Swab protocol for rapid laboratory diagnosis of cutaneous anthrax

Abstract

The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as culture and directly straining smears of clinical specimens. However, these methods rely on recovery of viable Bacillus anthracis cells from swabs of cutaneous lesions and often yield negative results. This study developed a rapid protocol for detection of B. anthracis on clinical swabs. Three types of swabs, flocked-nylon, rayon, and polyester, were evaluated by 3 extraction methods, the swab extraction tube system (SETS), sonication, and vortex. Swabs were spiked with virulent B. anthracis cells, and the methods were compared for their efficiency over time by culture and real-time PCR. Viability testing indicated that the SETS yielded greater recovery of B. anthracis from 1-day-old swabs; however, reduced viability was consistent for the 3 extraction methods after 7 days and nonviability was consistent by 28 days. Real-time PCR analysis showed that the PCR amplification was not impacted by time for any swab extraction method and that the SETS method provided the lowest limit of detection. When evaluated using lesion swabs from cutaneous anthrax outbreaks, the SETS yielded culture-negative, PCR-positive results. This study demonstrated that swab extraction methods differ in their efficiency of recovery of viable B. anthracis cells. Furthermore, the results indicated that culture is not reliable for isolation of B. anthracis from swabs at ≥ 7 days. Thus, we recommend the use of the SETS method with subsequent testing by culture and real-time PCR for diagnosis of cutaneous anthrax from clinical swabs of cutaneous lesions.

Fig 1

Box-and-whisker plots showing the distribution of C_T values with the chromosomal target of the B. anthracis real-time PCR assay (16) for polyester (A) and rayon (B) swabs spiked with virulent B. anthracis Ames strain cells at a concentration of 10⁷ CFU/ml and then processed by the use of the three swab recovery methods. The bottom, middle, and top lines of each box correspond to the 25, 50, and 75% cumulative frequencies of the observed values, respectively. The endpoints of the whiskers show the 2.5 and 97.5 percentiles. For both swab materials, the differences in mean C_T values between the three methods were found to be significant by one-way ANOVA (P < 0.001; n = 24). For polyester swabs, Tukey's multiple-comparison test revealed significant differences between all three methods (P < 0.001; n = 24). For rayon swabs, significant differences were identified between the SETS and the sonication method and the SETS and the vortex method (P < 0.001; n = 24); however, there was no significant difference between the sonication and vortex methods (P = 0.09; n = 24).