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).

A two-arm analysis of the immune response to heterologous boosting of inactivated SARS-CoV-2 vaccines.

Several vaccine programs were introduced during the COVID-19 pandemic, which included inactivated virus, DNA viral vectors and mRNA vaccines. Booster programs are recommended, especially for those in high-risk groups. However, many of these booster programs involve heterologous vaccines. This study enrolled volunteers who first received two full-dose CoronaVac vaccinations before receiving heterologous boosters with DNA- and/or mRNA-vaccines for an additional 2 doses (n = 40) or an additional 3 doses (n = 16). Our results showed no difference in side effects, neutralizing antibodies, or T-cell responses for any of the heterologous vaccination programs. However, the neutralizing capacity and IFN-γ responses against the Omicron variant in volunteers who received 4 or 5 doses were improved. Polarization of peripheral memory T cells after stimulation in all booster groups with Omicron peptide showed an increased trend of naïve and central memory phenotypes of both CD4+ and CD8+ T cells, suggesting that exposure to Omicron antigens will drive T cells into a lymphoid resident T cell phenotype. Our data support a continuous vaccination program to maximize the effectiveness of immunity, especially in people at high risk. Furthermore, the number of boosting doses is important for maintaining immunity.

Association of MICA Gene Polymorphism in Opisthorchis viverrini-Induced Periductal Fibrosis in Northeastern Thais.

OBJECTIVE: Chronic Opisthorchis viverrini (OV) infection is the cause of advanced periductal fibrosis (APF), subsequently leading to cholangiocarcinoma (CCA). Natural killer (NK) cells can kill hepatic stellate cells (HSCs), the initiating cells for fibrosis formation, by using the interaction between the natural killer group 2 member D (NKG2D) receptor and its ligand on the HSCs. This can inhibit the fibrosis formation. Major histocompatibility complex class I chain-related A (MICA) is the ligand of the NKG2D receptor and has highly polymorphic characteristics that are involved in NKG2D binding and NK cell activation. This study aimed to investigate the polymorphism of MICA in OV-induced fibrosis. METHOD: MICA typing was performed by polymerase chain reaction- sequence specific primer (PCR-SSP) and sequencing in two groups: OV infection without fibrosis (N = 99) and with fibrosis (N = 290). RESULT: Six alleles were identified and the MICA*010 allele had the highest frequency in both groups. The MICA*00201-02 allele was a protective factor for fibrosis (OR= 0.508, 95%CI= 0.34-0.76, Pc <0.05), while the MICA*019 allele was suggested to be a risk allele for fibrosis (OR=1.95, 95%CI=1.25-3.03, Pc<0.005). In addition, two motifs, glycine (G) at position 14 and glutamine (Q) at position 251, were negatively associated with fibrosis (G14: OR=0.508, 95%CI=0.34-0.76, Pc <0.05 and Q251: OR=0.586, 95%CI=0.41-0.84, Pc <0.05). Moreover, the distribution of the MICA-129 genotype also showed the protective genotype (Pc<0.05, OR=0.319, 95%CI= 0.12-0.54) for fibrosis. The MICA*00201-02 allele encoded all these motifs, and this suggested that it might lead to strong NK cell activation to kill HSCs, subsequently preventing fibrosis formation. CONCLUSION: This study described initial evidence suggesting that the polymorphism of the MICA gene might be a marker for OV-derived periductal fibrosis.

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.

Phenotypic and functional changes of T cell subsets after CoronaVac vaccination.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection. METHODS: This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot). FINDINGS: Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4+ T cell-, but not CD8+ T cell-responses. Among the CD4+ T cells, CoronaVac activated mainly Th2 (CD4+ T) cells. Serum antibody levels significantly declined three months after the second dose. INTERPRETATION: CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

High rate of reinfection and possible transmission of Mycobacterium avium complex in Northeast Thailand.

The Mycobacterium avium complex (MAC) includes two main species of non-tuberculous mycobacteria (NTM), M. avium and Mycobacterium intracellulare. These can cause serious disease, especially in immunocompromised patients. Little information is available concerning genetic diversity of NTM. We used multilocus sequence typing (MLST) based on a highly discriminative gene set to analyze MAC serially isolated from patients to determine the rate of MAC reinfection. Genomic DNA was sequenced from 49 MAC isolates (15 cases comprised of 11 true infections and 4 instances of colonization). More than half of the MAC isolates tested were found to be multidrug resistant. The discriminatory power was assessed of 24 house-keeping genes (fusA, atpD, pheT, glnA, topA, secA, argH, glpK, murC, cya, pta, rrl, rrs, hsp65, rpoB, 16S-23S rRNA ITS, recF, lipT, pepB, gnd, aspB, groEL, sodA and est) previously used for genotyping of MAC and other NTM. Seven genes (fusA, secA, rpoB, hsp65, 16S rRNA, 23S rRNA, 16S-23S rRNA ITS) had a discriminatory power index higher than 0.9 and were included in the optimized set that we used. This set was significantly better for genotyping and diagnosis of MAC than previously used 4-gene, 5-gene and 9-gene sets. MLST using our 7-gene set indicated that the rate of reinfection was 54.55% (6/11 cases). Persistent infections (n = 5 cases, 45.45%) were found. A changing of clone in the same patient was found in 1/4 (25%) of the colonization cases. Two small clusters of possible MAC transmission between humans were found. Our study demonstrated that the high frequency of apparent treatment failure of MAC might be artefactual, as a consequence of a high rate of MAC reinfection in Thai population. Our useful highly discriminative gene set for MAC species and clonal strain analysis could be further applied for the diagnosis and patient management.

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.

Traditional Thai Massage Promoted Immunity in the Elderly via Attenuation of Senescent CD4+ T Cell Subsets: A Randomized Crossover Study.

The beneficial physiological effects of traditional Thai massage (TTM) have been previously documented. However, its effect on immune status, particularly in the elderly, has not been explored. This study aimed to investigate the effects of multiple rounds of TTM on senescent CD4+ T cell subsets in the elderly. The study recruited 12 volunteers (61-75 years), with senescent CD4+ T cell subsets, who received six weekly 1-h TTM sessions or rest, using a randomized controlled crossover study with a 30-day washout period. Flow cytometry analysis of surface markers and intracellular cytokine staining was performed. TTM could attenuate the senescent CD4+ T cell subsets, especially in CD4+28null NKG2D+ T cells (n = 12; p < 0.001). The participants were allocated into two groups (low < 2.75% or high ≥ 2.75%) depending on the number of CD4+28null NKG2D+ T cells. After receiving TTM over 6 sessions, the cell population of the high group had significantly decreased (p < 0.001), but the low group had no significant changes. In conclusion, multiple rounds of TTM may promote immunity through the attenuation of aberrant CD4+ T subsets. TTM may be provided as a complementary therapy to improve the immune system in elderly populations.

Oral vitamin C treatment increases polymorphonuclear cell functions in type 2 diabetes mellitus patients with poor glycemic control.

This study investigated the effect of vitamin C on polymorphonuclear (PMN) cell functions in type 2 diabetes mellitus patients with poor glycemic control. We hypothesized that oral vitamin C treatment improves PMN cell functions. Patients (14) received either a vitamin C (1000 mg/d) or placebo (anhydrous calcium hydrogen phosphate) tablet for 6 weeks and were subjected to a 6-week washout period followed by a 6-week treatment crossover period. Blood samples were collected at pretreatment and posttreatment for PMN cell functions (by flow cytometry) and plasma vitamin C concentration. Phagocytosis was examined by incubating whole blood samples with fluorescein isothiocyanate-labeled Staphylococcus aureus, and oxidative burst was simultaneously evaluated by adding hydroethidine. In comparison with placebo, vitamin C increased both PMN cell phagocytosis (pretreatment: placebo, 17.8% ± 1.6% and vitamin C, 19.0% ± 3.4%, P = .70; posttreatment: placebo, 16.6% ± 1.7% and vitamin C, 27.1% ± 2.9%, P = .005) and oxidative burst (pretreatment: placebo, 6.4% ± 0.8% and vitamin C, 7.1% ± 1.2%, P = .60; posttreatment: placebo, 6.9% ± 1.3% and vitamin C, 12.1% ± 1.6%, P = .02). The plasma vitamin C concentration was elevated after vitamin C treatment as compared with that before treatment (P < .001) and was higher than that observed in the placebo treatment group (P < .01). Plasma vitamin C concentration and PMN cell functions were not significantly different before both treatments. We conclude that the 6-week 1000-mg/d vitamin C increased PMN phagocytosis and oxidative burst in type 2 diabetes mellitus patients with poor glycemic control.

IL-17 and IFN-γ Productions by CD4+ T cells and T cell Subsets Expressing NKG2D Associated with the Number of Risk Factors for Cardiovascular Diseases.

Cardiovascular diseases (CVD), which are major causes of morbidity and mortality worldwide, are characterized by complicated chronic inflammatory manifestation inducing from multi-risk factors. Previously, we have identified a pathological T cell subpopulation producing interleukin (IL)-17 in diabetes. We hypothesized that this T cell subpopulation could exist in the elderly with persistence low grade inflammation related to the risk factors for cardiovascular diseases. Thus, we investigated whether high levels of the natural group 2, member D (NKG2D) expression, IL-17 and interferon (IFN)-γ production by CD4 + T cells and T cell subsets were more prevalent in individuals who had age ≥ 60 years with > 2 risk factors for CVD (dyslipidemia, hypertension and/or diabetes mellitus) compared to subjects who had < 2 risk factors. Using flow cytometric analysis, we found that CD4 + T cells of subjects who had ≥ 2 risk factors had significantly higher NKG2D expression than those of subjects with < 2 risk factors (P = 0.023). Apparently, CD4+CD28null T subset of both two groups preferentially expressed NKG2D, and prominently produced IL-17 and IFN-γ compared to the CD4+CD28+ T subset. Expectedly, there was a statistical significance of IL-17 and IFN-γ production of CD4 + 28nullNKG2D + T cells (P = 0.037 and P = 0.042, respectively). We concluded that cumulative number of CVD risk factors associated with progressive alteration of CD4+ T cell phenotypes and their functions. Handling of metabolic risk factors may be an approach for healthcare of the elderly to prevent cardiovascular morbidity resulting from alteration of immunity.

IL-17 production by NKG2D-expressing CD56+ T cells in type 2 diabetes.

T cells expressing CD56 (identified as CD3+CD56+) play a potential role in activation or regulation of other immune cells by secreting various cytokines. We hypothesized that these cells expressing the natural group 2, member D (NKG2D) could produce high levels of interleukin (IL)-17 in type 2 diabetes (T2D). CD56 + T cells expressing NKG2D of T2D patients, particularly in poor glycemic control (PC) predominantly produced higher IL-17 compared to the NKG2D negative population. IL-17 production of CD56 + T cells with NKG2D + was positively correlated with the level of HbA1c (N = 22, R2 = 0.120 and P = 0.044). Interestingly, CD56+ T cells with NKG2DHi of T2D patients had significantly higher IL-17 production than those of CD56 + T cells with NKG2DLow (P = 0.027) and showed statistically significant with P-value < 0.001 compared to CD56 + T cells with NKG2DHi of non-diabetic individuals (ND). In summary, CD56 + T cells expressing NKG2D, especially in the NKG2DHi population may be involved in pathogenesis and severity of T2D via IL-17.

Aberrant NKG2D expression with IL-17 production of CD4+ T subsets in patients with type 2 diabetes.

Type 2 diabetes (T2D) is a systemic inflammatory disease. Although the natural killer group 2, member D (NKG2D) receptor, was not expressed normally on CD4+ T cells, the aberrant expression was found in pathological conditions such as in auto-immune diseases. However, the involvement of NKG2D in pathogenesis of T2D is unclear. We hypothesize that there is an inflammatory CD4+ T cell subpopulation expressing NKG2D and producing interleukin (IL)-17 in T2D. NKG2D expression on CD4+ T cells and their subsets were analyzed by multi-color staining using flow cytometry. Lymphocytes were activated by phorbol-12-myristate-13-acetate (PMA) and ionomycin, and were stained for intracellular IL-17. To investigate the mechanism of IL-17 production, patients' lymphocytes were stimulated using specific anti-T cell receptor (TCR) alone, anti-NKG2D alone or a combination of the two antibodies. CD4+ T cells and particularly, CD4+CD28nullT subset of T2D patients were highly expressed NKG2D and more prevalent compared to non-diabetic individuals (ND) (P=0.039 and P=0.022, respectively). Significantly higher percentages of CD4+CD28nullNKG2D+T cells of patients produced IL-17 when compared to those of ND (P=0.024) and were positively correlated with the level of glycated hemoglobin A1c (HbA1c) (R2=0.386, P=0.041). Additionally, this cell population could be stimulated by specific monoclonal anti-NKG2D to produce IL-17. In conclusion, CD4+CD28nullNKG2D+T cells were expanded in T2D, especially in patients with poor glycemic control. NKG2D may be one of the surrogate co-stimulatory receptors leading to irregular inflammatory function producing IL-17. An IL-17 producing CD4+CD28nullNKG2D+T cells may potentially be involved in pathogenesis and drive severity of the disease with the glycemic dependence. This particular cell type could be targeted for prognostic or therapeutic purposes.