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Background: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. Methods and principal findings: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97–100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. Conclusions: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts. Author summary: The Gram-negative environmental pathogen Burkholderia pseudomallei, causes the severe disease melioidosis. It is highly endemic in southeast Asia and northern Australia, but recent studies suggest that it is also present in many other parts of the world where it is severely underreported. The latter results from the extremely variable and non-specific clinical manifestations of the disease, lack of clinical recognition, and the global scarcity of good quality laboratories to allow diagnosis from microbiological culture. This is even more unfortunate, as early diagnosis of the disease is indispensable for an effective therapy, since B. pseudomallei is intrinsically resistant to many antibiotics used for empirical treatment in endemic areas. Therefore, the development of new, standardized and sensitive tools is of high importance for both diagnostics and epidemiology. We focused on the development of a dipstick assay, which is based on the detection of serum antibodies against four B. pseudomallei specific protein antigens. Here we present a cost effective, simple and rapid melioidosis assay with improved sensitivity that does not depend on sophisticated laboratory equipment and therefore addresses most of the before mentioned obstacles and is easy to manufacture in large scales. [ABSTRACT FROM AUTHOR]

Introduction: The prevalence of bacteremia caused by Gram negative non-fermentative (GNNF) bacteria has been increasing globally over the past decade. Many studies have investigated their epidemiology but focus on the common GNNF including Pseudomonas aeruginosa and Acinetobacter baumannii. Knowledge of the uncommon GNNF bacteremias is very limited. This study explores invasive bloodstream infection GNNF isolates that were initially unidentified after testing with standard microbiological techniques. All isolations were made during laboratory-based surveillance activities in two rural provinces of Thailand between 2006 and 2014. Methods: A subset of GNNF clinical isolates (204/947), not identified by standard manual biochemical methodologies were run on the BD Phoenix automated identification and susceptibility testing system. If an organism was not identified (12/204) DNA was extracted for whole genome sequencing (WGS) on a MiSeq platform and data analysis performed using 3 web-based platforms: Taxonomer, CGE KmerFinder and One Codex. Results: The BD Phoenix automated identification system recognized 92% (187/204) of the GNNF isolates, and because of their taxonomic complexity and high phenotypic similarity 37% (69/187) were only identified to the genus level. Five isolates grew too slowly for identification. Antimicrobial sensitivity (AST) data was not obtained for 93/187 (50%) identified isolates either because of their slow growth or their taxa were not in the AST database associated with the instrument. WGS identified the 12 remaining unknowns, four to genus level only. Conclusion: The GNNF bacteria are of increasing concern in the clinical setting, and our inability to identify these organisms and determine their AST profiles will impede treatment. Databases for automated identification systems and sequencing annotation need to be improved so that opportunistic organisms are better covered. [ABSTRACT FROM AUTHOR]

Bloodstream infection surveillance conducted from 2008 to 2014 in all 20 hospitals in Sa Kaeo and Nakhon Phanom provinces, Thailand, allowed us to look at disease burden, antibiotic susceptibilities, and recurrent infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae . Of 97,832 blood specimens, 3,338 were positive for E. coli and 1,086 for K. pneumoniae . The proportion of E. coli isolates producing ESBL significantly increased from 19% to 22% in 2008-2010 to approximately 30% from 2011 to 2014 ( P -value for trend = 0.02), whereas ESBL production among K. pneumoniae cases was 27.4% with no significant trend over time. Incidence of community-onset ESBL-producing E. coli increased from 5.4 per 100,000 population in 2008 to 12.8 in 2014, with the highest rates among persons aged ≥ 70 years at 79 cases per 100,000 persons in 2014. From 2008 to 2014, community-onset ESBL-producing K. pneumoniae incidence was 2.7 per 100,000, with a rate of 12.9 among those aged ≥ 70 years. Although most (93.6% of E. coli and 87.6% of K. pneumoniae ) infections were community-onset, hospital-onset infections were twice as likely to be ESBL. Population-based surveillance, as described, is vital to accurately monitor emergence and trends in antimicrobial resistance, and in guiding the development of rational antimicrobial therapy recommendations.

Melioidosis is a fatal infectious disease caused by the environmental bacterium Burkholderia pseudomallei It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations, and definitive diagnosis requires bacterial culture, which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by enzyme-linked immunosorbent assay (ELISA) that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects, including the following: (i) 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; (ii) 202 healthy donors from northeast Thailand; (iii) 90 U.S. healthy donors; and (iv) 207 patients infected with other organisms. Compared to culture results as a gold standard, the sensitivity of ICT for all hospitals was 88.3%. The specificities for Thai donors and U.S. donors were 86.1% and 100%, respectively, and the specificity for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA results with a kappa value of 0.829, indicating that the method is much improved compared with the current serological method, the indirect hemagglutination assay (IHA) (69.5% sensitivity and 67.6% specificity for Thais). The Hcp1-ICT represents a potential point-of-care (POC) test and may be used to replace the IHA for screening of melioidosis in hospitals as well as in resource-limited areas., (Copyright © 2018 Phokrai et al.)