CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
The steps usually include methanol washes, bead beating for inactivation, and then formic acid and acetonitrile protein extraction and isolation. This does add to the workflow time so it’s not as quick as MALDI-TOF for bacteriology. There’s about a 90-minute addition to workflow time if you’re coming from liquid culture, about 45 minutes if you’re coming from solid culture.
For solid culture ID, it starts with a mechanical disruption with glass beads and bead beating for five minutes or vortex mixing for 15 minutes, and then incubation in 70 percent ethanol for 10 minutes, followed by protein extraction using 70 percent formic acid and acetonitrile. At this point it’s similar to what goes on in the bacteriology laboratory, where a microliter of that protein extract can be applied to a spot on the target slide, allowed to dry, and then overlaid with one microliter of matrix.
For liquid culture ID, you can take a 3-mL aliquot of liquid culture, centrifuge it for 10 minutes, decant the supernatant to remove the residual media, resuspend in 70 percent ethanol with beads, mix on a horizontal position vortexer for 15 minutes or a bead beater for five minutes, incubate 10 minutes at room temperature, then centrifuge, decant the supernatant, and resuspend the pellet in formic acid and acetonitrile. In addition, you take one microliter of that protein extract, apply it to a spot on the target slide, allow it to dry, and overlay it with one microliter of matrix. From that point on, functionally it looks very much the same.
We don’t see all of the more than 190 species of mycobacteria in our patients, and all species do not cause disease. Take note of what FDA cleared on the Vitek MS (Fig. 4), meaning what sample identifications can come off and be released without extra testing by the laboratory. I find it to be a fairly comprehensive group of organisms, and I will talk about how the list compares with the UCLA isolates.We looked at the specimens collected between November 2015 and February 2018 and we identified by probe 1,100 mycobacteria isolates. At UCLA the most common isolate is M. avium complex, accounting for 55 percent of what we see. We isolate only about four percent M. tuberculosis complex. The rapid-growing mycobacteria account for 30 percent of our organisms, and the slow-growing mycobacteria account for another four percent. And these groups are the groups we’ve been doing rpoB gene sequencing on and the groups we want to be able to target when we use MALDI-TOF.
If we were using MALDI-TOF for the identification of mycobacteria for all of the mycobacteria we looked at, more than 96 percent of our mycobacteria isolates would be in the FDA-cleared database. Four percent would not be in that database. And any identification of nonclinically validated organisms must be performed with an alternative laboratory method. In our laboratory we will still do rpoB gene sequencing on those organisms to be able to confirm the identity that comes off the MALDI-TOF.
Their data were strong. Of the 651 clinical mycobacteria isolates tested, 94 percent were correctly identified to the species, complex, or group level, run on the Vitek MS: 100 percent of the MTB, 92 percent of the slow-growing NTM, and 98 percent of the rapid growers. Thirty-three isolates, or five percent, could not be identified, meaning the MALDI-TOF provided no ID. This typically means there weren’t enough spectral peaks or there was not enough to compare it with to give a reliable ID in the database.
Of the 33 isolates, there were 27 slow growers. Some of those isolates like M. paraffinicum were species that were not in the database. Some of those isolates that belonged to the M. avium complex but were not M. avium or M. intracellulare were also species not found in the database. And there were five rapid growers, two of which were isolates of M. mucogenicum that could not be identified.
Four isolates—less than one percent—were misidentified, and the misidentification can be dangerous because if isolates are misidentified as an FDA-approved isolate, then that ID would just go out. They found that the low percentage of organisms that were misidentified were not misidentified on that particular list, so from a solid media perspective the data are good.
Does MALDI-TOF work for a liquid culture? A paper was published on the performance of the Vitek MS v3.0 for identifying species from patient samples by use of automated liquid media systems (Miller E, et al. J Clin Microbiol. 2018;56[8]e00219-18). LabCorp (using VersaTrek mycobacteria bottles) and Memorial Sloan Kettering (using Bactec MGIT 960 tubes) were the testing laboratories. They again tested for mycobacteria identification directly from the liquid media. They used either a seed and recovery or clinical isolate model, and they compared to reference sequencing ID.
In the first test they did, they asked whether the media found in the liquid cultures—and liquid cultures can have various supplements, antimicrobials, residual NALC-NaOH—created interfering peaks. They ran those supplements, and not enough peaks were shown to be able to interfere with identification. Then they did a seeded simulated sputum culture—a seed and recovery—and this is with 383 liquid cultures covering 77 strains and 21 species, and the data were very good. They had 99 percent result in correct identification to the species, complex, or group level. Only four isolates resulted in a no ID.
What’s more interesting in this paper is how this ID system performs out of liquid cultures. They tested 73 clinical liquid cultures, of which 64, or 87.7 percent, were identified correctly to species, complex, or group level. But about 10 percent resulted in a no ID (three M. avium, two M. intracellulare, one M. lentiflavum, one M. tuberculosis complex). Thus the data are starting to show that the system seems to work very well out of solid culture but may have some issues in being able to give an identification from liquid culture. But we’re still below 10 percent.
Now for the UCLA data. You will recall the importance of inactivation. The MALDI-TOF as an instrument is not in my AFB laboratory, so if I’m going to put something on the MALDI-TOF, I want to be sure everything is nonviable. We ran a test where from solid media we did ethanol, bead beating and inactivation, and extraction, and then cultured that out for six weeks. Six weeks in, of the 35 isolates we tested, there was no growth whatsoever. One hundred percent of the isolates didn’t show growth, and we tried to cover as much as possible. We of course ran some MTB, we ran slow growers including M. avium complex, and we ran rapid growers. This gave us confidence in the FDA approval, in that if you follow the protocols correctly you will have inactivated and you can use this safely outside of your BSL2 with BSL3 practices or BSL3 facility for identification.
The UCLA validation data (Fig. 5) are based on MH711 biplate media, and our gold standard to compare the identification from the MALDI-TOF was either the DNA probe for M. tuberculosis and M. avium complex or rpoB gene sequencing. Of 46 M. tuberculosis isolates tested, we tested five MTB and got 100 percent identification. With the rapid growers, we saw a very high identification to the species or to the complex. We had one M. chelonae that gave a no ID, one M. neoaurum that was called M. arupense, and one M. smegmatis complex that was identified as Citrobacter koseri. You immediately say, “Wow, a Citrobacter when we’re working in a mycobacteria laboratory; you’re doing an AFB stain.” And in doing an AFB stain, you’re getting other information. MALDI-TOF is a fantastic tool for the clinical laboratory. It is an identification scheme; it is not there to replace the expertise of the technologists. Just like everything else in the microbiology laboratory, it relies on the expertise of bench technologists to be able to put out correct IDs for patient care. No system is going to be 100 percent.
We did 22 slow growers, and of those 22, we had about 100 percent identification to species or complex.
We see a lot of the rapid growers on blood agar plates, so we did a blood agar plate validation as compared to rpoB, and again the data were good. Whether M. abscessus, M. chelonae, M. fortuitum group, or any of these isolates growing on blood agar plate, we had a reliable identification. There was one that gave a no ID.
More on liquid culture and a bit about biomass. For MALDI-TOF mass spec to work, there has to be enough organism or enough protein extracted to be able to get a spectral ID, and there cannot be interfering substances. For solid media, the suggestion for Mycobacterium is to use a one microliter loopful. We found it works better for a large loopful. For isolations out of liquid, a positive tube test needs to be done as soon as practical. The suggestion is either 1.8 mL or 3 mL. In our liquid culture validation, we got more reliable identification using 6 mL of the liquid culture medium, and then we would let it grow six days post that initial positive. Once we let it grow six days post, that gave us a sufficiently reliable ID. Is that necessary for all mycobacteria? It’s hard to know. We looked at that study again, and for the rapid growers we could get it to work much sooner, in the 48-hour range. But when a liquid culture grows in mycobacteria, what you don’t know is whether it’s a rapid grower or a nonrapid grower. All you know is that it’s acid-fast bacteria. For our setting we’re going to do six days post liquid culture for identification. This is still a dramatic improvement in time as compared with solid culture identification.Six days post culture we had reliable identification of M. tuberculosis at 100 percent, and across the board we saw 100 percent in the rapid growers and slow growers for identification. You will recall that earlier study and those no IDs—were they a question of biomass, and if they were able to wait a little longer could they functionally get an identification? Our study potentially could show that, and that MALDI-TOF, if your biomass is appropriate, is a reliable system for identification.
How do we then put MALDI-TOF into our system? The MGIT is going to flag positive, you’ll see growth on the primary plates, and at UCLA we’re going to use the probes because we still have them. Probes can be used day one from liquid culture, so it’s going to be our fastest way from liquid cultures to still identify MTB or M. avium. If the probes are negative, then we can do the six days post liquid culture MALDI-TOF or directly from solid media. The rapid growers grow up fast on the solid media and then we can run MALDI-TOF. If the MALDI-TOF identification is FDA approved, we can release that result. If it’s not FDA approved, then we’ll still move to our rpoB gene sequencing.
What are the estimated improvements in the laboratory if you’re able to put in MALDI-TOF mass spec? The improvements I will point to are specific to UCLA. If we look at the number of days to identification from a pure isolated culture, we estimate that our one- to eight-day range for rpoB sequencing would be cut to zero to one day, with an average of a half-day turnaround time, allowing us on average to give an identification of those rapid and slow growers for which we were doing gene sequencing three and a half days earlier than before. This will apply in 96.6 percent of our non-M. tuberculosis, non-M. avium complex patient isolates.What do we save in FTE time so technologists can do other important work? RpoB gene sequencing is intensive. It’s four hours per day and takes two days. At that one test per week it probably “costs” 0.2 FTE. If we put in MALDI-TOF, it’s about 1.25 hours. We can run it three times a week and we’re at a savings of at least 0.1 FTE in these general settings. These are estimates only; we have not done a full workup to see what the savings are. There are also potential cost savings in reagents and QC. The savings do not include equipment. If you don’t have a MALDI-TOF already, it’s going to be expensive to buy one for mycobacteria.
But if you already have a MALDI-TOF and send-out costs are expensive, you can potentially recoup costs. If we look at rpoB testing for the year (before mycobacteria), for QC we were spending about $9,000. For patient testing we’re spending about $18,000—just in reagents. The total is $27,000. After MALDI-TOF, looking back over that year, if we just did MALDI-TOF, now our rpoB gene sequencing has gone way down. Most of these isolates have moved over to MALDI-TOF. We’re only spending $700 there, $1,500 for QC, for a total of $2,200. Patient testing on the MALDI-TOF, generally with all the necessary reagents, is about $9,000. So ultimately this could save UCLA in excess of $16,000 a year.
The time and cost savings at UCLA are not a guarantee of results at other laboratories, and, again, we haven’t done a full cost analysis at UCLA so this isn’t even a guarantee of cost savings we could see. But I think we’re in the ballpark. As all lab directors know, it’s getting harder for us to justify our tests and new testing to the hospital, so these are ways that you can think about it, not even considering shortening patient turnaround time and length of stay. But just in the laboratory how you can accrue savings if you bring this in.
MALDI-TOF for mycobacterial identification will definitely allow for species identification faster. The faster we can get species identification, the higher our clinical impact. We’ve just begun this and our infectious disease service has already noticed how much faster it’s getting identifications for nontuberculous mycobacteria.
MALDI also allows for new opportunities. It is adding more clinically important tests for Mycobacterium, and as we have more and more immunocompromised patients who are living longer—thanks to transplants and new cancer therapies—more and more people will be subject to these types of diseases, making it more critical for laboratories to be able to identify them.