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.
Bartlett’s Q scoring of sputum samples
| Score | Average Number of Neutrophils per LPF | Score | Average of Squamous Epithelial cells per LPF |
| 0 | 0 (Null) | 0 | 0 (Null) |
| +1 | 1-9 (Few) | -1 | 1-9 (Few) |
| +2 | 10-24 (moderate) | -2 | 10-24 (moderate) |
| +3 | ≥25 (many) | -3 | ≥25 (many) |
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
| Group | Epithelial cells per LPF | Leukocytes per LPF |
| 1 | >25 | <10 |
| 2 | >25 | 10-25 |
| 3 | >25 | >25 |
| 4 | 10-25 | >25 |
| 5 | <10 | >25 |
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.
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