Burkholderia pseudomallei (Melioidosis) - Culture and molecular diagnosis (PCR)

Information 15/10/07.

Burkholderia pseudomallei is a Gram - negative intracellular bacteria of the genus Burkholderia, family Burkholderiaceae, which causes melioidosis, a disease that is associated with high fatality rates in people and animals. Melioidosis occurs mainly in countries of Southeast Asia and northern Australia , where the bacteria is endemic , although sporadic cases were reported worldwide in patients from these areas. Currently it is increasing the incidence of cases in places like Bangladesh, India, East and West Africa, Middle East, Central America, South America and even North America.

Burkholderia pseudomallei has a size 02.05 .mu.m x 0.4-0.8 of microns, and small bacillus morphology rounded ends. This bacterium is found in endemic areas like saprófita soil (land) and freshwater in tropical and subtropical regions between 20 degrees north latitude and 20 degrees south latitude, where it can survive in harsh environmental conditions, such as prolonged nutrient deficiency , acidic and alkaline environments, and a wide range of temperatures and dehydration. This is the case, among others, rice fields endemic regions. The bacteria infect a host does not need an intermediary to survive, but is considered a saprophyte, considered an "accidental pathogen". Several factors can influence the distribution of Burkholderia pseudomallei and telluric water environment such as rain, humidity, UV radiation, temperature, composition of the soil, vegetation and use of fertilizers.

The strains causing disease in humans differ from those that cause disease in animals, because they have certain genomic pathogenicity islands. In this sense, they may have the ability to cause disease in humans because the DNA has acquired other microorganisms. In addition, the mutation rate of this organism is high, because it continues to evolve even after infecting the host.

There are other species such as Burkholderia thailandensis, less virulent, which rarely causes human infections and can not be differentiated by 16S rRNA sequencing by antigenic differences or by biochemical identification systems such as Vitek or API.

Burkholderia pseudomallei The group includes B. pseudomallei, B. thailandensis, B. mallei (glanders / glander), and B. oklahomensis. Burkholderia cepacia complex group includes 17 species as B. multivorans, B. cenocepacia, B. cepacia, B. arboris, B. contaminans, anthina B. and B. pyrrocinia, among others. Differentiation is required for sequencing groEL, 16S rRNA genes and several conserved genes species (Tap protein to B. pseudomallei) 70kDa protein (B. thailandensis), 12 kDa protein (B. cepacia complex).

In humans, Burkholderia pseudomallei infection usually occurs by traumatic inoculation or contamination of skin traumatic lesions and rarely, by inhalation or ingestion. Once reached a host, Burkholderia pseudomallei invades cells. This bacterium synthesizes a polysaccharide capsule that allows the formation of microcolonies which protect phagocytosis and antibiotics. Likewise, it is able to polymerize actin and spread from cell to cell, causing cell fusion and the formation of multinucleated giant cells. The bacterium also expresses a toxin called lethal factor 1.

Farmers, for their repeated exposure are a high risk group, especially rice farmers for their frequent contact with soil and water. Infection should be suspected clinically in non - endemic countries with compatible manifestations in patients who have traveled to endemic areas or originating in those regions. Mortality is high (14-40%), but can reach 70% if not properly treated. The acute form of the disease can manifest as septicemia rapidly evolving, with death within 48 hours in the 80 to 90% of cases. Only, appropriate antimicrobial therapy, mortality reduces by half. This form of septicemia is similar signs and symptoms to those caused by many other bacteria, so clinical suspicion must be based on the origin of the patient from an endemic area. From the bacteremic infection the bacteria can be found in various organs. Clinical manifestations of melioidosis are extremely variable, from abscesses benign and localized to a pneumonia or septicemia fatal. On many occasions, and in endemic areas, are cases of asymptomatic patients who are diagnosed On performing chest x-ray for another reason. Other times its presentation varies from mild bronchitis to pneumonia necrotizing, which manifests as a radiologically upper lobe consolidation or thin - walled cavities. Pulmonary involvement can be the focus of hematogenous spread to cause an acute septicemic form of melioidosis.

The infection also affects captive animals, including marine mammals such as dolphins, sea lions and whales.

Recommended tests for diagnosis:

The reference method (Gold Standard) is culture isolation. However, cultivation and identification can take several days (2 to 7 days), and in cases of septicemic infections, results can be obtained when the patient has died. Moreover, rapid biochemical identification systems sometimes provide misleading results (p. Eg. API 20NE). Increasing the number of new species and other species phylogenetically related, difficult diagnosis and identification, even in endemic regions, and even in non - endemic areas when imported.

Several methods have been developed to detect antibodies (hemagglutination inhibition -IHA-, enzimoinmunoanálisis - ELISA, ...), and to detect antigens, but for obvious reasons due to the speed of evolution of the infection, are not useful in to make a diagnosis only considered useful in endemic areas to meet seroprevalence rates in exposed patients who have not developed the disease or infection affects chronic evolution.

For this reason, several strategies have been developed for molecular diagnosis (PCR) to try to get a faster result. Currently, molecular diagnosis as the most sensitive and specific for identification of Burkholderia pseudomallei method is recommended and, therefore, for the diagnosis of melioidosis. There are several target genes which prevent cross with other species of Burkholderia reactions.

In bacteremic acute forms is possible to obtain results of culture isolation, or molecular diagnostic PCR from blood, preferably buffy coat, but subacute and chronic forms, both blood cultures, such as PCR can be negative and biopsies are required.

Tests in IVAMI:

• Culturing of clinical or environmental samples (land and fresh water).

• Molecular diagnosis (PCR) human clinical specimens or animal and environmental (land and fresh water).

Recommended sample:

• Septicemic infections: whole blood collected with EDTA (2 to 5 mL) to obtain the buffy coat.

• Localized infections (skin): lesion exudate.

• Deep infections: biopsy of the affected organ.

• Land: 100 to 200 g of soil obtained 20 to 30 cm deep.

• Freshwaters: take 1 liter of water, 50 cm deep from the banks of rivers or lakes. Filter through a membrane of 0.2 to 0.45 .mu.m pore diameter. For shipments from a remote location, if you have a filter device, you can send the filter into a sterile container.

Preservation and shipment of sample:

• Refrigerated (preferred) for less than 2 days.

• Frozen: over 2 days.

Delivery term:

• Cultivation: 12 to 14 working days.

• Molecular diagnosis (PCR): 4 to 5 days (48 hours broth culture enrichment, 24 to 48 hours more PCR).  

Cost of the test:

• Consult ivami@ivami.com