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This study focuses on enhancing the sensitivity of lateral-flow strips (LFSs) based on gold nanoparticles (AuNPs) for the detection of Neutrophil Gelatinase-Associated Lipocalin (NGAL) protein in urine samples. Several sizes of AuNP-based LFS biosensors were tested to optimize colorimetric signals for NGAL detection based on improved conjugation conditions. AuNPs of 39.8 nm diameter at pH 8 were the most sensitive for the detection of NGAL. Through systematic enhancements to the AuNP-based LFS, the study significantly improves the sensitivity, enabling the reliable detection of NGAL protein in urine samples at a level as low as 12.5 ng mL -1 . These advances contribute to the refinement of diagnostic tools for the early detection of kidney injury, specifically in cases associated with the presence of NGAL protein, offering a more precise and effective screening approach.

The incidence of kidney disease is increasing worldwide. Rapid and cost-effective approaches for early detection help prevent this disease. Neutrophil gelatinase-associated lipocalin protein (NGAL) is a novel biomarker for acute kidney injury (AKI) and chronic kidney disease (CKD). We aimed to develop a lateral flow strip (LFS) based on a lateral flow immunoassay method (LFIA), using latex microspheres (LMs) as a color labeling to detect NGAL in urine. The performance and potential of the developed LMs-LFS at a point-of-care (POC) testing were evaluated. The results showed that LMs-LFS successfully detected urinary NGAL within 15 min with high specificity without cross-reactivity to or interference from other endogenous substances in urine. The visual limit of detection (vLOD) was 18.75 ng/mL, and the limit of detection (LOD) was 1.65 ng/mL under the optimum condition. The LMs-LFS developed in this study showed a high correlation with the enzyme-linked immunosorbent assay (ELISA) method ( R 2 = 0.973, n = 60 urine specimens) for detecting NGAL in urine. The LMs-LFS remained stable for at least six months at room temperature. The LMs-LFS can be a rapid, sensitive, and specific tool for the diagnosis and follow-up of renal disorders at the POC., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

In this study, the covalently fixed "end-on" orientation of a monoclonal Listeria monocytogenes antibody (mAb- Lis ) to amino terminated oligo (ethylene glycol)-capped gold nanoparticles (NH 2 -TEG-AuNPs) was used to fabricate an in-house lateral flow strip (LFS), namely, the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS. The aim was to evaluate the performance of the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS in detecting L. monocytogenes . The proposed LFS enabled the sensitive detection of L. monocytogenes in 15 min with a visual limit of detection of 10 2 CFU/mL. Quantitative analysis indicated an LOD at 10 CFU/mL. The fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS showed no cross-reactivity with other pathogenic bacteria and practical performance across different food matrices, including human blood, milk, and mushroom samples. Furthermore, the clinical performance of the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS for detecting L. monocytogenes was evaluated by using 12 clinical samples validated by the hemoculture method. It demonstrated excellent concordance with the reference methods, with no false-positive or false-negative results observed. Therefore, the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS serves as a promising candidate for a point-of-care test (POCT), enabling the rapid, precise, and highly sensitive detection of L. monocytogenes in clinical samples and contaminated food.

Background: Dioscorea bulbifera Linn. has been used for wound care in Thailand. However, a comprehensive evaluation of its antibacterial activity is required. This study aimed to investigate the antibacterial efficacy of D. bulbifera extract against skin-associated bacteria and isolate and characterize its active antibacterial agent, flavanthrinin., Methods: Air-dried bulbils of D. bulbifera were pulverised and extracted with hexane, dichloromethane, ethyl acetate, methanol, ethanol, and distilled water; vacuum filtered; concentrated; freeze-dried; and stored at -20 ºC. Antibacterial activity of the extracts was assessed using microdilution techniques against several skin-associated bacteria. Thin-layer chromatography (TLC) bioautography was used to identify the active compounds in the extract, which were fractionated by column chromatography and purified by preparative TLC. The chemical structures of the purified compounds were analysed using nuclear magnetic resonance (NMR). The cytotoxicity of the extract and its active compounds was evaluated in Vero cells., Results: The ethyl acetate extract exhibited distinct inhibition zones against bacteria compared to other extracts. Therefore, the ethyl acetate extract of D. bulbifera in the ethyl acetate layer was used for subsequent analyses. D. bulbifera extract exhibited antibacterial activity, with minimum inhibitory concentrations (MICs) of 0.78-1.56 mg/mL. An active compound, identified through TLC-bioautography, demonstrated enhanced antibacterial activity, with MICs of 0.02-0.78 mg/mL. NMR analysis identified this bioactive compound as flavanthrinin. Both D. bulbifera extract and flavanthrinin-containing fraction demonstrated potent antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and S. epidermidis. The flavanthrinin containing fraction demonstrated low cytotoxicity against Vero cells, showing CC 50 values of 0.41 ± 0.03 mg/mL. These values are lower than the MIC value, indicating that this fraction is safer than the initial ethyl acetate extract., Conclusions: Dioscorea bulbifera extract and its bioactive component flavanthrinin demonstrated significant antibacterial activity against the skin-associated bacteria Staphylococci, including MRSA. Flavanthrinin has potential as a complementary therapeutic agent for managing skin infections owing to its potent antibacterial effects and low cytotoxicity., (© 2024. The Author(s).)

Staphylococcus aureus is one of the most common pathogens. The conventional workflow for identifying this organism is time-consuming and takes up to several days. Therefore, we developed a colloidal gold-based lateral flow immunoassay (LFIA) using human IgG as a conjugated antibody to detect S. aureus . One hundred and thirty-eight clinical isolates, including 79 S . aureus and 59 non- S . aureus were spiked in blood samples, and incubated at 37°C for 24 h. The bacterial antigens were simply extracted before being tested by the developed LFIA strips. The results were read by the naked eye within 15 min. Conventional PCR was used as a reference method. The sensitivity and specificity of the developed LFIA were 100% (95% CI: 94.2%-100.0% and 92.4%-100.0%, respectively) in spiked blood culture samples. The detection limits of the LFIA for the purified protein A and bacterial colonies were 10 -3 µg/mL and 10 7 CFU/mL, respectively. The performance of the LFIA testing in 221 bacterial colony isolates and 118 positive blood culture bottles from three hospitals by their medical technologists showed 98.1% (95% CI: 94.1%-99.5%) and 89.7% (95% CI: 79.3%-95.4%) sensitivity, respectively. The LFIA is a quick, easy, and sensitive method for detecting S. aureus without expensive equipment. It might have the potential for early diagnosis of routine service in low-resource laboratories, leading to a rapid and effective treatment.IMPORTANCEIn this study, we modified our previously developed lateral flow immunoassay (LFIA) test for the detection of Staphylococcus aureus by using an in-house human IgG as a conjugated antibody instead of the specific commercial antibody. It gave comparable results to the former developed-LFIA test and helped cost reduction., Competing Interests: The authors declare no conflict of interest.

Importance: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination., Competing Interests: N.S., P.K., N.C., and M.S.-L. were listed as inventors in patents and patent applications involving the mature virus-like particles of flaviviruses. Other authors declare that they do not have conflicts of interest.

Background: Human respiratory syncytial virus (hRSV) is an important cause of acute respiratory infection, especially in children. Few studies have investigated molecular epidemiology of hRSV infection in Thailand. The aims of this study were to investigate the prevalence and genotype diversity of hRSV in children with acute respiratory infection (ARI) in Thailand., Methods: A total of 383 nasopharyngeal swabs collected from children with ARI from October 2020 to September 2021 were screened for hRSV and nucleotide sequences of the hypervariable region 2 (HVR2) of G gene of the detected hRSV were analysed., Results: Of 383 nasopharyngeal swabs, 104 (27.2 %) were positive for hRSV, of which 51 (49.0 %), 43 (41.3 %), and 10 (9.6 %) were hRSV-A, hRSV-B, and untypeable strains, respectively. All hRSV-A and hRSV-B were ON1 genotype and BA9 genotype, respectively. Most of the hRSV strains were detected in the cool months, November 2020 to February 2021. Phylogenetic analysis of the HVR2 sequence of G gene revealed three clusters of hRSV-A (ON1 genotype) and two clusters of hRSV-B (BA9 genotype). The hRSV-A strains in cluster 1 and 3 were closely related to the hRSV-A reference strains reported previously from other regions of Thailand whereas those in cluster 2 were closely related to the hRSV-A reference strains reported previously from Europe and Africa. For the hRSV-B strains, both clusters 1 and 2 were closely related to the hRSV-B reference strains reported previously from Europe, Australia, and Taiwan. The predicted N- and O-linked glycosylation sites were found along the length of HVR2 of G protein, mostly in the hRSV-B strains., Conclusions: The ON1 and BA9 were the only two hRSV genotypes that were co-predominant and solely detected in this study. The findings indicated that the ON1 and BA9 are the only two hRSV genotypes currently circulating in children with ARI in northern Thailand., Competing Interests: Declaration of Competing Interest There is no conflict of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Humans infected with dengue virus (DENV) acquire long-term protection against the infecting serotype, whereas cross-protection against other serotypes is short-lived. Long-term protection induced by low levels of type-specific neutralizing antibodies can be assessed using the virus-neutralizing antibody test. However, this test is laborious and time-consuming. In this study, a blockade-of-binding enzyme-linked immunoassay was developed to assess antibody activity by using a set of neutralizing anti-E monoclonal antibodies and blood samples from dengue virus-infected or -immunized macaques. Diluted blood samples were incubated with plate-bound dengue virus particles before the addition of an enzyme-conjugated antibody specific to the epitope of interest. Based on blocking reference curves constructed using autologous purified antibodies, sample blocking activity was determined as the relative concentration of unconjugated antibody that resulted in the same percent signal reduction. In separate DENV-1-, -2-, -3-, and -4-related sets of samples, moderate to strong correlations of the blocking activity with neutralizing antibody titers were found with the four type-specific antibodies 1F4, 3H5, 8A1, and 5H2, respectively. Significant correlations were observed for single samples taken 1 month after infection as well as samples drawn before and at various time points after infection/immunization. Similar testing using a cross-reactive EDE-1 antibody revealed a moderate correlation between the blocking activity and the neutralizing antibody titer only for the DENV-2-related set. The potential usefulness of the blockade-of-binding activity as a correlative marker of neutralizing antibodies against dengue viruses needs to be validated in humans. IMPORTANCE This study describes a blockade-of-binding assay for the determination of antibodies that recognize a selected set of serotype-specific or group-reactive epitopes in the envelope of dengue virus. By employing blood samples collected from dengue virus-infected or -immunized macaques, moderate to strong correlations of the epitope-blocking activities with the virus-neutralizing antibody titers were observed with serotype-specific blocking activities for each of the four dengue serotypes. This simple, rapid, and less laborious method should be useful for the evaluation of antibody responses to dengue virus infection and may serve as, or be a component of, an in vitro correlate of protection against dengue in the future., Competing Interests: The authors declare no conflict of interest.

Background: Evidence shows poor adherence to strategies for reducing morbidity and mortality in intensive care unit (ICU) patients receiving mechanical ventilation globally. Best practice management relies on training all members of the interprofessional ICU team, each with complementary roles in patient management., Objectives: To develop and evaluate a novel two-phase, train-the-trainer, interprofessional and multicultural "Best Practice Management of the Ventilated ICU Patient" multimodality, simulation-enhanced curriculum for Thai education leaders in critical care., Methods: In phase 1 (Oregon Health and Science University cohort), two groups of nine ICU nurses and one critical care physician representing experts in critical care and education from a large hospital system in Thailand participated in a weeklong, immersive course consisting of didactic, simulation, and in situ immersive sessions focused on best practice management of mechanically ventilated ICU patients, as well as training in our educational techniques. Outcomes were assessed with pre- and postcourse knowledge assessments and overall course evaluation. In phase 2 (Thai cohort), participants from phase 1 returned to Thailand and implemented a lower fidelity curriculum in two hospitals, using the same pre- and posttest knowledge assessment in 41 participants, before the onset of the coronavirus disease (COVID-19) 6 pandemic., Results: In the Oregon Health and Science University cohort, the mean pretest knowledge score was 58.4 ± 13.2%, with a mean improvement to 82.5 ± 11.6% after completion of the course ( P  , 0.05). The greatest improvements were seen in respiratory physiology and advanced/disease-specific concepts, which demonstrated absolute improvements of 30.4% and 30.6%, respectively ( P  < 0.05). Participants had a high degree of satisfaction, with 90% rating the course as "excellent" and .90% reporting that the course "greatly improved" their understanding of best practices and comfort in managing mechanical ventilation. The Thai cohort had a mean baseline score of 45.4 ± 15.0% and a mean improvement to 70.3 ± 19.1% after training ( P  < 0.05). This cohort also saw the greatest improvement in respiratory physiology and advanced/disease-specific concepts, with 26.2% and 26.3% absolute improvements, respectively ( P  < 0.05)., Conclusion: A novel, two-phase, interprofessional, multicultural, simulation-enhanced train-the-trainer curriculum was feasible and effective in improving education in best practice management of mechanically ventilated patients and may be a useful model for improving the care of ICU patients across the world., (Copyright © 2022 by the American Thoracic Society.)

Carbapenemase-producing Gram-negative bacteria have been increasingly reported. Simple and sensitive methods for carbapenemase detection are still needed. In this study, a gold nanoparticle (AuNP) solution was modified by the addition of zinc sulfate (ZnSO 4 ) for improving the conventional GoldNano Carb (cGoldC) test, and the modified GoldC (mGoldC) test was then evaluated for phenotypic detection of carbapenemase production in Gram-negative bacilli clinical isolates. ZnSO 4 was added to give final concentrations of 0.25, 0.5, 0.75, and 1 mM. The performance of the mGoldC test was evaluated in Enterobacterales, Acinetobacter spp., and Pseudomonas aeruginosa isolates from six hospitals in different regions using polymerase chain reaction (PCR) as a gold standard. The AuNP solution with 0.25 mM ZnSO 4 was used for the mGoldC test. Evaluation of the mGoldC test in 495 Enterobacterales, 212 Acinetobacter spp., and 125 P. aeruginosa isolates (including 444 carbapenemase producers and 388 non-carbapenemase producers) revealed sensitivity, specificity, a positive likelihood ratio, and a negative likelihood ratio of 98.6%, 98.2%, 54.7, and 0.01, respectively. This test is fast, easy to perform, cost-effective (~0.25 USD per test), and highly sensitive and specific for routine carbapenemase detection, thus leading to effective antimicrobial therapy and infection control measures.

We modified rapid polymyxin Nordmann-Poirel (RPNP) test, called rapid colistin disk elution (RCDE) test, for detecting colistin resistance in Gram-negative bacilli and evaluated its performance compared with colistin broth disk elution (CBDE) test recommended by Clinical and Laboratory Standards Institute (CLSI). The RCDE test was performed by using a 10-μg colistin disk in 2.7 mL volume (final colistin concentration of 3.7 μg/mL) of either cation-adjusted Mueller-Hinton broth or phenol red broth base media with bacterial inoculum of 1-μL loop, and 1-4 and 16-20 hr incubation for Enterobacteriaceae and Acinetobacter baumannii isolates, respectively. Both tests were evaluated in 236 Enterobacteriaceae and 49 A. baumannii isolates using broth microdilution as reference method. Among the Enterobacteriaceae isolates, categorical agreement and very major error (VME or false intermediate susceptibility) rate were 98.3% and 5.4%, respectively, for the RCDE test, compared with 97.9% and 7.1%, respectively, for the CBDE test. Both tests had major error (ME or false resistance) rate of 0.6%. For the A. baumannii isolates, the RCDE and CBDE tests gave high VME rates of 8.3% and 16.7%, respectively. The RCDE test showed good performance comparable with the CBDE test but is cheaper and more rapid (3 hr) and convenient, thus suggesting as an alternative for detecting colistin resistance among Enterobacteriaceae in low-income countries.

Aims: Attenuated Total Reflection Fourier Transform Infrared (ATR-FT-IR) Spectroscopy and chemometric modelling, including soft independent modelling by class analogy (SIMCA), partial least squares discriminant analysis (PLS-DA) and support vector machine (SVM), were applied to attempt to discriminate 60 clinical isolates of Enterococcus faecium and Enterococcus faecalis and hence evaluate the performance of the spectroscopic approach in identifying enterococci infections., Methods and Results: The bacterial samples were identified by polymerize chain reaction (PCR) amplification and their ATR-FT-IR spectra acquired. Spectra were processed to the second derivative using the Savitzky-Golay algorithm and normalized using extended multiplicative signal correction employing the UnscramblerX (CAMO, Norway) software package. Multivariate classification models and their performance were evaluated using Cohen's Kappa coefficient. Principal component analysis (PCA) score plots showed separate clusters of spectra related to membership to E. faecium and E. faecalis, with this explained by bands assigned to PO 2 (1230 cm -1 ), P-O-C (1114 cm -1 ), monosubstituted alkene (997, 987 cm -1 ) and C-O (1070, 1055, 1036 cm -1 ) corresponding to teichoic acids, polysaccharides and peptidoglycan from the cell wall in PCA and PLS-DA loading plots. The best classification model for E. faecium and E. faecalis is SVM, indicating via highest Kappa score. The classification coefficient between SIMCA, PLS-DA, SVM and PCR as reference method were 0·59, 0·9 and 1, respectively, shown as the Kappa scores., Conclusions: The main spectral differences observed between the two clinically relevant enterococci species were associated with changes in the teichoic acid content of cell walls. With regard to the binary classification method, SVM was found to be the best performing classification model, providing the highest correlation with the PCR results., Significance and Impact of the Study: The study shows that ATR-FT-IR spectroscopy in combination with chemometric modelling can be applied for the phenotypic identification and discrimination of clinically relevant and similar enterococcal species., (© 2020 The Society for Applied Microbiology.)

The global emergence of colistin resistance in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates is a serious public health concern. We therefore aimed to investigate colistin resistance mechanisms in 5 colistin-resistant (COL-R) CRAB isolates collected from Thai patients in 2016 by whole genome sequencing (WGS) compared with those of 5 colistin-intermediate (COL-I) CRAB isolates from the same period. All isolates were subjected to antimicrobial susceptibility testing, efflux pump inhibitor-based test and WGS. Mutations in known genes associated with colistin resistance were analyzed and deleterious mutations were then predicted by PROVEAN tool. The 10 CRAB isolates carried bla OXA-23 with the addition of bla OXA-58 in 1 isolate. All COL-R isolates exhibited colistin MICs of 4 μg/mL except for 1 isolate with that of 16 μg/mL. They belonged to ST2, ST16, ST23, ST164 and ST215, whereas the COL-I isolates with colistin MICs of ≤0.25-1 μg/mL were ST2, ST164 and ST215. Neither increased efflux pump activity nor mcr gene was found in any COL-R isolate. Three COL-R isolates contained different PmrB variants: a novel 10-amino acid (aa) repeat sequence insertion, VILGCILIFS between positions 27 and 28 (S27&#95;A28insVILGCILIFS) in transmembrane domain (TM); a 1-aa insertion, alanine between positions 162 and 163 (A162&#95;I163insA) in TM; and a 1-aa substitution, A226T in histidine kinase domain. One COL-R isolate possessed PmrA variant with A80V substitution. These alterations were predicted as deleterious. Mechanisms of colistin resistance in the remaining COL-R isolate were still unknown. In conclusion, the alterations in both PmrB and PmrA were predicted and suggested as initial mutations responsible for low-level colistin resistance in our CRAB isolates. Under selective pressure, these isolates may exhibit higher level colistin resistance by the additional mutations, leading to more therapeutic difficulties., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5-2 µg/mL, with MIC 50 and MIC 90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3-8 μg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC 50 and MIC 90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried bla OXA-23-like . The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.

Twelve nonreplicate carbapenemase-negative ertapenem (ETP)-nonsusceptible (CNENS) Escherichia coli isolates obtained at a Thai university hospital between 2010 and 2014 were characterized and compared with 2 carbapenemase-producing E. coli isolates from the same hospital. Eight unique pulsed-field gel electrophoresis patterns were obtained. All the isolates produced CTX-M-15 β-lactamase and 2 either coexpressed CMY-2 cephalosporinase or showed increased efflux pump activity. Amino acid sequence analysis revealed that an OmpF defect (in 7 isolates) due to mutations generating truncated proteins or an IS1 insertion was more prevalent than a defect in OmpC was (no truncated proteins detected). Seven out of 10 isolates possessing OmpC variants with any OmpF defect were weakly ETP-resistant (minimum inhibitory concentrations [MICs] of 1-4 μg/mL) and imipenem (IPM)- and meropenem (MEM)-susceptible (MICs 0.125-0.5 μg/mL). Two isolates with ompC PCR-negative results and an OmpF defect showed higher carbapenem MICs (8-32, 1-8, and 1-4 μg/mL for ETP, IPM, and MEM, respectively) with the highest MICs associated with the additional efflux pump activity. Both carbapenemase producers possessing CTX-M-15 and a porin background identical to that in the CNENS isolates showed ETP, IPM, and MEM MICs of 128-256, 8, and 2-32 μg/mL, respectively. These findings suggest that a porin defect combined with CTX-M-15 production is the major mechanism of low carbapenem susceptibility among our CNENS isolates, which have potential to become strongly carbapenem-resistant because of additional carbapenemase or efflux pump activities.

Objectives: To develop a simple gold nanoparticle (AuNP)-based colorimetric test, GoldNano Carb (GoldC), for detecting carbapenemase production in Gram-negative bacteria, compared with updated Carba NP (CNP) and CarbAcineto NP (CAcNP) tests by using PCR methods as gold standard., Methods: Ninety-nine carbapenemase-producing Enterobacteriaceae (CPE), Pseudomonas spp. and Acinetobacter spp. isolates and 89 non-CPE isolates were tested by the GoldC and CNP. Additionally, the CAcNP was performed in the Acinetobacter spp. isolates. The final imipenem (imipenem/cilastatin form) concentration was 5 mg/mL for all three tests. For the GoldC, the imipenem powder was added directly to bacterial cell suspension in distilled water prior to detection of acid product by the citrate-capped AuNP solution. An AuNP change from red to purple, blue or green indicates carbapenemase activity., Results: The GoldC detected all carbapenemase producers except one OXA-23-like producer (99.0% sensitivity), whereas 11 carbapenemase producers (10 Acinetobacter and 1 P. aeruginosa) were CNP negative (88.9% sensitivity). However, the GoldC and CNP provided 100% and 98.6% sensitivity, respectively, for the CPE and Pseudomonas spp. Both tests gave one false positive from CTX-M-1-like-producing Enterobacter spp. (98.9% specificity). The GoldC and CAcNP detected 96.7% and 93.3% of the Acinetobacter spp. isolates, respectively. Interestingly, times to positivity by the GoldC were markedly shorter than those by the CNP (76.8% versus 36.2% positive at 5 min) and CAcNP (43.3% at 5 min versus 20% within 30 min)., Conclusions: The GoldC is fast, easy, highly sensitive and inexpensive (∼$0.25 per test), suggesting that it may be suitable for routine carbapenemase detection in low-resource settings for infection control or epidemiological purposes., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of bla NDM , bla OXA-48 , bla VIM , bla IMP-14 and bla KPC groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla NDM-1 , 9 bla IMP-14a , 2 bla IMP-48 , 1 bla IMP-1 , 1 bla IMP-4 , 1 bla IMP-9 , 1 bla IMP-15 , 4 bla VIM-2 , 1 bla VIM-1 , 1 bla IMP-14a & bla VIM-2 , 7 bla KPC-2 , 3 bla OXA-48 and 3 bla OXA-181 ) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the bla NDM , bla OXA-48 , bla VIM , bla IMP-14 and bla KPC groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes.

Carbapenemase-producing Enterobacteriaceae (CPE) isolates have now emerged worldwide. We therefore modified the phenotypic Carba NP test by use of a filter paper strip for easily and rapidly identifying CPE in routine laboratory. A collection of 56 CPE and carbapenemase-producing Pseudomonas spp. isolates (including 28 NDM-1, 11 IMP-14a, 1 IMP-1, 1 IMP-4, 1 IMP-9, 1 IMP-15, 4 VIM-2, 1 VIM-1, 1 IMP-14a with VIM-2, 3 OXA-48, 3 OXA-181 and 1 KPC-2 producers) and 41 non-CPE isolates (including 19 ESBL, 7 pAmpC, 3 AmpC, 9 ESBL with pAmpC and 3 non-ESBL & non-AmpC producers) as confirmed by the PCR methods were tested by the paper strip method using pharmaceutical imipenem/cilastatin as a substrate. Bacterial colonies of each isolate were applied directly on filter paper strips dropped with either imipenem-phenol red (test strip) or phenol red solution alone (control strip). The reaction was read within 5 min. This test failed to detect 3 OXA-181, 2 OXA-48 and 3 IMP-14a producers (85.7 % sensitivity), whereas no false positives were seen (100 % specificity). Further evaluation of the paper strip test in 267 CPE screening-positive isolates from three hospitals by their medical technologists showed 92.0 % sensitivity (100 % for NDM producers) and 100 % specificity compared with the PCR methods. Because of its ease, rapidness and cost effective, the paper strip test has a potential for routine CPE testing in low-resource laboratories particularly in areas with high prevalence of NDM enzymes, leading to appropriate antimicrobial therapy and infection control strategy.

Five blaOXA-48-like-carrying Enterobacteriaceae isolates collected from two Thai patients in December 2012 were characterized. Three Klebsiella pneumoniae isolates giving two different pulsed-field gel electrophoresis patterns and sequence types (ST11 and ST37) from patient 1 harbored blaOXA-48 locating on Tn1999.2, whereas two Escherichia coli isolates with the same pulsotype and ST5 from Patient 2 carried ISEcp1-associated blaOXA-181. One K. pneumoniae strain had blaSHV-12, blaDHA-1, qnrB, and qnrS, while another strain harbored blaCTX-M-15, qnrS and aac(6')-Ib-cr. The E. coli strain contained blaCTX-M-15, blaCMY-2, qnrS, and aac(6')-Ib-cr. Interestingly, the OXA-48 producers with a novel OmpK36 variant by a substitution of Gly to Asp in the L3 loop-borne PEFXG motif exhibited high-level resistance to ertapenem, imipenem, and meropenem. In contrast, the OXA-181 producer with non-porin-deficient background showed low-level resistance to ertapenem only. Both patients died because of either septic shock or pneumonia. This study showed the impact of OXA-48-like carbapenemases in porin-defective clinical isolate background, which may lead to serious therapeutic problems in the near future., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: To detect carbapenemases in clinical isolates of Enterobacteriaceae collected from patients in a university hospital in Thailand between October 2010 and August 2011., Methods: A total of 4818 Enterobacteriaceae isolates were screened for the presence of carbapenemases by ertapenem and imipenem disc diffusion tests. All positive screening isolates were subjected to modified Hodge test, phenylboronic acid- and EDTA-carbapenem combined disc tests and two multiplex PCRs of bla(IMP), bla(VIM), bla(SPM), bla(SIM) and bla(GIM), and of bla(KPC), bla(NDM) and bla(OXA-48). Carbapenemase-producing isolates were typed by PFGE and then characterized by antimicrobial susceptibility tests. Conjugation was performed using a broth culture mating method., Results: Two isolates each of Escherichia coli, Klebsiella pneumoniae and Citrobacter freundii produced NDM-1, whereas two other isolates of K. pneumoniae produced IMP-14a. DNA fingerprints revealed that the metallo-β-lactamase (MBL)-producing isolates were of different strains except for clonal strains of C. freundii. In vitro transfer of carbapenem resistance was successful for the eight MBL-producing isolates. All MBL producers were susceptible to colistin and tigecycline. The six NDM-producing isolates were recovered from the urine of three patients, who had no history of travel outside Thailand. Interestingly, one patient had chronic urinary tract infections caused by a K. pneumoniae strain and two strains of E. coli producing NDM-1., Conclusions: Surveillance of carbapenemases, particularly NDM-1, in Enterobacteriaceae is urgently needed to control and prevent the spread of these resistance determinants in our country.

Background: Acute Coronary Syndrome (ACS) occurs when a vulnerable plaque ruptures and induces platelet aggregation and coagulation process at the rupture. Thrombogenesis is the final process that forms a clot in the coronary lumen causing myocardial injury. Plasma D-dimer, a primary degradation product and circulating marker offibrin turnover, serves as a direct marker of ongoing fibrinolysis in site of coronary artery occlusion., Objective: To determine the correlation between plasma D-dimer levels and severity of coronary artery obstruction based on angiographic data that is composed of the number of coronary arteries affected and the percentage of maximum stenosis of coronary artery lumen in non-ST elevation ACS (NSTE-ACS) patients., Material and Method: NSTE-ACS patients who admitted in Siriraj hospital duringJune 2009 and March 2010 were enrolled. Conditions that increased plasma D-dimer other than NSTE-ACS were excluded. Demographic characteristics were assessed by a standardized questionnaire. Plasma D-dimer was measured and coronary angiography was performed to evaluate severity of coronary artery stenosis., Results: Total of 74 NSTE-ACS patients were enrolled (29 in unstable angina and 45 in non-ST elevation myocardial infarction). Mean age of these patients (54.1% in female and 45.9% in male) were 66 years. D-Dimer was significantly increased with the number of coronary arteries affected (p = 0.03). In non-significant and single coronary artery disease (CAD) patients, median D-dimer was 406 (178-2,788) mcg/L. In multivessel CAD, median D-dimer was 941 (131-7,110) mcg/L. D-dimer levels had a trend to be increased with percentage of maximum stenosis of coronary artery lumen; atheromatosis, (p = 0.30). In mild and moderate atheromatosis (coronary artery stenosis < 70%), median D-dimer was 479 (182-5902) mcg/L while median D-dimer was 789 (131-7110) mcg/L in severe atheromatosis (coronary artery stenosis > 70%). Moreover plasma D-dimer levels correlated with complication of NSTE-ACS (Congestive heartfailure; p < 0.001, arrhythmia; p = 0.007 and death; p = 0.009) and was increased in patients who underwent treatment with CABG more often than those who received PCI and medication treatment alone. D-dimer also correlated with serum creatinine (r = 0.517, p < 0.001), creatinine clearance (r = -0.463, p < 0.001), troponin-T level (r = 0.381, p < 0.001) and left ventricular ejection fraction (r = -0.368, p = 0.002)., Conclusion: D-dimer is useful coagulation marker use to evaluate extent of coronary affected and may predict in-hospital CV complication. However, other conditions that increased plasma D-dimer also excluded.

Objectives: To investigate the prevalence of HPV, HPV16, and HPV16 variants in scraped cervical cells cytologically diagnosed as normal cervical cell and in formalin-fixed, paraffin-embedded tissues of cervical intraepithelial neoplasia II-III and squamous cervical carcinoma in Northeast Thailand., Methods: All samples were subjected to PCR using consensus GP5+/GP6+ primers. HPV16 was genotyped by Southern blot hybridization and reverse line blot hybridization. The HPV16 E6 gene was amplified and sequenced., Results: HPV infections were found in 33.8% of normal cervical cells, 97.3% of cervical intraepithelial neoplasia II-III, and 100% of squamous cervical carcinomas. The prevalence of HPV16 increased significantly with histological grade (normal cervical cell, 16.7%; cervical intraepithelial neoplasia II-III, 38.9%; squamous cervical carcinoma, 75%). The most common variant found was the Asian (As) (58.7%) followed by the European (E) lineage (41.3%). The HPV16 As lineages showed a risk association in 73.9% of squamous cervical cancer and 57.1% of cervical intraepithelial neoplasia II-III, while no increased risk was observed in the E lineages., Conclusion: Our study demonstrates that HPV16, in particular the As variant, was the major causative agent associated with cervical cancer in Northeast Thailand, and our study suggests that some mutations of the E6 gene in this variant, which leads to amino acid changes, may be more carcinogenic.