Adventures in culturing—five micro lab tales

Ann Griswold, PhD

October 2013—Culturing organisms is not always easy. Some microbes are fastidious. Others are below the limits of detection. And still others are just difficult to culture, for no discernible reason.

But set aside baffling cultures of the past because clinical microbiology is about to get more interesting than ever before.

“Clinical microbiologists should be aware that their laboratories are going to be increasingly asked to culture samples they’re not used to working with, samples that are not coming from patients,” says Matthew Arduino, MS, DrPH, FSHEA, lead microbiologist and chief of the Clinical and Environmental Microbiology Branch of the Division of Healthcare Quality Promotion at the Centers for Disease Control and Prevention’s National Center for Emerging and Zoonotic Infectious Diseases.

“They’re being asked to do more and more environmental microbiology.”

Those cultures will not always be straightforward, he says. “It may be a medication. It may be someone asking you how to sample a device that might be causing a problem with a patient. It might be performing QA for the dialysis center. It might be performing QA for the pharmacy.”

Whatever it is, Dr. Arduino says, the key to success lies in a careful approach. Here are the lessons learned by five clinical laboratorians who have faced these challenges head on.

Clinical laboratories have always played a critical role in patient safety. But focusing on patient safety no longer means focusing on specimens obtained directly from patients, says Alice Weissfeld, PhD, D(ABMM), president, CEO, and laboratory director of Microbiology Specialists Inc. (MSI), Houston. “We’re going to have to stretch and examine things that are not natural fits for the clinical laboratory to be able to prevent infections and really help the patients.”

As a long-standing American Society for Microbiology delegate to the United States Pharmacopeial Convention, Dr. Weissfeld is leading the push for mandatory environmental testing of hospital compounding pharmacies. Frequent recalls of sterile products have shed light on this important issue. “Monitoring the pharmacy environment is as important as any patient culture,” she says. “We cannot let this fall by the wayside.”

Contamination in the pharmacy is a bigger problem than most people think, she notes. An article by Dr. Weissfeld and colleagues in the October issue of the Journal of Clinical Microbiology (2013;51[10]:3172–3175) examines five years of environmental sampling data from 30 hospital pharmacies and two freestanding pharmacies, and found that, on average, 35 percent of the pharmacies failed to meet USP standards every year. “When I ask pharmacists that I know how much contamination they actually think there is in the pharmacy, some say, ‘Oh, probably about one and a half percent.’ But that’s certainly not what we found.”

Dr. Weissfeld cites the recent outbreak of fungal meningitis that was traced to contaminated epidural steroid injections produced at the private NECC compounding pharmacy in Massachusetts. “Even though it was a freestanding pharmacy, some of that medication was administered in hospitals. We need to be prepared to help limit the damage from this type of problem.”

The USP has established recommendations for monitoring the pharmacy environment. “Under USP chapter 797, there are five activities that a clinical microbiologist can participate in with the pharmacy,” she notes. One is media-fill testing, which mimics the process of making sterile compounded preparations but uses tryptic soy broth (TSB) instead of the compound. The broth is then incubated for 14 days to check for contamination. Other activities include gloved fingertip testing, direct impaction air sampling, and surface sampling of hoods and compounding surfaces.

Dr. Weissfeld’s company, MSI, performs this work on a contract basis for hospitals. But to be most effective, she says, these activities should be performed on site by the hospital’s own laboratories, reserving the use of private labs for bioremediation and consulting. Most hospitals are open 24/7, and the test results would be available more rapidly from a hospital lab than from an environmental laboratory, which may only be open Monday through Friday, from nine to five.

“There’s a perception amongst the community that doing environmental sampling will somehow interfere with work on clinical cultures,” Dr. Weissfeld notes. “But this is not rocket science. These activities can be easily added. Yes, the media is slightly different [for environmental testing], but it’s not media that people who are clinical microbiologists would be unable to read.”

Private laboratories could continue to handle the challenging aspects—interpreting and addressing test results that exceed the limits described in the USP chapter. “I’m not asking clinical microbiologists to become bioremediators,” she says. “But I think the daily, weekly, and monthly sampling should be done in-house.”

Under increasing pressure to do more with less, hospital laboratories may find it difficult to add environmental testing to their plates. Environmental testing often falls into the category of things that do not have a direct impact on patient care, particularly in clinical laboratories that are dealing with cuts in staff, hiring freezes, and other constraints. “But when you’re looking down a list of things that you need to do, pharmacy testing is not one that should be thrown away,” she cautions. “Because if you are injecting a nonsterile product, the patient is either going to die or get very sick.”

Dr. Weissfeld eschews the idea that pharmacists are in denial about the widespread problems with environmental contamination. “The disconnect is that they are out there by themselves. As with anything, this should be a team approach.”

One problem, she notes, is that most pharmacists have only a basic microbiology background and are not aware of the implications of pathogens for certain patient populations. That’s where close collaboration with a clinical laboratory would come in. “For example, if a hospital sees a lot of cystic fibrosis patients, the presence of Burkholderia spp may carry implications far beyond that of a gram-negative rod and source of endotoxin. In fact, infection with Burkholderia spp can have a profound effect on survival both pre- and post-lung transplantation. Clinical microbiologists know their patients and therefore would be able to respond quickly, if necessary.”

Performing environmental testing in-house could circumvent this problem by harnessing the clinical laboratory’s knowledge of its own patient population, and of the hospital’s resident pathogens. “It makes for a much more dynamic situation than if the pharmacy or an outside lab just counts numbers of colonies and identifies things, with no idea what any of it means. Microbiologists know what the results mean and can help the pharmacy interpret them.”

The next time Dr. Weissfeld surveys environmental sampling data from hospital pharmacies, she hopes to see a much lower failure rate. And that means finding better ways to support the hospital pharmacy. Organizations like the CDC, she says, are considering bringing together groups of clinical microbiologists and other stakeholders to establish guidelines for environmental testing in compounding pharmacies. Other efforts could be natural outgrowths of existing antibiotic stewardship programs, for example.

“If we can show pharmacists what they need to do—if our hospitals can better support them—then we can better protect patients as well as the hospital itself.”

Culturing unusual objects is simply part of the job for the Clinical and Environmental Microbiology Branch of the CDC. Microbiologists in the branch perform outbreak support, environmental surveillance, preparedness research, and susceptibility testing of resistant organisms such as carbapenem-resistant Enterobacteriaceae and methicillin-resistant Staphylococcus aureus.

“If it’s epidemiologically linked, we will sit here and figure out how to do it,” Dr. Arduino says. Though he has faced many daunting tasks, one of the most memorable involved a large outbreak in a neonatal ward.

The outbreak was traced back to instruments used to humidify air for the infants’ CPAP machines. When one of the suspect devices was shipped to the CDC for inspection, Dr. Arduino and his colleagues discovered that Ralstonia pickettii—a water-borne organism with a penchant for nutrient-poor environments—had formed a biofilm on the inside of the device. Sure enough, cultures from the instrument matched those from the infants. But how the pathogen made its way from the device to the patients’ airway remained a mystery. “There was a membrane, and we didn’t know how [the pathogen] was crossing through to the patient. It should have been a separate circuit,” Dr. Arduino recalls. Finally, the team unearthed an important clue: “We found out that the company manufactured this device overseas, and that one of the final steps before they shipped it was rinsing it with tap water.”

From this and countless other experiences, Dr. Arduino has learned an important lesson about human nature: “You can invent a better mousetrap but people will always find a way to circumvent it,” he says. Most problems occur when workers in manufacturing and clinical settings eschew microbiologists’ appreciation for cleanliness. He has seen a number of examples over the course of his career.

On Dr. Arduino’s first day of work at the CDC, he investigated an outbreak linked to a dialysis facility in California. Multiple patients had developed systemic infections with abscesses.

“When I first began investigating dialysis outbreaks, more than half of the facilities were performing manual reuse on the artificial kidneys,” he recalls. “There was somebody in a back room, usually a low-paid technician, who was rinsing the residual blood from the dialyzer, doing a pressure test, and then filling it with germicide so that it could be used on that same person again when they came in for the next session.”

The investigation revealed that the dialysis water was contaminated with a mycobacterium (Mycobacterium abscessus). When the CDC team dug further, they learned that the facility had recently switched from formaldehyde as its germicide to peracetic acid—but they were only using half the required strength. “The salesperson had said, ‘You know, this stuff is really good. The label says to use this concentration, but you can use half,’” he recalls. But that wasn’t the only precipitating factor. Dr. Arduino and his colleagues observed the facility’s cleaning process and realized that the technicians were filling the dialyzers with only half the recommended amount of germicide. The residual water remaining in the dialyzer diluted the germicide further, and the water used to rinse the dialyzers and prepare the disinfectant contained mycobacteria, which were not killed by the disinfectant placed into the dialyzer. The resulting outbreak was another byproduct of misinformation and inadequate training.

Communication between all workers in the clinical environment, including janitorial staff, is crucial, he says. In another outbreak he investigated—this one unrelated to dialysis—the only common exposure was a portable x-ray machine. The CDC obtained a sponge sample from various parts of the machine and recovered Acinetobacter baumannii from the surfaces that touched the patient.

“And then, the question was asked, ‘Who cleans the machine?’ Nursing said, ‘We don’t clean. That’s housekeeping.’ Housekeeping said, ‘No, no. That’s an instrument. We don’t touch those. That’s nursing.’ So the machine was never disinfected between uses,” he says.

A career’s worth of experiences like this one have taught Dr. Arduino a few things about culturing challenging objects. Most importantly, he says, a negative result doesn’t always mean the culture is negative. It’s essential to use techniques and media appropriate to the object being cultured, and to realize when an outdated or inappropriate technique might be raising the limit of detection.

As evidence, he points to the gradual evolution of standards for testing the water used in hemodialysis. History has shown that the ability to detect contaminants depends in large part on the methods and reagents used—and on the willingness to break from tradition in favor of techniques better suited to a novel task.

Today, most dialysis samples are sent to an environmental laboratory or a dialysis company laboratory for testing. But when clinical microbiology laboratories were first faced with the challenge of testing dialysis fluids in the early 1980s, Dr. Arduino says, standard clinical culture methods hindered the detection of dialysate contaminants, leading to many false-negatives.

In one outbreak, Dr. Arduino recalls, several dialysis patients developed fever, chills, and clear signs of bacteremia despite negative dialysate cultures. It turned out that the samples were cultured on blood agar, the standard substrate for patient specimens. “But microbes that you find in water or dialysate tend to be nutrient-poor,” Dr. Arduino says. “So when they inoculated a chocolate plate or a blood agar plate, the organisms would actually die because of the richness of the media.”

In addition to the rich agar, many clinical microbiologists at the time were using calibrated loops, which are typically used for urine analysis, to plate the dialysate samples. As a result, they missed the cutoffs for detection, Dr. Arduino says.

Because of this long and complicated history, he worries that some pathogens will continue to evade detection by routine screening due to inconsistencies in the practices various labs use. “Some laboratories are still doing membrane filtration; some are doing spread plates,” he notes.

Part of the problem may stem from discrepancies between the relatively stringent recommendations for testing hemodialysis fluids set by the International Organization for Standardization (ISO), and the older, more liberal standards established in 2004 by the Association for the Advancement of Medical Instrumentation and required of labs that are reimbursed by the Centers for Medicare and Medicaid Services. The detection limits in the U.S. are somewhat less stringent than those used abroad, Dr. Arduino notes, and not all labs adhere to the same standards. Labs that go the extra mile by adhering to ISO standards are more likely than other labs to identify contaminants in dialysis fluids.

Outbreak investigators like Dr. Arduino typically know what organism they’re looking for—the challenge lies in locating the contaminant on medical devices. But many clinical laboratories face the opposite challenge: identifying the underlying pathogen in the first place. And sometimes it’s unclear whether there’s even a pathogen at all. That’s the frontier explored by Amy Leber, PhD, D(ABMM), clinical assistant professor of pathology and pediatrics at The Ohio State University and director of clinical microbiology and immunoserology, Nationwide Children’s Hospital, Columbus.

When patients are treated for injuries that involve foreign objects, Dr. Leber says, the challenge lies in determining what objects are appropriate to culture. In some cases, the patient does not have an infection and the culture is a preemptive measure. In these cases, there’s little evidence to guide the decisionmaking.

“There’s a movement toward evidence-based medicine to verify the utility of our practices in the laboratory,” says Dr. Leber. “But in a lot of instances, there is no evidence. Sometimes things are done a certain way because we’ve just been doing it that way for many, many years.”

Over the course of her career, Dr. Leber has been asked many times to culture a foreign object, but none compare to the time she was asked to culture a pencil that had been extracted from a child’s brain tissue. The child had fallen onto the sharpened pencil, and during the surgery, a sterile baggie containing the pencil was sent to the pathology laboratory.

“Would you have cultured it? Are there any benefits to culturing that pencil, in terms of guiding the physician?” In most cases, Dr. Leber says, the activity is similar to a fishing expedition. In the case of the pencil, her concerns were multifold. “No. 1, if it’s cultured, the organisms we grow are not necessarily those that will take seed and cause infection. No. 2, the pencil was not handled sterilely by the EMS and everybody who handled the child on the way to surgery, so the organisms that grow may not represent what actually was in the brain. And third, there is no way the surgeons were going to be able to routinely access that site again to get cultures. So this was an irretrievable sample.”

After mulling it over, the third concern gave Dr. Leber pause. Conversations with the clinician revealed his concern about the presence of highly resistant organisms on the pencil that might alter that therapy, so Dr. Leber reluctantly agreed to culture it. “We had a one-time ability to culture something that might potentially relate to a deep-seated infection in the brain. So we went against what would otherwise be the guidance,” she says. In the end, the culture didn’t alter the patient’s therapy and she’s still not convinced it was a good use of resources.

“But this is the dilemma,” she says. “Often what happens is that laboratory technicians or managers are forced to either acquiesce to clinicians’ demands and culture things that don’t make clinical sense, or they have to have some kind of evidence or starting point to explain why it’s not a good idea.”

Most object-related injuries are treated empirically based on the flora at the body site and what’s known about the injury, she says. “If you have a penetrating wound into the gut, you’re going to treat based on the enteric flora in the gut. If you get injured in water, you’re going to cover for water-related organisms, like Pseudomonas.”

But the answer is not always obvious. Consider a child who gets a wood splinter embedded in her hand. The child’s pediatrician might try to remove the entire splinter, but suppose a small part remains and the child develops a slow, smoldering abscess. Once the rest of the thorn is removed, the site of infection will be cultured—but should the foreign material be cultured as well? “Sometimes you look at an injury, and there will be a part of the object that is left behind. So it’s more of a chronic injury. In those cases, there’s more evidence to suggest that it might be useful to culture that object.”

In the case of the pencil, she says, there were no evidence-based guidelines to light the path. “It’s all based on historical practice,” Dr. Leber notes. “You can’t predict if someone will get an infection. So there might be labs out there that just routinely would take these objects and culture them.”

The lesson, she says, is that sometimes it’s necessary to say no. “If you have a very strong microbiology lab where the physicians respect their expertise, the microbiologists might say, ‘No, we don’t culture those things,’ and they won’t. Other times, clinicians might throw their weight around a little and get the lab to culture these objects. But the message is that you can say no to these requests, because they’re not always a good use of resources.”

Evidence-based medicine and strong communication between laboratories and clinicians are crucial not just when culturing foreign objects but also when it comes to autopsy cultures.

“Autopsy cultures are a standard activity, but there is so much variety among facilities,” says Carol Rauch, MD, PhD, associate professor of pathology, microbiology, and immunology; associate medical director of clinical laboratories; associate medical director of Vanderbilt Pathology Laboratory Services; and medical director of the One Hundred Oaks Diagnostic Laboratories at Vanderbilt University School of Medicine.

The usefulness of autopsy cultures depends in large part on the quality of the specimen.

“In an autopsy environment, unless you have a good specimen, it’s very easy to grow things that have nothing to do with what infected the patient,” she notes. “You might grow what was in a contaminated environment, or what came out of a patient site that already had a lot of bacteria and fungi living there. Reporting that information isn’t helpful, and it may be dangerous if it’s misinterpreted.”

Dr. Rauch shares the concerns of Dr. Leber and others that laboratory directors are charged with the important task of helping clinicians determine what cultures might yield meaningful results, and what cultures might be a waste of resources, either because the results would have negligible impact or because the results would be extremely difficult to obtain.

Microbiologists have well-stocked toolboxes: Specialized media can be used to grow fastidious organisms; incubation times can be extended to allow slowly growing organisms to be identified in culture. But the request to perform a culture must be based on a solid clinical question. If not, the answers will be meaningless or misleading. “Microbiology laboratories answer clinical questions. To do that, we need a good specimen and ideally some information about what the clinician is thinking so that we can frame what we do in the laboratory.”

Dr. Rauch recalls when a technologist in her lab was once asked to work up 11 organisms discovered during an autopsy, at the request of a trainee who seemed to not understand the issues or extensive work involved. “That is tremendously laborious, and the growth of many organisms very likely reflects a contaminated specimen rather than organisms related to disease in the patient,” she notes. “Excessively manipulating tissue or the body can increase the chances of false-positive results, and having 11 organisms is a red flag for not reflecting an actual disease process.”

Often this type of request reflects what Dr. Rauch calls a “worry about it later” approach by the person ordering the cultures—an approach that is increasingly difficult to justify in today’s resource-constrained environment.

With strong teamwork between laboratories and clinicians, Dr. Rauch argues, autopsy cultures can provide important clues that can improve patient care overall. Last year, Vanderbilt’s clinical microbiology team illustrated just how valuable autopsy microbiology can be, when they cared for the index case in a large fungal meningitis outbreak. “Our patient’s diagnosis was made during life and the infection was treated, but unfortunately this particular infection was fatal,” she says. The patient’s illness had challenged clinicians and laboratory personnel at Vanderbilt to think out of the box. When the patient died, the team was still evaluating the smoking gun.

In a New England Journal of Medicine article (Pettit AC, et al. 2012; 367[22]:2119–2125), Dr. Rauch and colleagues described the index case as an immunocompetent man with persistent neutrophilic meningitis but no evidence of sinopulmonary or cutaneous disease. An autopsy was performed to help confirm and analyze unusual antemortem findings that included growth of a mold, Aspergillus fumigatus, from cerebrospinal fluid.

The early stages of the outbreak investigation were filled with challenges that pushed the boundaries of routine autopsy culture. “We needed to obtain many more specimens and higher-volume specimens. We needed to hold our cultures for longer incubation periods. We were sending a lot of specimens through the public health system to the CDC, where new tests were being developed rapidly, including a new fungal PCR test to identify a broad group of fungi.”

The CDC Infectious Diseases Pathology Branch examined many cases, further aiding the outbreak investigation and management. A clinical microbiology fellow at Vanderbilt assisted in obtaining high-quality autopsy specimens for use in multiple laboratory tests, and state and federal public health partners quickly applied communication systems, new laboratory tools, guidance documents, expert panels, and other tools.

During the search for agents involved in the outbreak, autopsy culture findings were front and center. “Given the often ho-hum attitude toward autopsy, it was refreshing to see that it also continues to play an important role in important problems,” Dr. Rauch says. Although the initial case was an infection with Aspergillus fumigatus, most of the cases in the outbreak attributed to contaminated medication injections have been infections by Exserohilum rostratum.

Above all, the experience underscored the importance of teamwork. “Our efforts are strongest when we all work together and connect our different areas of expertise to serve others,” Dr. Rauch says.

Of the various types of infection that require clinical culture, prosthetic joint infections are among the most severe. “The stakes are high,” says Aaron Tande, MD, a clinical and research fellow in the Mayo Clinic’s Division of Infectious Diseases. “A prosthetic joint infection typically requires at least one—usually more than one—major orthopedic surgery, and then a prolonged period of IV and possibly oral antibiotics.”

When such an infection occurs, the prosthetic joint may need to be surgically removed and cultured to diagnose the infection and determine the most appropriate therapy. But isolating organisms from these devices is no easy task, particularly when patients have already been treated with one course or more of antibiotics.

There is some consensus in this area: The Infectious Diseases Society of America’s guidelines for diagnosis and management of prosthetic joint infection have clearly stated what constitutes a prosthetic joint infection, for example. In cases of suspected infection, the guidelines recommend assessing inflammatory markers, culturing tissue from the area around the prosthesis, and obtaining fluid from inside the joint before the operation to measure white blood cell counts and see if the fluid can be cultured.

But several uncertainties remain. In particular, Dr. Tande points to the controversy over the role of sonication to dislodge any bacteria from the device into the surrounding fluid, which is then cultured to check for contaminants. The method is particularly useful for recovering microbes from the prosthetic joints of patients who have been treated with antibiotics before surgery.

“It’s a newer technique. Most studies have shown that it is more sensitive than tissue culture, but it takes some time for these things to make it into the guidelines,” he notes. The real controversy is about whether the use of sonication is worth the extra hassle. Culturing prosthetic joints is much more labor-intensive than culturing periprosthetic tissue, and sonication only increases the complexity of the culturing process. “The question is, how much more advantageous is it to use sonication?”

Another uncertainty in culturing prosthetic tissues and periprosthetic tissues relates to incubation times. “Some bacteria that cause prosthetic joint infections, like Propionibacterium acnes, are classically described as slow growers,” Dr. Tande says. “This forces microbiologists to extend incubation times, which carries the risk of environmental contaminants and false-positives.”

These challenges can be overcome, at least in part by close collaboration between laboratories and clinicians. “A team-based approach can prove essential if something doesn’t smell right from the lab’s standpoint,” he says. “Clinicians want to know if the microbiology lab has suspicions about a sample, or if clinicians need to provide additional information that might better help to frame a result.

“In the end,” he adds, “it’s all about trying to help patients through improved methods of diagnosing and treating infections.”

Ann Griswold is a writer in Annapolis, Md.