Burkholderia pseudomallei Is Spatially Distributed in Soil in Northeast Thailand

Melioidosis is a severe infection caused by the environmental bacterium Burkholderia pseudomallei. Soil sampling is important to identify geographic regions where humans and animals are at risk of exposure. The purpose of this study was to examine a factor that has a major bearing on the accuracy of soil sampling: the spatial distribution of B. pseudomallei in soil of a specified sampling site. Soil sampling was performed using a fixed-interval grid of 100 sampling points in each of two sites (disused land and rice field) in northeast Thailand, and the presence and amount of B. pseudomallei determined using culture. Mapping of the presence and B. pseudomallei count demonstrated that samples taken from areas adjacent to sampling points that were culture positive (negative) for B. pseudomallei were also likely to be culture positive (negative), and samples taken from areas adjacent to sampling points with a high (low) B. pseudomallei count were also likely to yield a high (low) count (spatial autocorrelation). These data were used as the basis for highlighting several pitfalls in current approaches to soil sampling, together with a discussion of the suitability of a range of sampling strategies in different geographical locations and for different study objectives

Defining the True Sensitivity of Culture for the Diagnosis of Melioidosis Using Bayesian Latent Class Models

BACKGROUND: Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests. METHODS/PRINCIPAL FINDINGS: Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard. CONCLUSIONS/SIGNIFICANCE: Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections

Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis

Abstract Background Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium. Methods We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed. Results 39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species). Conclusion Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

The soil dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis, a serious human infection that occurs in Southeast Asia and northern Australia. The purpose of this study was to evaluate the population genetic structure of B. pseudomallei in the environment. To achieve this, we undertook soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within an area of disused land in northeast Thailand, and undertook detailed genotyping of primary plate colonies isolated from three independent sampling points. Our results demonstrated that multiple B. pseudomallei genotypes were present within a single soil sample, and that different genotypes were present at independent but nearby sampling points. The B. pseudomallei genetic population was unevenly distributed within a given sample, with a predominant genotype co-existing with several genotypes present as a minority population. We discuss the implications of this structuring of genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies

Burkholderia pseudomallei Is Genetically Diverse in Agricultural Land in Northeast Thailand

Burkholderia pseudomallei is the cause of melioidosis, a serious human infection most commonly diagnosed in southeast Asia and northern Australia. The organism lives in the soil in a specific geographical distribution and infection results from bacterial inoculation, inhalation or ingestion. The purpose of this study was to define the distribution and genetic diversity of B. pseudomallei in agricultural land where most human infections probably occur. We performed soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within a rice paddy in northeast Thailand, and undertook genotyping of primary culture plate colonies from 11 sampling points. We identified 7 different genotypes, with relatively limited overlap between different sampling points. Two samples contained more than one B. pseudomallei genotype, in which a numerically dominant genotype coexisted with one or more additional genotypes present as a minority population. We conclude that genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings inform future efforts to define B. pseudomallei in the environment, and should be considered during the design stage of studies comparing B. pseudomallei isolated from the environment and from patients with invasive disease

Persistent Gastric Colonization with Burkholderia pseudomallei and Dissemination from the Gastrointestinal Tract following Mucosal Inoculation of Mice

Melioidosis is a disease of humans caused by opportunistic infection with the soil and water bacterium Burkholderia pseudomallei. Melioidosis can manifest as an acute, overwhelming infection or as a chronic, recurrent infection. At present, it is not clear where B. pseudomallei resides in the mammalian host during the chronic, recurrent phase of infection. To address this question, we developed a mouse low-dose mucosal challenge model of chronic B. pseudomallei infection and investigated sites of bacterial persistence over 60 days. Sensitive culture techniques and selective media were used to quantitate bacterial burden in major organs, including the gastrointestinal (GI) tract. We found that the GI tract was the primary site of bacterial persistence during the chronic infection phase, and was the only site from which the organism could be consistently cultured during a 60-day infection period. The organism could be repeatedly recovered from all levels of the GI tract, and chronic infection was accompanied by sustained low-level fecal shedding. The stomach was identified as the primary site of GI colonization as determined by fluorescent in situ hybridization. Organisms in the stomach were associated with the gastric mucosal surface, and the propensity to colonize the gastric mucosa was observed with 4 different B. pseudomallei isolates. In contrast, B. pseudomallei organisms were present at low numbers within luminal contents in the small and large intestine and cecum relative to the stomach. Notably, inflammatory lesions were not detected in any GI tissue examined in chronically-infected mice. Only low-dose oral or intranasal inoculation led to GI colonization and development of chronic infection of the spleen and liver. Thus, we concluded that in a mouse model of melioidosis B. pseudomallei preferentially colonizes the stomach following oral inoculation, and that the chronically colonized GI tract likely serves as a reservoir for dissemination of infection to extra-intestinal sites

BurkDiff: A Real-Time PCR Allelic Discrimination Assay for Burkholderia Pseudomallei and B. mallei

A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei

Evolution of Burkholderia pseudomallei in Recurrent Melioidosis

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis

Causes of acute undifferentiated fever and the utility of biomarkers in Chiangrai, northern Thailand

Tropical infectious diseases like dengue, scrub typhus, murine typhus, leptospirosis, and enteric fever continue to contribute substantially to the febrile disease burden throughout Southeast Asia while malaria is declining. Recently, there has been increasing focus on biomarkers (i.e. C-reactive protein (CRP) and procalcitonin) in delineating bacterial from viral infections.A prospective observational study was performed to investigate the causes of acute undifferentiated fever (AUF) in adults admitted to Chiangrai Prachanukroh hospital, northern Thailand, which included an evaluation of CRP and procalcitonin as diagnostic tools. In total, 200 patients with AUF were recruited. Scrub typhus was the leading bacterial cause of AUF (45/200, 22.5%) followed by leptospirosis (15/200, 7.5%) and murine typhus (7/200, 3.5%), while dengue was the leading viral cause (23/200, 11.5%). Bloodstream infections contributed to 7/200 (3.5%) of the study cohort. There were 9 deaths during this study (4.5%): 3 cases of scrub typhus, 2 with septicaemia (Talaromyces marneffei and Haemophilus influenzae), and 4 of unknown aetiologies. Rickettsioses, leptospirosis and culture-attributed bacterial infections, received a combination of 3rd generation cephalosporin plus a rickettsia-active drug in 53%, 73% and 67% of cases, respectively. Low CRP and white blood count were significant predictors of a viral infection (mainly dengue) while the presence of an eschar and elevated aspartate aminotransferase and alkaline phosphatase were important predictors of scrub typhus.Scrub typhus and dengue are the leading causes of AUF in Chiangrai, Thailand. Eschar, white blood count and CRP were beneficial in differentiating between bacterial and viral infections in this study. CRP outperformed procalcitonin although cut-offs for positivity require further assessment. The study provides evidence that accurate, pathogen-specific rapid diagnostic tests coupled with biomarker point-of-care tests such as CRP can inform the correct use of antibiotics and improve antimicrobial stewardship in this setting

Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

Melioidosis is a severe disease affecting humans and animals in the tropics. It is caused by the bacterium Burkholderia pseudomallei, which lives in tropical soil and especially occurs in southeast Asia and northern Australia. Despite the recognition that melioidosis is an emerging infectious disease, little is known about the habitat of B. pseudomallei in the environment. We performed a survey in the Darwin area in tropical Australia, screening 809 soil samples for the presence of these bacteria using molecular methods. We found that environmental factors describing the habitat of these bacteria differed between environmentally undisturbed and disturbed sites. At undisturbed sites, B. pseudomallei was primarily found in close proximity to streams and in grass- and roots-rich areas. In disturbed soil, B. pseudomallei was associated with the presence of animals, farming or irrigation. Highest B. pseudomallei counts were retrieved from paddocks, pens and kennels holding livestock and dogs. This study contributes to the elucidation of the habitat of B. pseudomallei in northern Australia. It also raises concerns that B. pseudomallei may spread due to changes in land management