Diagnostic Testing for Fungal Infections

New technology is being developed to help professionals detect and identify yeasts and molds

Fungal infections-on the rise due to an increase in immunocompromised patients-pose a critical threat to hospitalized populations. The laboratory has witnessed a shift away from the hands and scrutiny of the individual to more molecular and proteomic based methods in processing clinical specimens to isolate and identify fungi.

“In recent years, clinical laboratories in the United States have been challenged to detect and identify the increasing diversity of fungal pathogens,” explained Michael A. Pfaller, MD, is Emeritus Professor of Clinical Microbiology, Department of Pathology at University of Iowa College of Medicine and College of Public Health in Iowa City, Iowa and Chief Medical Officer for T2Biosystems located in Lexington, MA.

Classical mycology requires skill and experience to identify the structural characteristics of fungi, according to Pfaller, a physician, educator and researcher in the areas of antifungal agents and resistance, and the role of the clinical microbiology laboratory in hospital infection control.

According to Pfaller, there have been improvements in the lab’s ability to identify fungi to the species level facilitated by molecular testing based on DNA sequence analysis and by the development of proteomic methods such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). These methods are more frequently employed in larger institutions where the organisms are a bigger problem than in community hospitals.

“The classical mycologic expertise is being lost among clinical laboratory personnel due to the lack of training in this area,” Pfaller observed. “The newer molecular and proteomic approaches can help mitigate this lack of training but not all hospitals can afford to employ these newer methods so in general the laboratory struggles with the identification of all but the most common fungi.”

According to Pfaller, more uncommon fungi are seen causing infections as the population of immunocompromised patients in the United States grows. The patients at highest risk for invasive fungal infections are the recipients of both stem cell and solid organ transplants as well as those patients hospitalized in intensive care units. “The recipients of stem cell and bone marrow transplants are often placed on antifungal prophylaxis which works well against the common fungi such as Candida and Aspergillus species,” Pfaller explained. “Unfortunately we are seeing the development of opportunistic infections due to less common organisms such as Fusarium and the Mucormycetes in these patients.”

Invasive aspergillosis (IA), an opportunistic fungal infection, leads to high mortality rates in immunocompromised patients. Along with medical breakthroughs in the areas of cancer, and bone marrow and solid organ transplants, the industry has witnessed a rise in patients developing the often-fatal infection. Traditional laboratory methods that are often invasive, time-consuming and insensitive have made the infection difficult to diagnose.

Candidemia, the most common fungal bloodstream infection, has been subject to insensitive and slow diagnostic testing. According to Pfaller, the mortality rate for candidemia has not changed in the last 20 years despite the introduction of new and effective antifungal agents because infections are being diagnosed too late.

With new technology being developed to help professionals detect and identify yeasts and molds, Pfaller is hopeful that mortality rates can improve. “Improvements in the sensitivity, specificity and timeliness of fungal diagnosis are directly dependent on the application of new technology in the field of medical mycology,” he said.

Diagnostic Process

The laboratory provides a range of diagnostic tests for the detection and identification of superficial and invasive fungal infections, including cultures, molecular and proteomic methods, serologic tests, and antifungal susceptibility testing. The downfall of most of these approaches, according to Pfaller, is that most depend on the growth of fungi in culture, a process that is both slow and insensitive.

“One of the keys to successful treatment is getting the correct treatment on board early,” Pfaller shared. “We don’t do that with most patients with invasive fungal infections today and that is a problem.”

Antifungal susceptibility testing, an in vitro approach of determining the ability of antifungal agents to inhibit or lill the infecting fungal pathogens, has been around since the late1980s. The industry has finally standardized these methods so labs performing the tests correctly will achieve consistent results and be better able to determine which drugs can work for infections, according to Pfaller.

About 400 labs in the U.S. perform this testing on fungal isolates infecting high-risk solid organ transplant and stem cell patients as well as trauma patients. Antifungal susceptibility testing methods-available from one of three commercial suppliers in the U.S. -are mainly  used in large tertiary care medical centers, Pfaller told ADVANCE.

The commercially available antifungal susceptibility testing products include the Sensititre YeastOne (Thermofisher), the Vitek2 Yeast panel (bioMerieux) and the ETest (bioMerieux). In each case, the test method allows the laboratory to determine the minimum inhibitory concentration (MIC) of one or more antifungal agents against the pathogen of interest. Presently all three methods are FDA cleared for testing Candida species.

New Technology

T2 Biosystems’ T2Candida, a recently FDA cleared molecular diagnostic test can be performed without culture, sample manipulation or nucleic acid extraction and gives rapid results, according to Pfaller. Results can be achieved within 3 hours directly from whole blood without culture.

“This test can both detect and identify the major species of Candida directly from the patient sample with a combination of speed, sensitivity and specificity unparalleled in the field of diagnostic microbiology,” he said. “It has the potential to make a big difference in the timely and appropriate administration of antifungal therapy with potentially significant impacts on mortality, length of hospital stay and antifungal drug costs.”

In Europe, according to Pfaller, clinicians employ molecular testing for Candida and other organisms (primarily bacterial organisms) on serum or whole blood following DNA extraction. One widely employed method outside of the U.S. is the SeptiFast real-time PCR system by Roche Molecular Systems Inc. This system has not been FDA cleared and is not available in the U.S. “Its performance has been difficult to evaluate because of a variety of different study designs that makes specificity and sensitivity hard to define,” Pfaller said.

New non-culture-based serological and PCR assays are in development and show promise. The Lateral-Flow Device (LFD) test is a rapid (15 minute) single-sample point-of-care test that is based on the detection of an Aspergillus extracellular glycoprotein antigen by monoclonal antibody JF5. “These tests improve our ability to diagnose patients with aspergillosis and coupled with mold active anti-fungal agents have the potential to decrease mortality overall,” Pfaller stated.

Another frequently employed test is the FDA-approved Fungitell ß-D Glucan assay, indicated for the presumptive diagnosis of invasive fungal disease through detection of elevated levels of (1,3)- ß-D-glucan in serum. “This test detects the presence of cell wall carbohydrates of several different fungi in the serum of infected patients,” Pfaller explained.

The non-specific test for fungi has been used to help identify patients who do not need antifungal agents by virtue of a relatively high negative predictive value, Pfaller shared. “It’s primarily a send-out test that is conducted at a reference laboratory,” he said. “It is rarely performed in clinical labs because it’s tedious and difficult to perform. The down side of the test is that it has many false positives.”

Pfaller believes molecular tests in development hold promise in identifying organisms missed by blood culture. “Unfortunately, aside from the T2Candida test, most methods for identification of Candida from clinical specimens require a culture step prior to performing the test resulting in a significant delay in providing the information to the clinician.”

When new tests that are well validated and have good detection of organisms are introduced, clinicians want to know the resistance markers or sensitivity results. “We need to develop molecular markers that would be helpful for clinicians,” Pfaller observed. “Tests presently under development have the potential to provide such information in the near future.”

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