Yersinia pestis: Culture; Molecular diagnosis (PCR).

Information 26-12-2019.

Yersinia pestis is the etiologic agent of plague, a disease that is transmitted from infected rodents to humans, mainly through flea bites. The disease can also occur when inhaling contaminated aerosols or by direct contact with tissues of infected animals. Y. pestis has caused three plague pandemics in the modern era (in the middle of the 6th century, in the middle of the 14th century - known as the Black Death -, and at the beginning of the 20th century), and the bacteria still persists endemically among the rodent populations from many countries.

Y. pestis was discovered by the bacteriologist of the Pasteur Institute Alexandre Yersin, and almost at the same time by the Japanese bacteriologist Shibasaburo Kitasato, during an outbreak of plague in Hong Kong in 1894. It is a gram-negative bacillus or cocobacillus, facultative anaerobic, which has a set of virulence factors that facilitate the infection of fleas and the evasion of the immune response of infected mammals, leading to the rapid death of the host in the absence of adequate treatment.

Among the virulence factors are the antifagocyte Factor 1 (F1) and V-antigen (LcrV) antigens. The bacterium releases F1, which is assembled in a capsule-like structure that increases the resistance of Y. pestis to phagocytosis by macrophages. LcrV also increases resistance to phagocytosis, as well as the negative regulation of the inflammatory response. In addition, the type-III secretion system (T3SS) allows the bacteria to inject proteins into macrophages and other immune cells. These proteins, called Yersinia outer proteins (Yops), include on the one hand Yop B/D, which form pores in the host cell membrane and have been linked to cytolysis, and on the other hand YopO, YopH , YopM, YopT, YopJ and YopE, which are injected into the cytoplasm of the host cells by T3SS by the pore created by YopB/D, and limit phagocytosis and cell signaling pathways important for innate immune defense.

Y. pestis is present in several animal reservoirs, particularly in rodents, on all continents except Oceania. However, it remains a relatively rare problem in most countries, and 90% of cases occur in Africa. The three most endemic countries in the world are the Democratic Republic of the Congo, Madagascar and Peru. Madagascar is the most affected country, with reports of plague outbreaks among the population every year.

Contrary to popular belief, rats did not directly start the spread of bubonic plague. It is mainly a disease in fleas (Xenopsylla cheopis, or rat flea) that infested rats making them the first victims of the plague. Infection in a human occurs when a person is bitten by a flea that has been infected by biting a rodent, which had also been infected by the bite of a flea that carried the bacteria. The microorganisms multiply inside the flea, joining to form a plug that blocks its stomach and makes it unable to suck and feed, so it changes from animal or person in search of food and injecting Y. pestis when trying to suck. Severe outbreaks of human plague are usually initiated by other disease outbreaks in rodents, or by an increase in the rodent population.

People infected by this bacterium usually present, after an incubation period from one to seven days, an acute febrile syndrome along with other nonspecific symptoms such as sudden onset of fever, chills, headaches and weakness, vomiting and nausea. Infection can occur naturally in three different ways:

• Bubonic plague: it is the most common form of plague and is acquired through the bite of an infected flea. The bacterium enters the body through the bite and travels to the lymph nodes, where it replicates. Patients develop sudden fever, headaches, chills and weakness, and one or more swollen, tender and painful lymph nodes (called buboes). In advanced stages of infection, these buboes can become purulent ulcerations. When the infection progress it can spread to the lungs and produce a more serious type of plague called pneumonic plague. The transmission of bubonic plague from human to human is rare.

• Pneumonic plague: it is the result of infection with Y. pestis in the lungs. It is the most virulent form of infection and the incubation period can be very short, even less than 24 hours. Transmission between humans can occur through respiratory secretions or aerosols, but this type of presentation can also develop from an untreated bubonic or septicemic plague, after the bacteria have spread to the lungs. It is a rapidly developing pneumonia with shortness of breath, chest pain, cough and sometimes bloody respiratory secretions, eventually leading to respiratory failure and shock.

• Septicemic plague: it is the result of the bacteria multiplying in blood and spreading throughout the body. It usually occurs as a result of an untreated bubonic or pneumonic plague. Patients develop fever, chills, extreme weakness and abdominal pain. Bacterial endotoxins can cause disseminated intravascular coagulation (DIC), producing small clots throughout the body that can cause ischemic necrosis. In addition, the DIC causes a depletion of the clotting factors of the organism, so hemorrhages are difficult to control. Consequently, skin and other organ hemorrhages occur, which can cause hemoptysis and hematemesis. Without treatment, septicemic plague is usually fatal.

• Pharyngeal plague: it is a rare type of infection, which occurs in people who have eaten raw or undercooked meat from animals infected by Y. pestis. Although it can produce symptoms similar to the other forms of plague, it also causes specific symptoms such as pharyngitis, dysphagia, submandibular lymphadenitis, tonsillitis and abdominal pain. This type of infection is more common in areas of the Middle East, North Africa and Central Asia, and most of the time it has been produced by eating meat from infected camels.

• Other forms of infection: cases of gastrointestinal, skin, meningeal and endophthalmic plague have been reported.

The plague is first diagnosed by the symptoms and by a history of possible exposure to rodents. Because the symptoms may be similar to those observed in other processes such as tularemia, dengue or pneumonia, the diagnosis should include the identification of Y. pestis in a laboratory test. The isolation of the bacterium by the culture of clinical samples constitutes the "gold standard" of diagnosis. Samples should be obtained from appropriate tissues to isolate the bacteria and depend on the clinical presentation: lymph nodes aspirates, blood cultures (in case of septicemia), or sputum, bronchial or tracheal lavage and lung biopsy (in case of pneumonic plague).

In addition to culture, molecular techniques can also be used. Virulence genes located in three well characterized plasmids (pFra, pYV and pPst) and in the chromosomal "pathogenicity island" of Y. pestis can be used as a target for the identification and characterization of this bacterium. In cases where living organisms are not cultivable (as in autopsy samples), samples of lymphoid tissue, spleen, lung, liver or bone marrow can show evidence of Y. pestis infection by methods such as direct immunofluorescence (DIF) or PCR.

Y. pestis is an organism classified in Category A by the CDC. This implies that it is considered as a high priority agent that represents a risk to public safety due to its ease to be transmitted person to person and because the infection can produce a high mortality rate and generate panic among the population. Currently, under natural conditions, the plague is quite well controlled and remains a rare disease, even in most areas of the world where it is endemic. However, one of the greatest future dangers may be the threat of this microorganism used as a biological weapon.

Tests performed in IVAMI: 

• Culture.
• Molecular diagnosis (PCR). 

Sample recommended: 

• For culture and molecular detection by PCR: lymph node aspiration, sputum, bronchial/tracheal lavage, pleural fluid, blood, ulcer exudate, biopsy/necropsy samples (lymph node, lung, liver, spleen).

Storage and shipment the sample: 

• Refrigerated (preferred) for less than 2 days.
• Frozen: more than 48 hours. 

Delivery of results: 

• Culture: 4 to 5 working days.
• Molecular diagnosis (PCR): 48 to 72 hours. 

Cost of the test: 

• Culture: Consult to ivami@ivami.com.
• Molecular diagnosis (PCR): Consult to ivami@ivami.com.