Electrochemical Immunoassay for Capturing Capsular Polysaccharide of Burkholderia pseudomallei: Early Onsite Detection of Melioidosis.

This study presented the detection and quantification of capsular polysaccharide (CPS) as a biomarker for the diagnosis of melioidosis. After successfully screening four monoclonal antibodies (mAbs) previously determined to bind CPS molecules, the team developed a portable electrochemical immunosensor based on antibody-antigen interactions. The biosensor was able to detect CPS with a wide detection range from 0.1pg/mL to 1 µg/mL. The developed biosensor achieved high sensitivity for the detection of CPS spiked into both urine and serum. The developed assay platform was successfully programmed into a Windows app, and the sensor performance was evaluated with different spiked concentrations. The rapid electro-analytical device (READ) sensor showed great unprecedented sensitivity for the detection of CPS molecules in both serum and urine, and results were cross-validated with ELISA methods.

Evaluation of the Active Melioidosis Detect™ test as a point-of-care tool for the early diagnosis of melioidosis: a comparison with culture in Laos.

BACKGROUND: Melioidosis is difficult to diagnose clinically and culture of Burkholderia pseudomallei is the current, imperfect gold standard. However, a reliable point-of-care test (POCT) could enable earlier treatment and improve outcomes. METHODS: We evaluated the sensitivity and specificity of the Active Melioidosis Detect™ (AMD) rapid test as a POCT and determined how much it reduced the time to diagnosis compared with culture. RESULTS: We tested 106 whole blood, plasma and buffy coat samples, 96 urine, 28 sputum and 20 pus samples from 112 patients, of whom 26 (23.2%) were culture-positive for B. pseudomallei. AMD sensitivity and specificity were 65.4 and 87.2%, respectively, the latter related to 10 weak positive reactions on urine samples, considered likely false positives. The positive predictive value was 60.7%, negative predictive value was 89.3% and concordance rate between operators reading the test was 95.7%; time to diagnosis decreased by a median of 23 h. CONCLUSIONS: Our findings confirm that a strongly positive AMD result can reduce the time to diagnosis of melioidosis. However, the AMD currently has a disappointing overall sensitivity, especially with blood fractions, and specificity problems when testing urine samples.

Rapid Filter-Based Detection and Culture of Burkholderia pseudomallei from Small Volumes of Urine.

Clinical outcomes of melioidosis patients improve when the infecting agent, Burkholderia pseudomallei, is rapidly detected and identified by laboratory testing. Detection of B. pseudomallei DNA or recovery of the pathogen by culture from urine can support a diagnosis of melioidosis and guide patient care. Two new methods, designated filter-capture DNA isolation (FCDI) and filter cellular recovery (FCR), were developed to increase the sensitivity of detection and recovery of viable B. pseudomallei cells from small volumes (0.45 ml) of urine. DNA from eight strains of B. pseudomallei that were spiked into synthetic urine at low concentrations (1 × 102 CFU/ml) was detected in FCDI cell lysates using real-time PCR with greater consistency than with preparations from a QIAamp DNA Blood minikit. The FCR method showed greater B. pseudomallei detection sensitivity than conventional urine culture methods and resulted in typical colony growth at 24 h from as few as 1 × 102 CFU/ml. In addition, the FCR method does not rely on precipitation of a urine pellet by centrifugation and requires a smaller volume of urine. The FCDI and FCR methods described here could improve time-to-results and decrease the number of negative B. pseudomallei reports that are currently observed from urine culture as a consequence of samples containing low or variable bacterial cell concentrations.

Lack of correlation of Burkholderia pseudomallei quantities in blood, urine, sputum and pus.

We evaluated the correlation of Burkholderia pseudomallei quantities in blood versus urine, sputum or pus. Correlations between bacterial counts in blood and other samples were not found. It is likely that an initial seeding event to extracellular organs is followed by independent growth of B. pseudomallei, and that bacteria in the urine were not passively filtered from the bloodstream.

Quantitation of B. Pseudomallei in clinical samples.

We undertook a prospective study to quantitate Burkholderia pseudomallei in blood, urine, respiratory secretions, and pus [corrected] obtained from 414 patients with melioidosis. The median was count 1.1, 1.5 x 10(4), 1.1 x 10(5), and 1.1 x 10(7) CFU/mL in these sample types, respectively. This provides important insights into the likely feasibility of future studies such as expression microarray analysis using clinical material.

Role and significance of quantitative urine cultures in diagnosis of melioidosis.

Melioidosis is associated with significant mortality in countries in which it is endemic. Previous studies have demonstrated that quantitative Burkholderia pseudomallei counts in blood are predictive of mortality. Here we examine the relationship between outcomes and quantitative B. pseudomallei counts in urine. A total of 755 patients presenting to Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand (in the northeast part of the country), with melioidosis between July 1993 and October 2003 had quantitative urine cultures performed within 72 h of admission. Urine culture results were divided into the following groups: (i) no growth of B. pseudomallei from a neat sample or pellet, (ii) positive result from a centrifuged pellet only (< 10(3) CFU/ml), (iii) detection of between 10(3) CFU/ml and 10(5) CFU/ml from a neat sample, or (iv) detection of > or = 10(5) CFU/ml from a neat sample. The overall in-hospital mortality rate was 45%. Patients with negative urine cultures had the lowest death rate (39%). Mortality rates rose with increasing B. pseudomallei counts in urine, from 58% for those with positive spun pellets only to 61% for those with between 10(3) CFU/ml and 10(5) CFU/ml and 71% for those with > or = 10(5) CFU/ml. This was independent of age, presence of bacteremia, known risk factors for melioidosis such as diabetes, and the prior administration of antibiotics. The presence of B. pseudomallei in urine during systemic infection is associated with a poor prognosis.

Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis.

A latex agglutination test for the detection of Pseudomonas pseudomallei antigen in urine was evaluated for the rapid diagnosis of melioidosis. With unconcentrated urine, antigen was detected in only 18% of patients with melioidosis overall. However, when urine was concentrated 100-fold, antigen was detected in 47% overall and in 67% of patients with septicaemia or disseminated infection, in whom a rapid diagnosis is most important. The specificity of the test was 100%. These results compared favourably with an enzyme immunoassay. This latex agglutination test is a simple, rapid and highly specific method of diagnosing melioidosis, and will be particularly useful in areas with limited laboratory facilities.

Immunofluorescence microscopy for the rapid diagnosis of melioidosis.

A direct immunofluorescent antibody test (DIF) was developed for the rapid diagnosis of melioidosis, a potentially fatal infection caused by Pseudomonas pseudomallei. In a clinical evaluation of 369 sputum, pus, or urine specimens from 272 patients with suspected melioidosis, the DIF had a sensitivity of 73% and a specificity of 99% compared with culture. Using this DIF, a confident diagnosis of melioidosis can now be made within two hours of admission to hospital, compared with the delay of two to four days required for culture results. Consequent early institution of specific antimicrobial therapy may help to save lives.