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Exploring MALDI-TOF mass spec for mycobacteria

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The Food and Drug Administration in 2017 approved MALDI-TOF mass spectrometry for the identification of mycobacteria. What is the protocol? How is workflow affected? Are there cost savings and turnaround time improvements? Omai Garner, PhD, D(ABMM), answered these and other questions and shared his laboratory’s validation data in a Dec. 13, 2018 CAP TODAY webinar supported by Bio­Mérieux (captoday​online.com). Here is an edited transcript of what he said.

Dr. Garner is an assistant clinical professor, section chief of clinical microbiology, and director of point-of-care testing, Department of Pathology and Laboratory Medicine, University of California Los Angeles. His laboratory began to use MALDI-TOF mass spec for mycobacteria in fall 2018.

March 2019—The family of Mycobacteriaceae includes the genus Mycobacterium, which includes more than 190 species. These organisms have an unusual cell wall. Those of us who work in mycobacteria laboratories know that the cell wall has a high lipid content that makes these organisms acid fast. Thus they’re acid-fast bacteria.

Dr. Garner

They have to put together this complicated cell wall and thus it can take from two to 60 days post culture in the organism on a plate to get that organism to grow. This is why we divide this group of organisms into slow-growing mycobacteria, which appear in culture after seven days, and this is typically related to solid culture, and rapid-growing mycobacteria, which grow in fewer than seven days.

The most clinically relevant mycobacteria species is Mycobacterium tuberculosis, which is a complex of organisms, including of course M. tuberculosis but also M. bovis/BCG, M. africanum, M. caprae, M. canettii, and others. We separate all the rest of the mycobacteria by calling them nontuberculous mycobacteria, or NTM, and they can be divided further into groups (Fig. 1).

These organisms can be in our drinking and bathing water and in water used for recreation, and this is because often they’re found in almost all natural water sources. So we are regularly exposed to nontuberculous mycobacteria, but they exclusively or almost exclusively cause infections in immunocompromised patients.

Mycobacterium or nontuberculous mycobacterial infections in North America are on the rise. Between 1998 and 2010 the incidence of M. tuberculosis declined overall, though there are populations where this is not true, such as in Los Angeles. The opposite is happening for nontuberculous mycobacteria, in part because all hospitals are serving a larger variety of immunocompromised patients.

Laboratories are getting better at being able to identify NTM to species. And MALDI-TOF mass spectrometry may make it possible for labs that do not identify NTM to species to be able to do so. From the clinical perspective of whether to treat, it’s critically important to identify these organisms to species. Furthermore, some species of NTM require antimicrobial susceptibility testing for appropriate therapy, and AST relies on accurate species identification for interpretive criteria or for the use of breakpoints.

A level one mycobacteria laboratory performs no mycobacteriological procedures and would send the testing out. A level two laboratory would do acid-fast stains of exudates, effusions, and body fluids, and it may inoculate but then refer cultures to a reference laboratory.

Level three laboratories are able to isolate mycobacteria and have identification schemes in place for M. tuberculosis complex and perhaps preliminary identification of NTM. It is the level three laboratory that can take most advantage of MALDI-TOF MS identification of mycobacteria.

Level four laboratories are able to do definitive identification of mycobacteria. They can isolate them and identify them to the extent required to establish a correct clinical diagnosis; they may even do some level of susceptibility testing. Our mycobacteria laboratory at UCLA is a level four laboratory. From a level four laboratory perspective, there are distinct advantages to moving to MALDI-TOF.

At UCLA in our mycobacteria laboratory, we have direct tests so we’re able to offer AFB smears and microscopy. We also offer M. tuberculosis complex PCR, for which we use GeneXpert (Cepheid). We’re able to do culture and identification, including liquid culture and solid media culture. Once that culture is positive, we use a set of DNA probes, a hybridization scheme to be able to immediately identify M. tuberculosis or M. avium complex.

We also have in-house Sanger sequencing, and this allows us to do gene sequencing for the rpoB gene to fully identify nontuberculous mycobacteria to species. In addition, we do in-house some rapid-growing Mycobacteria drug susceptibility testing by broth microdilution.

For M. tuberculosis complex and M. avium complex drug susceptibility testing, we use a reference laboratory. Other nontuberculous mycobacteria that are slow growers are sent to specialized reference laboratories for susceptibility testing, to include even the newest drugs available for mycobacteria treatment.

How would MALDI-TOF fit into and improve this? We receive a number of different specimens for mycobacteria culture as selected by the physician. These diseases can show up anywhere; they are not restricted to pulmonary illnesses. If sterile specimens come in that are non-blood—surgical tissue, bone marrow, sterile fluids—we can grind and vortex and centrifuge and then move on to the direct AFB smear and inoculation of the sample.

If a nonsterile sample comes in, and this is a typical sample for mycobacteria—it could be respiratory swabs, gastric, urine, or stool—we go through the process of N-acetyl-L- cysteine -NaOH liquefaction and decontamination. We neutralize and centrifuge, and then move that into the AFB smear process. We do a fluorochrome stain and a Ziehl-Neelsen stain, and then we inoculate solid and liquid media for all specimens. The liquid media system we use is the Mycobacteria Growth Indicator Tube, or MGIT, system.

We do both systems because we look for that liquid media to come up with clinically relevant results possibly faster than the solid media, especially for the slow-growing mycobacteria. But there’s still an advantage to using the solid media because potentially you could have polymicrobic mycobacterial infections, and those can be worked up better on solid media.

Once that liquid culture flags as positive, or we see growth on the primary plates, the next important step is to confirm whether it’s acid-fast bacteria. Once we do that, we move into our probes because they can provide a specific answer directly off the liquid media. We run a DNA probe for the M. tuberculosis complex and for M. avium complex.

If either one of those is positive, we have a confirmed ID and can send it out for susceptibility testing by culturing onto solid media. Additionally, though, if the MGIT cultures are negative and if the DNA probes are negative, we’ll subculture to solid media, and all probe-negative organisms that grow on solid media in greater than seven days will be called slow growers. If they grow in fewer than seven days, we’ll call them rapid growers. Ultimately, either way, as soon as we get solid media growth, we’re able to move on to Sanger sequencing—rpoB gene sequencing identifying the region of the rpoB gene that can reliably be called to identification for species-level ID of mycobacteria. In our laboratory we are using a greater than 98 percent match to identify these organisms. It is a long, labor-intensive process, but at the end we have an ID that can guide clinical treatment.

The majority of our isolates that are outside of M. avium complex and M. tuberculosis are identified by rpoB gene sequencing. How long does that take? We looked at the number of days to identification from a pure isolated culture of a Mycobacterium, and this is in the most ideal setting, meaning that the rpoB gene sequencing works on the first try and there are no repeats. We’re able to offer this only once per week because of the strenuousness of the assay, so the turnaround time can be one to eight days post culture for an identification; the average is four days. In microbiology we always ask: Can we do better?

The FDA in 2017 approved MALDI-TOF for the identification of mycobacteria. What’s the process? In the bacteriology laboratory, a sample can be taken and put onto a target plate, and the first thing that happens is that matrix is put onto that sample. The matrix is able to, with typical bacteria, kill the bacteria and ionize the proteins to get them ready for the time-of-flight tube.

Then a laser hits on the spot where the bacteria are. The proteins that are ionized within that section desorb or float up above that section where the laser hits. They’re then ready to enter the time-of-flight tube, in which they fly to the detector. The smaller proteins will get to the detector first and show up on the spectrum first; the larger proteins from that bacteria will take a longer time to get through and show up later on the spectra. Then you have a proteomical ray of the different sizes of proteins found in that bacteria.

The y-axis of the spectrum is about the quantity of proteins that were there, so not only do you separate proteins by size, but you also get a read of how many proteins are there. That gives you an identifiable spectra that you can compare with other organisms that can lead to an identification.

When I first heard about MALDI mass spec, my questions were what proteins are being analyzed and what proteins are flying through. And what’s interesting about MALDI-TOF is that you’re only interested in the proteins that identify the difference between, say, Escherichia coli and Staphylococcus aureus. Those proteins are structural proteins found in the window between 2,000 and 20,000 daltons. The reason we look only in this window for MALDI-TOF is that if you look below this window, those are going to be metabolites and matrices that are not associated with the specific bacteria. Above that window will be the proteins that are enzymes and enzyme complexes.

The challenge of looking at enzymes and enzyme complexes can be laid out in the Gram-negative identification by MALTI-TOF. Let’s say I have an E. coli growing on a blood agar plate. It’s going to produce a certain protein profile of enzymes. If I take that same E. coli and grow it on a MacConkey plate, it needs to produce a different set of enzymes to be able to survive the crystal violet and bile salts on that plate. Thus my profile would end up looking very different even though I had the same E. coli. That’s not good for microbial ID.

Functionally we put almost horse blinders on just to look in this area where you can identify organisms to species, and ultimately it works well. So you see different species, different pattern MALDI-TOF profiles for Streptococcus pneumoniae, E. coli, and Pseudomonas aeruginosa (Fig. 2). They’re all distinct. It’s not a pattern for a technologist to memorize, but it is a pattern that a computer algorithm is able to identify. The variabilities seen in the protein peaks between members of the same species don’t fool the MALDI-TOF into calling it something different. That’s critical.

Let’s look at two genetically similar organisms of coagulase-negative Staph and S. aureus (Fig. 3). The peaks are sufficiently different to determine the difference between these two, but the small variations seen among the five S. aureus isolates still do not make the computer make a mistake and call that S. aureus by genus and species something else.

The Vitek MS specifically does this by Bin Matrixing, a methodology proprietary to BioMérieux and the Vitek MS. Bin Matrixing looks at each peak of the spectrum and asks whether the presence or absence of that peak is related to the species ID. If it’s peak one and it’s a high peak, does that peak relate or not relate to S. aureus? And it does that 1,300 times across the spectra, thereby providing a reliable ID if it comes out at the end. So the spectra will be collected and analyzed and then the identification will be delivered, and there will be a competence factor associated with that identification. In addition to providing a reliable ID, MALDI-TOF is a simple workflow.

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