Name: diagnosing pulmonary tuberculosis with the xpert mtb/rif test
Uploaded: 2012-04-09T12:00:00.000+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="GeneXpert MTB/RIF test result; fluorescence vs. cycles graph; diagnostic software screenshot." src="/files/ftp_upload/3547/3547fig1A.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1A"> 
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.remotexs.ntu.edu.sg/files/ftp_upload/3547/3547fig1A.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="GeneXpert Dx software interface displaying PCR test results graph for tuberculosis detection." src="/files/ftp_upload/3547/3547fig1B.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1B"> 
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.remotexs.ntu.edu.sg/files/ftp_upload/3547/3547fig1Blarge.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Gene testing interface showing PCR assay results; fluorescence vs. cycles graph, MTB not detected." src="/files/ftp_upload/3547/3547fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. MTB target DNA was not detected; both controls, SPC and PCC, meet the assigned acceptance criteria. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/3547/3547fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="GeneXpert Dx System interface, PCR analysis results, fluorescence graph, diagnostic application." src="/files/ftp_upload/3547/3547fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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.remotexs.ntu.edu.sg/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><tbody><tr><td>Xpert MTB/RIF test</td><td>Cepheid</td><td>GXMTB/RIF-10</td><td></td></tr><tr><td>Transfer pipettes</td><td>Sarstedt Ltd</td><td>86.1172.001</td><td></td></tr><tr><td>Conical tubes, 15 ml, 120 x 17 mm, PS</td><td>Sarstedt Ltd</td><td>62.553.542</td><td></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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

80.4K Views.  University of Bern. De Xpert MTB / RIF-test integreert monster ontsmetting, hands-free bediening, on-board steekproef verwerking, en ultra-gevoelige hemi-geneste PCR voor de simultane detectie van Mycobacterium tuberculosis En rifampicine weerstand, zowel in opgehoest sputum of geconcentreerde sputum sedimenten, in ongeveer twee uur. Testen is gestandaardiseerd en vereist slechts een matige laboratorium infrastructuur en opleiding.

Video: Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections.  Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains.  The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB.  The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB.  In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

Biology

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

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

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

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

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

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

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

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

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 ...

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 ...

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 ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Medicine

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 ...

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

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