PCR combined with lateral flow dipstick assay (PCR-LFD) for a rapid diagnosis of melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Septicemic melioidosis patients have a high mortality rate within 48 hours. OBJECTIVE: To develop a polymerase chain reaction (PCR) combined with a lateral flow dipstick (LFD) assay for detection of B. pseudomallei in blood samples. METHODS: The PCR with wcbG gene primers and a PCR-LFD test were developed. The specificity and sensitivity were determined using the B. pseudomallei and other bacterial DNAs. They were evaluated using 43 B. pseudomallei positive blood samples and another 43 blood samples positive for other microbial infections. RESULTS: The detection limit of the PCR-LFD test was 50 fg of bacterial gDNA or 1.0 CFU per 200 µl of blood. All B. pseudomallei were positive while B. thailandensis and selected gram-negative bacterial strains were negative. The PCR-LFD gave all positives with all 43 B. pseudomallei culture positive patient blood samples and all negative with 43 blood samples that were culture positive for K. pneumoniae, E. gallinarum, E. faecium, E. coli, S. aureus, A. baumannii, A. hydrophila, S. haemolyticus, S. pneumoniae, P. aeruginosa, E. cloacae, S. hominis, E. aerogenes, P. mirabilis, C. neoformans, C. albicans, A. caviae, E. faecalis and K. variicola. CONCLUSION: The developed PCR-LFD assay provided 100% sensitivity and 100% specificity compared to the conventional blood culture. The technique took only 1.5 hours that is easy and quick to perform compared to the 3-7 days of culture method. The new method of PCR with LFD could facilitate the detection to be a semi-point-of-care testing (POCT).

Comparative in vitro activity of sitafloxacin against bacteria isolated from Thai patients with urinary tract infections and lower respiratory tract infections.

OBJECTIVE: To determine comparative in vitro activity of sitafloxacin against clinical isolates of bacteria from Thai patients with urinary tract infection and those with lower respiratory tract infection. MATERIAL AND METHOD: 1,255 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus spp, Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis isolated from different Thai patients with urinary tract infection and those with lower respiratory tract infection in 2010 were included. The minimum inhibitory concentrations (MICs) of sitafloxacin, ciprofloxacin, levofloxacin, moxifloxacin, imipenem, amikacin, ampicillin, ceftazidime, ceftriaxone, penicillin, piperacillin/tazobactam, vancomycin, azithromycin and trimethoprim/sulfamethoxazole were determined by standard agar dilution method. RESULTS: The MIC50 and MIC90 values of sitafloxacin against all tested bacteria were lowest when compared with those of levofloxacin, ciprofloxacin and moxifloxacin. Sitafloxacin was active against 51% of methicillin-resistant S. aureus (MRSA) isolates. The activity of sitafloxacin against multidrug-resistant (MDR) Gram-negative bacteria, such as, extended spectrum beta-lactamase (ESBL)-producing E. coli and K. pneumomiae, P. aeruginosa and A. baumannii was comparable to or more than that of some beta-lactam/beta-lactamase inhibitors, cephalosporins or carbapenems. CONCLUSION: Sitafloxacin is more active than levofloxacin, ciprofloxacin and moxifloxacin against isolated bacteria from Thai patients with urinary tract and lower respiratory infections including antibiotic resistant bacteria, such as MRSA, ESBL-producing Gram-negatives, carbapenem-resistant A. baumannii.

Evolution of the Staphylococcus argenteus ST2250 Clone in Northeastern Thailand Is Linked with the Acquisition of Livestock-Associated Staphylococcal Genes.

Staphylococcus argenteus is a newly named species previously described as a divergent lineage of Staphylococcus aureus that has recently been shown to have a global distribution. Despite growing evidence of the clinical importance of this species, knowledge about its population epidemiology and genomic architecture is limited. We used whole-genome sequencing to evaluate and compare S. aureus (n = 251) and S. argenteus (n = 68) isolates from adults with staphylococcal sepsis at several hospitals in northeastern Thailand between 2006 and 2013. The majority (82%) of the S. argenteus isolates were of multilocus sequence type 2250 (ST2250). S. aureus was more diverse, although 43% of the isolates belonged to ST121. Bayesian analysis suggested an S. argenteus ST2250 substitution rate of 4.66 (95% confidence interval [CI], 3.12 to 6.38) mutations per genome per year, which was comparable to the S. aureus ST121 substitution rate of 4.07 (95% CI, 2.61 to 5.55). S. argenteus ST2250 emerged in Thailand an estimated 15 years ago, which contrasts with the S. aureus ST1, ST88, and ST121 clades that emerged around 100 to 150 years ago. Comparison of S. argenteus ST2250 genomes from Thailand and a global collection indicated a single introduction into Thailand, followed by transmission to local and more distant countries in Southeast Asia and further afield. S. argenteus and S. aureus shared around half of their core gene repertoire, indicating a high level of divergence and providing strong support for their classification as separate species. Several gene clusters were present in ST2250 isolates but absent from the other S. argenteus and S. aureus study isolates. These included multiple exotoxins and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus, consistent with a livestock reservoir for S. argenteus These genes appeared to be associated with plasmids and mobile genetic elements and may have contributed to the biological success of ST2250.IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen.

Bacterial loads and antibody responses in BALB/c mice infected with low and high doses of Burkholderia pseudomallei.

Profiles of Burkholderia pseudomallei persistence and antibodies in blood, spleen, liver, and lungs of infected BALB/c mice were investigated. Animals were infected intraperitoneally with low (6 colony-forming units) or high (230 colony-forming units) doses of B. pseudomallei. In the high-dose infected group, bacteria were found by culture in 100% of blood, liver, spleen and lung samples at 24 and 48 hours after infections; blood samples were 100% positive by polymerase chain reaction after 60 hours. Antibody responses in the high-dose infected group were low. These responses were detected in the low-dose infected group after 5 days, peaked at 7-14 days, and showed persistence until 28 days post-infection. Bacterial loads and antibody profiles varied according to the level of bacterial infections. This kinetic study, although in animals, provides crucial knowledge that might be useful for the development of a sensitive and specific diagnostic assay for patients with melioidosis.