Rapid diagnostic testing on patient samples—ranging from the humble Gram stain to more sophisticated molecular and genetic tests—is expected to increase in the coming years. These tests can help guide treatment decisions in a fraction of the time it takes to get the results of a traditional culture or other more specific tests.

The results can help shorten the time until appropriate care is given, and also potentially help decrease inappropriate antibiotic prescribing. Because broad-spectrum antibiotics are often used for empiric treatment pending culture results, rapid diagnostic testing can help to narrow the field of suspect pathogens—and by extension narrow the field of appropriate treatment.

A key recommendation of general antimicrobial stewardship program recommendations, including those by the Infectious Diseases Society of America and the U.S. Centers for Disease Control and Prevention, is to narrow the use of antimicrobials to the most appropriate drugs for the minimum amount of time.

Dr. Eric C. Feucht of Metro Health in Ann Arbor, Michigan, recently told Bedside Bronchoscopy that sterile, disposable bronchoscopes have combined well with Gram testing—a fairly simple and go-to test of specimens for more than 100 years now—to determine appropriate therapies for their ICU patients with pneumonia.

These tests can be particularly helpful during flu season.

“We are frequently getting patients coming in with severe pneumonia, bilateral infiltrates from the community, that look and sound like the flu,” Dr. Feucht said. “But we also wonder if they have a secondary bacterial infection. So, it’s our practice when those patients come to the ICU intubated to use the bronchoscope for sampling.”

A Century of ‘Gram’s Method’

Depending upon a laboratory’s resources and staffing, some of the rapid tests currently used to guide antimicrobial prescribing include:

  • Gram stains
  • Rapid viral testing by rapid antigen, rapid immunoassay, or direct fluorescent antigen testing
  • Rapid molecular assays and mass spectrometry to identify bacterial species and susceptibility, such as:
    • Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)
    • Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)
    • Rapid multiplex PCR (rmPCR)
  • Rapid fungal testing by galactomannan (GM), (1,3)-β-D-glucan (BDG), or single- or multipathogen fungal PCR
  • Serial measurement of PCT to guide discontinuation of antibiotic treatment

A Gram stain is a simple means of staining, fixing, rinsing, and counterstaining a slide for visualization under a microscope. Staining makes bacteria easier to visualize and also helps convey information about the shape, arrangement, and cell wall characteristics of the bacteria.

The Gram staining process, also known as “Gram’s method,” was introduced by Danish bacteriologist Hans Christian Gram in 1884.  The Gram stain has often been one of the first tests to be performed on a specimen for more than a century.

How is it done? A sample slide is first stained with a compound called crystal violet. This violet dye will be retained by the thick peptidoglycan layer, found in the cell wall of the bacteria referred to as “Gram-positive.” The sample is then fixed with iodine and the violet is rinsed away with a compound such as alcohol or acetone.

Because of the thinner peptidoglycan wall of the “Gram-negative” bacteria, the violet does not retain after rinsing, and so is counterstained with a pink dye called safranin. Some bacteria stain unpredictably, and they are known as “Gram-variable” or “Gram-indeterminate.”

What the Tests Can Tell Microbiologists

Gram stains can tell microbiologists a few important pieces of information, such as:

  • Is the sample valid? Does it contain too much of what looks like saliva or like skin cells?
  • Does the sample seem to have a consistent bacterial presence? Or is it possible that the infection is more likely viral in origin?
  • Are the bacteria present Gram+ or Gram-? What is the structure of the cell wall, and which antibiotics are likely to be effective against it?

If the pathogen is found to be viral pending preliminary tests, antibiotics can possibly be avoided altogether, with the patient starting antiviral medications instead.

On the other hand, if the pathogen is thought to be bacterial, knowing whether the bacteria are Gram+ or Gram- can help to guide prescribing decisions. This minimizes exposure to the side effects and emerging drug resistance found in the context of broad-spectrum antibiotics. It may also help decrease the financial and human costs of broad-spectrum antibiotics and their adverse effects (including C. difficile and Candida infections).

Narrowing Down Antibiotics ‘Very Effectively’

Community-associated pneumonias (CAP) can be caused by a vast number of pathogens, so narrowing the field helps immensely in guiding treatment.

“We will get Gram stains back within an hour or two, and if they have no organisms from our guide BAL [bronchoalveolar lavage] Gram stains, then we will hold the antibiotics and only treat them with antivirals,” said Dr. Feucht. “We can narrow down our antibiotics very quickly and very effectively.”

The downside? Not all facilities have qualified staff available to prep and read Gram stains during off hours. Facilities without proper staffing may need to send samples out, making a stat Gram stain impossible.

“We are neurotic enough with our antimicrobial stewardship that our lab does Gram stains 24/7,” Dr. Feucht added.

Editor’s Note: We’ve hyperlinked throughout this piece to the source materials that guided our research but thought we’d group all of the citations here for you to peruse and explore in more detail if you’d like. Thanks for reading.

1. Barlam et al. “Implementing an Antibiotic Stewardship Program: Guidelines by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America.” CID 2016: 62, e51–e77: https://www.ncbi.nlm.nih.gov/pubmed/27080992

2. Bassetti et al. “Treatment of Gram-negative pneumonia in the critical care setting: is the beta-lactam antibiotic backbone broken beyond repair?” Critical Care 2016:20:19. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731981/pdf/13054_2016_Article_1197.pdf

3. U.S. Centers for Disease Control and Prevention. “Antibiotic Prescribing and Use.” Accessed from: https://www.cdc.gov/antibiotic-use/?s_cid=NCEZID-AntibioticUse-005

4. Jain et al. “Community-Acquired Pneumonia Requiring Hospitalization among U.S. Adults.” N Engl J Med 2015:373: 415-427. https://www.nejm.org/doi/pdf/10.1056/NEJMoa1500245?articleTools=true