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Burkholderia pseudomallei—a causative agent of melioidosis that is endemic in Southeast Asia and Northern Australia—is a Gram-negative bacterium transmitted to humans via inhalation, inoculation through skin abrasions, and ingestion. Melioidosis causes a range of clinical presentations including skin infection, pneumonia, and septicemia. Despite skin infection being one of the clinical symptoms of melioidosis, the pathogenesis of B. pseudomallei in skin fibroblasts has not yet been elucidated. In this study, we investigated B. pseudomallei pathogenesis in the HFF-1 human skin fibroblasts. On the basis of co-culture assays between different B. pseudomallei clinical strains and the HFF-1 human skin fibroblasts, we found that all B. pseudomallei strains have the ability to mediate invasion, intracellular replication, and multinucleated giant cell (MNGC) formation. Furthermore, all strains showed a significant increase in cytotoxicity in human fibroblasts, which coincides with the augmented expression of matrix metalloproteinase-2. Using B. pseudomallei mutants, we showed that the B. pseudomallei Bsa type III secretion system (T3SS) contributes to skin fibroblast pathogenesis, but O-polysaccharide, capsular polysaccharide, and short-chain dehydrogenase metabolism do not play a role in this process. Taken together, our findings reveal a probable connection for the Bsa T3SS in B. pseudomallei infection of skin fibroblasts, and this may be linked to the pathogenesis of cutaneous melioidosis. [ABSTRACT FROM AUTHOR]

Background: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. Methods and principal findings: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97–100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. Conclusions: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts. Author summary: The Gram-negative environmental pathogen Burkholderia pseudomallei, causes the severe disease melioidosis. It is highly endemic in southeast Asia and northern Australia, but recent studies suggest that it is also present in many other parts of the world where it is severely underreported. The latter results from the extremely variable and non-specific clinical manifestations of the disease, lack of clinical recognition, and the global scarcity of good quality laboratories to allow diagnosis from microbiological culture. This is even more unfortunate, as early diagnosis of the disease is indispensable for an effective therapy, since B. pseudomallei is intrinsically resistant to many antibiotics used for empirical treatment in endemic areas. Therefore, the development of new, standardized and sensitive tools is of high importance for both diagnostics and epidemiology. We focused on the development of a dipstick assay, which is based on the detection of serum antibodies against four B. pseudomallei specific protein antigens. Here we present a cost effective, simple and rapid melioidosis assay with improved sensitivity that does not depend on sophisticated laboratory equipment and therefore addresses most of the before mentioned obstacles and is easy to manufacture in large scales. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation. Author Summary: Burkholderia pseudomallei is an environmental bacterium and the cause of melioidosis. Culture of patient samples is the "gold standard" diagnostic test, but may take up to 7 days to complete. Melioidosis has a 10–40% case fatality rate depending on the geographic location. Delays in diagnosis could lead to administration of ineffective antimicrobial therapy, since B. pseudomallei is resistant to empiric antibiotic regimens. Therefore, we have developed a lateral flow immunoassay that can be used in the clinical setting to diagnose melioidosis in 15 minutes. The test promises to provide improved management of patients with melioidosis. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is one of the most important virulence and antigenic components of Burkholderia pseudomallei, the causative agent of melioidosis. LPS diversity in B. pseudomallei has been described as typical, atypical or rough, based upon banding patterns on SDS-PAGE. Here, we studied the genetic and molecular basis of these phenotypic differences. Bioinformatics was used to determine the diversity of genes known or predicted to be involved in biosynthesis of the O-antigenic moiety of LPS in B. pseudomallei and its near-relative species. Multiplex-PCR assays were developed to target diversity of the O-antigen biosynthesis gene patterns or LPS genotypes in B. pseudomallei populations. We found that the typical LPS genotype (LPS genotype A) was highly prevalent in strains from Thailand and other countries in Southeast Asia, whereas the atypical LPS genotype (LPS genotype B) was most often detected in Australian strains (∼13.8%). In addition, we report a novel LPS ladder pattern, a derivative of the atypical LPS phenotype, associated with an uncommon O-antigen biosynthesis gene cluster that is found in only a small B. pseudomallei sub-population. This new LPS group was designated as genotype B2. We also report natural mutations in the O-antigen biosynthesis genes that potentially cause the rough LPS phenotype. We postulate that the diversity of LPS may correlate with differential immunopathogenicity and virulence among B. pseudomallei strains. Author Summary: Burkholderia pseudomallei is an environmental Gram-negative bacterium and the cause of melioidosis, an often life-threatening disease affecting people in Southeast Asia and northern Australia. Melioidosis is usually contracted by bacterial inoculation, ingestion or inhalation. Effective vaccines for melioidosis are currently unavailable. This organism contains a large genome, which varies greatly among strains due to a high frequency of genetic recombination. We report here on diversity of lipopolysaccharides (LPS) in this species, a major component of the bacterial outer membrane and a known immunogenic virulence factor. We developed LPS genotyping techniques to study frequency of two major LPS types, known as typical and atypical LPS, in B. pseudomallei strains collected from two endemic regions: Southeast Asia and Northern Australia. LPS genotype variation differed among B. pseudomallei populations. During the investigation, we discovered a new LPS genotype in a sub-population group of B. pseudomallei in Australia. We postulate that such differences are likely to be associated with variable immunopathogenicity and clinical presentation in the human host. [ABSTRACT FROM AUTHOR]