or Leishmania, immunometabolism at sites of infection, and the relationship between local tissue metabolic perturbation and clinical disease symptoms, leading to comprehensive insight into trypanosomatid disease pathogenesis.

This protocol describes the steps to generate a 3D model of metabolite distribution during trypanosomatid infection, including sample collection, metabolite extraction, an overview of liquid chromatography-tandem mass spectrometry data acquisition, 3D model generation, and finally, data visualization.

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

Accelerating Tuberculosis Determination Using the Micro-CFU Method

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Author Spotlight: Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

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Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis