Un caso de melioidosis en la Argentina / A case of melioidosis in Argentina

Se describe el caso de un varón de 17 años oriundo de República Dominicana, con antecedente de linfoma de Hodgkin, que presenta tumoraciones blandas con supuración espontánea. En sus cultivos desarrolló Burkholderia pseudomallei, agente etiológico de la melioidosis. El paciente recibió tratamiento antibiótico con imipenem y luego con amoxicilina-ácido clavulánico con muy buena evolución clínica del proceso infeccioso. En razón de la baja incidencia de Burkholderia pseudomallei en nuestro continente el diagnóstico de melioidosis pudo haber sido subestimado. Su diagnóstico definitivo depende del aislamiento e identificación del agente causal en la muestra clínica.

We describe a case of 17-year- old man native of Dominican Republic, with Hodgkin´s lymphoma, who presented soft espontaneous draining nodules. In the clinical samples grew Burkholderia pseudomallei; the etiological agent of melioidosis. He received antimicrobial treatment with imipenem and amoxicillin/clavulanic with very good clinical evolution of the infectious process. Melioidosis diagnosis could be underestimated due to the low incidence of Burkholderia pseudomallei in our continent. The definitive diagnosis depends of the isolation and identification in the clinical sample.

False-positive widal in melioidosis.

Enteric fever is endemic in this part of the world, and Widal test is one of the time-honored laboratory tests that are being used for years to diagnose the disease. On the other hand, melioidosis is a newly emerging disease from this region, which is most often misdiagnosed or underdiagnosed by clinicians. It is well accepted that false-positive Widal reactions following certain non-typhoid Salmonella infections may occur commonly. Three cases of high titers of Widal test are described, where melioidosis was the actual diagnosis in every occasion and was never suspected until diagnosed microbiologically. All the patients had shown a partial response to ceftriaxone. Blood and pus cultures grew Burkholderia pseudomallei, whereas Salmonella typhi was not isolated from blood in any patient. With appropriate antibiotics, the patients showed clinical and microbiological improvement with lowering of Widal titers. These 3 cases show that high Widal titer in any patient may mislead the diagnosis of melioidosis, and further laboratory workup should always be done to rule out melioidosis, especially in cases with nonresponsiveness to treatment.

PCR-based identification of Burkholderia pseudomallei / Identificação de Burkholderia pseudomallei baseada em PCR

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

As técnicas de amplificação de DNA estão sendo cada vez mais utilizadas em laboratórios clínicos para a confirmação da identificação de bactérias que têm importância médica. Um método de identificação de Burkholderia pseudomallei baseado em PCR tem sido usado em nosso centro há 10 anos e foi utilizado para confirmar a identificação de bactérias isoladas de casos de melioidose no Ceará desde 2003. Este método particular tem sido usado como padrão ouro para métodos menos discriminatórios. Nesse estudo, avaliamos três métodos de identificação de B. pseudomallei baseados em PCR e usamos seqüenciamento de DNA para solucionar discrepâncias entre os resultados baseados em PCR e os métodos de identificação fenotípica. O estabelecido protocolo de PCR semi-nested para a região espacial 16-23s da B. pseudomallei produziu um consistente resultado negativo para um de nossos 100 isolados testados (BCC#99), mas identificou corretamente todos os outros 71 isolados de B. pseudomallei. Uma variação anômala da seqüência foi detectada na região interna do sítio de ligação do primer reverso para este método. Métodos de PCR foram desenvolvidos para a detecção de outros dois genes bacterianos metabólicos de B. pseudomallei. O protocolo de PCR IpxO convencional teve sensibilidade de 0,89 e especificidade de 1,0, enquanto que o PCR em tempo real mostrou-se ainda melhor, com sensibilidade de 1,0 e especificidade de 1,0. Este método identificou todos os isolados de B. pseudomallei, incluindo o isolado discrepante que teve o PCR negativo. O protocolo de PCR phaC detectou o gene de todos os B. pseudomallei e em todos exceto três isolados de B. cepacia, tornando este método de identificação de B. pseudomallei baseado em PCR inadequado. Esta experiência com métodos de identificação de B. pseudomallei baseados em PCR indica que devemos ter precaução quando estes forem utilizados sozinhos para identificação dessa bactéria e que eles necessitam ser interpretados em conjunto com métodos fenotípicos e moleculares alternativos, tais como seqüenciamento genético.

In vitro motility of a population of clinical Burkholderia pseudomallei isolates.

Melioidosis, a serious infection caused by Burkholderia pseudomallei, is a leading cause of community-acquired sepsis in Northeast Thailand, and the commonest cause of death from community-acquired pneumonia in the Top End of Northern Australia. The causative organism is a Gram-negative, motile bacillus that is a facultative intracellular pathogen. B. pseudomallei flagella have been proposed as a possible vaccine candidate and putative virulence determinant. Flagella expression was highly conserved for 205 clinical B. pseudomallei isolates, as defined by in vitro swim and swarm motility assays. No association was found between motility and clinical factors including bacteremia and death.

Phenotypic characterization of three clinical isolates of Burkholderia pseudomallei in Ceará, Brazil

Burkholderia pseudomallei, the causative agent of melioidosis was found in a small cluster of cases in Tejuçuoca, Ceará, Brazil. Tests were carried out to determine its phenotypic characteristics: colony morphology on Ashdown agar and MacConkey agar, biochemical profile in conventional biochemical tests and API 20NE, arabinose assimilation and susceptibility testing by disk diffusion, comparing with data in the literature. This study confirms the presence of B. pseudomallei in Brazil and describes its characteristics.

Searching for virulence Burkholderia pseudomallei genes by immunoscreening the lambda ZAPII expressed genomic library.

The lambdaZAP II expressed genomic library of B. pseudomallei was screened with pooled melioidosis serum preabsorbed with E. coli host cell. The positive clones were detected by using protein A-CDP-star chemiluminescence. All of 14 positive clones reacted with only the pooled absorbed melioidosis serum and not the pooled absorbed normal serum when tested with the plaque dot blot analysis. The expressed genes were detected by using a combination of immunoscreening, bioinformatics and molecular biology. At least six in vivo expressed genes were identified by this approach. Two were well known virulent genes, gmhA (a capsule biosynthetic gene) and bipD (type III secretion protein gene). Another two were genes coded for conserved hypothetical protein. The last two isolated genes were groEL (a chaperonine protein gene), and a gene encoding transmembrane protein.

Severe community-acquired pneumonia and sepsis caused by Burkholderia pseudomallei associated with flooding in Puerto Rico

Burkholderia pseudomallei (melioidosis) is usually found in endemic areas of Southeast Asia and Northern Australia. However, a few cases of confirmed melioidosis indigenous to Puerto Rico and the Americas have been reported previously. We describe the occurrence of a B. pseudomallei infection in a female with insulin-dependent diabetes mellitus exposed to flood waters in Puerto Rico. We conclude that B. pseudomallei should be considered a potential pathogen in high-risk patients with severe community-acquired pneumonia and sepsis in Puerto Rico especially in individuals exposed to flood waters during rainy seasons. A more thorough epidemiologic and microbiologic surveillance with environmental sampling may be warranted in the island

Burkholderia pseudomallei--abscess in an unusual site.

Meloidosis in a unusual site has been reported in a child. Complete identification of the organism has been presented.

Evolution of cell-surface acid phosphatase of Burkholderia pseudomallei.

Acid phosphatase active fractions were obtained from cell-free extract, outermembrane fraction and culture filtrate of Burkholderia pseudomallei by column chromatography with sepharose 6B and DEAE cellulose. The comparison of the elution patterns of protein, sugar and enzymatic activity among these three components suggested that the enzyme is a glycoprotein evolving from premature proteins through glycosylation and that the enzyme is translocated during glycosylation from the cytoplasm to the outer membrane and finally excreted into the environment. When tunicamycin, a glycosylation inhibitor, was added to the culture, the peaks of sugar and enzymatic activity were lowered concomitantly leaving the protein peak unchanged in the elution pattern of the culture filtrate. The affinity of the bacterial surface to antienzyme sera was demonstrated by immuno-fluorescence microscopy.

Multifactorial pathogenic mechanisms of Burkholderia pseudomallei as suggested from comparison with Burkholderia cepacia.

With the purpose to elucidate the pathogenesis of disease due to Burkholderia pseudomallei some biological and biochemical properties of this species were studied in comparison with B. cepacia, since the difference in the level of virulence between the two species is remarkable despite of their toxonomic closeness. B. pseudomallei was distinct from B. cepacia in the capability to grow under anaerobic conditions, with positive nitrate respiration, excretion of high-molecular polysaccharides into liquid culture, and cytotoxicity against cultured tissue cells. From these observations together with our previous finding that B. pseudomallei can grow and survive in an acidic environment, we suggest multifactorial mechanisms for the pathogenesis of melioidosis due to B. pseudomallei.

Pseudomonas pseudomallei: II. Laboratory and experimental studies in animals.

The cultural, biochemical characteristics and antibiotic sensitivity of strains of Pseudomonas pseudomallei isolated from four cases of melioidosis admitted to Ramathibodi Hospital are described. The organisms were gram-negative bacilli often with bipolar staining. The colonies were wrinkled when incubated for long periods. The characteristic non-specific uptake of dye from media into the colonies and their musty or earthy odour rendered them easily distinguishable from other organisms. All strains were sensitive to chloramphenicol and all but one were sensitive to tetracycline. All strains were resistant to colimycin and gentamicin. The pathogenicity of the strain isolated from a fatal case of peritonitis was studied in guinea pigs. The findings showed that following a large inoculation intraperitoneally, the animal developed acute septicaemia and died shortly afterwards. Only a few micro-abscesses were found on the surface of the liver. Chronic infection of longer duration occurred when a small number of organisms were introduced through a cutaneous abrasion. The lesions included pneumonitis and multiple abscesses of various organs including subcutaneous tissue, liver, spleen and mediastinum.