Name: diagnosing pulmonary tuberculosis with the xpert mtb/rif test
Uploaded: 2012-04-09T12:00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test at JoVE.com

The authors acknowledge W. J. Looney for carefully reviewing the manuscript.

Standard 2 of the International Standards for Tuberculosis Care states that all patients suspected of having pulmonary TB should submit at least two sputum specimens for bacteriological examination. When possible, at least one early-morning specimen should be obtained, as sputum collected at this time has the highest yield.11 The Xpert MTB/RIF assay can be applied to sputum samples or concentrated sputum sediments prepared from induced or expectorated sputa that are either acid-fast bacilli (AFB) smear positive or negative.6, 12, 13 The assay is intended for use with specimens from patients for whom there is clinical suspicion of pulmonary TB who have: 1) not received anti-tuberculosis therapy, 2) had &lt; 7 days of therapy, or 3) have not received therapy in the last 60 days. Do note that it should not be used for monitoring the effects of drug therapy7 because bacterial DNA might persist following antimicrobial therapy.
Treat clinical specimens, including used cartridges, as if capable of transmitting infectious agents. Use proper precautions, such as wearing protective disposable gloves, laboratory coats and eye protection, and follow your institution's safety procedures and guidelines.
1. Expectorated Sputum Samples
Note: Process only as many specimens at one time, as there are GeneXpert Dx System modules available to run the test!
<ol>
 <li>Label each Xpert MTB/RIF cartridge with the corresponding specimen ID.</li>
 <li>Transfer 1.0 ml expectorated sputum to a conical, screw-capped tube using a sterile transfer pipette. Alternatively, the entire specimen may be processed in the original leak-proof sputum collection container.</li>
 <li>Add 2.0 ml Xpert MTB/RIF Sample Reagent (2:1; v/v) to the expectorated sputum using a sterile transfer pipette.</li>
 <li>Replace the lid, and shake the tube vigorously 10-20 times. Alternatively, the mixture may be vortexed (30 sec).</li>
 <li>Allow the tube to stand upright for 5 min at room temperature.</li>
 <li>Shake the tube vigorously 10-20 times. Alternatively, use a vortex (30 sec).</li>
 <li>Allow the tube to stand upright for another 10 min at room temperature.</li>
 <li>Inspect the specimens: samples should be liquefied with no visible clumps of sputum.</li>
</ol>
2. Concentrated Sediments
Note: Process only as many specimens at one time as there are GeneXpert Dx System modules available to run the tests!
<ol>
 <li>Label each Xpert MTB/RIF cartridge with the corresponding specimen ID.</li>
 <li>Transfer 1.5 ml Xpert MTB/RIF Sample Reagent to a conical, screw-capped tube using a sterile transfer pipette.</li>
 <li>Add at least 0.5 ml concentrated sediment to the Sample Reagent containing tube using a sterile transfer pipette.</li>
 <li>Replace the lid, and shake the tube vigorously 10-20 times. Alternatively, use a vortex (30 sec).</li>
 <li>Allow the tube to stand upright for 5 min at room temperature.</li>
 <li>Shake the tube vigorously 10-20 times. Alternatively, use a vortex (30 sec).</li>
 <li>Allow the tube to stand upright for another 10 min at room temperature.</li>
 <li>Inspect the specimens: samples should be liquefied with no visible clumps of sputum.</li>
</ol>
3. Loading the Xpert MTB/RIF Cartridge
Note: Start the test within 30 minutes of adding the sample to the cartridge!
<ol>
 <li>Check the labels on the Xpert MTB/RIF cartridge and the specimen ID.</li>
 <li>Open the cartridge lid.</li>
 <li>Using the sterile transfer pipette provided, aspirate the liquefied specimen into the transfer pipette until the meniscus is above the minimum mark and transfer the sample into the open port of the Xpert MTB/RIF cartridge.</li>
</ol>
Note: Dispense slowly to minimize the risk of aerosol formation!
<ol start="4">
 <li>Close the cartridge lid.</li>
</ol>
4. Starting the Test
Note: Before starting the test, ensure that the GeneXpert Dx System is equipped with the GX2.1 software, and the Xpert MTB/RIF assay is imported into the software! 9
<ol>
 <li>Turn on the computer, and then turn on the GeneXpert Dx instrument.</li>
 <li>On the Windows desktop, double-click the GeneXpert Dx shortcut icon.</li>
 <li>Log on to the GeneXpert Dx System software using your user name and password.</li>
 <li>In the GeneXpert Dx System window, click Create Test. The Scan Cartridge Barcode dialog box appears.</li>
 <li>In the Sample ID box, scan or type the sample ID.</li>
 <li>Scan the barcode on the Xpert MTB/RIF cartridge. The Create Test window appears.</li>
 <li>Click Start Test, and enter your password in the dialog box that appears.</li>
 <li>Open the instrument module door with the blinking green light, and load the cartridge.</li>
 <li>Close the door. The test starts and the light stops blinking and remains constantly green. When the test is finished, the light turns off.</li>
 <li>Wait until the system releases the door lock at the end of the run, then open the module door and remove the cartridge.</li>
 <li>Dispose of used cartridges in the appropriate waste containers according to your institution's standard practices.</li>
</ol>
5. Representative Results
Each Xpert MTB/RIF cartridge includes reagents for the detection of MTB complex and RIF resistance as well as a sample processing control (SPC) to control for adequate processing of the target bacteria and to monitor the presence of inhibitor(s) in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.9 The primers in the Xpert MTB/RIF assay amplify a portion of the rpoB gene containing the 81 base pair "core" region. Five differently colored fluorogenic nucleic acid hybridization probes, called molecular beacons, interrogate the entire 81-bp core.14 Each molecular beacon was designed to be so specific that it does not bind to its target if the target sequence differs from the wild-type rpoB sequence by as little as a single nucleotide substitution. Since molecular beacons fluorescence only when they are bound to their targets, i.e. wild-type rpoB sequence, the absence of any one of the five colors in the assay differentiate between the conserved wild-type sequence and mutations in the core region that are associated with RIF resistance.6
The SPC should be positive in a negative sample and can be negative or positive in a positive sample. The test result will be "Invalid" if the SPC is not detected in a negative test. Before the start of the PCR reaction, the GeneXpert Dx System measures the fluorescence signal from the probes to monitor bead rehydration, reaction-tube filling, probe integrity and dye stability. The PCC passes if the fluorescence signal from the probes meets the assigned acceptance criteria. The results are interpreted by the GeneXpert Dx System from measured fluorescence signals and embedded calculation algorithms and are displayed in the View Results window9, as indicated below:
MTB Detected: MTB target DNA is detected; both controls, SPC and PCC, meet the assigned acceptance criteria. Lower Ct values represent a higher starting concentration of DNA template; higher Ct values represent a lower concentration of DNA template. In MTB DETECTED results "RIF Resistance DETECTED" (Figure 1A), "RIF Resistance NOT DETECTED" (Figure 1B), or "RIF Resistance INDETERMINATE" will display on a separate line.
MTB Not Detected: MTB target DNA is not detected; both controls, SPC and PCC, meet the assigned acceptance criteria (Figure 2).
Invalid: Presence or absence of MTB cannot be determined: SPC does not meet acceptance criteria, i.e. the sample was not properly processed, or PCR was inhibited (Figure 3). Note: repeat test with extra specimen!
Error: One or more of the PCC results failed (FAIL). Both MTB and SPC display NO RESULT. Note: repeat test with extra specimen! If the PCC passed (PASS), the error is caused by a system component failure.
Figure 1: MTB target DNA is detected; both controls, SPC and PCC, meet the assigned acceptance criteria. Lower Ct values represent a higher starting concentration of DNA template; higher Ct values represent a lower concentration of DNA template. In MTB DETECTED results "RIF Resistance NOT DETECTED" (Figure 1A), "RIF Resistance DETECTED" (Figure 1B), or "RIF Resistance INDETERMINATE" will display on a separate line.
<img alt="Figure 1A" src="/files/ftp_upload/3547/3547fig1A.jpg" /> 
Figure 1A. MTB DETECTED; RIF Resistance NOT DETECTED, i.e. only the wild-type nucleic acid sequence of the 81-bp RIF resistance-determining region (RRDR) of the rpoB gene was present.6 <a href="https://www.jove.com/files/ftp_upload/3547/3547fig1A.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 1B" src="/files/ftp_upload/3547/3547fig1B.jpg" /> 
Figure 1B. MTB DETECTED; RIF Resistance DETECTED. Complete drop out of Probe B (arrow), which covers the positions 513 to 517 of the RRDR of the rpoB gene.6 In the example, partial sequencing revealed that the rpoB gene mutation D516V was detected.8 <a href="https://www.jove.com/files/ftp_upload/3547/3547fig1Blarge.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3547/3547fig2.jpg" /> 
Figure 2. MTB target DNA was not detected; both controls, SPC and PCC, meet the assigned acceptance criteria. <a href="https://www.jove.com/files/ftp_upload/3547/3547fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/3547/3547fig3.jpg" /> 
Figure 3. Invalid test result, i.e. presence or absence of MTB cannot be determined: SPC does not meet acceptance criteria, because the sample was not properly processed, or because PCR was inhibited. <a href="https://www.jove.com/files/ftp_upload/3547/3547fig3large.jpg" target="_blank">Click here to view larger figure</a>.

<ol>
	<li>Sudre, P., Dam, t. e. n., Chan, G., C, A., Kochi, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis+in+the+present+time:+a+global+overview+of+the+tuberculosis+situation.">Tuberculosis in the present time: a global overview of the tuberculosis situation.</a> World Health Organisation Document WHO/TUB/91. 158, 1-47 (1991).</li><li>World Health Organisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+tuberculosis+control:+epidemiology,+strategy,+financing.">Global tuberculosis control: epidemiology, strategy, financing.</a> WHO report 2010. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The global plan to stop TB, 2006-2015. , (2006).</li><li>Morris, S., 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=Molecular+mechanisms+of+multiple+drug+resistance+in+clinical+isolates+of+Mycobacterium+tuberculosis.">Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis.</a> J. Infect. Dis. 171, 954-960 (1995).</li><li>Traore, H., Fissette, K., Bastian, I., Devleeschouwer, M., Portaels, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampicin+resistance+in+Mycobacterium+tuberculosis+isolates+from+diverse+countries+by+a+commercial+line+probe+assay+as+aninitial+indicator+of+multidrug+resistance.">Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as aninitial indicator of multidrug resistance.</a> Int. J. Tuberc. Lung Dis. 4, 481-484 (2000).</li><li>Helb, D., 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=Rapid+Detection+of+Mycobacterium+tuberculosis+and+Rifampin+Resistance+by+Use+of+On-Demand+Near-Patient+Technology.">Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand Near-Patient Technology.</a> J. Clin. Microbiol. 48, 229-237 (2010).</li><li>Blakemore, 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=Evaluation+of+the+Analytical+Performance+of+the+Xpert+MTB/RIF.">Evaluation of the Analytical Performance of the Xpert MTB/RIF.</a> Assay. J. Clin. Microbiol. 48, 2495-2501 (2010).</li><li>Telenti, 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=Detection+of+rifampicin-resistance+in+Mycobacterium+tuberculosis.">Detection of rifampicin-resistance in Mycobacterium tuberculosis.</a> Lancet. 341, 647-650 (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GeneXpert Dx System Operator Manual. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Standards for Tuberculosis Care (ISTC). , (2009).</li><li>Boehme, C. C., 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=Rapid+Molecular+Detection+of+Tuberculosis+and+Rifampin+Resistance.">Rapid Molecular Detection of Tuberculosis and Rifampin Resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li><li>Marlowe, E. 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=Evaluation+of+the+Cepheid+Xpert+MTB/RIF+assay+for+the+Direct+Detection+of+Mycobacterium+tuberculosis+Complex+from+Respiratory+Specimens.">Evaluation of the Cepheid Xpert MTB/RIF assay for the Direct Detection of Mycobacterium tuberculosis Complex from Respiratory Specimens.</a> J. Clin. Microbiol. 49, 1621-1623 (2011).</li><li>El-Hajj, H. H., Marras, S. A. M., Tyagi, S., Kramer, F. R., Alland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampin+resistance+in+Mycobacterium+tuberculosis+in+a+single+tube+with+molecular+beacons.">Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons.</a> J. Clin. Microbiol. 39, 4131-4137 (2001).</li><li>Hillemann, D., Ruesch-Gerdes, S., Boehme, C., Richter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+extrapulmonary+tuberculosis+by+automated+GeneXpert+MTB/RIF+system.">Rapid molecular detection of extrapulmonary tuberculosis by automated GeneXpert MTB/RIF system.</a> J. Clin. Microbiol. 49, 1202-1205 (2011).</li></ol>

No conflicts of interest declared.

The Xpert MTB/RIF assay integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of MTB and RIF resistance, either in expectorated sputum or concentrated sediments, in a single, easy to use system. The assay's analytical limit of detection was found to be 131 CFU/ml of sputum.6 The log-linear relationship between Ct values and the number of MTB cells present holds true from 102 to 107 CFU/ml of sputum. As clinically relevant MTB concentrations all fall within the linear range of the assay, the assay provides semi-quantitative estimates of the bacterial load.7 The assay requires only moderate laboratory infrastructure and training. The limit of detection lies at least two orders of magnitude lower than that of conventional microscopy.6 Specificity is excellent. Furthermore, the simultaneous detection of RIF-resistant MTB is a significant benefit in the setting of point-of-care testing in the era of drug-resistant TB.3
To date only limited published data on the assay's performance for diagnosing pulmonary TB in clinical settings are available.12; 13 The Xpert MTB/RIF assay's overall clinical sensitivity of detecting MTB in sputum of patients with suspected pulmonary TB was 97.6% when compared to culture as the reference. These were the findings of a prospective multi-center study that involved five study sites (Lima, Peru; Baku, Azerbaijan; Cape Town &amp; Durban, South Africa; Mumbai, India) and a total of 1462 patients. The overall pre-test probability of culture-confirmed TB in the study population was 50.7%. The sensitivity was 99.8% for smear- and culture-positive cases and 90.2% for smear-negative, culture-positive cases. Among HIV-positive patients with pulmonary TB, the sensitivity of the MTB/ RIF test was 93.9%, as compared with 98.4% in HIV-negative patients.12 In a setting of low TB prevalence, the overall agreement for detecting MTB was 89% when compared to culture; the sensitivity was 98% for smear-positive and 72% for smear-negative specimens, respectively, and the specificity was excellent.13 There was no significant difference in sensitivity between tests on untreated sputum and those on concentrated sediments.12 In the same study, the MTB/RIF test correctly detected RIF resistance in 209 of 211 patients (99.1% sensitivity) and in all 506 patients with RIF susceptibility (100% specificity), when sequencing results were taken into account.12 The authors found that performance for both case detection, and discrimination of RIF resistance, was similar across diverse sites, suggesting that the findings are likely to be widely applicable.12 Furthermore, concentrated sediments of respiratory specimens other than sputum, such as broncho-alveolar lavage and bronchial secretions, were tested with comparable sensitivity and specificity.13 Data on the assay's performance when testing extra-pulmonary TB is emerging.15
Global TB control urgently needs effective tools that permit rapid diagnosis of new TB cases and detection of RIF-resistant MTB in universal-access point-of-care settings.3 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to these settings, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions for the Xpert MTB/RIF test. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Xpert MTB/RIF test</td>
			<td>Cepheid</td>
			<td>GXMTB/RIF-10</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sarstedt</td>
			<td>86.1172.001</td>
		</tr>
		<tr>
			<td>Conical tubes, 15 ml, 120 x 17 mm, PS</td>
			<td>Sarstedt</td>
			<td>62.553.542</td>
		</tr>
	</tbody>
</table>

diagnosing pulmonary tuberculosis with the xpert mtb/rif test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

Watch this Scientific Journal Video about Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test at JoVE.com

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

diagnosing-pulmonary-tuberculosis-with-the-xpert-mtbrif-test

Research

JoVE Journal

Immunology and Infection

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

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

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

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 respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

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

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

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

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

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 ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

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 fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

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

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...