Francisella tularensis (tularemia) - Culture and molecular diagnosis (PCR)

Francisella tularensis is a Gram - negative intracellular bacterium, Francisella genus, family Francisellaceae. It has a worldwide distribution, but more often in North America, parts of Europe and Asia. This microorganism is mainly considered an animal pathogen, but can affect people, which causes the disease tularemia (rabbit fever called). Francisella tularensis has been described in birds, reptiles, fish, invertebrates, rodents and other mammals.

This bacterium is a pleomorphic short bacillus, gram - negative, of 0.2 to 0.7 microns x 0.2 microns and intracellular localization, fastidious from the viewpoint of cultivation slowly grown as small colonies and require agar media blood glucose and cysteine. For these requirements most cases are of clinical diagnosis.

Francisella tularensis and Francisella philomiragia: since 2000 two species allowed. The first species, F. tularensis, is classified into 4 subspecies tularensis F. tularensis (type A), holarctica F. tularensis (PALEARCTIC, type B), and F. novicida F. tularensis tularensis mediasiatica. Of these four subspecies, F. tularensis tularensis subspecies is the most virulent. F. tularensis subspecies tulsarensis, holarctica F. tularensis (palarctica) and F. tularensis mediasiatica cause human and animal infections. F. tularensis novicida has been found in few human cases. This organism is able to survive outside a host for weeks, in water more than three months at temperatures of 13-15ºC, and persist in animal carcasses up to four months depending on the ambient temperature. Tularensis F. tularensis (type A), is located in North America and is related to rabbits and ticks, causing fulminant disease that before introduction of the antibiotic had a 10% fatality. Holarctica F. tularensis (PALEARCTIC) (type B) is found in Asia, Europe and North America and part is related to rodents, rabbits , and in Europe it can be transmitted by mosquitoes.

The life cycle of this microorganism involves an intermediate host acting as vector transmission, generally ticks and flies (horseflies), but found some cases attributed to mosquitoes that transmit infection through bites during feeding and a where multiplies definitive host. Animals that keep the infection in nature are rodents and lagomorphs (Leporidae -liebres and rabbits, and Ochotonidae -pikas-). Once the definitive host, Francisella tularensis mainly infects macrophages, although it has the ability to infect most cell types. Entering the macrophage phagocytosis and is produced by the bacteria would be found within the infected cell in a phagosome. Then he leaves the phagosome into the cytoplasm, where mushrooms. Eventually, the infected cell undergoes apoptosis, and the bacteria are released to initiate infection of new cells.

Francisella tularensis owe much of their pathogenicity to their ability to survive within macrophages unstimulated. Macrophages engulf microorganisms easily, but these resist death compared to radicals derived oxidants oxygen . It is likely that resistance to intracellular killing due to its superoxide dismutase . Once activated macrophages, microorganisms die of nitrous oxide intraleucocitario . A local neutrophilic response counteracts infection by this organism in its initial stage, but fails to contain its progress. Neutrophilic response forms an ulcer skin localized later becomes an injury scar when neutrophils are replaced by macrophages. Subsequently, microorganisms spread distance where forming granulomas . Microorganisms tend to accumulate in lymphoid tissue, and patients show splenomegaly and bubones. Tularemia can be confused with plague when buboes are observed.

Transmission Francisella tularensis can occur via different routes, with two main: (.. Horseflies as Chrysops spp, or ticks as spp Dermacentor) by the bite of an arthropod or direct contact with infected animals causing ulceroglandular form, or by inhalation of contaminated, causing pneumonic respiratory form, potentially fatal powder. Other transmission routes include oropharyngeal airway that occurs after ingestion of meat poorly cooked a sick animal or ingestion of water contaminated with feces or urine of infected animals, as well as the conjunctival route due to inoculation of bacteria in the eye or skin contamination by skin lesions.

Infection occurs approximately 3 to 5 days after exposure. Associated signs and symptoms include chills, eye irritation (in the case that infection has occurred through the conjunctiva), fever, headache, stiffness, muscle pain, skin ulcers, difficulty breathing, sweating and loss weight. Some of the associated clinical complications include osteomyelitis, pericarditis, meningitis and pneumonia.

Recommended tests for diagnosis:

Tularemia diagnosis can be established by culturing the bacteria responsible for antibody detection (ELISA), or by molecular diagnostic methods (PCR).

Culture isolation is difficult due slowly grown as small colonies and require blood agar media with glucose and cysteine. For these requirements most cases are of clinical diagnosis. It has been also grown in chocolate agar media, Thayer-Martin medium, agar yeast extract-cysteine, or in liquid media blood culture bottles. Two of his subspecies, Francisella tularensis and Francisella philomiragia novicida can develop in unenriched usual means. In any case, crops tend to be avoided because of the risk of infection in the laboratory (is considered a pathogen of Class 3, and must be handled under these conditions), in addition to the difficulties of growing to be fastidious bacteria growth and slow .

Antibody detection is typically the method used by being more accessible to many laboratories when antigens are used commercially. However, encounters the drawback that the antibodies are not detectable by 2 weeks or more after infection. The most commonly used tests are direct agglutination using bacterial suspensions, and enzyme immunoassay (ELISA). Because of the time required to develop antibodies and the fact that sometimes the antibody tests are negative, the detection of antibodies is not considered the most suitable method. With methods should be noted antibodies that can give cross Brucella, Yersinia and Proteus reactions.

The molecular diagnostic methods (PCR), obviates the problems noted for crop and detecting antibodies, so today be considered the first choice for diagnosis.

Tests in IVAMI:

• Detection by isolation by culturing in selective media.

• Molecular diagnosis (PCR), to detect DNA of Francisella tularensis.

Recommended sample:

• Whole blood collected with EDTA (2 to 5 mL).

• Exudate from skin lesion.

• Exudate obtained by puncturing affected lymph node.

Preservation and shipment of sample:

• Refrigerated (preferred) for less than 2 days.

• Frozen: over 2 days (for molecular diagnostic tests only).

Delivery term:

• Cultivation: 3 to 4 days.
• Molecular diagnosis (PCR): 24 to 48 hours.

Cost of the test:

Consult ivami@ivami.com