The rapid identification of microorganisms is often of paramount importance in human health. One such organism when this is especially true is Yersinia pestis, the causative agent of plague.

Earlier, confirmatory tests for the diagnosis of infection included the isolation and identification of bacterial colonies or the demonstration of a 4-fold increase in antibody titers against its capsule antigen F1.¹

Culture identification can take as long as 48 to 72 hours and poses health risks to laboratory scientists, and it could take several weeks to demonstrate a significant rise in antibody titer.

Later, the polymerase chain reaction was adapted for the identification of many bacterial species, but that too had its share of problems including the need to run gel electrophoresis.

New developments in the field of molecular biology have demonstrated that it is possible to reduce the time required for accurate identification of pathogenic microorganisms. In this article we will discuss relatively new methods that can identify rapidly Y. pestis, specifically modified real-time polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF).

Lingering Threat
Y. pestis is a Gram-negative rod and a member of the family Enterobacteriaceae. It is mostly a zoonotic microorganism transmitted from rodents to human through flea bites.¹

The organism can also be transmitted from person to person in the pneumonic form. It has been known to cause bubonic and pneumonic plague and is responsible for as many as 200 million human deaths spanning a total of three pandemic events.¹

Even in this day and age, it still lingers in almost all the major continents, and according to the World Health Organization causes about 2,000 infections annually.¹

The pneumonic form in humans is easily transmissible to others through respiratory droplets and aerosols causing it to be classified by the CDC as a category A biothreat agent.

Of the 15 species within the genus Yersinia, only three are considered human pathogens. The rest reside in soil and water and are regarded as harmless or weakly pathogenic.²

However, among the three pathogens, Yersinia pseudotuberculosis, Yersinia enterocolitica and Y. pestis, the latter causes the most lethal infections and can be used as a biowarfare agent potentially causing heavy casualties. For this reason, and because it can spread easily and has a high mortality rate in naturally acquired infections, it is important to be able to rapidly identify Y. pestis with high specificity and sensitivity.

Current Laboratory Identification
Plague is usually suspected amongst patients with fever and lymphadenopathy and those with a history of recent travels to a plague-endemic area.³

The specimen for laboratory tests is selected from sites based on the patient's condition. Sputum, bronchial wash or transtracheal aspirates are collected from patients with respiratory symptoms, whereas blood is drawn from patients who appear septic. For patients with buboes, a saline wash should be injected into the bubo before lesion fluid is aspirated.⁴

A direct Gram-stained smear might reveal small, Gram-negative coccobacilli. Clinical material stained with the Wright stain should display the organism's bi-polar staining characteristic. This is characterized by more intense staining at the ends of the cells and lighter staining in the center.

Preliminary testing for Y. pestis yields nonlactose fermenting growth on MacConkey agar and positive for catalase but negative for oxidase and urease activity.⁴

Currently, confirmatory test for Y. pestis as recommended by the CDC involves lysis of the bacteria using lytic bacteriophage, like fA1122, specific to Y. pestis. Genomic amplification by the bacteriophage is then monitored using quantitative polymerase chain reaction (qPCR).

However, Sergueev et al. showed that this particular phage also has the ability to lyse some strains of Y. pseudotuberculosis.¹ Instead, the authors suggest a rapid, parallel two-phage qPCR assay that is both sensitive and specific.

In this assay, L-413C, a mutant bacteriophage, is used which has a high homology to coliphage P2 and a mosaic structure of its tail fiber protein H which makes it very specific to Y. pestis and not to any of the 2000 strains of Y. pseudotuberculosis.¹

Comparatively, fA1122 was found to be less specific but more sensitive with a requirement of only one cell/µL of sample for detection. Conversely, L-413C is more specific but less sensitive with a requirement of approximately 100 cells/sample.¹ Phage typing is performed primarily by state health and reference laboratories.

Newer Identification Methods
The relatively new matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is now seen as the next generation method for microbial identification. There is a slow but steady transition of such assays from the research laboratory into the clinical laboratories.

Already companies such as Germany-based Bruker Daltonics have introduced bench-top mass spectrometers for clinical applications. MALDI-TOF MS provides protein profiles for microorganisms at the genus and species level by separating molecules according to their mass to charge ratio by ionizing them via laser pulses.

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