Name: a tuberculosis molecular bacterial load assay (tb-mbla)
Uploaded: 2020-04-30T13:00:00.000+00:00
Duration: 10 min 41 s
Description: Watch this Scientific Journal Video about A Tuberculosis Molecular Bacterial Load Assay TB-MBLA at JoVE.com

The study was made possible by funding from the European and Developing Countries Clinical Trials Partnership (EDCTP) &#8211; Pan African Biomarker Expansion program (PanBIOME) grant SP.2011.41304.008. Support was also obtained the University of St Andrews School of Medicine research grant. The publication of this manuscript and TB-MBLA protocol as a visual resource has been made possible by the funding from Scottish Funding Council and Global Challenges Research Fund to the University of St Andrews. Thanks to the Maputo Maternal hospital and Mavalane Health Centre which provided the clinical sputum specimens, and the Maputo Tuberculosis Treatment Unit team who helped with the heat inactivation experiments of clinical sputum specimens.

1. Sample Preparation
<ol>
	<li>Culture
	<ol>
		<li>Working on a clean bench or class 1 cabin, harvest 1 mL aliquots of exponential phase Bacillus Calmette-Gu&#233;rin (BCG) culture into 15 mL plastic centrifuge tubes. Tightly close the tubes. 
		NOTE: To process a whole 5 mL sample, five 15 mL centrifuge tubes are required. 
		CAUTION: A biosafety cabinet is required when working with any TB culture.</li>
	</ol>
	</li>
	<li>Patient sputum specimen
	<ol>
		<li>Working in a well-ventilated space and wearing a nasal mask, carefully open the specimen cup, pipette 1 mL aliquots into 15 mL plastic centrifuge tubes and tightly close the tubes. 
		NOTE: A wide mouth tip is recommended to pipette sputum. Using a pair of scissors, clip off the fine part of the 1 mL tip mouth to create a wider mouth.</li>
	</ol>
	</li>
</ol>
2. Heat Inactivation
<ol>
	<li>Prior to sample preparation, set the water bath to 95 &#176;C. 
	NOTE: The 95 &#176;C temperature increases the chance of reducing RNase activity and thus preserve more RNA for downstream TB-MBLA.</li>
	<li>Transfer the sample tubes to a holding rack immersed in the water bath. Ensure that three-fourths of each sample tube is immersed in the water.</li>
	<li>Boil at 95 &#176;C for 20 min, then transfer the tubes to the bench to cool at room temperature for 5 min before starting the RNA extraction. 
	NOTE: Complete heat inactivation of M. tuberculosis bacilli and BCG was verified by incubating heat inactivated samples and controls at 37 &#176;C for 42 days to verify growth. An optical density measurement at 600 nm (OD600) was taken at baseline and then weekly for the 42 day incubation period.</li>
</ol>
3. RNA Extraction
NOTE: The RNA extraction process described here is for the RNA kit listed in the Table of Materials. Other suitable RNA extraction kits by different manufacturers can be used.
<ol>
	<li>Extraction control (EC) addition
	<ol>
		<li>Transfer 1 mL aliquots of the heat inactivated samples to 1.5 mL tubes. Spike 100 &#181;L of the EC into each sample, close the tube, and mix by inverting the tube upside down 3x. 
		NOTE: The EC is supplied with the TB-MBLA vital bacteria kit (Table of Materials).</li>
	</ol>
	</li>
	<li>Cell sedimentation
	<ol>
		<li>Using a benchtop microcentrifuge, centrifuge the tubes at 20,000 x g for 10 min at room temperature. Pipette off the supernatant, leaving 50 &#181;L of sediment.</li>
		<li>Suspend the sediment in 950 &#181;L of lysis buffer by pipetting up and down, and transfer the whole suspension into the lysing matrix tube supplied with the RNA extraction kit (Table of Materials). Ensure the tubes are tightly closed and label both the lid and the side of the tube.</li>
	</ol>
	</li>
	<li>For cell lysis, transfer the tubes from step 3.2.2 to a homogenizer. Homogenize the samples for 40 s at 6,000 rpm.</li>
	<li>Nucleic acid purification
	<ol>
		<li>Centrifuge the lysate from step 3.3 at 12,000 x g for 5 min at room temperature.</li>
		<li>Prepare fresh 1 mL tubes and add 300 &#181;L of chloroform into each tube.</li>
		<li>Using a 1 mL tip carefully pipette off the supernatant without touching the lysing matrix.</li>
		<li>Transfer the supernatant to the chloroform containing tubes and vortex for 5 s. Leave the tube to settle for 5 min or longer until three phases (upper, middle, and bottom) are clearly visible.</li>
		<li>Centrifuge at 12,000 x g for 5 min at room temperature. Carefully pipette the upper phase and transfer into fresh 1.5 mL tubes.</li>
		<li>To the tubes in step 3.4.5, add 500 &#181;L of ice-cold 100% ethanol, close the tubes, and mix by gently inverting upside down 3x. Incubate the tubes at -80 &#176;C for 15 min or -20 &#176;C for 30 min and continue the extraction, or leave at -20 &#176;C overnight to complete the extraction the following day.</li>
		<li>Set the microcentrifuge to 4 &#176;C and leave to chill to at least 12 &#176;C before commencing centrifugation. Load the tubes into the microcentrifuge and centrifuge for 20 min at 13,000 x g. Discard the supernatant, replace with 70% ice-cold ethanol, and centrifuge for another 10 min at 13,000 x g. 
		NOTE: The 70% ethanol should be made with molecular grade nuclease free water.</li>
		<li>Discard all the supernatant from step 3.4.7 and transfer the tubes to an incubator set at 50 &#176;C. Incubate for 20 min to dry the RNA/DNA pellet. Keep the tubes partially open to enable evaporation of all ethanol.</li>
		<li>Add 100 &#181;L of nuclease free water to the dry pellet and incubate for 5 min at room temperature. Vortex for 3 s to mix the contents. 
		NOTE: At this stage the extract may be stored 2&#8722;3 days in the fridge or longer at -80 &#176;C until section 3.5 is performed.</li>
	</ol>
	</li>
	<li>DNA removal 
	NOTE: This step is crucial because the presence of DNA in the extract invalidates the MBLA result. This section is based on a DNA removal kit (Table of Materials).
	<ol>
		<li>Prepare a mix of the enzyme DNase I 10x buffer and DNase I enzyme for the number of samples (10 &#181;L of buffer and 1 &#181;L of DNase per sample) plus 10% extra to cover any loss from pipetting. Mix by vortexing and then pipette 11 &#181;L into each tube containing the RNA extract.</li>
		<li>Mix by vortexing 3 s and then spin briefly (10 s at 13,000 x g) to remove any droplets on the walls. Incubate at 37 &#176;C for 30 min in the hot block or incubator. Add an additional 1 &#956;L of DNase I enzyme directly into each tube, mix well by vortexing, and incubate for a further 30 min at 37 &#176;C.</li>
		<li>Thaw the DNase inactivation reagent 10 min prior to the end of the DNase incubation. Vortex 20 s to ensure a homogenous, milky suspension and then add 10 &#956;L of DNase inactivation reagent into each RNA extract from step 3.5.2.</li>
		<li>Incubate the mixture at room temperature for 5 min. Vortex 3x during the 5 min incubation step.</li>
		<li>Centrifuge the mixture at 13,000 x g for 2 min. Carefully transfer the supernatant to 1.5 mL RNase free tubes without touching any of the inactivation matrix.</li>
		<li>Store the RNA extract in the fridge if running the RT-qPCR on the same day or at -80 &#176;C for long-term storage.</li>
	</ol>
	</li>
</ol>
4. Reverse Transcriptase qPCR
<ol>
	<li>For unknown samples, dilute all RNA extracts to be used in a 1:10 ratio in RNase free water. Mix well by vortexing for 5 s and briefly spin down to remove any droplets or air bubbles.</li>
	<li>For standard samples for a standard curve, take the Mtb and EC RNA standards from the -80 &#176;C freezer and thaw at room temperature. Make seven and six 10-fold dilutions of Mtb and EC standard samples respectively. Change the tips before transferring the mixture from one tube to another. 
	NOTE: Standard samples are supplied with the TB-MBLA kit.</li>
	<li>Master mix preparation 
	NOTE: Master mix (MM) is a solution of PCR reagents sufficient to amplify all samples, standards, and water for a no template control (NTC). The water used as NTC should be the same water used in the extraction and for preparing the MM. Ensure that the standards, each RNA sample, and its decimal dilution are amplified 2x for the reverse transcriptase positive (RT+) reaction and 1x for the reverse transcriptase negative (RT-) reaction. The RT- reaction is a control to determine the efficiency of DNA removal (Table 1).
	<ol>
		<li>Transfer 16 &#181;L of MM into each PCR reaction tube.</li>
		<li>Add 4 &#181;L of RNA extract into each RT+ and RT- reaction tube and water into the NTC reaction tubes.</li>
		<li>Load the reaction tubes into a real time PCR machine and set the PCR conditions as follows: 50 &#176;C for 30 min, 95 &#176;C for 15 min, 40x cycles at 94 &#176;C for 45 s, and 60 &#176;C for 1 min with acquisition with fluorophores that absorb in green and yellow channels. 
		NOTE: The green channel is the Mtb detection fluorophore and the yellow is the extraction control detection fluorophore.</li>
	</ol>
	</li>
	<li>Result interpretation 
	NOTE: Ensure that the duplicate reactions of the same sample do not differ by more than 1 standard deviation. Mtb and EC Cq values higher than 30 are considered negative. See further interpretation details in Table 2.
	<ol>
		<li>To interpret the treatment response, convert the Cq values into bacterial load (eCFU/mL) using the standard curve. Read the treatment response as the change in bacterial load over the treatment follow-up period. 
		NOTE: The fall in bacterial load following treatment signifies a positive response (i.e., anti-TB drugs killing the TB bacteria) while no change or rise in bacterial load implies a negative response, which may mean resistance of TB bacteria to anti-TB drugs or the patient not appropriately adhering to their treatment dose. The fall in bacterial load measured by TB-MBLA correlates with the increase in MGIT time to culture positivity (TTP).</li>
	</ol>
	</li>
</ol>
5. Transmission Electron Microscopy
<ol>
	<li>Transfer 2 mL aliquots of heat inactivated cultures and controls into 2 mL microcentrifuge tubes. Centrifuge for 10 min at 20,000 x g.</li>
	<li>Discard the supernatant and suspend the pellet in 700 &#181;L of cell fixation buffer and incubate at room temperature for 5 min to fix the cells.</li>
	<li>Centrifuge the suspension for 30 min at 16,000 x g to obtain a hard pellet. Discard the supernatant and replace with 1% sucrose in phosphate-buffered saline (PBS). Store the pellets in sucrose at 4 &#176;C until sectioning for electron microscopy.</li>
	<li>Sectioning and TEM 
	NOTE: The protocol below is adapted from Griffiths et al.28.
	<ol>
		<li>Cryoprotect the cell pellet by embedding it in 2.1 M sucrose in PBS overnight at 4 &#176;C. Wash 3x with ice-cold water.</li>
		<li>Cool the cryoprotected cell pellets in liquid nitrogen and mount them cryomicrotomy stubs. Using the cryomicrotome, cut ultrathin sections at 90 nm.</li>
		<li>Retrieve the cut sections using the tungsten wire loops of 1:1 mixture of 2% methyl cellulose and 2.1 M sucrose in PBS.</li>
		<li>Transfer the sections to pioloform coated 150 mesh hexagonal copper TEM supports (grids) and store at 4 &#176;C or proceed to step 5.4.5.</li>
		<li>Contrast the grids in uranyl acetate and air-dry in a film of methyl acetate. Wash the grids with ice-cold water followed by two drops of PBS over 5 min and dry for 15&#8722;30 min. Examine the sections under TEM following the manufacturer&#8217;s guidelines. 
		NOTE: All labelling steps were performed at ice temperature (liquid temperature) except for the washing steps, which were performed at ambient temperature.</li>
	</ol>
	</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Mycobacterium+tuberculosis+for+DNA+typing+analysis.">Inactivation of Mycobacterium tuberculosis for DNA typing analysis.</a> Journal of Clinical Microbiology. 37, 2350-2351 (1999).</li><li>McKillip, J. L., Jaykus, L. A., Drake, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rRNA+stability+in+heat+killed+and+UV-irradiated+enterotoxigenic+Staphylococcus+aureus+and+Escherichia+coli+O157:H7.">rRNA stability in heat killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.</a> Journal of Appied Environmental Microbiology. 64, 4264-4268 (1998).</li><li>Blank, A., Dekker, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleases+of+Human+Serum,+Urine,+Cerebrospinal+Fluid,+and+Leukocytes.+Activity+Staining+following+Electrophoresis+in+Sodium+Dodecyl+Sulfate-Polyacrylamide+Gels.">Ribonucleases of Human Serum, Urine, Cerebrospinal Fluid, and Leukocytes. Activity Staining following Electrophoresis in Sodium Dodecyl Sulfate-Polyacrylamide Gels.</a> Biochemistry. 20, 2261-2267 (1981).</li><li>O'Leary, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+the+impact+of+endogenous+ribonucleases+on+reverse+transcription-PCR+assay+systems.">Reducing the impact of endogenous ribonucleases on reverse transcription-PCR assay systems.</a> Clinical Chemistry. 45, 449-450 (1999).</li><li>Yamada, H., Yamaguchi, M., Chikamatsu, K., Aono, A., Mitarai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structome+analysis+of+virulent+Mycobacterium+tuberculosis,+which+survives+with+only+700+ribosomes+per+0.1+fl+of+cytoplasm.">Structome analysis of virulent Mycobacterium tuberculosis, which survives with only 700 ribosomes per 0.1 fl of cytoplasm.</a> PLoS One. 10, 1-14 (2015).</li><li>Yang, K., 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=Structural+insights+into+species-specific+features+of+the+ribosome+from+the+human+pathogen+Mycobacterium+tuberculosis.">Structural insights into species-specific features of the ribosome from the human pathogen Mycobacterium tuberculosis.</a> Nucleic Acids Research. 45, 10884-10894 (2017).</li></ol>

The authors have nothing to disclose.

This article shows that heat treatment for 20 min at 80 &#176;C, 85 &#176;C, and 95 &#176;C inactivates tuberculosis specimens effectively, making it possible for TB-MBLA to be performed in a non-CL3 facility without risk of infection to laboratory workers. The findings confirm observations made in previous studies while contrasting with some on the effectiveness of heat inactivation of Mtb25,30. For instance, some reports indicate that heating at 80 &#176;C is not effective on high bacillary load samples25,31,32,33. The high-density inoculum effect was avoided in our study by ensuring that all sputum and pure cultures were heated at a 1 mL volume per 15 mL centrifuge tube providing adequate space to expose every part of the sample to boiling27.
RNA preservation following heat inactivation makes it possible for TB-MBLA to be performed. This finding concurs with two studies that demonstrated RNA preservation after heat inactivation12,34. We showed that the RNA in heat inactivated samples is stable at 37 &#176;C for 4 days, implying that laboratories could batch tests by maintaining inactivated samples at room temperature for a week. By applying RNA extraction kits that require refrigeration or freezing, the ability to maintain heat inactivated samples at room temperature obviates the need for both cold chain and Category 3 laboratories to perform TB-MBLA in resource limited settings.
Less than 1log bacterial load was lost using the 16S rRNA as a marker. Although there was a difference between live and heat inactivated sample, the amount lost to heat inactivation is too small to compromise downstream results. Increasing temperature did not increase the amount of RNA lost, implying that the observed loss is independent of the heat treatment. Heat treatment at high temperatures most likely causes cell lysis, exposing RNA to degradation by RNases. Indeed, exogenous addition of RNase A to the heat inactivated fraction increased the rate of RNA degradation. Boiling did not reduce the activity of RNase, implying that it is a very resilient protein.
It is important to note that an average RNA degradation of 1.5 log10&#160;eCFU/mL is not a large loss. To this end we hypothesize that heating lyses a small proportion of Mtb bacilli, thus exposing a small amount of RNA to RNase. Using TEM we showed that Mtb cell morphology and integrity of the cell walls is hardly affected by heating at 95 &#176;C. This means that it may require various physiological factors and sufficient supply of RNases such as in the host to significantly degrade RNA35,36. Furthermore, being a structural ribosome, 16S rRNA is potentially less susceptible to RNase37,38. A single Mtb cell contains ~700 ribosomes/0.1 mm3 of cytoplasm37, implying that there are higher quantities of rRNA per cell. Thus, smaller quantities of RNase may have a smaller impact38. The existence of high numbers of ribosomes gives an advantage to TB-MBLA in terms of sensitivity and ability to detect low burden TB patients.
There was a strong correlation between bacterial load measured by TB-MBLA and MGIT culture TTP. This confirms bacterial load as the driver of culture positivity to some extent. However, the advantage of TB-MBLA is that it directly quantifies bacillary load present in the sample and does not require Mtb cell proliferation before detection. This contrasts with culture whose time to positivity depends on the level of bacterial load and rate of Mtb cell proliferation. Future studies will evaluate the TB-MBLA workflow, including heat inactivation, in routine clinical settings. The study will also explore samples with a range of bacterial loads to understand the number that might change from positive to negative (i.e., those with fewer bacteria following heat inactivation).
The TB-MBLA protocol for molecular quantification of bacterial load is the first of its kind in bacteriology. The method directly quantifies Mtb bacillary load from patient sputum and requires no culture to do so. This makes it faster and increases its potential to inform a clinical decision about patient progress. The heat inactivation step reduces the risk of infection and increases applicability of TB-MBLA in settings that do not have a category 3 laboratory. Following heat inactivation of the sample, there are three protocol steps to achieve TB-MBLA results: RNA extraction, reverse transcriptase (RT)-qPCR, and qPCR results analysis.
The higher the efficiency of isolating Mtb RNA from a patient sample, the higher the quality of the results. It is important to note that the quality of the sputum sample affects the amount of RNA isolated. For instance, salivary sputum is considered low quality and has been associated with low bacillary load. This means that training the patient for quality sputum expectoration is important. To assess the efficiency of the extraction process, an extraction control (i.e, the known number of non-Mtb cells) is spiked into the sample prior to RNA extraction. Retrieval of the extraction control confirms the efficiency of the RNA extraction process. The RNA isolation process cannot be valid unless the extraction control has been retrieved. Given the fact that Mtb is a resilient organism makes mechanical lysis a crucial part of the process. Homogenization of the sample at high speed (600 rpm) in the presence of beads (i.e., lysing matrix) effectively lyses the cells. Purification of the lysate yields an extract containing both RNA and DNA. Removal of DNA is a crucial last step of the RNA extraction. TB-MBLA aims to measure viable bacilli by quantifying RNA. Thus, failure to remove genomic DNA means that the results will have a signal from the DNA, which is not a good marker for cell viability6.
The RT-qPCR is a duplex running dual labelled probes for Mtb and extraction control. It involves three steps: 30 min reverse transcription by reverse transcriptase at 50 &#176;C, 15 min denaturation at 95 &#176;C, and 40 cycles of amplification at 94 &#176;C and 60 &#176;C. Acquisition of fluorescence from the probes occurs at 60 &#176;C (i.e., the fragment elongation stage). It is important to note that the TB-MBLA has been optimized using a particular qPCR machine, so operators using other qPCR platforms should optimize the conditions for their equipment. The efficiency of DNA removal is controlled for by running a single reaction per sample in the absence of RT. A positive result from this reaction signifies incomplete removal of DNA. High burden samples that have high amounts of DNA may require double the amount of DNase enzyme to completely remove DNA. Fortunately, in high bacillary load samples, the presence of small amounts of DNA is less likely to affect the result from the RNA. Ribosomal RNA, the TB-MBLA target, naturally occurs in twice the amount of DNA37. In the PCR, a no template control (NTC), which is the water used to dissolve the PCR reagents, controls for cross contamination with exogenous DNA or RNA. A positive signal in the NTC implies cross contamination and the result considered invalid. This means all solutions constituted using this water have to be discarded and new ones made using a fresh vial of water. It is advisable to keep PCR water in separate aliquots to avoid contamination of all the water. A positive control (Mtb RNA) is used to control for the overall efficiency of PCR.
Result analysis involves the conversion of PCR Cqs into bacterial load (i.e., the estimated colony forming units per mL) using the standard curve. Setting and optimising the standard curve is crucial for this step. A standard curve efficiency of 0.95&#8211;1 is recommended. Standard curves for MTb and extraction control should be set up and optimized before patients or other test samples are run on the machine. Standard samples are provided with the TB-MBLA kit. A ten-fold dilution of the RNA extract is recommended for PCR. This implies that the bacterial load result has to be multiplied by a factor of 10 to obtain the final bacterial load result per mL. It is important to note that Cqs above 30 are considered negative for TB-MBLA. A minimum of two prospective bacterial load results measured at different time points is required to make an inference on treatment response. It is strongly recommended that one of the two results should be baseline, before initiation of treatment. However, if the patient initiated bacterial load assessment occurs midway through treatment, there must be a second time point bacterial load measurement to evaluate the treatment response. TB-MBLA can distinguish bacterial load in a space of 3 days on treatment but the ideal is two bacterial load measurements taken 7 days apart.
While the protocol generates informative quantitative results for treatment response, it is still largely manual and demands substantial hands on time for RNA extraction. Technicians in busy labs may not have this time. Arrangements are underway to automate the RNA extraction and PCR processes.

Caused by Mycobacterium tuberculosis (Mtb), over 7 x 106 new cases of tuberculosis (TB) are reported globally of which over 1 x 106 die per year1,2. To reverse the trend, the World Health Organization (WHO) launched a three-pillar approach including developing effective diagnostic and treatment tools3. Classified as a dangerous biological substance B by the UN, working with samples presumed positive for Mtb requires a containment level (CL) of 3. CL3 laboratories are expensive to build and maintain. Consequently, most countries have centralized TB culture services at regional or national levels. This means that smear microscopy is the most available diagnostic tool in the peripheral healthcare facilities.
There is WHO approval to implement rapid molecular tests like Xpert MTB/RIF at level 4 healthcare facilities, mostly situated at district levels4,5. Some districts are quite large and less accessible to some people. While beneficial, Xpert MTB/RIF works by detecting the Mtb DNA. DNA is a stable molecule that survives long after cells have died and thus is not a good standard for measuring viable cells critical for monitoring treatment response5,6. RNA-based assays offer an alternative for accurate measurement of viable cells7,8,9,10,11,12,13. RNA exists in different species of varying stability: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). Messenger RNA is associated with gene expression and thus the most closely associated with cell activity and viability14. It is important to note that absence of gene expression is not equivalent to cell death because pathogens like Mtb are known to exist in inactive (dormant) but viable states15,16. Stable RNA species such as rRNA are therefore better markers of both active and inactive states of viable cells.
Using Escherichia coli, Sheridan showed that 16S rRNA proportionally increased with bacterial growth measured by colony forming units (CFU) counts17. There was a concurrent decline in CFU counts and 16S rRNA when E. coli bacteria were exposed to antibiotics. The fall in rRNA following cell death was an indicator that it could be used as a marker for cell viability13,17. Drawing from this principle, the TB molecular bacterial load assay (TB-MBLA) was developed for targeting M. tuberculosis 16S rRNA to measure viable TB bacillary load as a marker of treatment response for patients on anti-TB therapy11,18,19. We have further developed and optimized the TB-MBLA to incorporate a cellular extraction control that reflects lysis of M. tuberculosis bacilli and is robust in different environmental settings20. The TB-MBLA procedure requires the first steps of RNA isolation from Mtb to be conducted in a CL3 laboratory until Mtb cells are completely lysed to ensure the safety of the workers. It also includes sample preservation for retrospective batched analysis to be maintained at -80 &#176;C in guanidine thiocyanate, a level 4 toxic substance. To this end, we have used heat to inactivate Mtb and render samples safe for TB-MBLA to be performed in smear microscopy level laboratories.
Use of heat in laboratory and clinical applications has been around for centuries21,22. However, some microorganisms like Mtb are tough to kill, and shorter exposure to heat is insufficient to kill all the cells23,24. A study revealed that 20 min heating of TB cultures at 80 &#176;C killed all Mtb bacilli without destroying the DNA needed for PCR25. Subsequently, a number of laboratory DNA extraction techniques currently heat to 95 &#176;C. We have applied the same principle to show that boiling TB samples at either 80 &#176;C, 85 &#176;C, or 95 &#176;C inactivates Mtb while preserving sufficient RNA for TB-MBLA to be performed. Inactivated culture or sputum can be maintained in tightly closed containers at room temperature or refrigerated for 7 days without reducing the amount of quantifiable rRNA.
The TB-MBLA, currently used as a research use only (RUO) test is adaptable and has been applied to different sample types including sputum, lung tissue, and cerebral spinal fluid. It is yet to be applied on bronchial alveolar fluid, blood, and other sample types. Using sputum as the sample, results from multisite evaluation in Africa (unpublished data) and previous publications18,26 show that the sensitivity of MBLA is consistent with mycobacterium growth indicator tube (MGIT) liquid culture. However, TB-MBLA is faster, giving results in hours as opposed to days or weeks of culture, specific, not affected by non-TB microorganisms in the sample, and gives a quantitative measure of disease severity. The WHO has recently recognized TB-MBLA as a candidate to replace smear microscopy and culture for monitoring TB treatment2.
In this article we describe in detail the heat inactivation and TB-MBLA protocol published in Sabiiti et al.27. This detailed protocol will provide a one-stop visual resource for the TB-MBLA users across the globe.

<table><tbody><tr><td>&lt;strong&gt;Heat inactivation:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 mL Centrifuge tubes</td><td>Fisher-Scientific</td><td>10136120</td><td>50 mL tubes may be used if larger sample volumes are involved</td></tr><tr><td>15 mL Wire Tube rack</td><td>Fisher-Scientific</td><td>11749128</td><td>Other brands with similar make can be used</td></tr><tr><td>Water bath</td><td>Fisher-Scientific</td><td>15700619</td><td>Other brands with similar make can be used</td></tr><tr><td>&lt;strong&gt;RNA extraction:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>372978-1L</td><td>Not necessary in other RNA extraction kit brands</td></tr><tr><td>Ethanol, absolute 99-100%</td><td>Sigma</td><td>51976-500ML-F</td><td>Larger volume available</td></tr><tr><td>Extraction control</td><td>SOI group St Andrews</td><td>VitalbacteriaEC</td><td>Supplied with the TB-MBLA Vitalbacteria kit</td></tr><tr><td>FASTRNA Pro blue Kit</td><td>MP Biomedicals</td><td>116025050</td><td>Supplied with lysing matrix tubes</td></tr><tr><td>Molecular grade water (RNase free)</td><td>Sigma</td><td>W4502-1L</td><td>Qiagen, Fisherscientific can also supply same product</td></tr><tr><td>Precellys 24 homogeniser</td><td>VWR</td><td>432-3750</td><td>FastPrep MP Biomedicals can be used too</td></tr><tr><td>Refrigerated micro-centrifuge, Fresco 21, Heraeus</td><td>Fisher-Scientific</td><td>15352117</td><td>Other brands with similar capacity can be used</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>BLD-455-010C</td><td>Works with 1-2 mL tubes</td></tr><tr><td>TURBO DNA-free</td><td>Fisher-Scientific</td><td>AM1907M</td><td>Takes longer but more effective than shorter DNA removal procedures.</td></tr><tr><td>&lt;strong&gt;RT-qPCR:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Primers and Taqman probes</td><td>SOI group St Andrews</td><td>VitalbacteriaPP</td><td>Supplied with the TB-MBLA Vitalbacteria kit. Probe fluorophores are FAM (green) for Mtb and HEX (yellow) for Extraction control (EC). Applied Biosystems qPCR platforms use VIC instead of HEX.</td></tr><tr><td>QuantiTect Multiplex RT-PCR NR Kit</td><td>Qiagen</td><td>204845</td><td>PCR mix plus the RT enzyme</td></tr><tr><td>RotorGene Q 5plex machine</td><td>Qiagen</td><td>9001580</td><td>Other qPCR machines: Vii7, Quantistudio, Steponelus etc. can be used</td></tr><tr><td>Strip Tubes and Caps, 0.1 mL</td><td>Qiagen</td><td>981103</td><td>Other tube sizes available depending on the rotor size. For other PCR platforms 96 well PCR plates are needed.</td></tr><tr><td>&lt;strong&gt;Non-heat inactivation Sample preservation materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 M Tris-HCl pH 7.5</td><td>Sigma</td><td>93313</td><td>Supplied ready to use</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>63689</td><td>Many competent suppliers</td></tr><tr><td>Guanidine thiocyanate (GTC)</td><td>Promega</td><td>V2791</td><td>Promega brand recommended for quality</td></tr><tr><td>Molecular grade water (RNase free)</td><td>Sigma</td><td>W4502-1L</td><td>Ensures that GTC solution is free of nucleases</td></tr><tr><td>&lt;strong&gt;General materials (used but not specific to TB-MBLA)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>500 mL plastic containers</td><td></td><td>734-5087</td><td>Nalgene brand recommeded</td></tr><tr><td>Biological waste discard jars</td><td></td><td></td><td>Many competent suppliers</td></tr><tr><td>Chemical waste discard jars</td><td></td><td></td><td>Many competent suppliers</td></tr><tr><td>Disposable gloves, chemical resistant</td><td></td><td></td><td>Many competent suppliers</td></tr><tr><td>Freezer (-20 &amp;deg;C)</td><td></td><td></td><td>Many competent suppliers</td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td></td><td></td><td>Many competent suppliers</td></tr><tr><td>Fridge (0-8 &amp;deg;C)</td><td></td><td></td><td>Many competent suppliers</td></tr><tr><td>Fume hood</td><td></td><td></td><td>For safe handling of toxic reagents</td></tr><tr><td>Laboratory scales</td><td></td><td></td><td>For accurate measurement of reagents</td></tr><tr><td>Measuring cylinders, plastic</td><td></td><td></td><td>To ensure accurate measurement of reagents</td></tr><tr><td>PCR reaction tubes</td><td></td><td></td><td>Should be suitable to the PCR instrument used</td></tr><tr><td>Pipettes and matching sterile filtered pipette tips,</td><td></td><td></td><td>DNAse and RNAse-free, range: P1000, P200, P10, P2 recommended</td></tr><tr><td>Qiagility loading plates</td><td>Qiagen</td><td></td><td>5-well master mix plate, 16-well reagent plate, 32-well sample plate, 72-well and 96-well reaction plates</td></tr><tr><td>QIAgility Pipetting Robot</td><td>Qiagen</td><td></td><td>Important for high throughput pipetting</td></tr><tr><td>Racks for 1.5 mL and 2 mL microtubes and for 15 mL and 50 mL Falcon tubes</td><td></td><td></td><td>Chemical-resistant and autoclavable recommended</td></tr><tr><td>RNase Away (Removes RNases enzymes from working space)</td><td>Fisher Scientific</td><td>10666421</td><td>Important to ensure services and devices are free from RNase enzyme</td></tr><tr><td>Safety goggles, chemical resistant</td><td>Fisher Scientific</td><td></td><td>Can also be got from Sigma. Protect eyes from toxic reagents.</td></tr><tr><td>Sterile Pasteur pipettes, 1.5-3 mL</td><td></td><td></td><td>Many competent suppliers</td></tr><tr><td>Sterile RNAse-free microtubes</td><td></td><td></td><td>1.5 mL tubes suitable for freezing at -80 &amp;deg;C recommended</td></tr><tr><td>TB disinfectant, e.g. Tristel Fuse</td><td></td><td></td><td>Ensure it is freshly prepared or prepared within a week</td></tr><tr><td>Vortex</td><td></td><td></td><td>Genie 2 brand recommended</td></tr><tr><td>&lt;strong&gt;Transmission electron microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glutaldehyde (8%)</td><td>Sigma</td><td>G7526-10ML</td><td>Part of the Cell fixation buffer (HEPES buffer)</td></tr><tr><td>HEPES (pH 7.4, 100 mM)</td><td>Sigma</td><td>H4034-25G</td><td>Part of the Cell fixation buffer (HEPES buffer)</td></tr><tr><td>Leica FCS ultracryo</td><td>Leica</td><td></td><td>Cryomicrotome for tissue sectioning</td></tr><tr><td>Methyl cellulose</td><td>Agar Scientific</td><td>AGG6425</td><td>Cold mounting resin</td></tr><tr><td>Paraformaldehyde (4% in PBS)</td><td>Sigma</td><td>P6148-500G</td><td>Part of the Cell fixation buffer (HEPES buffer)</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>84097-250G</td><td>Preservation and mounting medium</td></tr><tr><td>Uranyl acetate</td><td>Agar Scientific</td><td>AGR1260A</td><td>Universal Electron microscopy stain</td></tr></tbody></table>

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.

Heat inactivates all M. tuberculosis bacilli 
The optical density (OD) of controls (live cells) increased over time, (0.04OD&#8211;0.85OD) and no OD change was observed in the heat inactivated samples, signifying growth and no growth respectively (Figure 1)27. Similarly, control clinical sputum grew positive by day 3 in MGIT while heat inactivated clinical samples did not flag positive until the end of incubation. Growth of Mtb in MGIT was confirmed by Ziehl-Neelsen smear microscopy and the antigen MPT6429.
RNA in inactivated samples is stable at 37 &#176;C for 4 days 
Heat inactivated samples were incubated at 37 &#176;C to determine whether RNA degrades following heat inactivation of cells. No difference was found between the RNA harvested at Day (D) 0 immediately after heat inactivation and the RNA isolated at D1, 2, 3, and 4 in both BCG cultures (Figure 2A-C) and TB positive sputum (Figure 2D).
Exogenous RNase increases the rate of RNA degradation in the heat inactivated fractions 
To determine why RNA was not degrading, RNase A enzyme was exogenously added at 1,000 U/mL before and/or after heat inactivation. This caused RNA loss equivalent to bacterial load 1.5 &#177; 0.3 -, 1.8 &#177; 0.2 -, and 1.3 &#177; 0.1 &#8211; log10&#160;eCFU/mL at 80 &#176;C, 85 &#176;C, and 95 &#176;C respectively across 4 days of incubation. There was a difference in the RNA degraded in samples where RNase was added before and/or after heat inactivation (Figure 3A-C).
Sufficient RNA is preserved for TB-MBLA using 16S rRNA as reference marker 
The effect of heat inactivation on rRNA was measured in BCG cultures and sputum from TB positive patients. The measured bacterial load of control BCG culture, 5.3 &#177; 0.2 log10&#160;eCFU/mL, was 0.2 &#177; 0.1 log10&#160;eCFU/mL higher than the combined 5.1 &#177; 0.3 log10&#160;eCFU/mL of heat inactivated culture (ANOVA p &#60; 0.0001) at 80 &#176;C, 85 &#176;C, and 95 &#176;C (Figure 4A)27. Similarly, the bacterial load of control patient sputum, 7.1 log10&#160;eCFU/mL, was 0.8 &#177; 0.1 log10&#160;eCFU/mL higher than the combined 6.3 &#177; 0.41 log10eCFU/mL at 80 &#176;C, 85 &#176;C, and 95 &#176;C (Figure 4B)27. The bacterial load reduction was &#60;1log in the two types of samples tested.
Sidak&#8217;s multiple comparisons test revealed a significant difference between the bacterial load at 95 &#176;C versus that of 80 &#176;C and 85 &#176;C (p = 0.001). No difference was found in TB samples at all temperatures, p = 0.8.
Cell wall integrity is not destroyed by heat in the majority of Mtb cells 
Using transmission electron microscopy (TEM) we investigated whether cells were lysed by heat inactivation. Thin sections of paraformaldehyde fixed pellets of cells were made and embedded in an electron rich medium prior to examination by TEM. Inspection of the cells at lower and higher magnification revealed intact cell wall and visible intracellular lipid bodies. Cells morphologically appeared elongated but not lysed. Figure 5 illustrates the morphology of mycobacterium cells at different magnifications. The top two panels reveal intracellular lipid bodies and the unhampered rope-like cording, a morphological characteristic typical of mycobacterium species. The lower two panels are higher magnification expanding the view of lipid bodies and revealing some micro-intracellular structures.
Bacterial load measured by TB-MBLA is inversely correlated to MGIT culture time to positivity 
For patients responding to therapy, the bacterial load falls at an average 1 log10&#160;eCFU/mL per week over the course of treatment. Fast responders clear faster, converting to negative (zero bacterial load) by 2 weeks of treatment. The fall in bacterial load measured by TB-MBLA corresponded to the rise in MGIT culture time to positivity (TTP). Figure 6 demonstrates the inverse correlation found between bacterial load and MGIT TTP. The difference, however, is that the TB-MBLA results are available in 4 h and time-to-result is independent of the level of bacterial load. This is in contrast with 5&#8211;25 days for MGIT culture tests. Contamination with non-TB bacteria further compromise the results from culture tests.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60460/60460fig1v3.jpg" /> 
Figure 1: Verification of BCG inactivation at 80 &#176;C (dot pattern curve), 85 &#176;C (dot-dash pattern curve), and 95 &#176;C (dash pattern curve). The control (black curve) was live (unheated) BCG culture inoculated into same growth medium. Growth in the control was confirmed by the increase in the OD of the culture over the incubation period. This figure was modified from Sabiiti et al.27. <a href="https://www.jove.com/files/ftp_upload/60460/60460fig1large.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/60460/60460fig2v3.jpg" /> 
Figure 2: Stability of quantifiable RNA in heat inactivated sample incubated at 37 &#176;C for 4 days (D0&#8211;D4). (A), (B), and (C) show BCG cultures inactivated at 80 &#176;C, 85 &#176;C, and 95 &#176;C respectively. (D) shows the TB sputum inactivated at 80 &#176;C. The control is the untreated (live) fraction of the same sample. Error bars = standard deviation. Three repeats, nine replicates per run. <a href="https://www.jove.com/files/ftp_upload/60460/60460fig2large.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/60460/60460fig3v2.jpg" /> 
Figure 3: Higher RNA degradation after exogenous addition of RNase A enzyme. (A) Heat inactivation (HI) at 80 &#176;C, (B) HI at 85 &#176;C, and (C) HI 95 &#176;C. Error bars = standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/60460/60460fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60460/60460fig4v2.jpg" /> 
Figure 4: The preservation of sufficient RNA for TB-MBLA bacterial load measurement using 16S rRNA as a marker. (A) Bacterial load estimated from in vitro BCG cultures. (B) Bacterial load estimated from tuberculosis positive sputum. Error bars = standard error of the mean (n = 18 and 20 replicates for A and B respectively). This figure was modified from Sabiiti et al.27. <a href="https://www.jove.com/files/ftp_upload/60460/60460fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60460/60460fig5.jpg" /> 
Figure 5: Electron micrographs of intact Mtb bacilli after inactivation at 95 &#176;C for 20 min. Clear intact cell wall and enumerable lipid bodies were observed with TEM. Top: low magnification images of a group of cells revealing unhampered mycobacterial rope-like cording morphology and intracellular lipid bodies. Bottom: high magnification of the top panels to expand the view of lipid bodies and revealing some micro-intracellular structures. <a href="https://www.jove.com/files/ftp_upload/60460/60460fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60460/60460fig6.jpg" /> 
Figure 6: The 12 week treatment response curve of a patient on anti-TB therapy showing an inverse correlation of TB-MBLA measured bacterial load and MGIT TTP. The bacterial load (blue curve) falls while TTP rises (red curve) as the patient responds to treatment. <a href="https://www.jove.com/files/ftp_upload/60460/60460fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td rowspan="2">Master mix</td>
			<td>RT positive reaction</td>
			<td>RT negative reaction</td>
		</tr>
		<tr>
			<td>Volume per reaction x no. of reactions + 5</td>
			<td>Volume per reaction x no. of reactions + 5</td>
		</tr>
		<tr>
			<td>Quantitect mix</td>
			<td>10.0 &#956;L</td>
			<td>10.0 &#956;L</td>
		</tr>
		<tr>
			<td>Mtb16S primer mix (F + R)</td>
			<td>0.4 &#956;L</td>
			<td>0.4 &#956;L</td>
		</tr>
		<tr>
			<td>Mtb16S probe</td>
			<td>0.2 &#956;L</td>
			<td>0.2 &#956;L</td>
		</tr>
		<tr>
			<td>EC primer mix (F + R)</td>
			<td>0.4 &#956;L</td>
			<td>0.4 &#956;L</td>
		</tr>
		<tr>
			<td>EC probe</td>
			<td>0.2 &#956;L</td>
			<td>0.2 &#956;L</td>
		</tr>
		<tr>
			<td>RT enzyme</td>
			<td>0.2 &#956;L</td>
			<td>-------</td>
		</tr>
		<tr>
			<td>RNase free water</td>
			<td>4.6 &#956;L</td>
			<td>4.8 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>16 &#181;L</td>
			<td>16 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 1: Master mix preparation guide for the TB-MBLA qPCR.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Type</td>
			<td>MTB channel</td>
			<td>EC channel</td>
			<td>Interpretation</td>
		</tr>
		<tr>
			<td>Sample</td>
			<td>Positive</td>
			<td>Positive</td>
			<td>Valid</td>
		</tr>
		<tr>
			<td>Sample</td>
			<td>Positive</td>
			<td>Negative</td>
			<td>Indeterminate</td>
		</tr>
		<tr>
			<td>Sample</td>
			<td>Negative</td>
			<td>Positive</td>
			<td>Valid</td>
		</tr>
		<tr>
			<td>Sample</td>
			<td>Negative</td>
			<td>Negative</td>
			<td>Invalid</td>
		</tr>
		<tr>
			<td>MTB + Control</td>
			<td>Positive</td>
			<td>Negative</td>
			<td>Valid</td>
		</tr>
		<tr>
			<td>Extraction Control (EC)</td>
			<td>Negative</td>
			<td>Positive</td>
			<td>Valid</td>
		</tr>
		<tr>
			<td>DNA control</td>
			<td>Negative</td>
			<td>Negative</td>
			<td>Valid</td>
		</tr>
		<tr>
			<td>Negative Control (NTC)</td>
			<td>Negative</td>
			<td>Negative</td>
			<td>Valid</td>
		</tr>
	</tbody>
</table>
Table 2: TB-MBLA results interpretation guide.

Watch this Scientific Journal Video about A Tuberculosis Molecular Bacterial Load Assay TB-MBLA at JoVE.com

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.

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 ...

a-tuberculosis-molecular-bacterial-load-assay-tb-mbla

Research

JoVE Journal

Immunology and Infection

13.8K Views.  University of St Andrews. Deze tuberculose moleculaire bacteriële test, beantwoordt drie belangrijke vragen. Een: Wat is de omvang van de ziekte van de patiënt last? Twee: Hoe reageert de patiëntenziektelast op de antituberculosemedicatie?En drie: Wat is de relatie tussen ziektelast en behandelingsrespons? Deze test levert een kwantitatief resultaat op van de tuberculoselast van de patiënt en meet hoe deze last in korte tijd verandert met de behandeling. Met modificatie kan deze methode worden toegepast op ...

Video: A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

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). ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

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

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 ...

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

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

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 ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

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 ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

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