In this study, a younger sample populace might have underestimated the true level of human exposure toB. 13.8) for polyvalent immunoglobulins, 9.8% (95% CI: 7.7, 11.9) Rabbit polyclonal to AGAP1 for IgG, and 1.7% (95% CI: 0.8, 2.6%) for IgM. The seroprevalence was not significantly different by gender (P= 0.16), but increased significantly (P< 0.001) with age, yielding an estimated annual seroconversion rate of 1 1.05% (95% CI: 0.81, 1.3). The detection of both recent (IgM+) and previous (IgG+) exposure toB. pseudomalleiprovides serological evidence that melioidosis is usually endemic in Haiti. == INTRODUCTION == Originally discovered in 1911, melioidosis has gained attention over the past decade because of its possible use as a weapon of bioterrorism.13The etiological agent,Burkholderia pseudomallei, is a nonsporulating, Gram-negative betaproteobacterium that occurs STO-609 acetate naturally in surface water and soils throughout tropical and subtropical climates.4Melioidosis is a febrile illness characterized by abscess formation and subsequent hematogenous spread of bacteria to virtually any host organ, initiating most commonly in the lungs.5The clinical manifestations arise days to years after inhalational or percutaneous exposure and include acute suppurative soft tissue infections, acute pulmonary infections, chronic localized infections, and often fulminant septicemia.6Although serological studies suggest most infections are symptomatic, the wide range of clinical manifestations and inherent antibiotic resistance complicate timely diagnosis and effective treatment, leading to mortality rates that can exceed 80%.7Historically, Southeast Asia and Northern Australia were considered the main endemic foci; however, case reports from the Indian subcontinent, Africa, and the Americas have demonstrated a much wider geographic STO-609 acetate range.8The recent discovery of autochthonous cases in Puerto Rico has led to the hypothesis that melioidosis could be endemic throughout the Caribbean, including the impoverished nation of Haiti.9Nevertheless, because of the protean clinical manifestations, presence of asymptomatic infections, and minimal medical diagnostic capacity, the identification of active cases remains challenging. Therefore, a large serological study was conducted to investigate the potential for human exposure toB. pseudomalleiin Haiti. == MATERIAL AND METHODS == == Development ofB. pseudomalleilipopolysaccharide (LPS) STO-609 acetate enzyme-linked immunosorbent assay (ELISA). == == Burkholderia pseudomallei LPS antigen preparation. == An indirect ELISA was developed to detect antibodies toward LPS from the outer membrane ofB. pseudomallei. Type A LPS was purified from a derivative ofB. pseudomalleiBp82, an attenuated strain,10using a protocol altered from Lam as well as others.11Strain Bp82 wcb, a capsular polysaccharide (CPS) mutant STO-609 acetate was grown on 10 plates of tryptic soy agar (TSA) agar for 72 hours. Bacterial lawns were scraped with a plate spreader and suspended in phosphate buffered saline (PBS). Bacterial suspensions were incubated at 110C for 15 minutes in 2 mL gasketed microcentrifuge tubes. Phenol was added to the lysed treatment for a final concentration of 50% and 10% volume of the resulting mixture was plated on TSA to ensure sterility. After sterility verification, samples were gently stirred for 30 minutes at 60C on a hot plate before dialysis with 1214 kDa molecular weight cutoff tubing against distilled water for 35 days to completely remove phenol. Dialyzed samples were treated with DNase I for 2 hours, RNase H for 2 hours, and Proteinase K overnight, then further purified as previously described.12After lyophilization, dry weights were determined, the residues were resuspended in 2 mL of endotoxin-free water, and diluted to measure endotoxin units using the Pierce limulus amebocyte lysate assay according to manufacturers instructions. The resulting crude preparation was separated using a liquid chromatography system (AKTA Purifier; GE Healthcare, Pittsburgh, PA), with a HiPrep Sephacryl S-300 high-resolution column and refractive index detector (Agilent) to fractionate the LPS. The purified LPS was lyophilized, reconstituted in endotoxin-free water to a stock concentration of 100 mg/mL, and frozen at 80C for future use. == Validation of LPS ELISA using previously uncovered nonhuman primate serum. == Positive-control.