Melioidosis is a major cause of morbidity and mortality in Southeast Asia, where the causative organism (Burkholderia pseudomallei) is present in the soil. In the Lao People's Democratic Republic (Laos), B. pseudomallei is a significant cause of sepsis around the capital, Vientiane, and has been isolated in soil near the city, adjacent to the Mekong River. We explored whether B. pseudomallei occurs in Lao soil distant from the Mekong River, drawing three axes across northwest, northeast, and southern Laos to create nine sampling areas in six provinces. Within each sampling area, a random rice field site containing a grid of 100 sampling points each 5 m apart was selected. Soil was obtained from a depth of 30 cm and cultured for B. pseudomallei.
Abstract.Melioidosis is a major neglected tropical disease with high mortality, caused by the Gram-negative bacterium Burkholderia pseudomallei (Bp). Microbiological culture remains the gold standard for diagnosis, but a simpler and more readily available test such as an antibody assay is highly desirable. In this study, we conducted a serological survey of blood donors (n = 1,060) and adult melioidosis patients (n = 200) in northeast Thailand to measure the antibody response to Bp using the indirect hemagglutination assay (IHA). We found that 38% of healthy adults (aged 17–59 years) have seropositivity (IHA titer ≥ 1:80). The seropositivity in healthy blood donors was associated with having a declared occupation of rice farmer and with residence in a nonurban area, but not with gender or age. In the melioidosis cohort, the seropositivity rate was higher in adult patients aged between 18 and 45 years (90%, 37/41) compared with those aged ≥ 45 years (68%, 108/159, P = 0.004). The seropositivity rate was significantly higher in people with diabetes (P = 0.008). Seropositivity was associated with decreased mortality on univariable analysis (P = 0.005), but not on multivariable analysis when adjusted for age, diabetes status, preexisting renal disease, and neutrophil count. This study confirms the presence of high background antibodies in an endemic region and demonstrates the limitations of using IHA during acute melioidosis in this population.
g Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO 2 incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO 2 for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC 90 values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 g/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.
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