Primary Source Article Review

Example of a
Bacterial Culture of Y. pestis (2)

In the article "Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague" Julia Riehm et al. investigate a tool to help identify bubonic plague extremely quickly. The idea is that because the plague is highly infectious and has the potential to be used as a biological weapon if made into an aerosol, it is a good idea to have a test that can identify affected individuals quickly. The standard method of detecting Y. pestis consists of culturing bacteria, which can take up to 5 days to reveal the diagnosis. Riehm et al. suggest real-time PCR as an alternative method which can achieve the same result in twenty minutes. They test their proposed method against cell culture and ICT methods. 

Example of a Lateral Flow Dipstick assay (3)

Riehm et al. tested their method in the field, investigating 149 patients in high risk areas who presented symptoms of bubonic plague. Samples were drawn from buboes (infected and swollen lymph nodes) and extra samples were taken to Institut Pasteur in Madagascar. A fieldable Y. pestis-specification  fraction 1 capsular (F1)-antigen detection using an immunochromatographic lateral flow dipstick assay test (ICT)  was done, cells were cultured on agar, and real time PCR was repeated in a research setting. Negative controls used in their experiment were lymph nodes removed from 31 German patients. DNA of samples was then extracted through centrifugation and prepared for PCR. PCR was performed on the samples as well as negative controls. Positive controls were also included, consisting of matrix containing Y. pestis. finally, blank samples containing water were introduced into the PCR cycle.

The distribution of patients was roughly 1/3 female and 2/3 male. Although all patients were given antibiotic treatment, there was still a 4.7% death rate. The data from cultured cells concluded that 47 of the 149 patients were positive for Y. pestis, 88 of the 149 patients tested positive using the ICT method, and real-time PCR provided 120 positive samples. Multiple forms of real-time PCR were done, and the most effective test (yielding the 120 positive samples) was the 5'-N pla. Clinical diagnosis suggested that all 149 of the patients had infection due to Yersinia pestis. 

Previous PCR methods tested only the F1 antigen, but Riehm's real-time PCR method uses three plasmids commonly found in the Y. pestis, as well as the bacterial chromosome increase the likelihood of detection. When tested against other methods of detection, real-time PCR was found to be by far the most reliable, recognizing 120/149 cases while traditional methods were only able to detect 47 and 88 cases respectively. Not only was 5'-N real-time PCR found to be the most precise method of determining the respective pathogen, but it is also the fastest and can be done in the field. Culturing bacteria takes days, which greatly lowers the chance of survival of the afflicted, but real-time PCR takes 20 minutes and can be done remotely with much higher accuracy. 

Results of the methods used to test for Y. pestis bacteria (1)

5'-N pla real-time PCR has been found to be an effective tool to determine presence of bacterial infection, more so than traditional methods, but is not perfect. One aspect that future research can focus on is finding other plasmids or genetic markers that can increase the sensitivity of the test. While this test is much better than other available methods, it does not detect disease in all patients who are infected with the bacteria. More genetic markers will allow the real-time PCR to be more accurate and help earlier detection of disease.  

References:

(1) : Riehm JM, et al., Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague, Molecular and Cellular Probes (2010), doi:10.1016/j.mcp.2010.09.002

(2) http://www.bacteriainphotos.com/yersinia_pestis_blood_agar.html

(3) https://groeptms1316.wordpress.com/2013/02/10/lateral-flow-technology/