Sputum gram stain

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Gram staining of expectorated sputum is a simplistic, easy-to-perform, commonly available, and low-cost diagnostic for pneumonia patients. Sputum Gram stain may detect many infections at the same time, and the test has a quick turnaround time. With adequate specimens, the sputum Gram stain can aid in establishing the right bacterial diagnosis and guiding suitable antibiotic therapy in community-acquired pneumonia (CAP).

Microscopic grading

Microscopic evaluation can also help identify if a specimen is likely to be useful in diagnostic or culture procedures. Nonrepresentative specimen processing and culture interpretation may result in delayed treatment due to a false-negative culture or improper antimicrobial therapy due to a false-positive culture from growth of normal biota or antibiotic-altered biota.

Several grading or classification systems have been developed to assist laboratory scientists in making decisions about culturing specimens or interpreting growth from culture of specimens. Sputum collection is impacted by contamination with biota from the throat and mouth, and culture alone can be deceptive. Before culture or culture interpretation, the representative sample must be separated from the contaminated sample.

Sputum Gram stain

Bartlett’s Q scoring of sputum samples

ScoreAverage Number of Neutrophils per LPFScoreAverage of Squamous Epithelial cells per LPF
00 (Null)00 (Null)
+11-9 (Few)-11-9 (Few)
+210-24 (moderate)-210-24 (moderate)
+3≥25 (many)-3≥25 (many)
Bartlett’s Q scoring of sputum samples

Interpretation:

Q score = points of average number of neutrophils + points of average number of squamous cells.

Minimum score = -3

Maximum score = +3

The better the specimen, the higher the score. Culture the specimen that has a composite score of ≥+1. A sputum specimen from a leukopenic patient having ciliated respiratory epithelial cells should also be cultured. A composite Q score of 0 or negative indicates a shallow sample that may not be a reliable specimen and should not be cultured.

Murray Washington method

GroupEpithelial cells per LPFLeukocytes per LPF
1>25<10
2>2510-25
3>25>25
410-25>25
5<10>25
Murray Washington method of scoring

Only samples from groups 4 and 5 are ideally appropriate for culture. Immunocompromised people, on the other hand, will have less leukocytes in their secretions as their cell count decreases. As a result, the criteria were updated such that the quantity of epithelial cells (>25/LPF) is a stronger predictor of mucosal or saliva contamination.

Sputum Gram Stain & Pneumonia Diagnosis FAQ
What is the role of sputum Gram staining in pneumonia diagnosis?
Gram staining of expectorated sputum is a simple, low-cost, and widely available diagnostic tool for pneumonia. It provides rapid results and can detect multiple infections simultaneously. When performed on a good-quality specimen, it helps identify the causative bacteria and guides appropriate antibiotic therapy in community-acquired pneumonia (CAP).
Why is microscopic grading of sputum important?
Microscopic grading helps determine whether a sputum sample is suitable for culture and diagnosis. Poor-quality or contaminated samples can lead to false-negative or false-positive results, resulting in delayed treatment or inappropriate antimicrobial therapy.
What challenges exist with sputum sample collection?
Sputum samples are often contaminated with normal flora from the mouth and throat, making culture results misleading. Therefore, it is essential to distinguish representative samples from contaminated ones before performing culture or interpreting results.
What is Bartlett’s Q scoring system?
Bartlett’s Q score evaluates sputum quality by combining scores for neutrophils and squamous epithelial cells. The score ranges from -3 to +3. A score of ≥+1 indicates a good specimen suitable for culture, while scores of 0 or below suggest poor-quality samples that should not be cultured.
How is Bartlett’s Q score interpreted?
The Q score is calculated by adding points for neutrophils and subtracting points for squamous epithelial cells. Higher scores indicate better specimen quality. Samples with ≥+1 should be cultured, while those with 0 or negative scores are considered unreliable.
What is the Murray Washington method?
The Murray Washington method classifies sputum samples into five groups based on epithelial cell and leukocyte counts. Only groups 4 and 5 are considered appropriate for culture, as they indicate high leukocyte presence and low contamination.
How does immunocompromised status affect sputum analysis?
Immunocompromised patients may have fewer leukocytes in their sputum. In such cases, epithelial cell count becomes a more reliable indicator of contamination, and modified criteria are used to assess specimen quality.

References:

  • Del Rio-Pertuz, G., Gutiérrez, J.F., Triana, A.J. et al. Usefulness of sputum gram stain for etiologic diagnosis in community-acquired pneumonia: a systematic review and meta-analysis. BMC Infect Dis 19, 403 (2019). https://doi.org/10.1186/s12879-019-4048-6
  • Huang, W. Y., Lee, M. S., Lin, L. M., & Liu, Y. C. (2020). Diagnostic performance of the Sputum Gram Stain in predicting sputum culture results for critically ill pediatric patients with pneumonia. Pediatrics and neonatology, 61(4), 420–425. https://doi.org/10.1016/j.pedneo.2020.03.014
  • Ogawa, H., Kitsios, G. D., Iwata, M., & Terasawa, T. (2019). Sputum Gram stain for diagnosing causative bacterial pathogens and guiding antimicrobial therapies in community-acquired pneumonia: a systematic review and meta-analysis protocol. Fujita medical journal, 5(3), 79–84. https://doi.org/10.20407/fmj.2018-019
  • Boerner DF, Zwadyk P. The Value of the Sputum Gram’s Stain in Community-Acquired Pneumonia. JAMA. 1982;247(5):642–645. doi:10.1001/jama.1982.03320300046021

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