The Evaluation of a Lateral Flow Strip Based on the Covalently Fixed "End-On" Orientation of an Antibody for Listeria monocytogenes Detection.

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 (NH2-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 102 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.

Rapid detection of Staphylococcus aureus in blood culture samples using human IgG-based lateral flow assay.

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 107 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.

Examination of Diagnostic Performance of New IgG4 Rapid Test Compared with IgG- and IgG4-ELISAs to Investigate Epidemiology of Strongyloidiasis in Northeast Thailand.

Strongyloidiasis, caused by Strongyloides stercoralis, is a neglected tropical disease with a global distribution. The infection can be fatal in immunocompromised individuals, and accurate diagnosis leading to timely treatment can save lives. Serodiagnosis is a sensitive method for diagnosis and is recommended for screening high-risk individuals. A point-of-care rapid test will facilitate the screening activities, especially in low-resource settings. This study aims to apply a new IgG4 immunochromatographic test using S. stercoralis recombinant antigen (SsRapid® cassette test) and to compare it with in-house IgG and IgG4 enzyme-linked immunosorbent assays (IgG- and IgG4-ELISAs) using native Strongyloides ratti antigen to investigate the epidemiology of strongyloidiasis in northeast Thailand. A total of 300 people participated, with 136 males and 164 females of a similar mean age. The reference tests were fecal examinations using the formalin-ethyl acetate concentration technique and an agar plate culture technique. The prevalence of S. stercoralis determined by SsRapid (81.7%) was significantly higher than that by fecal examinations (43.3%) or by antibody detection by IgG-ELISA (53.0%) or IgG4-ELISA (44.0%). The diagnostic sensitivities of SsRapid, IgG-ELISA, and IgG4-ELISA were found to be 93.9%, 77.7%, and 63.1%, respectively. The rate of positive tests by the SsRapid was significantly correlated to the levels of Strongyloides-specific IgG4 and IgG antibodies. By all diagnostic methods, male participants had a significantly higher prevalence of strongyloidiasis than females. Age was significantly associated with the concentration of specific serum IgG but not with the SsRapid grading score. In conclusion, SsRapid was shown to be a sensitive and valuable diagnostic test for the epidemiology study of strongyloidiasis.

Diagnostic performance of Strongyloides-specific IgG4 detection in urine for diagnosis of human strongyloidiasis.

BACKGROUND: Detection of parasite-specific IgG in urine is a sensitive method for diagnosis of strongyloidiasis and gives similar accuracy to serum IgG. However, there are no data concerning detection of IgG subclass in urine. To further explore the utility of diagnosis from urine samples, we evaluated the diagnostic performance of IgG4 in urine compared with parasitological and other immunological methods. METHODS: The urine and sera included proven strongyloidiasis (group 1, n = 93), other parasitic infections (group 2, n = 40) and parasite negatives (group 3, n = 93). The performance of Strongyloides-specific IgG4 in urine for diagnosis of strongyloidiasis using fecal examinations as the reference standard was assessed. RESULTS: With fecal examination as a gold standard, Strongyloides-specific IgG4 in urine had 91.4% sensitivity and 93.2% specificity while serum IgG4 had 93.6% sensitivity and 91.0% specificity. IgG4 in both urine and serum had almost perfect diagnostic agreements with fecal examination (Cohen's kappa coefficient was > 0.8). Cross-reactivity to Opisthorchis viverrini and Taenia spp. of IgG4 in urine were 7.5% and 12.5% in serum. Concurrent analyses of total IgG in urine and serum showed that the sensitivities (97.9-100%) and specificities (88.7-91.0%) were similar (P > 0.05). The sensitivity for parasitological examination by the formalin-ethyl acetate concentration technique (FECT) was 49.5% and that for agar plate culture technique (APC) it was 92.6%. CONCLUSION: Our findings showed that specific IgG4 detection in urine yielded similar diagnostic performance to the same biomarkers in serum. This suggests that accurate diagnosis of strongyloidiasis can be performed using urine samples and IgG4 is a valid choice of diagnostic marker. Further assessment is required to assess the utility of urine IgG4 for measuring the response treatment in strongyloidiasis.

Potential of jackfruit inner skin fibre for encapsulation of probiotics on their stability against adverse conditions.

The aim of this study was to investigate the impact of jackfruit inner skin fibre (JS) incorporated with whey protein isolate (WPI) and soybean oil (SO) as a wall material for probiotic encapsulation to improve probiotic stability against freeze-drying and gastrointestinal (GI) tract conditions. Bifidobacterium bifidum TISTR2129, Bifidobacterium breve TISTR2130, and Lactobacillus acidophilus TISTR1338 were studied in terms of SCFA production and the antibiotic-resistant profile and in an antagonistic assay to select suitable strains for preparing a probiotic cocktail, which was then encapsulated. The results revealed that B. breve and L. acidophilus can be used effectively as core materials. JS showed the most influential effect on protecting probiotics from freeze-drying. WPI:SO:JS at a ratio of 3.9:2.4:3.7 was the optimized wall material, which provided an ideal formulation with 83.1 ± 6.1% encapsulation efficiency. This formulation presented > 50% probiotic survival after exposure to gastro-intestinal tract conditions. Up to 77.8 ± 0.1% of the encapsulated probiotics survived after 8 weeks of storage at refrigeration temperature. This study highlights a process and formulation to encapsulate probiotics for use as food supplements that could provide benefits to human health as well as an alternative approach to reduce agricultural waste by increasing the value of jackfruit inner skin.

Changes in the chemical compositions and biological properties of kombucha beverages made from black teas and pineapple peels and cores.

Several raw materials have been used as partial supplements or entire replacements for the main ingredients of kombucha to improve the biological properties of the resulting kombucha beverage. This study used pineapple peels and cores (PPC), byproducts of pineapple processing, as alternative raw materials instead of sugar for kombucha production. Kombuchas were produced from fusions of black tea and PPC at different ratios, and their chemical profiles and biological properties, including antioxidant and antimicrobial activities, were determined and compared with the control kombucha without PPC supplementation. The results showed that PPC contained high amounts of beneficial substances, including sugars, polyphenols, organic acids, vitamins, and minerals. An analysis of the microbial community in a kombucha SCOBY (Symbiotic Cultures of Bacteria and Yeasts) using next-generation sequencing revealed that Acetobacter and Komagataeibacter were the most predominant acetic acid bacteria. Furthermore, Dekkera and Bacillus were also the prominent yeast and bacteria in the kombucha SCOBY. A comparative analysis was performed for kombucha products fermented using black tea and a fusion of black tea and PPC, and the results revealed that the kombucha made from the black tea and PPC infusion exhibited a higher total phenolic content and antioxidant activity than the control kombucha. The antimicrobial properties of the kombucha products made from black tea and the PPC infusion were also greater than those of the control. Several volatile compounds that contributed to the flavor, aroma, and beneficial health properties, such as esters, carboxylic acids, phenols, alcohols, aldehydes, and ketones, were detected in kombucha products made from a fusion of black tea and PPC. This study shows that PPC exhibits high potential as a supplement to the raw material infusion used with black tea for functional kombucha production.

Ca2+/Calmodulin-dependent Protein Kinase II Inhibitor KN-93 Enhances Chondrogenesis of Bone Marrow Mesenchymal Stem Cells and Delays Chondrogenic Hypertrophy.

BACKGROUND/AIM: Cartilage tissue engineering has been popularly applied in the treatment of articular cartilage defect because it is more effective in generating functional engineered cartilage than traditional methods. Although the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) is well established, it is often accompanied by undesired hypertrophy. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator in the ion channel pathway which is known to be involved in chondrogenic hypertrophy. Therefore, this study aimed to reduce the hypertrophy of BM-MSCs by inhibiting CaMKII activation. MATERIALS AND METHODS: BM-MSCs were cultured in three-dimensional (3D) scaffold under chondrogenic induction with and without CaMKII inhibitor, KN-93. After cultivation, markers of chondrogenesis and hypertrophy were investigated. RESULTS: KN-93 at a concentration of 2.0 µM had no effect on the viability of BM-MSCs, while the activation of CaMKII was suppressed. A long period of KN-93 treatment significantly up-regulated the expression of SRY-box transcription factor 9 and aggrecan on day 28 compared to untreated BM-MSCs. Furthermore, KN-93 treatment significantly down-regulated the expression of RUNX family transcription factor 2 and collagen type X alpha 1 chain on days 21 and 28. Immunohistochemistry showed increased expression of aggrecan and type II collagen while the expression of type X collagen was reduced. CONCLUSION: A CaMKII inhibitor, KN-93 is able to enhance chondrogenesis of BM-MSCs and suppress chondrogenic hypertrophy, suggesting its potential applicability in cartilage tissue engineering.

Three-dimensional silk fibroin-gelatin/chondroitin sulfate/hyaluronic acid-aloe vera scaffold supports in vitro chondrogenesis of bone marrow mesenchymal stem cells and reduces inflammatory effect.

A limited self-healing ability of injured articular cartilage results in osteoarthritis and a joint dysfunction afterward. Cartilage tissue engineering is a promising approach to increase the treatment efficiency. Moreover, host response to implanted biomaterial has been increasingly concerned. Thus, this study aimed to establish three-dimensional (3D) scaffold that could support cartilage tissue engineering and reduce inflammatory. The various ratios of silk fibroin (SF), gelatin (G), chondroitin sulfate (C), hyaluronic acid (H), and aloe vera (A) were used to fabricate 3D scaffolds by lyophilization, designated as SF, SF-A, SF-gelatin/chondroitin sulfate/hyaluronic acid (GCH)-A-411, and SF-GCH-A-111. The physical and biological characteristics of the scaffolds were investigated. All scaffolds possessed interconnected porous structures, which the highest pore size of 209 µm was found in SF and SF-GCH-A-411 scaffolds. Moreover, high porosity, high water uptake, and good mechanical strength were observed in the SF-GCH-A-411 scaffold. The SF, SF-A, and SF-GCH-A-411 scaffolds could retain their structures up to 21 days, while SF-GCH-A-111 was rapidly degraded. The proliferation of human bone marrow mesenchymal stem cells (BM-MSCs) was significantly higher in SF-A and SF-GCH-A-411 than in the SF scaffold. Besides, the SF-A and SF-GCH-A-411 revealed significantly lower expression of pro-inflammatory cytokine, interleukin-1 beta than the SF scaffold, suggesting the beneficial role of aloe vera in anti-inflammatory effect. Furthermore, the SF-GCH-A-411 scaffold could support chondrogenic differentiation of BM-MSCs. In conclusion, based on its superior physical and biological characteristics that support chondrogenesis of BM-MSCs, the SF-GCH-A-411 scaffold is recommended for cartilage tissue engineering.

Prebiotic, Antipathogenic Bacteria and Hypocholesterolemia Properties of Fermented Rice Bran Extracts Derived from Black Rice and Germinated Brown Rice.

Rice bran is a rich source of health-promoting nutrition and bioactive compounds; nevertheless, the properties of rice brans depend on cultivars, ages, and preparation methods, drawing the potential of raw materials for health benefits. Therefore, this research aimed to investigate the health-promoting properties of fermented rice bran extracts from cultivar black rice (H7F) and germinated brown rice (G13F), focusing on their prebiotic, antipathogenic bacteria activity and safety demonstrated in vitro and in vivo study models, respectively. Here, the screening of metabolites' change after rice bran fermentation by ATR-FTIR spectra revealed specific peaks corresponding to the composited components of protein, carbohydrate, and lipid. Then, in the in vitro study, the prebiotic capability of H7F and G13F extracts was demonstrated by a growth-promoting effect on Lactobacillus delbrueckii subsp. lactis under specific acidic conditions. Furthermore, antipathogenic bacterial activity against Escherichia coli and Staphylococcus aureus was presented at 25 mg/mL of MIC values and 50 mg/mL of MBC of both fermented rice bran extracts, eliminating the bacteria by interfering with the biofilm formation. For safety, an acute and chronic toxicity study using Wistar rats was conducted, in which changes in the body and organ weights, histopathology of organs, blood chemistry, and hematological parameters were observed after H7F and G13F treatment. Desirably, they showed no toxicity, with a significant reduction in blood cholesterol levels in the chronic treatment of H7F and G13F. Conclusively, the overall results evidenced the health benefits of H7F and G13F related to their prebiotic and antipathogenic bacteria properties and hypocholesterolemia potential with a high level of safety. Therefore, the fermented rice bran extracts were demonstrated as potential materials for the further development of functional ingredients and health products.

Anserine/Carnosine-Rich Extract from Thai Native Chicken Suppresses Melanogenesis via Activation of ERK Signaling Pathway.

Skin hyperpigmentation is an aesthetic problem that leads to psychosocial issues. Thus, skin whitening agents from agro- and poultry-industrial co-products are considered high economic value ingredients of interest for sustainable application. Therefore, this study aimed to determine the cosmeceutical potential of anserine/carnosine-rich chicken extract (ACCE) from the Thai native chicken Pradu Hang Dam Mor Kor 55 (PD) meat. The chemical composition was identified and quantified using the HPLC-UV method. Then, the antioxidation potential of the extract was compared to that of L-anserine and L-carnosine, using 1,1-diphenyl-2-picrylhydrazyl assay and shikonin-induced production of reactive oxygen species in CCD-986Sk cell models, and the anti-melanogenesis effect in the MNT-1 melanoma cell line model was investigated. Furthermore, related mechanisms were identified using colorimetric tyrosinase assay and the Western blot technique. The ACCE was composed of L-anserine and L-carnosine as two major constituents. In a dose-dependent manner, ACCE, L-anserine, and L-carnosine manifested significant antioxidation potential and significant reduction of melanin production. Activation of the extracellular signal-regulated kinase (ERK) signaling pathway and inhibition of tyrosinase activity of ACCE were demonstrated as the mechanisms of the anti-melanogenesis effect. In conclusion, ACCE has been revealed as a potential cosmeceutical agent due to its antioxidation and anti-melanogenic activity in association with L-anserine and L-carnosine composition and biomolecular regulating ability. Therefore, further studies and development should be considered to support the utilization of anserine/carnosine-rich chicken extract in the cosmetic industry for economic value creation and sustainability.

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Rapid Detection of OXA-48-like Carbapenemase Genes in Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) possessing various carbapenemases, particularly the OXA-48 group, are now rapidly spreading and becoming a major public health concern worldwide. Phenotypic detection of OXA-48-like carbapenemases is still suboptimal due to their weak carbapenemase activity, whereas highly sensitive and specific polymerase chain reaction (PCR)-based methods take at least 3-4 h. We, therefore, developed a recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip assay for the rapid detection of blaOXA-48-like in Enterobacterales. A total of 131 clinical isolates including 61 blaOXA-48-like-carrying Enterobacterales isolates and 70 Gram-negative bacilli isolates containing other bla genes were subjected to the RPA method performed under isothermal conditions at 37 °C within 10 min and visually inspected by LF strip within 5 min. The RPA-LF assay provided 100% sensitivity (95% confidence interval, 92.6-100%) and 100% specificity (93.5-100%) for detecting blaOXA-48-like genes from bacterial colonies. Its detection limit was 100 times less than that of the PCR method. This assay is rapid, easy to perform, and provides excellent performance without any special equipment. It may be applied for directly identifying the blaOXA-48-like genes in Enterobacterales obtained from blood culture. Rapid identification of carbapenemase types is essential for selecting appropriate antimicrobial options, particularly the ß-lactams combined with novel ß-lactamase inhibitors.

High prevalence of chronic kidney disease and its related risk factors in rural areas of Northeast Thailand.

In Thailand, chronic kidney disease (CKD) screening was reported in 2009 with an overall prevalence of 17.5% and the highest at 22.2% in the northeastern region. This study aimed to find out CKD prevalence of the Kidney Disease Improving Global Outcomes criteria and their related risk factors in the rural community. A population-based study was conducted in the rural sub-districts of northeastern Thailand. Data of socio-demographic status, lifestyle, underlying diseases, blood pressure, and body mass index were recorded. Blood and urine analysis was conducted along with ultrasonography of kidneys. Specimen collection and analyses were repeated after 3 months, and the factors associated with CKD were studied by logistic regression analysis. A total of 2205 participants with a mean age of 57.8 ± 11.7 years and female predominance (66.7%) completed the study. The prevalence of CKD was 26.8%, i.e., stages 1 (7.3%); stage 2 (9.0%); stage 3a (6.0%); stage 3b (2.8%); stage 4 (1.4%); and stage 5 (0.3%). Hypertension, diabetes mellitus, and renal stones were the major underlying diseases. Only 3.5% of the participants were aware of having CKD. An increase in age, male, unemployment, current smoking, diabetes, hypertension, underweight, anemia, hyperuricemia, and leukocytosis were significantly associated factors with the disease. The study revealed that CKD has developed as a significant public health problem in rural northeastern Thailand and one out of every four people has CKD. Therefore, early interventions are essential for the proper management and prevention of CKD.

Insight into the Covalently Oriented Immobilization of Antibodies on Gold Nanoparticle Probes to Improve Sensitivity in the Colorimetric Detection of Listeria monocytogenes.

In this work, the covalently oriented conjugation of monoclonal Listeria monocytogenes antibody (mAb-Lis) to amino-terminated oligo(ethylene glycol)-capped gold nanoparticles (NH2-TEG-AuNPs) was studied. After NH2-TEG-AuNPs were synthesized, the amino-terminated ligands on the particles were then linked to the carboxyl groups in the mAb-Lis through EDC/NHS chemistry. By maintaining the pH of the solution at ∼5, the Fc region of the antibody could preferably attach to the particle surface, providing a specific Fab region that was available for binding with the target pathogen. The resulting mAb-NH-TEG-AuNPs could act as a colorimetric probe for L. monocytogenes based on a particular antigen-antibody interaction, which resulted in a drastic aggregation of particles. This caused the color of the colloidal solution to transition from red-pink to purple or even gray depending on the pathogen concentration. To perform quantitative analysis, the absorbance ratio of A650/A534 was monitored as a function of L. monocytogenes concentration using a spectrophotometer. The detection limit was very low at 11 CFU/mL. Furthermore, a lateral flow strip (LFS) was fabricated as a portable device for onsite utilization. LFS detection could be completed by the naked eye and by a smartphone. The detection limit of LFS was estimated to be 103 CFU/mL. Our methods exhibited a substantial improvement in sensitivity compared to that of previous studies on immuno-based nanoparticles. The assay could be completed in 15 min, and no cross reactivity by any pathogen was found. Hence, the designed AuNPs exhibit great promise for use in monitoring L. monocytogenes for food safety and in other applications.

High expression of protein tyrosine phosphatase receptor S (PTPRS) is an independent prognostic marker for cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive tumor of the bile duct with a high rate of mortality. Lymph node metastasis is an important factor facilitating the progression of CCA. A reliable biomarker for diagnosis, progression status, or prognosis of CCA is still lacking. To identify a novel and reliable biomarker for diagnosis/prognosis of CCA, liquid chromatography-mass spectrometry and tandem mass spectrometry (LC-MS/MS) in combination with bioinformatics analysis were applied for the representative serum samples of patients with CCA. The proteome results showed that protein tyrosine phosphatase receptor S (PTPRS) had the highest potential candidate. Then, a dot blot assay was used to measure the level of serum PTPRS in patients with CCA (n = 80), benign biliary disease patients (BBD; n = 39), and healthy controls (HC; n = 55). PTPRS level of CCA sera (14.38 ± 9.42 ng/ml) was significantly higher than that of BBD (10.7 ± 5.05 ng/ml) or HC (6 ± 3.73 ng/ml) (P < 0.0001). PTPRS was associated with serum albumin (P = 0.028), lymph node metastasis (P = 0.038), and the survival time of patients (P = 0.011). Using a log-rank test, higher serum PTPRS level was significantly (P = 0.031) correlated with a longer overall survival time of patients with CCA, and PTPRS was an independent prognostic marker for CCA superior to carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA) or alkaline phosphatase (ALP). High expression of PTPRS could be a good independent prognostic marker for CCA.

Tamarind Seed Coat: A Catechin-Rich Source with Anti-Oxidation, Anti-Melanogenesis, Anti-Adipogenesis and Anti-Microbial Activities.

Tamarindus indica L. or tamarind seed is an industrial by-product of interest to be investigated for its potential and value-added application. An ethanolic tamarind seed coat (TS) extract was prepared using the maceration technique and used to determine the phytochemical composition and bioactivities. The total phenolic and flavonoid contents were determined using colorimetric methods; moreover, chemical constituents were identified and quantified compared to the standard compounds using the HPLC-UV DAD technique. Bioactivities were investigated using various models: antioxidative activity in a DPPH assay model, anti-melanogenesis in B16 melanoma cells, anti-adipogenesis in 3T3-L1 adipocytes, and anti-microbial activity against S. aureus, P. aeruginosa, E. coli, and C. albican using agar disc diffusion and microdilution methods. The results manifested a high content of catechin as a chemical constituent and multiple beneficiary bioactivities of TS extract, including superior antioxidation to ascorbic acid and catechin, comparable anti-melanogenesis to deoxyarbutin, and significant anti-adipogenesis through inhibition of pre-adipocyte differentiation and reduction of lipid and triglyceride accumulation, and a broad spectral anti-microbial activity with a selectively high susceptibility to S. aureus when compared to 1% Parabens. Conclusively, TS extract has been revealed as a potential bioactive agent as well as an alternative preservative for application in food, cosmetic, and pharmaceutical product development.

Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip.

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.

Disinfection efficiency test for contaminated surgical mask by using Ozone generator.

BACKGROUND: Ozone (O3) is an effective disinfectant agent that leaves no harmful residues. Due to the global health crisis caused by the COVID-19 pandemic, surgical masks are in high demand, with some needing to be reused in certain regions. This study aims to evaluate the effects of O3 for pathogen disinfection on reused surgical masks in various conditions. METHODS: O3 generators, a modified PZ 2-4 for Air (2000 mg O3/L) and a modified PZ 7 -2HO for Air (500 mg O3/L), were used together with 1.063 m3 (0.68 × 0.68 × 2.3 m) and 0.456 m3 (0.68 × 0.68 × 1.15 m) acrylic boxes as well as a room-sized 56 m3 (4 × 4 × 3.5 m) box to provide 3 conditions for the disinfection of masks contaminated with enveloped RNA virus (105 FFU/mL), bacteria (103 CFU/mL) and fungi (102 spores/mL). RESULTS: The virucidal effects were 82.99% and 81.70% after 15 min of treatment with 2000 mg/L O3 at 1.063 m3 and 500 mg/L O3 at 0.456 m3, respectively. The viral killing effect was increased over time and reached more than 95% after 2 h of incubation in both conditions. By using 2000 mg/L O3 in a 1.063 m3 box, the growth of bacteria and fungi was found to be completely inhibited on surgical masks after 30 min and 2 h of treatment, respectively. Using a lower-dose O3 generator at 500 mg O3/L in 0.456 m3 provided lower efficiency, although the difference was not significant. Using O3 at 2000 mg O3/L or 500 mg O3/L in a 56 m3 room is efficient for the disinfection of all pathogens on the surface of reused surgical masks. CONCLUSIONS: This study provided the conditions for using O3 (500-2000 mg/L) to reduce pathogens and disinfect contaminated surgical masks, which might be applied to reduce the inappropriate usage of reused surgical masks.

Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy Discriminates the Elderly with a Low and High Percentage of Pathogenic CD4+ T Cells.

In the aging process, the presence of interleukin (IL)-17-producing CD4+CD28-NKG2D+T cells (called pathogenic CD4+ T cells) is strongly associated with inflammation and the development of various diseases. Thus, their presence needs to be monitored. The emergence of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy empowered with machine learning is a breakthrough in the field of medical diagnostics. This study aimed to discriminate between the elderly with a low percentage (LP; ≤3%) and a high percentage (HP; ≥6%) of pathogenic CD4+CD28-NKG2D+IL17+ T cells by utilizing ATR-FTIR coupled with machine learning algorithms. ATR spectra of serum, exosome, and HDL from both groups were explored in this study. Only exosome spectra in the 1700-1500 cm-1 region exhibited possible discrimination for the LP and HP groups based on principal component analysis (PCA). Furthermore, partial least square-discriminant analysis (PLS-DA) could differentiate both groups using the 1700-1500 cm-1 region of exosome ATR spectra with 64% accuracy, 69% sensitivity, and 61% specificity. To obtain better classification performance, several spectral models were then established using advanced machine learning algorithms, including J48 decision tree, support vector machine (SVM), random forest (RF), and neural network (NN). Herein, NN was considered to be the best model with an accuracy of 100%, sensitivity of 100%, and specificity of 100% using serum spectra in the region of 1800-900 cm-1. Exosome spectra in the 1700-1500 and combined 3000-2800 and 1800-900 cm-1 regions using the NN algorithm gave the same accuracy performance of 95% with a variation in sensitivity and specificity. HDL spectra with the NN algorithm also showed excellent test performance in the 1800-900 cm-1 region with 97% accuracy, 100% sensitivity, and 95% specificity. This study demonstrates that ATR-FTIR coupled with machine learning algorithms can be used to study immunosenescence. Furthermore, this approach can possibly be applied to monitor the presence of pathogenic CD4+ T cells in the elderly. Due to the limited number of samples used in this study, it is necessary to conduct a large-scale study to obtain more robust classification models and to assess the true clinical diagnostic performance.

Anti-Proteus Activity, Anti-Struvite Crystal, and Phytochemical Analysis of Sida acuta Burm. F. Ethanolic Leaf Extract.

Proteus mirabilis is a significant cause of urinary tract infection that may contribute to struvite stones. Anti-infection of this bacterium and anti-struvite formation must be considered. Sida acuta Burm. F. (SA) has been used for the treatment of diseases related to kidneys. Therefore, we investigated the effects of the SA leaf ethanolic extract (SAEE) on growth and on virulent factors (swarming motility and urease activity) of Proteusmirabilis isolated from kidney stone formers. We also evaluated anti-struvite crystal formation and phytochemical constituents of SAEE. The minimum inhibitory concentrations (MICs) of SAEE against three clinical P. mirabilis isolates were 8 mg/mL. Intriguingly, the 1/2MIC of SAEE had significant inhibitory effects on the swarming motility and urease activity of clinical P. mirabilis isolates when compared with the condition without SAEE. The SAEE at the various concentrations significantly inhibited the average weights of struvite crystals in a dose-dependent manner, compared with the control. The phytochemical analysis revealed that SAEE contained catechin, chlorogenic acid, rutin, and ferulic acid. This study indicated that SAEE has anti-P. mirabilis and anti-struvite crystal activities via its bioactive compounds. For this reason, SAEE may be developed as a new agent for the treatment of struvite stone induced by P. mirabilis.

Effect of Vancomycin on Cellular Fatty Acid Profiles of Vancomycin-Susceptible and Nonsusceptible Staphylococcus aureus.

Vancomycin is widely used for treatment of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) leading to an increasing appearance of low-level vancomycin-resistant isolates called heterogeneous vancomycin-intermediate S. aureus (hVISA). The mechanism of vancomycin tolerance in hVISA is still unclear. This study aimed to investigate the fatty acid compositions of S. aureus isolates under the stress environment with vancomycin. The different responses of hVISA and vancomycin-susceptible S. aureus (VSSA) may lead to more understanding the mechanism. The bacterial lipid profiles were tested three times from three extractions of each isolate cultured on tryptic soy agar (TSA) and TSA with vancomycin. Of the 30 MRSA isolates studied, 13, 12, and 5 isolates were VSSA, hVISA, and VISA, respectively. The analysis of bacterial lipid profiles showed that under vancomycin stress, there was a reduction of straight chain fatty acids (SCFAs) in VSSA isolates but an increase in branched chain fatty acids (BCFAs). In contrast, the hVISA group exhibited an increase only in the BCFAs but not in SCFAs. Of interest, vancomycin had no effect on either BCFAs or SCFAs of the VISA cells. This study provided information of bacterial adaptation during stress with vancomycin that may be helpful to overcome the resistant bacteria.