Comparison of culture and real-time PCR for anthrax detection

Abstract

Anthrax, a serious zoonotic disease caused by the bacterium, Bacillus anthracis, is a threat to the livelihood of many people living in Africa. In Zambia, anthrax is endemic in the Western and Eastern provinces and control is mainly focused on limiting environmental contamination and human exposure as much as possible through vaccination of animals and rapid response to animal disease outbreaks. Anthrax outbreak response is largely dependent on laboratory confirmation of the disease which is a part of the surveillance system for the country. The definitive method for confirmation of B. anthracis used in Zambia is culture, which contributes significantly to delayed response since it is lengthy and needs adequate logistical and technical support. This study aimed to compare culture and the non-culture methods from different host and sample types, semi-quantitative / real-time PCR (qPCR), using samples from different provinces in Zambia. A total number of 91 samples and 18 B. anthracis strains were used in the study. The samples were cultured using the standard microbiological method of identifying the causative bacterium and the obtained isolates and B. anthracis strains were confirmed by qPCR using a chromosomal marker Ba-1, lethal factor gene, lef on pXO1 and capsule B gene on pXO2, capB using TaqMan qPCR assay as well as chromosomal marker on small acid soluble proteins (sasp) using ANT probe, protective antigen gene, pagA using the probe BAPA on pXO1 and capsule C gene, capC on pXO2 using SYBR green qPCR assay. DNA was extracted from the samples and tested using TaqMan and SYBR green qPCR assays. Eighteen B. anthracis strains isolated were confirmed on SYBR green qPCR assay. With culture, the 91 samples yielded 41 (10.9%) B. anthracis isolates that were confirmed on SYBR green qPCR assay. The TaqMan qPCR assay detected 82.4% (75/91) of samples as B. anthracis using Ba-1 positive with lef and/or capB gene markers criteria. The SYBR green assay detected 63.7% (58/91) positive B. anthracis specimen using the criteria of positivity on BAPA and/or capC and ANT gene markers. Skin samples and buffalo host samples yielded the highest positive B. anthracis results with the Western Province with the highest anthrax incidence. The study demonstrated that culture infrequently detects anthrax positives from specimen and that qPCR is a more accurate method to use with TaqMan being more sensitive than SYBR green assay. Further, the study uncovered that isolate DNA was more reliable to use than specimen DNA for detection of B. anthracis using qPCR with specific samples type and species yielding better results.