Background: Gram-positive coccus (GPC) in cluster bacteremia is associated with high mortality and morbidity. Although laboratory technologists note morphological characteristics such as the size and number of cells help distinguish S. aureus (SA) and Coagulase negative staphylococci (CoNS), there are few studies that explored the optimal findings to distinguish between them by Gram stain. Here, we analyze some findings in Gram stain contributing to identify SA or CNS.
Methods: This study was conducted at St. Luke`s International Hospital from November 2016 and September 2017. Broths for which a Gram stain showed GPC cluster were included in our study. Two infectious diseases fellows examined direct Gram stains of blood culture within 24 hours of positivity. We defined the sign as follows: four-leaf clover (FLC) sign if 4 GPCs gather like four-leaf clover (Figure 1) and three-dimensional (3D) sign if GPCs resemble big grapes. They counted the number of fields that have FLC and 3D signs out of 10 fields per each slide. We performed logistic regression analysis to assess whether these signs are the contributable factors to identify SA or CNS. The predictive ability of these signs was evaluated on the basis of the sensitivity (Sen), specificity (Spe), positive predictive value (PPV) and negative predictive value (NPV) for CoNS with receiver operating curve (ROC) analysis.
Results: In total, 106 blood cultures in which a direct Gram stain showed GPC in cluster were examined. Cultures revealed 46 (43%) were SA and 66 (57%) were CoNS. In multivariate logistic analysis showed FLC sign was statistically significant variable of CoNS with odds ratio (OR) 1.20 (95%CI 1.09-1.35, p<0.05). In aerobic bottles, Sen, Spe, PPV, NPV were 0.67, 0.91, 0.92, and 0.65, respectively (Table 1) and area under the curve (AUC) was 0.79 (95%CI 0.67-0.91) (Figure 2). Cut-off fields were 6.5 out of 20.
Conclusion: This is the first study to show FLC sign could be a rapid and useful finding of Gram stain to identify CoNS rather than SA in aerobic bottles. In the presence of FLC sign, clinicians should highly suspect of CoNS with PPV of 92% before the final identification.
Figure 1. Four-leaf clover (FLC) sign
Table 1. Logistic regression analysis of FLC sign
Figure 2. ROC analysis of FLC sign in aerobic bottles
N. Mori, None