The Gram stain goes molecular – quickly!

July 1, 2012

According to a recent joint policy statement on antimicrobial stewardship by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA), antimicrobial resistance has emerged as a significant healthcare quality and patient safety issue in the twenty-first century that, combined with a rapidly dwindling antimicrobial armamentarium, has resulted in a critical threat to the public health of the United States.1 The Centers for Disease Control and Prevention (CDC) provides educational resources to healthcare workers to learn how to use antibiotics prudently.2

What can the Clinical Microbiology Lab do to help with the goal of reducing or avoiding unnecessary antibiotic use? We can start with understanding how information supplied by the Gram stain is reported on the Critical Values Telephone Call when we report initial positive blood cultures.

The Critical Values Call is a standard reporting mechanism in most hospitals, used to report proactively on patient cases with initial blood culture positivity results. This phone report may directly reach the attending physician, physician assistant, nurse, or pharmacist. The intention of the call is to alert for potential bloodstream infection as evidenced by a positive blood culture. The positive bottle is usually immediately tested using a Gram stain, with Gram positive or Gram negative organisms reported, including also information on cell morphology—usually rods, cocci, clusters, or chains.

For more than 128 years, the Gram stain has served the microbiology lab and clinicians as an immediate test performed on a positive blood culture. It is the first evidence utilized by clinicians when supporting an interventional pathway. For about three million blood cultures that turn positive annually in the United States, the Gram stain is a high volume, on-demand test. However, do Gram stain results alone help or hinder?

Consider those cases where a patient does not have a bloodstream infection, but the positive blood culture results are indicative of Gram Positive Cocci in Clusters (GPCC). In these cases, the Critical Values Call may trigger clinical teams to start an unnecessary antibiotic regimen. Why does this happen? The most common blood culture contaminant is usually Coagulase Negative Staph (CoNS). When viewed under the brightfield microscope and using Gram staining, CoNS are indistinguishable from Staphylococcus aureus.

If S. aureus is identified as a suspect pathogen, it is rarely a blood culture contaminant, and it must be immediately treated. Clinicians need the earliest alert for such true pathogens. Time is critical for getting a head start on treating S. aureus bloodstream infections. It is therefore important to be able to separate S. aureus blood culture cases that need immediate initiation of treatment from contamination cases that may not need treatment.

How big a problem is this for an individual hospital? Let’s consider a theoretical facility that draws 15,000 blood cultures a year and has a blood culture contamination rate of 3%. Therefore, 450 patients a year could be unnecessarily treated with antibiotics due to the initial Critical Values Call reporting GPCC+ results! Those 450 patients are likely to be treated with a regimen of intravenous vancomycin dosed at 2 to 4 grams per day for 72 hours and then reviewed. If the case is considered to be CoNS blood culture contamination, antibiotics are stopped and the patient often may be discharged the next day. If the same hospital has about 150 annual cases of S. aureus bloodstream infections, about three quarters of the patient cases with GPCC+ positive blood cultures may be unnecessarily treated.

What test results could the Clinical Microbiology Lab report to clinical teams to avoid unnecessary vancomycin treatment for those 450 patients, and avoid using hundreds of grams of vancomycin? What additional harm could be avoided to these patients with regard to risk of sepsis over the next year as a result of unnecessary vancomycin exposure?3 Previous studies have shown that patient CoNS contamination cases with earlier vancomycin discontinuance can actually have a shorter hospitalization by 2 to 4 days, including ICU cases.4-6 In our hospital example, avoiding initiation of vancomycin and achieving earlier discharges for 450 cases could free up 900 patient bed days.

The solution is for the Lab to identify, with certainty, those cases with GPCC+ blood cultures that are due to S. aureus as distinct from those cases that are CoNS, and also to report species ID with the Critical Values Call. In vitro diagnostic kits which have recently received FDA approval can provide the accuracy and speed that is required. Slides are read with fluorescence microscopy. The kits’ platform uses peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) to identify S. aureus (green) and CoNS (red) in a single 15-minute incubation step. There is no need for buffer makeup, washing slides, and testing separate controls, and results are reportable in 20 minutes.7

With the ability to identify CoNS and rule out S. aureus, the Lab can help avoid initiation of unnecessary antibiotics. The Critical Values Call script would be “Patient # X has a positive blood culture. It is Gram Positive Cocci in Clusters. FISH results show that this case is coagulase negative staph and not S. aureus, with only one blood culture bottle of four positive. The blood culture contamination rate in our facility is recently 3%.” This gives clinicians sufficient evidence to consider NOT starting vancomycin, or to discontinue vancomycin if it has been already initiated. Clinicians can also justify continuing antibiotics for exceptional cases where CoNS could be considered a potential bloodstream infection and not contamination based on patient symptoms, histories, and other factors.

Thus, the Lab can provide vital and useful information on the Critical Values Telephone Call to achieve hospital goals of reduction in unnecessary antibiotics and their associated risks and costs. By adding the certainty of species identification to the Gram stain report, the Lab becomes a more valued partner in Antibiotic Stewardship Quality Improvement programs.


  1. Fishman N, Patterson J, Salman L, et al. Policy statement on antimicrobial stewardship by the Society for Healthcare Epidemiology of America (SHEA), the Infectious Diseases Society of America (IDSA), and the Pediatric Infectious Diseases Society(PIDS). Infect Control Hosp Epidemiol. 2012;33(4):322-327.
  2. Centers for Disease Control and Prevention. Antimicrobial stewardship for the community hospital: practical tools and techniques for implementation. Accessed June 2012.
  3. Mascitti KB, Edelstein PH, Fishman NO, et al. Prior vancomycin use is a risk factor for reduced vancomycin susceptibility in methicillin-susceptible but not methicillin-resistant Staphylococcus aureus bacteremia. Infect Control Hosp Epidemiol. 2012;33(2):160-166.
  4. Forrest GN, Mehta S, Weekes E, et al. Impact of rapid in situ hybridization testing on coagulase-negative staphylococci positive blood cultures. J Antimicrob Chemother. 2006;58(1):154-158.
  5. Ly T, Gulia J, Pyrgos V, Waga M, Shoham S. Impact upon clinical outcomes of translation of PNA FISH-generated laboratory data from the clinical microbiology bench to bedside in real time. Ther Clin Risk Manag. 2008;4(3):637-640.
  6. Brandon HB, Wallace MR, DeRyke CA. Peptide nucleic acid fluorescence in-situ hybridization (PNA-FISH) for rapid diagnosis of staphylococcal bacteremia—outcomes in a large teaching hospital. IDSA 2010, Vancouver, BC poster #1023.
  7. Deck MK, Anderson ES, Buckner RJ, et al. Multicenter evaluation of the staphylococcus QuickFISH method for simultaneous identification of staphylococcus aureus and coagulase-negative staphylococci directly from blood culture bottles in less than 30 minutes. J Clin Microbiol. 2012;50(6):1994-1998.

Philip Onigman is a director of Massachusetts-based AdvanDx Inc., developer of the QuickFISH rapid pathogen tests. AdvanDx 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.