Quantitation of B. Pseudomallei in Clinical Samples

Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Wongsuvan, Gumphol; Chierakul, Wirongrong; Teerawattanasook, Nittaya; Teparrukkul, Prapit; Day, Nicholas P. J.; Peacock, Sharon J.

doi:10.4269/ajtmh.2007.77.812

Cited by 46 publications

(52 citation statements)

References 11 publications

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“…pseudomallei from blood cultures from two septicemic patients in Australia [29]. The simulated blood culture experiment in which 10 CFU was used as the starting inoculum reflects the bacterial load in blood during human infection, which has been reported previously to be a median count of 1.1 CFU/ml [48]. Our observation that identification of B .…”

Section: Discussionmentioning

confidence: 99%

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species

et al. 2017

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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species.

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Section: Discussionmentioning

confidence: 99%

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species

et al. 2017

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“…Culture is routinely performed on multiple sample types (blood, urine, pus, sputum, etc.) and isolation of B. pseudomallei from any one of these cultures is diagnostic for melioidosis [5], [6]. However, recent modeling data has confirmed that culturing is an imperfect gold standard [7].…”

Section: Introductionmentioning

confidence: 99%

Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

et al. 2014

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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.

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“…Methods that achieve this detection include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and antibody-based immunofluorescence (IFA), which can be applied to a range of sample types, including sputum, pus, urine, respiratory secretion, and body fluids 3–6. Around one-half of patients with melioidosis have positive blood cultures,7 and an important drawback for all antigen assays is that the median B. pseudomallei count in blood is 1 cfu/mL,8 which is far below the achievable level of detection by the IFA. Pre-incubation of blood cultures would be predicted to increase the sensitivity of IFA, and despite this extra step would still reduce the time to diagnosis compared with conventional culture and biochemical identification.…”

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confidence: 99%

Rapid Detection of Burkholderia pseudomallei in Blood Cultures Using a Monoclonal Antibody-Based Immunofluorescent Assay

Chantratita¹,

Tandhavanant²,

Wongsuvan³

et al. 2013

The American Society of Tropical Medicine and Hygiene

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Melioidosis is a severe bacterial infection caused by Burkholderia pseudomallei. Rapid antimicrobial therapy is necessary to improve patient outcome, which is aided by direct detection of B. pseudomallei in clinical samples. A drawback for all antigen assays is that the number of B. pseudomallei in blood usually falls below the achievable level of detection. We performed a prospective cohort study of 461 patients with 541 blood cultures to evaluate the utility of a pre-incubation step prior to detection of B. pseudomallei using a monoclonal antibody-based immunofluorescent assay (Mab-IFA). The Mab-IFA was positive in 74 of 76 patients with melioidosis (sensitivity = 97.4%), and negative in 385 patients who did not have blood cultures containing B. pseudomallei (specificity = 100%). The Mab-IFA could be a valuable supplementary tool for rapid detection. We recommend the use of the Mab-IFA to test blood cultures that flag positive in regions where melioidosis is endemic.

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Quantitation of B. Pseudomallei in Clinical Samples

Cited by 46 publications

References 11 publications

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species

Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

Rapid Detection of Burkholderia pseudomallei in Blood Cultures Using a Monoclonal Antibody-Based Immunofluorescent Assay

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