Multicenter Assessment of Gram Stain Error Rates

Abstract

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories. C linical microbiology laboratories have undergone dramatic changes with the implementation of novel technologies, yet traditional techniques, such as the Gram stain, still play key roles throughout the diagnostic process (1-3). Gram stains are initially used as a preanalytical indicator of specimen quality and acceptability for culture. They also give the clinician preliminary information regarding the nature of potential pathogens present in the patient specimen and thus serve to guide empirical therapy. Although the Gram stain has been the staple of clinical microbiology laboratories for over a century, it is still considered a high-complexity procedure by the Clinical Laboratory Improvement Amendments (CLIA) program. The manual nature of the staining process and the subjectivity of Gram stain interpretation contribute to the incidence of errors (4-7). Inappropriate specimen sampling, specimen processing, smear preparation, and prior antibiotic therapy are all factors that can have an adverse impact on Gram stain result. The inherent nature of some organisms may also produce misleading results; for example, Acinetobacter spp. may stain Gram positive, while Bacillus spp. species may appear Gram negative. In addition, staining practices, such as use of cytospin and ...

Internal quality assurance in diagnostic microbiology: A simple approach for insightful data

“…Gram stain reading is well established as a complex procedure that has number of pitfalls and its poor reliability has been described at multiple occasions [43][44][45][46][47]. It is still worrisome to observe that almost 40% of the compared results included at least one discrepancy of more than two relative units, which we categorized as major ( Table 2).…”

“…This rate cannot be directly compared to any other data available in literature, the only similar experiment being the study of Constantine et al (14), which had only 2% of discrepancies for microscopy, but using a very different definition of discrepancy which allowed much larger differences in the semi-quantitative results. The other studies on the reproducibility of Gram stain use very different methodologies, such as comparison of the concordance between Gram stain and culture results or between different observers reading identical smears, thus providing non-comparable results [44][45][46][47].…”

Targeting errors in microbiology: the case of the Gram stain

“…The process of performing Gram stains may be manual or automated and the methods involved vary between laboratories but ultimately the challenge remains in accurately reading and reporting Gram stains. This can complicated by a number of variables such as quality of the specimen, method of fixation, organism viability and inherent variations in staining of the organisms present in the specimen as documented by Samuel et al in their multicenter study on the incidence of Gram stain errors [7]. The growing trend of consolidation of microbiology laboratories mean that many hospitals are left without dedicated microbiology staff [8].…”

Commentary: One Small Step for the Gram Stain, One Giant Leap for Clinical Microbiology

“…In this age of personalized medicine, there is no more personalized test than the Gram stain for those with an infection. This is how we build on the contributions by Samuel et al (7). As a profession, we can standardize the Gram stain procedure by defining specimen assessment, i.e., what portion of the specimen is used to prepare the smear, defining smear preparation resulting in a monolayer of cells and not clumped material, defining how to recognize and perform microscopic examination of the best areas in the smear, including the minimum number of low-and high-power fields to be examined, and defining terms used for quantitation for microorganism morphology and inflammatory cells.…”

“…Samuel and colleagues have taken a small but very important step to improve the Gram stain through their multicenter assessment of Gram stain error rates (7). In a simple but effective approach, they compared Gram stain and culture results for over 6,000 specimens processed at 4 different tertiary care medical centers.…”