Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36−43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25°C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.

In Thailand from 1996 to 2003, 171 strains of pathogenic aerobic actinomycetes from clinical specimens were isolated. Of those strains, 134 were mycolic acid containing actinomycetes, including 96 strains of Nocardia species. Others included 10 strains of Gordonia, 14 strains of Rhodococcus, and 22 strains of Mycobacterium. One strain each of the genera Tsukamurella and Corynebacterium were also isolated. Also identified were 27 strains of non-mycolic acid containing actinomycetes. Our identification studies of 96 strains of Nocardia species showed that significant pathogens in Thailand were N. beijingensis (18 strains), N. cyriacigeorgica (13 strains), and N. farcinica (34 strains); the most prevalent species was N. farcinica (35.4%). We also isolated four strains of N. asiatica, five strains of N. asteroides sensu stricto, four strains of N. nova, seven strains of N. otitidiscaviarum, eight strains of N. transvalensis, and two strains of N. pseudobrasiliensis.

PurposeColistin is a drug of last resort for treating multidrug-resistant Acinetobacter baumannii infections. Unfortunately, colistin-resistant A. baumannii (CoR-AB) has been reported. Here, we examined the in vitro effect of mono- and combined antimicrobials against CoR-AB strains and their resistance mechanism, and evaluated the clinical outcomes of CoR-AB-infected patients.Patients and methodsSeventeen clinical CoR-AB strains were isolated from patients at Phramongkutklao hospital, 2011–2015. The mono- and synergistic activities of colistin, tigecycline, sulbactam, imipenem, meropenem, amikacin, fosfomycin, and cotrimoxazole were examined by minimum inhibitory concentration (MIC) and fractional inhibitory concentration index. Clonal relationship and resistance genes were determined by repetitive extragenic palindromic polymerase chain reaction with specific primers. The effect of carbonyl cyanide 3-chlorophenylhydrazone combined with colistin was used to test efflux pump involvement. Patient treatment outcomes were also reported.ResultsThe most prevalent infection in CoR-AB patients was pneumonia (35.3%), and all patients were administered colistin combined with another agent. The 30-day mortality was 70.6%, and the colistin MIC range and MIC50 was 16–512 μg/mL and 64 μg/mL, respectively. All CoR-AB strains were sensitive to tigecycline. Sporadic isolates were susceptible to sulbactam, imipenem, meropenem, and cotrimoxazole. A synergistic or additive effect was observed for colistin plus imipenem or meropenem (16.7%), sulbactam (66.7%), or tigecycline (66.7%). The CoR-AB isolates could be divided into four different clones (A–D) with a high prevalence of group B (47.1%). Eight isolates harbored blaOXA23, blaIMP, blaKPC, and blaNDM, and one contained blaOXA23, blaIMP, and blaKPC, while the eight remaining isolates carried only blaOXA23. The MIC values of all strains were greatly reduced for colistin plus carbonyl cyanide 3-chlorophenylhydrazone.ConclusionCoR-AB clinical isolates exhibited very high colistin resistance and a high frequency of resistance genes. The mechanism of colistin resistance appears to be mediated via an efflux pump. Thus, certain antimicrobials could be used as salvage therapy for CoR-AB infection.

Purpose The incidence of infections with vancomycin-resistant enterococci (VRE) is increasing, with associated high mortality rates and limited therapeutic choices. We investigated the clinical characteristics and treatment outcomes of VRE infection and also determined the in vitro effect of monotherapy and combined antimicrobials against clinical VRE isolates. Patients and methods Clinical data and bacterial isolates obtained from patients with VRE infections between January 2014 and April 2018 at Phramongkutklao Hospital were reviewed. The clinical outcomes included in-hospital mortality, 30-day mortality, and microbiological eradication. Clonal relationships were assessed by random amplified polymorphic DNA analysis. In vitro activity of linezolid, tigecycline, fosfomycin, gentamicin, chloramphenicol, and ampicillin were determined by minimum inhibitory concentration (MIC) values. Tests of synergy of fosfomycin- or gentamicin-based combinations by the checkerboard method were reported with the fractional inhibitory concentration index or MIC reduction, respectively. Results Among 26 cases of VRE infection, nosocomial and gastrointestinal infections were the most common. There were various treatment regimens, but linezolid-containing regimens were generally used. In-hospital and 30-day mortality were 73.1% and 57.7%, respectively. Higher mortality was significantly associated with illness severity. The VRE isolates tested were universally susceptible to linezolid and tigecycline. A synergistic or additive effect was observed for fosfomycin combined with linezolid (100%) and with tigecycline (83.3%). Fourfold or greater MIC reduction was observed for linezolid or fosfomycin plus gentamicin at concentrations 1 (58.3%, 62.5%), 2 (83.3%, 62.5%), and 4 μg/mL (91.6%, 62.5%). Conclusion In-hospital mortality among patients with VRE infection was high. Linezolid remains a treatment of choice. However, combination therapy such as linezolid plus fosfomycin and linezolid plus gentamicin should be considered in cases of serious infection.

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