May 2011: Volume 37, Number 5
Rapid Identification using PNA FISH
By Donna M. Wolk, PhD, DABMM, and Linda D. Hilbert
Every year in the U. S., 350,000 patients acquire bloodstream infections while in the hospital, resulting in more than 90,000 deaths and significant costs to the healthcare system. Rapidly detecting and identifying the infectious pathogens is therefore critical for favorable patient outcomes. However, blood culture, the gold standard for diagnosis of bloodstream infections, often takes several days. Consequently, clinicians typically treat suspected bloodstream infections with broad-spectrum antibiotics while waiting for the patient’s blood culture results, a practice that, while necessary, has other undesirable effects.
Recently, new molecular assays have become available commercially that rapidly identify bloodstream pathogens, giving clinical laboratories an excellent opportunity to improve care of these patients. Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is an accurate fluorescent probe-based method that provides confirmation of genetic sequences from common microbes found in positive blood cultures, such asStaphylococcus aureus,Candida albicans,Enterococcus faecalis, andEscherichia coli. Based on this information, clinicians can select appropriate antimicrobial therapy for patients within hours—rather than days—of receiving confirmation of a positive blood culture result.
The PNA FISH assay fits the workflow of any clinical laboratory. Here we describe how the test works and how laboratories can use the test to speed up positive identification of pathogenic blood micro-organisms.
Traditional Blood Culture
To test for bloodstream infections, clinical microbiology laboratories typically inoculate liquid growth media with a blood sample from the patient, and an automated blood culture instrument monitors the culture for bacterial growth. The laboratory then performs a Gram stain on positive cultures and reports the results to the clinician. While the Gram stain helps determine the type of bacteria present, it does not definitively identify the specific microorganism causing the patient’s infection. After receiving the patient’s result, the clinician must continue to closely monitor additional test results over the next several days that identify the causative microbial species, as well as its antibiotic susceptibility.
Until recently, the importance of identifying the specific microbial species once the Gram stain result has been reported and its impact on switching from broad-spectrum antibiotics to more targeted species-specific antimicrobial therapy had largely gone unrecognized. However, studies have now shown that rapid targeting of antimicrobials and de-escalation from broad-spectrum antibiotics is critical to optimal recovery for patients with bloodstream infections (1–3), as well as to the efficient use of healthcare resources (4–8).
Advantages of the PNA FISH Method
The PNA FISH method has many advantages for identifying pathogens in positive blood cultures. Most importantly, it provides microbial species confirmation in as little as 90 minutes, whereas traditional methods used to definitively identify and differentiate between species take from 1–5 days after identifying a positive blood culture (Figure 1). Although Gram staining provides positive identification of staphylococci from blood cultures, the procedure typically takes overnight, and species confirmation also requires further subculture and biochemical analysis. In contrast, PNA FISH provides an accurate result shortly after a positive Gram stain. With PNA FISH, there also is no need to isolate colonies on agar media for additional testing. Not only is the method simple, but most laboratories already have the necessary tools in place that make adding the test to their menu easy. Studies from various laboratories demonstrate that the method is highly accurate, with high sensitivity and specificity (5–10).
Traditional vs. PNA FISH Identification of Bloodstream Pathogens
Courtesy of AdvanDx
The advantages of using PNA FISH to identify specific pathogens also have been documented in hospitals that use a system-based approach to optimize antimicrobial therapy, involving laboratory scientists, pharmacists, and physicians. For example, because the results of the test are available very soon after a positive blood culture, clinicians no longer need to second-guess a Gram-stain result or continue broad spectrum antibiotics for days while waiting for a confirmatory laboratory result. Patients also do better because they avoid the potential drug toxicity and resistance of broad spectrum antibiotics. When correlated with hospital antibiogram data and patient assessment, rapid species identification by PNA FISH enables pathogen-directed therapies that have been shown to reduce mortality, as well as length of hospital stays and treatment costs (4–8).
How the Assay Works
PNA FISH is based on complementary hybridization between a commercially available probe (AdvanDx and bioMérieux, Inc.) and the target microorganism. The assay uses fluorescently labeled PNA probes that target species-specific ribosomal RNA (rRNA). These probes contain the same nucleotide bases as DNA; however, the PNA hybridization probes contain a non-charged polyamide or peptide backbone rather than the negatively charged sugar-phosphate backbone of DNA probes.
The design of the PNA probes provides several unique advantages over traditional DNA probes used in whole-cellin situhybridization assays (11). First, PNA probes deliver superior hybridization characteristics because, unlike DNA probes, their net electronic charge is zero. This means that PNA probes are not subject to repulsion between two negatively charged species as are DNA probes. The net result is that PNA probes have faster hybridization kinetics that allows the small, dye-labeled probes of about 12–20 base pairs to hybridize faster to the target of interest. This unique chemistry also facilitates improved base discrimination for both DNA and RNA targets.
The hydrophobic character of PNA probes also enables them to penetrate the hydrophobic cell wall of bacteria more easily during the actual hybridization process. In addition, by using several PNA probes with different labels, one test can simultaneously identify multiple bacterial species.
Performing the Assay
To perform the test, the technologist prepares a blood smear on a microscope slide using a drop of fixation solution, followed by a drop of the positive blood culture broth (Figure 2). The hybridization step starts by adding a drop of the PNA probe to the microscope slide, which is then incubated at 55ºC for 30 minutes. Next, the technologist washes the slide with a preheated, prepared buffer solution, incubates it at 55ºC for an additional 30 minutes, and then places one drop of mounting medium on the smear before looking at it under a fluorescence microscope. The entire process, from set-up to reading the slide, takes approximately 90 minutes, most of which is incubation time.
Example of PNA FISH Method for Staphylococcal Species Identification
Courtesy of AdvanDx
The PNA FISH probes provide very distinctive results for identifying bloodstream pathogens. Under a fluorescent microscope,S. aureusappears as multiple bright-green fluorescent clusters of cocci with theS. aureus/CNS PNA FISH probe, while coagulase-negative staphylococci (CoNS) appear red (Figure 3). Similarly, with the PNA FISH test forE. faecalis/other enterocci,E. faecalisappears green with distinct fluorescing Gram-positive cocci in pairs and chains, while red fluorescing cells indicate other enterococci. TheC. albicans/C. glabrataassay produces green fluorescing cells forC. albicansand red fluorescing cells forC. glabrata. The absence of fluorescence on the slide indicates that some other micro-organism is present in the positive blood culture that does not react with the species-specific probes.
The Process for PNA FISH Assays
1. Prepare Smear
4. View and Interpret Results
Courtesy of AdvanDx
The most frequently isolated microorganisms from blood cultures are staphylococci, which are characterized by their clustered morphology and Gram-positive stain. S. aureus (SA) is coagulase positive, but more than 30 species of coagulase-negative staph (CoNS) exist, 15 of which are human pathogens.
After Gram staining reveals that a blood culture is positive for staphylococci, it is important to identify whether the organism is coagulase-positive or negative. Coagulase-positive results implicateS. aureusas the source of the infection. This highly virulent bacterium must be treated quickly with appropriate antibiotics. A finding of CoNS presents a different set of issues. With CoNS, clinicians must decide whether the positive culture indicates a true infection or contamination of the culture by skin flora from the blood collection (8). Further compounding diagnostic decision making is the fact that CoNS only produce a true bloodstream infection about 20% of the time. Consequently, while skin contamination of the blood culture may be the cause of the positive test result, clinicians often start patients on antibiotics while awaiting species confirmation. Unfortunately, this option results in extra days of vancomycin therapy and adds to an ever-growing population of patients whose bacterial flora has become vancomycin-resistant (5,8,12).
Enterococcus Species Identification
Another common cause of hospital-acquired bloodstream infections is enterococcal bacteremia (7). Two predominant species account for most enterococcal infections:Enterococcus faecalisandEnterococcus faecium. Standard detection methods for these pathogens involve a prolonged series of steps over the course of several days. As a result, clinicians typically initiate vancomycin therapy, a practice that has promoted the emergence of vancomycin-resistant enterococcal bacteremia (VRE). In one study, researchers showed that early, appropriate, and accurate diagnosis of enterococci markedly decreased length of stay in a hospital setting, as well as the intensive-care unit (7). With PNA FISH tests results for enterocci, clinicians either can quickly discontinue vancomycin and replace it with ampicillin whenE. faeciumis identified, or initiate other therapy when VRE is suspected.
Candida Species Identification
The traditional procedure to identify yeast from a Gram-positive stain is to subculture the organism on Sabouraud dextrose agar, a selective medium for fungi and yeasts, followed by biochemical testing, a process that takes as long as 4–7 days. Alternatively, microbiology laboratories also subculture on CHROMagar Candida (BD Diagnostics), a selective medium for the isolation and presumptive identification of yeast and filamentous fungi. This overnight process provides results faster, but still takes an additional 18–24 hours after a positive blood culture .
While fluconazole is the treatment of choice for yeast infections, the prevalence of other Candida species with various resistance patterns means that correctly identifying the causative species at the outset reduces improper therapy, as well as fluconazole resistance. For example,Candida kruseiis inherently resistant to fluconazole andC. glabratamay exhibit dose-dependent susceptibility or easily acquired resistance. Researchers have found that use of PNA FISH to identify Candida species not only decreases mortality, but also saves costs in some settings (4,6).
Setting Up PNA FISH Testing
Besides common laboratory equipment like a covered slide warmer and water bath, the PNA FISH assay requires a fluorescence microscope fitted with a dual FITC/Texas Red filter. Overall, start-up costs are minimal, and most laboratories have the necessary major equipment already in place.
In the beginning stages of training, it is a good idea to have technologists read the slides in tandem. This facilitates consistent procedures and gives lab staff additional practice using the technique. This training period is relatively short due to the high specificity and sensitivity of the assay, enabling technologists to work on their own after a fairly short period of time. The two-color format of the assays also eliminates the need for technologists to pursue additional costly and time-consuming workups, as accurate identification only requires reactions of both the green and red probe sets. Furthermore, technologists see both the organism’s familiar morphology and fluorescent color under the microscope. The cellular morphology remains intact during the assay protocol, adding an additional level of confidence for species differentiation.
Currently, nine FDA-cleared kits are available for hospital laboratory use in directly identifying Staphylococcus species, Candida species, Enterococcus species, and gram-negative bacilli from positive blood cultures. Specific needs of the local patient population dictate which of the various kits a laboratory should implement (Figure 4). Gram-stain results direct the kit selection. For a positive blood-smear with Gram-negative rods, laboratories can choose from three combination PNA FISH assays, including the latest to receive FDA clearance in December 2010, the GNR Traffic Light, which distinguishesE. coli(green),K. pneumonia(yellow), andP. aeruginosa(red) in a single test. As workflow is virtually identical for each kit and cross reactivity is not an issue. Laboratories frequently employ batch-testing once per shift to streamline sample processing.
Commercially Available PNA FISH Assays
- GBS PNA FISH from Lim Broth
- GNR—E. coli/P. aeruginosa
- GNR—EK/P. aeruginosa
- GPCC—S. aureus
- GPCC—S. aureus/CNS
- GPCPS—E. faecalis/OE
- Yeast—C. albicans
- Yeast—C. albicans/C. glabrata
- Yeast Traffic Light
Improving Patient Outcomes
Early and appropriate antimicrobial therapy is vital to the outcome of patients infected with bloodstream pathogens. Compared to traditional microbiology methods, PNA FISH provides more timely and definitive results from positive blood cultures, thereby helping clinicians improve antibiotic selection and outcomes for these patients. Furthermore, the assay is easy-to-perform and has high specificity and sensitivity. The accuracy and speed of this assay not only reduces length of hospital stays, but also shortens the duration of unnecessary antimicrobial therapy and the opportunity to develop resistance. In fact, a recent publication cites pharmacy intervention as a key factor to successful implementation (13). In short, accurate and rapid diagnosis of bloodstream infections has the potential to improve clinical outcomes, decrease length of hospital stay, and cut down on unnecessary healthcare costs. Close collaboration between the laboratory, pharmacy, and medical staff is an important aspect of successful implementation.
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Donna M. Wolk, PhD, DABMM is an associate professor in the Department of Pathology, and division chief of Clinical and Molecular Microbiology at the University of Arizona in Tucson.
Linda D. Hilbert is a volunteer science writer for the Infectious Disease Research Core at the University of Arizona in Tucson.
Disclosures: Dr. Wolk receives grant/research support from AdvanDx.