This work was supported by the National Institutes of Health (NIH) under grant RC3 AI089106.
MDR-TB nucleic acids were provided by the United Nations Children&#39;s Fund/United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, Switzerland.
We thank Dr. Tom Shinnick of the U.S. Centers for Disease Control and Prevention for guidance on the specific genes and mutations to include in the prototype assay.

For laboratories that follow universal PCR precautions, it is operationally more efficient to include several amplification microarrays and gaskets per substrate and wash all amplification microarrays simultaneously in a bulk container, as described here. Consumable formats are available for performing post-amplification microarray washing steps in an entirely sealed, closed amplicon test, as reported elsewhere30,33.
1. Setup
<ol>
	<li>Extract and purify nucleic acids from the sample under appropriate biosafety conditions with a method of choice. The requirement is that nucleic acids be of sufficient purity for asymmetric, multiplex PCR amplification.</li>
	<li>Prepare workspace by wiping surfaces and equipment with decontaminating solutions.</li>
	<li>Place amplification reagents on ice or cold block.</li>
	<li>Label a sterile 1.5 ml microfuge tube for the amplification master mix, and label a sterile 0.2 ml microfuge tube for each sample.</li>
	<li>After reagents are thawed, briefly vortex and collect contents to the bottom of the tube with a pulse-spin in a mini-centrifuge. Do not vortex Taq polymerase. Gently flick the tube to mix and follow with a pulse-spin in mini-centrifuge.</li>
	<li>Prepare an amplification master mix using the per-sample reaction volumes shown in Table 1. To account for possible pipetting inaccuracies, prepare at least one more reaction volume than the total number of samples being processed. Note that the master mix contains additional Taq polymerase, over and above what is provided with the muliplex PCR buffer. Only add the amplification/inhibition control to the master mix after all other reagents are combined, and the stock reagent tubes returned to storage.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Per- Sample Volume (&#181;l)</td>
			<td>Final [ ]</td>
		</tr>
		<tr>
			<td>Mulitplex PCR Buffer with HotStar Taq Plus</td>
			<td>25</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>0.55</td>
			<td>0.6 mg/ml</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>3.8</td>
			<td>7.6%</td>
		</tr>
		<tr>
			<td>Additional Taq polymerase</td>
			<td>0.8</td>
			<td>units/&#181;l (4 units total)</td>
		</tr>
		<tr>
			<td>MDR-TB primer mix</td>
			<td>15.75</td>
			<td>-</td>
		</tr>
		<tr>
			<td>RNase-free H2O</td>
			<td>2.1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Amplification/Inhibition Control</td>
			<td>1</td>
			<td>5.0 fg/&#181;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>49</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. MDR-TB amplification microarray master mix composition.
<ol start="7">
	<li>Vortex the master mix and collect contents to the bottom of the tube with a pulse-spin in a mini-centrifuge.</li>
	<li>Combine 49 &#181;l of master mix and 1 &#181;l of M. tuberculosis DNA to each of the sample tubes from step 1.4, above. For an external, no template control (NTC), use 1 &#181;l of molecular biology grade water in place of the M. tuberculosis DNA sample. Vortex and pulse-spin the tube(s) when finished.</li>
</ol>
2. Load Amplification Microarrays
<ol>
	<li>Add 48 &#181;l of each master mix/sample onto the center of their respective microarrays, being careful to not touch the microarray itself.</li>
	<li>Place a cover slip on top of the microarray gasket and seal, being careful not to trap any air bubbles in the microarray chamber. Air bubbles can negatively affect amplification efficiency and may create artifacts or noise in the subsequent microarray image.</li>
</ol>
3. Thermal Cycling
<ol>
	<li>Once all microarrays are loaded with sample, place substrates on the flat block thermal cycler. Make sure that the heated lid option is turned &#8220;Off&#8221;. Equipment obtained from Akonni Biosystems will already be prequalified for use. Otherwise, it is very important to verify that the flat block thermal cycler(s) provide(s) uniform, consistent heating across all microarray substrates.</li>
	<li>Open the thermal cycler software, select the appropriate program, enter &#8220;50 &#181;l&#8221; for the reaction volume, and initiate the run. The thermal cycling protocol described here consists of:
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Thermal Cycling Steps</td>
		</tr>
		<tr>
			<td>1</td>
			<td>88 &#176;C</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>2</td>
			<td>88 &#176;C</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td>3</td>
			<td>55 &#176;C</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>4</td>
			<td>65 &#176;C</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Repeat steps (2-4) for 50 cycles</td>
		</tr>
		<tr>
			<td>6</td>
			<td>65 &#176;C</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>7</td>
			<td>55 &#176;C</td>
			<td>3 hr</td>
		</tr>
	</tbody>
</table>
</li>
	<li>The total number of thermal cycles and 55 &#176;C post-amplification hold time are independent variables that the user may modify, as needed or appropriate for the specific experiment. Because the MDR-TB master mix creates asymmetric (predominantly single-stranded) amplicons, it is usually helpful to include more thermal cycles than might otherwise be utilized for a conventional, exponential PCR amplification protocol.</li>
	<li>When the thermal cycling and hybridization program is complete, end the program, remove the amplification microarrays, close the software, and turn off the thermal cycler(s).</li>
</ol>
4. Wash and Dry
<ol>
	<li>For washing up to 24 substrates simultaneously, prepare at least 250 ml 1x SSPE &#8211; 0.1% Triton X-100 wash buffer, and two containers with at least 250 ml deionized or Milli-Q grade water each. The wash buffer can be prepared in advance.</li>
	<li>Carefully remove the cover slip and gasket from each amplification microarray chamber using flat-end forceps. This step is the point at which there is the greatest risk of physically damaging gel element arrays. Use appropriate PCR contamination controls to prevent the spread of amplified nucleic acids within the work environment.</li>
	<li>Place microarray substrates in a histology slide holder, and place all slides into the wash bin containing the wash buffer. Cover the container with a lid.</li>
	<li>Wash microarrays for 10 min at room temperature with gentle agitation.</li>
	<li>Dip the microarrays three times each in two successive washes of deionized or Milli-Q grade water, and air dry.</li>
</ol>
5. Imaging
The asymmetric MDR-TB primer mix generates Cy3-labeled amplicons. Gel element microarrays can be imaged with any standard microarray imager capable of imaging Cy3. The following imaging procedure is specific for the MDR-TB master mix, Dx2100 field portable imager, and automated analysis software provided by Akonni.
<ol>
	<li>Ensure that the ethernet cable from the imager is connected to the computer.</li>
	<li>Turn on the imager. The power switch is located on the back panel of the instrument. Blue LEDs on the front of the imager will illuminate when imager is powered on.</li>
	<li>Turn on the computer.</li>
	<li>On the computer desktop, double click the software icon to launch the automated image analysis software.</li>
	<li>Wait for the software to establish a connection with the imager camera. Once the connection is established, the Analyze button becomes available, and the message &#8220;Connection Successful&#8221; will appear in the lower left corner of the screen.</li>
	<li>Insert the dried amplification microarray with the microarray facing away from the user, and toward the objective lens. If the microarray is improperly oriented with respect to the lens and camera, the automated analysis software will fail to place a grid on the fluorescence image.</li>
	<li>Close the access door. A preview image will be available on the computer screen.</li>
	<li>Select the MDR-TB analysis script from the drop-down menu and enter the desired Exposure Time (in milliseconds). A typical starting point for gel element amplification microarrays is 100-500 msec.</li>
	<li>Saturated pixels will be displayed on the Preview Image in red. Adjust the exposure time to minimize the number of saturated pixels within individual spots.</li>
	<li>Click Analyze to acquire and analyze the fluorescence image. The software will automatically place an MDR-TB assay-specific grid onto the image, extract integrated intensity and background values, and report drug resistance and genotyping results. The automated analysis will typically take a few seconds. For challenging images that contain scratches, dust, fluorescent artifacts, or physical damage to the microarray features, the software may need up to 1 min to complete the analysis.</li>
	<li>Once the analysis is complete, click on Save, select the destination folder, type in a filename, and click OK. Underlying microarray data are saved as an .xls file and can be exported for off-line analyses, if desired.</li>
	<li>Once the analysis is complete, remove the amplification microarray from the imager, and click on New to initiate the next analysis.</li>
	<li>When finished collecting data, close the software, shut down the computer, and turn off the imager.</li>
</ol>

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C., Nabeta, P., 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></ol>

The authors declare no competing financial interests.

The extent of multiplexing with an amplification microarray is ultimately dictated by the efficiency of multiplex asymmetric PCR, not the microarray. In our experience, 10-12 unique primer pairs can be readily multiplexed in an amplification microarray format. Conventional primer and probe design criteria therefore apply to new assays, except that one also needs to consider potential interactions between solution-phase nucleic acids and immobilized microarray probes, the thermal efficiency of the thermal cycler, and prob...

Early case detection and rapid treatment are considered the most effective control strategies to reduce Mycobacterium tuberculosis (MTB) transmission1, and there is now a broad consensus in the TB community that a point of care (POC) or near POC test to simultaneously diagnose TB and drug resistance (DR) is needed. Technologies such as Cepheid&#8217;s GeneXpert&#160;and other nucleic acid amplification tests reduce the time to diagnosis for many TB patients, and provide a rapid read-out indicating resistance to rifampin or selected mutations conferring resistance to other first or second line drugs2. Although real-time and isothermal nucleic acid amplification tests are designed to identify the drug resistance mutations that lead to MDR-TB, the spectrum of mutations they detect is often inadequate to design an individualized drug regimen corresponding to the drug resistance profile of the patient, and technical constraints related to optical cross-talk or the complexity of amplification and reporting chemistries3-7 may limit the number of loci or mutations that are detected. Thus, detection technologies with higher multiplexing capacity are required to address known gaps in MDR-TB POC diagnostics.
Microarrays and the WHO-endorsed Hain line probe assays can address the &#8220;multiple gene, multiple mutations&#8221; challenge of diagnosing MDR-TB8-29. Unfortunately, these hybridization-based, multiplexed detection platforms use multistep, complicated, and open-amplicon protocols that require significant training and proficiency in molecular techniques. The amplification microarray30 was designed to address some of these microarray work-flow and operational concerns. The simplifying fluidic principles are to amplify, hybridize, and detect nucleic acid targets within a single microfluidic chamber. The user introduces the nucleic acid and amplification master mix into a fluidic chamber with a pipette and starts the thermal cycling protocol. For the batch processing method shown here, microarrays are subsequently washed in bulk solution, dried, and imaged. This study demonstrates the functionality of an amplification microarray using an MDR-TB microarray test for rpoB (30 mutations), katG (2 mutations), inhA (4 mutations), rpsL (2 mutations), embB (1 mutation), IS1245, IS6110, and an internal amplification and hybridization control. At least one matched pair of microarray probes (wildtype (WT) and single-nucleotide mutant (MU)) is included for each mutation of interest. Purified nucleic acids from multi-drug resistant M. tuberculosis are from the TDR Tuberculosis Strain Bank31. Gel element microarrays are manufactured on glass substrates by copolymerization essentially as described elsewhere32, except that we use 4% monomer and 0.05 mM each probe in the polymerization mixture. Arrays are surrounded with a 50 ml gasket prior to use. After thermal cycling, hybridization, and wash steps, amplification microarrays are imaged on an Akonni portable analyzer. Background-corrected, integrated signal intensities are obtained from the raw .tif images using a fixed circle algorithm. Noise for each gel element is calculated as three times the standard deviation of the local spot backgrounds. Gene targets are typically considered detectable for signal to noise ratio (SNR) values &#8805;3. In order to determine the presence or absence of a specific mutation in each gene or codon, a discriminant ratio is calculated from the SNR values as (WT-MU)/(WT+MU). Discriminant ratios &#60;0 are indicative of a drug-resistance mutation at the locus, whereas ratios &#62;0 are indicative of the wild-type sequence.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;">MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus</td>
			<td>Qiagen</td>
			<td>#206143</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
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			<td height="26" style="height:26px;">20 mg mL-1 non-acetlyated bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#3B6917</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">5X concentrated MDR-TB primer mix</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">500 fg uL-1 amplification and inhibition control</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">20X SSPE</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP1328-4</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Triton X-100</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP151-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Disinfecting Spray&#160;</td>
			<td>Current Technologies, Inc.</td>
			<td>#BRSPRAY128</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">70% Isopropyl Alcohol</td>
			<td>Decon Labs, Inc.</td>
			<td>#8416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Equipment</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microarray imager, with automated image and data analysis software</td>
			<td>Akonni Biosystems</td>
			<td>100-20011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermal cycler with flat block insert</td>
			<td>Akonni Biosystems</td>
			<td>100-10021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">High-throughput wash station and slide holder</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting forceps</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#10-300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini-centrifuge</td>
			<td>VWR</td>
			<td>#93000-196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Airbrush Compressor Kit</td>
			<td>Central Pneumatic</td>
			<td>#95630</td>
			<td></td>
		</tr>
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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.

Qualitative image analysis can provide insight into sources of experimental noise or variability that are challenging to identify in data tables generated by automated image analysis software. Thus, it can be useful to visually ascertain that 1) all gel elements are intact and undamaged, 2) the global background is free from fluorescent artifacts that might affect individual signal to noise ratio (SNR) values, 3) there is no evidence for bubble formation or nonuniform amplification/hybridization across the array, and 4) ...

Watch this Scientific Journal Video about Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray at JoVE.com

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.

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 ...

demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

12.6K Aufrufe.  Akonni Biosystems, Inc..  Ein Verstärkungsmikroarray kombiniert asymmetrischen PCR-Amplifikation und Mikroarray-Hybridisierung in einer einzigen Kammer, die Mikroarray-Arbeitsprozess deutlich vereinfacht für den Endbenutzer. Vereinfachung der Microarray-Workflow ist ein notwendiger erster Schritt für die Erstellung von Microarray-basierte Diagnostik, die routinemäßig in der unteren-Ressource-Umgebungen eingesetzt werden kann. 

Serie: Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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). ...

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

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

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

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

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

Growth of Mycobacterium tuberculosis Biofilms

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

Growth of Mycobacterium tuberculosis Biofilms

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

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

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 ...