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Detection of Burkholderia mallei in Microbiological Culture: A Comparative Analysis of PCR Primer Sets
Jéssica Cristine K Moriya1, Paula Adas P Suniga2, Ana Clara L Araújo3
1CNPq Scholarship/Embrapa Beef Cattle, Campo Grande 79106-550, MS, Brazil.
Abstract:
Glanders is a highly contagious and often fatal zoonotic disease of equids caused by Burkholderia mallei, a pathogen of significant concern due to its potential for bioterrorism. In Brazil, glanders remains endemic, particularly among working equids in the Northeast region. Diagnostic confirmation typically involves serology, culture, and polymerase chain reaction (PCR), although false-negative PCR results have been increasingly reported. This study aimed to evaluate the diagnostic performance and analytical sensitivity of four B. mallei-specific PCR primer sets using samples from 30 seropositive equids. Microbiological cultures were obtained from various organs and swabs, followed by PCR targeting four genomic regions: fliP-IS407A(a), fliP-IS407A(b), Burk457, and Bm17. All animals were confirmed positive for B. mallei via culture, but PCR detection rates varied significantly across primer sets. The fliP-IS407A(b) primer set showed the highest sensitivity, detecting 86% of samples, while the WOAH-recommended fliP-IS407A(a) set had the lowest performance (13.4%). Analytical sensitivity assays confirmed that fliP-IS407A(b) and Bm17 primers detected DNA concentrations as low as 0.007 ng, outperforming the others. These findings suggest that certain widely used primer sets may lack sufficient sensitivity for reliable detection of B. mallei, especially in chronically infected animals with low bacterial loads. The study underscores the need for ongoing validation of molecular diagnostics to improve the detection and control of glanders in endemic regions.
Insights
Certain PCR primer sets for glanders detection in equids show low sensitivity, potentially leading to false negatives. The fliP-IS407A(b) primer set demonstrated superior performance in detecting Burkholderia mallei.
Area of Science:
- Veterinary Microbiology
- Zoonotic Diseases
- Molecular Diagnostics
Background:
- Glanders, a zoonotic disease caused by Burkholderia mallei, is endemic in Brazil, particularly affecting working equids.
- Current diagnostic methods like PCR can yield false negatives, complicating disease control.
- The potential for Burkholderia mallei as a bioterrorism agent necessitates reliable diagnostic tools.
Purpose of the Study:
- To evaluate the diagnostic performance and analytical sensitivity of four Burkholderia mallei-specific PCR primer sets.
- To identify the most effective primer set for accurate glanders diagnosis in equids.
- To address the issue of variable PCR detection rates and potential false negatives.
Main Methods:
- Samples from 30 seropositive equids were analyzed using microbiological cultures and PCR.
- Four distinct primer sets targeting different genomic regions of Burkholderia mallei were tested: fliP-IS407A(a), fliP-IS407A(b), Burk457, and Bm17.
- Analytical sensitivity assays were performed to determine the limit of detection for each primer set.
Main Results:
- All animals were confirmed positive for Burkholderia mallei by culture.
- PCR detection rates varied significantly among the primer sets, with fliP-IS407A(b) showing the highest sensitivity (86%).
- The WOAH-recommended fliP-IS407A(a) primer set exhibited the lowest performance (13.4%).
- fliP-IS407A(b) and Bm17 primers detected DNA concentrations as low as 0.007 ng.
Conclusions:
- Some commonly used PCR primer sets for Burkholderia mallei lack sufficient sensitivity for reliable glanders detection, especially in low bacterial load cases.
- The fliP-IS407A(b) primer set is recommended for improved diagnostic accuracy in endemic regions.
- Continuous validation of molecular diagnostic tools is crucial for effective glanders control programs.
