The Gram stain goes molecular.

Every year, 350,000 patients in the United States contract bloodstream infections, causing over 90,000 unnecessary deaths and significant costs to our healthcare system. The mainstream early diagnostic process continues to revolve around the Gram stain, which is performed in laboratories to provide diagnostic feedback to support strategies for patient treatments. (1) Unfortunately, the Gram stain, invented in 1884, is now 124 years old. Physicians still rely on basic Gram-stain results for the first 24 to 48 hours of antibiotic strategy for hospital patients who show a positive blood culture. To compound the issue, we are now over 60 years into the era of antibiotic usage, which is now largely characterized by rampant antibiotic resistance.

Before antibiotic resistance, it could be argued that the Gram stain actually served clinicians, physicians, and patients well, offering the first reliable initial guidepost on the road to antibiotic choice. "Is it Gram positive or Gram negative?" is the first question of the physician to the lab after a positive blood culture is reported. The genesis of antibiotic resistance started at the crossroads between when the diagnostic test was ordered and initial treatment, which often translates into providing the patient with "broad coverage."

Broad coverage refers to the practice of doctors prescribing antibiotics when they believe there is some type of infection--although they have not determined what the infection is (this practice is also known as "empiric therapy"). Broad coverage and related practices are no longer working--and one only needs to look at the brief history of antibiotics to understand why. Hospitals tend to be somewhat universal in their use of antibiotics when it comes to coverage for Gram-positive infections. (2) If you asked a physician in the 1960s or 1970s what his antibiotic of choice was, it was usually penicillin. Later, it became ampicillin or methicillin. Currently, it is vancomycin, a drug invented in the 1950s that was, for many decades, held in reserve as the drug of last resort. The penicillins were relatively non-toxic, had a wide therapeutic index, and--as generics--were considered inexpensive enough to be used without restriction. Therefore, any physician could traditionally prescribe a coverage drug for a Gram positive, while waiting for species identification. Unfortunately, this has led to the excess use of antibiotics over the years. The habit of broad-based coverage is not new. The selection of the coverage drug is based on individual hospital guidelines.

Rapid tests can speed lifesaving intervention

As a result, over 70% of bacteria that cause infections are now resistant to at least one of the drugs most commonly used to treat them, (3) according to the CDC. Moreover, using traditional diagnostic methods, 48 hours to 72 hours might elapse before physicians know exactly what a suspected infection is. In most cases, they will proceed to prescribe antibiotics anyway, based on the rationale that if there is a true Staphylococcus aureus infection and if the doctor waits too long, then there is the potential for a runaway infection. Traditionally, at the first sign of an infection, physicians usually treat patients empirically with broad-spectrum antibiotics. To complicate issues in the laboratory, over 30% of positive blood cultures are due to contamination with coagulase-negative Staphylococci (CNS) that are commonly found on the skin. (4) Because, however, these bacteria are closely related to the more dangerous S aureus, and look identical with the Gram stain, patients with CNS-contaminated blood cultures are often given antibiotics such as vancomycin for several days in an attempt to target any possible bacterial strains present.

If the bacterial turns out not to be S aureus, however, then the physician may have overprescribed for an infection, and if it is in fact S aureus, then the conventional antibiotic dosage that is prescribed may not be strong enough to kill the infection and could likely fuel resistance. Simply put, S aureus-positive blood cultures require aggressive intervention--and simple coverage with vancomycin is no longer considered to be completely effective. Moreover, S aureus necessitates the need for physicians to dose vancomycin right to the maximum borderline-toxicity level (15 pg/mL to 20pg/mL inserum) to be effective. (5) It is either highdose vancomycin or the physician needs to campaign with the pharmacy department and infectious disease for permission to use daptomycin or the physician needs to campaign with the pharmacy department and infectious disease for permission to use daptomycin, linezolid, or some of the newer approved therapies such as tigecycline. (6) Even with the newer, higher cost drugs, clinical success and optimal patient outcome associated with S aureus is not assured. This results not only in rising incidences of resistant strains of bacteria but also means that patients are not getting the targeted treatment that could lead to their more rapid recovery.

One point of agreement is certain: The sooner the physician knows it is S aureus, the sooner lifesaving intervention can be initiated. (7) Yet, this intervention cannot be delivered by using conventional processes and approaches. There is a need, therefore, for an accurate and rapid blood-culture test, which can inform physicians about the precise type of infection that a patient has.

The movement toward speed and accuracy

The 1980s brought the advent of instrumentation procedures resulting in the development of synthetic oligonucleotides. These were sophisticated instruments sold to government-funded research labs contributing to many facets of molecular biology, including the Human Genome Project. Bacterial and yeast genomes, smaller and simpler than the human genome, were characterized first. Scientists started planning the preparation of diagnostic kits using these probes in order to target known sequences that were developed as part of the genome projects.

In 1992, the capability for synthesizing peptide nucleic acid, or PNA, evolved. (8) A synthetic oligonucleotide was designed. Scientists could create a linear piece of PNA, which has a linear base sequence of interest. Scientists conceptualized the construction of living pieces of oligonucleotide with a fluorescent tag and incubated in bacteria or other microorganisms to identify its strain, thus leading to diagnostics. In a patient suffering from bacterial infection, identifying the particular pathogen species or strain becomes extremely difficult. PNA is an organic polymer made up of individual bases such as adenine (A), guanine (G), cytosine (C), and thymine (T) with a peptide backbone. The PNA molecules operate by biomimicry of the organic reaction pattern of a natural DNA molecule. Being constructed out of adenine, cytosine, guanine, and thymine by itself, PNA probes tend to bind (through hydrogen bonding) to a target gene segment, made up of a complementary linear sequence of A, T, G, or C base pairs, specific to the particular killer strains. As a result, the PNA probe can easily enter the in-situ environment of the cell. In the growth phase, bacteria and yeast cells produce copious amounts of ribosomal RNA, or rRNA, structures. These rRNAs are said to contain regions of highly conserved, species-specific sequences, which are, therefore, ideal targets for identification assays.

The PNA probe fragment is tagged/labeled with a fluorescence molecule that produces specific color, and hybridizes in situ to the target rRNA fragment belonging to the disease-causing killer microbial strain. This results in a simple, sensitive, and specific hybridization assay suited for rapid and accurate identification of the killer microbe. Simply stated, the organism that is identified can be clearly seen using fluorescence microscopy. (9)

The assay uses fluorescent-labeled PNA to target the species-specific rRNA in microbes (S aureus, (10-1) Candida albicans, (12-15) Candida glabrata, (16) Enterococsus facealis, and other Enterococci (17) including Enterococcus faecium [OE]), rendering the target cells fluorescent and easily observed using microscope equipment already present in most laboratories. Thus, the adaptation of the rapid test can be used by any hospital, without investment in new instrumentation. These new diagnostic procedures facilitate the dynamic and rapid testing of the blood sample without the need for the time-consuming repeated culture and cell-typing experimentation. Further, this technology speeds up treatment procedure and prevents the unnecessary use of drugs, thus minimizing the chances of creation of drug-resistant bacterial strains. These as says take about two hours in contrast to traditional methods that maytakeupto72hours,thus aiding attending physicians in charge of patients to make rapid diagnosis and, more importantly, saving patients' lives through early, appropriate, and effective antibiotic therapy. Going further, this technology can result in significant cost savings and development of new therapeutic guidelines. (18) As a result, rapid diagnostics have become a practical reality that can be easily implemented and used, thereby enabling the hospital to become less reliant on empirical coverage to provide a more targeted antibiotic therapy.

Laboratories that utilize PNA FISH (fluorescence in-situ hybridization) results have reported a high degree of physician satisfaction with these programs. Antibiotics and antifungals can be more prudently prescribed, patients are discharged sooner, fewer antibiotics are used, and mortality trends are improving. Speed when combined with accuracy in diagnostics really can work. Having a rapid diagnostic result within an hour (19) is a dramatic improvement compared to 2.5 hours, and has been achieved within only four years. But how does a multisite facility with thousands of physicians, nurses, and pharmacists use rapid-diagnostics information?

Consumer-electronics adaptation is running ahead of ability for most hospitals to fully utilize rapid-information systems for their healthcare workers. Think about how quickly conveniences such as cell phone, mobile e-mail, text messaging, and global-positioning systems, or GPSs, have moved from being a "cool technology" to a downright necessity. As large institutions integrate their patient databases to include lab results, pharmacy history, and patient-chart information, the "complete picture" of the patient will be readily available to the attending physician. Ultimately, within the next several decades, every physician will be able to catch every infection as soon as it is detected, with a specific, tailored therapy for every patient.

Philip Onigman is a director of AdvanDx Inc. (www.AdvanDx.com), located in Woburn,MA, a company that develops simple and easy-to-use diagnostic tests based on molecular technology platforms that utilize genomic information to identify specific gene or species-specific sequences in bacteria and yeast. Joe Romano is a consultant for the firm.

References

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(2.) Paladino JA, Sunderlin JL, Adelman MH, Singer ME, Schentag JJ.Observations on vancomycin use in U.S., hospitals. Am J Health Syst Pharm. 2007;64(15):1637-1641.

(3.) Campaign to prevent antimicrobial resistance in healthcare settings. Centers for Disease Control and Prevention, http://www.cdc.gov/drugresistance/healthcare/defaulthtm.Accessed April 16,2008.

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(6.) Daum RS. Skin and soft-tissue infections caused by methicillin-resistant Staphylococcus aureus. N Engl J Med. 2007;357(4):380-390.

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(9.) Stender H, Fiandaca M, Hyldig-Nielsen JJ, Coull J. PNA for rapid microbiology. J Micro Methods. 2002;48(1):1-17.

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(11.) Oliveira K, Brecher SM, Durbin A, et al. Direct identification of Staphylococcus aureus from positive blood culture bottles. J Clin Microbiol. 2003;41(2):889-891.

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(13.) Wilson DA, Joyce MJ, Hall LS, et al. Multicenter evaluation of a Candida albicans peptide nucleic acid fluorescent in situ hybridization probe for characterization of yeast isolates from blood cultures. J Clin Microbiol.2005;43(6):2909-2912.

(14.) Jabra-Rizk MA, Johnson JK, Forrest G, Mankes K, Meiller TF, Venezia RA. Prevalence of Candida dubliniensis fungemia at a large teaching hospital. Clin infect Dis. 2005:41(7):1064-1067.

(15.) Forrest GN, Mankes K, Jabra-Rizk MA, et al. Peptide nucleic acid fluorescence in-situ hybridization-based identification of Candida albicans and its impact on mortality and antifungal therapy costs. J Clin Microbiol. 2006;44(9):3381-3383.

(16.) Shepard JR, Addison RM, Alexander BD, et al. Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in-situ hybridization method for simultaneous dual-color identification of C aibicansand Cglabrata directly from blood culture bottles. J Clin Microbiol.2008:46(1):50-55.

(17.) Toombs L, Weekes E, Forrest G, Lincalis D, Johnson JK, Venezia RA. Impact of peptide nucleic acid (PNA) fluorescence in-situ hybridization (FISH) for Enterococcal blood stream infections. Presentation 131. http://www. advandx.com/uploads/documents/idsapresentationfinal.pdf. Accessed April 16,2008.

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(19.) TrnovskyJ, Merz W, Della-Latta P, Wu F, Arendrup MC, Stender H. Rapid and accurate identification of Candida albicans isolates by use of PNA FISH flow. J Clin Microbiol.2008;46(4):1537-1540.