Labs enter a MALDI-TOF state of mind

Karen Titus

October 2016—When MALDI-TOF mass spectrometry enters the microbiology lab, it’s a little like watching Sir John Falstaff settle his considerable girth onstage. Things happen. Characters fret and flee, scheme, opine, panic, and, in the case of Prince Hal, ascend to greatness. (And, if we’re honest, some just get drunk.) Both, in brief, are an upending presence.

Like Falstaff—also an inspirer of operas and a symphony as well as a beer—MALDI-TOF is complex as well as popular.

“It’s definitely come in to take its place as the preferred method of identification,” says Kevin Alby, PhD, assistant director of clinical microbiology, Hospital of the University of Pennsylvania, Philadelphia. “For academic medical centers and large community hospitals, MALDI-TOF is becoming the norm.” If such laboratories haven’t already begun using MALDI-TOF, they’re certainly thinking about it, says Dr. Alby, who is also an assistant professor of pathology and laboratory medicine at Penn’s Perelman School of Medicine.

Dr. Alby and several others spoke about MALDI-TOF with CAP TODAY as well as at the ASM Microbe 2016 conference in June, exploring what life has been like with MALDI-TOF front and center in microbiology laboratories.

At his institution, says John Branda, MD, MALDI-TOF  has been, among other things, a medical version of MythBusters.
To wit:

• Among clinicians—and sometimes even among pathologists and other lab personnel who work outside of microbiology—there rests the belief that MALDI-TOF can be used to directly identify a microorganism on a primary specimen. Not true, says Dr. Branda, associate director of clinical microbiology, Massachusetts General Hospital, and assistant professor of pathology, Harvard Medical School. “We still have to do a culture and isolate a microorganism as the first step—at least right now.”
• Within the lab, another misconception is that MALDI-TOF is a labor-saving device. Not only will it be a faster method, goes the thinking, but it will also greatly reduce technical hands-on time. “That’s not really how it works,” says Dr. Branda.
• Many laboratory personnel worry that when MALDI-TOF shows up, they, in turn, will be shown the door. “There’s apprehension: This is your replacement,” Dr. Branda says. “And that’s definitely not the case.”

When he and his colleagues began implementing MALDI-TOF, about five years ago, they reasonably expected a smooth sail. “On paper,” he says, “the technique of performing the analysis is very simple from a technical standpoint,” despite the instrument’s sophistication.

“For routine bacteria, you just have to find an isolated colony,” either on a primary culture plate or a subculture plate, Dr. Branda says. “You apply it to the target, you overlay it with a matrix, and off you go.” While yeast and mucoid bacteria usually require users to overlay the bugs with formic acid and then add the matrix, “that’s a very quick step.”

Those who add MALDI-TOF to their laboratories can use one of two approaches, he suggests: “Do you want to remove the Band-Aid slowly, or do you want to rip it off?” His lab chose the former, what he calls a staged rollout, moving from one section or group of organisms to another. It made sense, he says, for a complex lab like the one at MGH, and it allowed for incremental training of staff. (The downside, he notes, is that for a time some technologists will be identifying organisms by conventional methods and others will be using MALDI-TOF, which can create inconsistencies in reporting.)

Nevertheless, they were surprised by the learning curve, even with their unhurried approach.

Most of this related to preparing target slides, Dr. Branda says. Applying the isolate to the slide is neither automated nor standardized; the slide needs to contain an adequate amount of material but not so much that it’s counterproductive. “Some techs have a real knack for it and are good at it right from the beginning; other techs struggle to be able to do it reproducibly.”

Because of those difficulties, he recalls, when the lab had trouble with the instrument early on, it was hard to discern whether it was related to the instrument or to the user. “We soon realized that the first thing we needed to do was some troubleshooting with our expert ‘spotter’”—someone who excels at preparing target slides. “That removed a very important variable in the troubleshooting process.”

When the problem is with the instrument, fixes are less easy.

MALDI-TOF doesn’t lend itself to tinkering by existing lab staff—if you buy a Bentley, you don’t do your own oil changes. Instead, the manufacturers’ service engineers handle these problems. Unfortunately, Dr. Branda says, “Those difficulties arise—at this point, anyway—a little more than one would like, ideally.”

The problems can be even more complex depending on the target. Anna Lau, PhD, of the National Institutes of Health, is exploring inherent instrument variabilities and settings that labs need to consider when using MALDI-TOF for mold identification. In a study currently wrapping up, she and her colleagues have seen wide variability in instrument performance across eight U.S. academic medical centers. “This is unusual,” says Dr. Lau, co-director of bacteriology, parasitology, and molecular epidemiology, Department of Laboratory Medicine, Clinical Center, NIH. “It’s something that wasn’t seen with bacteria and yeast.” Those bacteria and yeast multicenter studies have shown great reproducibility, inter- and intralaboratory, regardless of the system being used. “So we’re trying to find the answer.”

She suspects instrument settings could be one culprit. “It’s also important to remember that each hospital’s instrument is going to be unique. It’s such a complicated instrument.” One hospital’s laser will be a different age than another’s, for example. “If you have a younger laser, it’s more powerful, so therefore your settings will need to be adjusted.”

When Dr. Branda and colleagues brought on MALDI-TOF at MGH, they discovered the instrument is quite sensitive to temperature. When the temperature exceeds a set point—“We’re talking about a few degrees above a normal room temperature”—the instrument will either stall or start to become unreliable.

Like most laboratories, the one at MGH doesn’t have space to spare. Once in the crowded room with other instruments and equipment, the device created enough heat on its own to lift the room temperature into a troublesome zone, Dr. Branda says. Dealing with that unexpected problem required “a whole lot of engineering work—we had to jury-rig a special exhaust system to siphon away the hot air.” The ultimate solution would have been to upgrade the HVAC system in the room, a massive and expensive undertaking.

Another surprise, as Dr. Branda alluded to, was that MALDI-TOF hasn’t led to labor savings. Technologists are still doing much of the work they previously did, plus the new procedure. In sections of the lab where staffing was already tight, he says, the additional workload required more staff. It’s not something they anticipated, he says, “particularly because there are articles in the literature that suggest the opposite. That just hasn’t been our experience.”

For those in the lab who worried their years of expertise now had a best-by date, Dr. Branda notes that there’s still a need to correlate MALDI-TOF results with colony morphology and Gram-stain morphology to ensure results are accurate. While MALDI-TOF results generally are reliable, he says, the potential for errors exists, as it does with any method. “So you still need a very well-trained, qualified medical technologist to look at the result and make a judgment about whether the result makes sense,” in the context of the specimen itself and the isolate’s phenotypic features.

Not that Dr. Branda and colleagues plan to evict MALDI-TOF from the laboratory. “The results, in general, are at least as accurate, and often more accurate, than the results we would have produced by more conventional means in the recent past,” he says.

But MALDI-TOF, for all its apparent simplicity, can, like a certain fictional fat man, try the patience of those who work in the lab. Knowing what he knows now, what would Dr. Branda do differently? What are he and his colleagues now doing better?

When they first introduced MALDI-TOF, he says, they followed a decentralized plan, in which bench technologists individually prepared their own target slides and target maps and did their own analysis on the instrument. But, like the Founding Fathers’ views on the federal government, things evolved in practice. “It turned out to be the wrong approach,” says Dr. Branda, “so we opted for a more centralized approach.” Now, each day one or two technologists are primarily responsible for the aforementioned tasks and, to some extent, ensuring the results are entered into the laboratory information system.

This has turned out to be much more practical for the large MGH lab, Dr. Branda says, with its many users and one MALDI-TOF instrument. “Also, it was inefficient for all these technologists to be getting up and down and going back and forth to the instrument to do procedures. It turned out to be far more efficient to have someone essentially making rounds to pick up cultures and do the analyses.”

They’ve also improved their technologist training, particularly in target slide preparation. Technologists need to demonstrate a level of success before they’re allowed to use the instrument clinically. Moreover, they’ve learned when to turn to the service engineers for help with troubleshooting. “We kind of know the warning signs when something’s not right.”

There’s one other myth Dr. Branda has seen busted, this one espoused by clinicians.

For the most part, feedback on MALDI-TOF analysis has been positive, he reports. Oftentimes clinicians are seeing identification results more quickly than they would have in the past. Moreover, the laboratory can precisely identify organisms that in the past it might have grouped or “lumped” (to use Dr. Branda’s word) together.

But as clinicians’ eyes have been opened to more precise identifications, their expectations may have also grown. “They also have misconceptions about the breadth of functionality,” Dr. Branda says, diplomatically. There are certain organisms that his lab, at least, has not yet tried to identify by MALDI-TOF, such as molds (though as Dr. Lau and others are showing, that capability certainly exists). “I think some of the clinicians either hear or read about these things and assume as long as you have this instrument that we can do almost anything with it,” Dr. Branda says.

The laboratory, for its part, tries not to provide overly precise identification when it’s not clinically meaningful, says Dr. Branda. Clinicians can benefit from knowing the identification of subspecies within the Strep bovis group, for example. But with a superficial wound culture, with four or five different commensal flora in the culture, “If you precisely identify each one of those, it signals to the clinician that those are somehow pathogenic or clinically relevant, and it may lead to misunderstanding and overtreatment.

“There are times when we need to pull back,” he continues. “Even though we know precisely what all those organisms are in the culture, we still use lumping terms, like ‘organisms resembling cutaneous flora.’”

MALDI-TOF identifications can create other muddles, such as split identifications. To sort through that, Dr. Branda asks whether the possible identifications are all from the same genus. If they’re not, he says, it’s time to troubleshoot and do supplemental testing to determine if the instrument is at fault or if the isolate is mixed or impure.

If there’s no identification, Dr. Branda recommends examining the spectrum itself. A poor-quality spectrum is, usually, the result of poor technique. “Spotting doesn’t come easily to everyone,” he says. “You’ve either added too much of the organism, or not enough. You forgot to apply the matrix, or you scooped up some medium with it.”

And if everything’s been handled well but no ID is forthcoming? “Usually that’s because the organism is not in your database,” he says.

In the future, Dr. Branda expects his lab to expand to mycobacteria, Nocardia, and, eventually, mold identification.

Beyond adding more organism classes, the laboratory continues adding to the list of specific organisms it can identify by MALDI-TOF. “There are a lot of uncommon or rare bacteria or yeasts that are present in the instrument database,” he acknowledges, but he wants the laboratory to gain more experience with MALDI-TOF before relying on results for clinical use.

Even longer term, he predicts a time when MALDI-TOF might be applied to areas that are only being investigated now, including antimicrobial susceptibility testing, strain typing, or even application to primary specimens, without the need for culture.

And in the near future, he notes, there’s other news: A CLSI document, M58, is likely to be published this year, offering guidance on MALDI-TOF use. “It’s a practical guideline for performing MALDI-TOF mass spec for microorganism identification.”

At times, an almost elegiac element slips into the discussions of MALDI-TOF and its role in the laboratory: Who among you is maintaining the custom of your ancestors?