MALDI-TOF mass spectrometry for sub-typing of Streptococcus pneumoniae.

BACKGROUND: Serotyping of Streptococcus pneumoniae is important for monitoring of vaccine impact. Unfortunately, conventional and molecular serotyping is expensive and technically demanding. This study aimed to determine the ability of matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry to discriminate between pneumococcal serotypes and genotypes (defined by global pneumococcal sequence cluster, GPSC). In this study, MALDI-TOF mass spectra were generated for a diverse panel of whole genome sequenced pneumococcal isolates using the bioMerieux VITEK MS in clinical diagnostic (IVD) mode. Discriminatory mass peaks were identified and hierarchical clustering was performed to visually assess discriminatory ability. Random forest and classification and regression tree (CART) algorithms were used to formally determine how well serotypes and genotypes were identified by MALDI-TOF mass spectrum. RESULTS: One hundred and ninety-nine pneumococci, comprising 16 serotypes and non-typeable isolates from 46 GPSC, were analysed. In the primary experiment, hierarchical clustering revealed poor congruence between MALDI-TOF mass spectrum and serotype. The correct serotype was identified from MALDI-TOF mass spectrum in just 14.6% (random forest) or 35.4% (CART) of 130 isolates. Restricting the dataset to the nine dominant GPSC (61 isolates / 13 serotypes), discriminatory ability improved slightly: the correct serotype was identified in 21.3% (random forest) and 41.0% (CART). Finally, analysis of 69 isolates of three dominant serotype-genotype pairs (6B-GPSC1, 19F-GPSC23, 23F-GPSC624) resulted in the correct serotype identification in 81.1% (random forest) and 94.2% (CART) of isolates. CONCLUSIONS: This work suggests that MALDI-TOF is not a useful technique for determination of pneumococcal serotype. MALDI-TOF mass spectra appear more associated with isolate genotype, which may still have utility for future pneumococcal surveillance activities.

Knowledge, attitudes, practices and prevalence of hepatitis B and C and hepatitis B vaccination coverage among public sector healthcare workers in Cambodia.

Healthcare workers (HCWs) are a key population at high risk for hepatitis B (HBV) and hepatitis C (HCV) infections. We aim to study HBV vaccination coverage, seroprevalence, knowledge, attitudes, and practices towards HBV and HCV infections among HCWs in public sector in Cambodia. A nationally representative cross-sectional study was implemented in 2019, among Cambodian HCWs. A standardized questionnaire was administered to randomly selected HCWs whose blood was then sampled. We used univariate and multivariate regression to determine predictors of outcomes. Among 755 participants, we found 4.9% positive HBsAg and 2.3% positive anti-HCV Ab. HBV vaccination coverage was 59.3%. Lack of knowledge was found on the route of transmission, HBV vaccination, diagnosis and treatment of HBV and HCV. 67% of HCWs thought that all patients should be screened for HBV and HCV and about 30% of them would refuse to take care of infected patients. 58% of HCWs always recapped the needle after use. In univariate analysis, older age-group (> 50 years) is more likely to have positive anti-HCV (OR: 9.48; 95% CI: 2.36-38.18). HCWs who were younger, female or having higher education or having ever been tested, were more likely to have gotten HBV vaccinated. Multivariate analysis reconfirmed these predictors of getting vaccinated. Study findings indicated an urgent need of a national policy for Cambodian HCWs given the high prevalence of hepatitis among this group. Policy should include an effective in-service training program to improve knowledge and practices, a testing and vaccination program for HCWs and it should emphasize stigma intervention towards people living with HBV/HCV.

Fatiguing stimulation of one skeletal muscle triggers heat shock protein activation in several rat organs: the role of muscle innervation.

We hypothesised that activation of muscle afferents by fatigue triggers a widespread activation of heat shock proteins (HSPs) in resting muscles and different organs. In anaesthetised rats, HSP25 and HSP70 levels were determined in both tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and in the diaphragm, kidney and brain by ELISA, which mostly identifies phosphorylated HSP, and western blotting. One TA muscle was electrically stimulated and tissues were sampled 10 or 60 min after the stimulation had ended. The nerve supply to the stimulated TA or its counterpart in the contralateral limb was left intact or suppressed. In control rats, no muscle stimulation was performed and tissues were sampled at the same time points (10 or 60 min). After TA stimulation, ELISA showed an increased HSP25 content in the contralateral TA, EDL and diaphragm at 10 min but not at 60 min, and HSP70 increased in all sampled tissues at 60 min. Western blotting did not show any changes in HSP25 and HSP70 at 10 min, while at 60 min HSP25 increased in all sampled tissues except the brain and HSP70 was elevated in all tissues. Denervation of the contralateral non-stimulated limb suppressed HSP changes in TA and after denervation of the stimulated TA the widespread activation of HSPs in other organs was absent. Our data suggest that fatigue-induced activation of skeletal muscle afferents triggers an early increase in phosphorylated HSP25 in muscles and a delayed elevation of non-phosphorylated HSP25 and HSP70 in skeletal and respiratory muscles, kidney and brain.

Fall in oxygen tension of culture medium stimulates the adenosinergic signalling of a human T cell line.

We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A(2A) receptor (A(2A)R) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3',5'-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A(2A)R expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A(2A)R agonists may have therapeutic relevance.

Expeditious synthesis and biological evaluation of new C-6 1,2,3-triazole adenosine derivatives A1 receptor antagonists or agonists.

The synthesis of new C-6 1,2,3-triazole adenosine derivatives via microwave assisted 1,3-dipolar cycloaddition as key step is described. The binding on membranes of cells that over express A(1) adenosine receptors (A(1)AR) was also evaluated. Among them, four compounds increased cAMP production, in a dose-dependent manner acting as antagonists of the A(1)AR, while two compounds act as agonists.

Head-up tilt induced syncope and adenosine A2A receptor gene polymorphism.

AIMS: High adenosine plasma levels and high expression of adenosine A(2A) receptors are observed in patients with unexplained syncope and a positive head-up tilt test (HUT). This study aimed to evaluate the single nucleotide polymorphism (SNP) (c.1364 T>C) which is the most commonly found polymorphism in the A(2A) receptor gene, in patients with unexplained syncope undergoing HUT. METHODS AND RESULTS: One hundred and five patients with unexplained syncope who underwent HUT were included. Fifty-two had a positive test. Receptor genotype determinations were performed in patients and in 121 healthy subjects. Genotype (TT, CC, TC) was determined from DNA leucocytes. The distribution of the polymorphism showed significant (P < 0.0001) difference when the results of HUT were analysed. Fifty-two per cent of patients with a positive HUT had a CC genotype and 34.6% a TC genotype, whereas 13.2% of the patients with a negative HUT had a CC genotype and 71.7% a TC genotype. Patients with a CC genotype had a higher incidence of spontaneous syncopal episodes. CONCLUSION: In patients with unexplained syncope, a significant association between high incidence of syncopal episodes, positive HUT, and the presence of the CC variant in the adenosine A(2A) receptor gene was elicited.

Production of an agonist-like monoclonal antibody to the human A2A receptor of adenosine for clinical use.

The second extracellular loop of the A(2A) receptor (A(2A)R) of adenosine was used to immunize mice for production of Adonis, an IgM monoclonal antibody. Adonis bound to the immunogen peptide and the native receptor in ELISA with K(D) values in 6.51-12.35 nM range. It recognized a linear epitope of 7 amino acids (LFEDVVP) at the C-terminal part of the external loop. Adonis revealed a 45-kDa band in lysate of human peripheral blood mononuclear cells in Western blotting in denaturing conditions. This served to monitor the up-regulation of the A(2A)R expression by caffeine. Adonis stimulated the cAMP production and inhibited the cell proliferation of an A(2A)R transfected stable cell line. These results confirm the immunogenicity and the functional relevance of the second extracellular loop of the A(2A)R. They suggest that Adonis may be of clinical use in various pathological situations to measure the regulation of the A(2A)R expression and to act as A(2A)R agonist drug.

Design, synthesis and biological evaluation of a bivalent micro opiate and adenosine A1 receptor antagonist.

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A(1) adenosine and mu opiate receptors with a K(i) of 0.8+/-0.05 and 0.7+/-0.03 microM, respectively. This hybrid molecule increases cAMP production in cells that over express the mu receptor as well as those over expressing the A(1) adenosine receptor and reverses the antalgic effects of mu and A(1) adenosine receptor agonists in animals.

Antimicrobial resistance in Cambodia: a review.

OBJECTIVES: Following the launch of the Global Antimicrobial Resistance Surveillance System (GLASS), antimicrobial resistance (AMR) rates in many countries remain poorly described. This review provides an overview of published AMR data from Cambodia in the context of recently initiated national human and food-animal surveillance. METHODS: PubMed and the Cochrane Database of Systematic Reviews were searched for articles published from 2000 to 2018, which reported antimicrobial susceptibility testing (AST) data for GLASS specific organisms isolated from Cambodia. Articles were screened using strict inclusion/exclusion criteria. AST data was extracted, with medians and ranges of resistance rates calculated for specific bug-drug combinations. RESULTS: Twenty-four papers were included for final analysis, with 20 describing isolates from human populations. Escherichia coli was the most commonly described organism, with median resistance rates from human isolates of 92.8% (n=6 articles), 46.4% (n=4), 55.4% (n=8), and 46.4% (n=5) to ampicillin, 3rd generation cephalosporins, fluoroquinolones, and gentamicin respectively. CONCLUSIONS: Whilst resistance rates are high for several GLASS organisms, there were insufficient data to draw robust conclusions about the AMR situation in Cambodia. The recently implemented national AMR surveillance systems will begin to address this data gap.

Serotype Distribution of Clinical Streptococcus pneumoniae Isolates before the Introduction of the 13-Valent Pneumococcal Conjugate Vaccine in Cambodia.

Childhood vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in Cambodia in January 2015. Baseline data regarding circulating serotypes are scarce. All microbiology laboratories in Cambodia were contacted for identification of stored isolates of Streptococcus pneumoniae from clinical specimens taken before the introduction of PCV13. Available isolates were serotyped using a multiplex polymerase chain reaction method. Among 166 identified isolates available for serotyping from patients with pneumococcal disease, 4% were isolated from upper respiratory samples and 80% were from lower respiratory samples, and 16% were invasive isolates. PCV13 serotypes accounted for 60% (95% confidence interval [CI] 52-67) of all isolates; 56% (95% CI 48-64) of noninvasive and 77% (95% CI 57-89) of invasive isolates. Antibiotic resistance was more common among PCV13 serotypes. This study of clinical S. pneumoniae isolates supports the potential for high reduction in pneumococcal disease burden and may serve as baseline data for future monitoring of S. pneumoniae serotypes circulation after implementation of PCV13 childhood vaccination in Cambodia.

Release of markers of myocardial damage evaluated in the coronary sinus during cardiac surgery.

Myocardial damage is a frequent complication of cardiac surgery by direct mechanical trauma during the surgical procedure and by myocardial ischemia, which occurs during the cardiopulmonary bypass (CBP). Because the concentrations of biomarkers in the blood collected from the coronary sinus are the best witness of the myocardial damages, we measured the levels of specific cardiac troponin I (c-TnI) and nonspecific (adenosine, myoglobin) markers of left ventricular damages in the coronary sinus of patients during cardiac surgery. Thirty patients who underwent aortic valve replacement for aortic stenosis were included. Blood samples were collected in the coronary sinus and in the radial artery at the beginning (T0), at the end of the CBP (T1), and then 24 hours later (T2). At T0 and T1, adenosine, c-TnI, and myoglobin levels were significantly higher in the coronary sinus than in the radial artery (T0: adenosine: mean +27%; c-TnI: +41%; myoglobin: +28%; T1: adenosine: mean +58%; c-TnI: +58%; myoglobin: +25%; p < .001). These parameters were significantly higher in the coronary sinus at T1 compared with T0 (adenosine: +50%; c-TnI: +229%; myoglobin: +94%; p < .01) and in the radial artery (adenosine: +21%; c-TnI: +194%; myoglobin: +98%; p < .01). At T2, c-TnI further increased. Damages to the myocardium during cardiac surgery are minimal in our surgical conditions but are probably underestimated when using markers measured at the peripheral level. However, most of the damages appear several hours after the surgery.

Biodiversity and health: Lessons and recommendations from an interdisciplinary conference to advise Southeast Asian research, society and policy.

Southeast Asia is an economic, biodiverse, cultural and disease hotspot. Due to rapid socio-economic and environmental changes, the role of biodiversity and ecosystems for human health ought to be examined and communicated to decision-makers and the public. We therefore summarized the lessons and recommendations from an interdisciplinary conference convened in Cambodia in 2014 to advise Southeast Asian societies on current research efforts, future research needs, and to provide suggestions for improved education, training and science-policy interactions. First, we reviewed several examples of the important role of ecosystems as 'sentinels' in the sense that potentially harmful developments for human health become first apparent in ecosystem components. Other ecosystem services which also benefit human well-being are briefly summarized. Second, we summarized the recommendations of the conference's roundtable discussions and added recent developments in the science-policy interface. The recommendations were organized along five themes: Ethical and legal considerations; implementation of the One Health approach; education, training, and capacity building; future research priorities; and potential science-policy interactions. While the role of biodiversity for human health needs further research, especially for zoonoses and emerging diseases, many direct and indirect benefits to human health are already apparent, but have yet to filter down to the science-policy interface in order to influence legislation and enforcement. Therefore, efforts to strengthen the interface in Southeast Asia should become a high priority in order to strengthen the health and resilience of Southeast Asian societies.

NF-κB enhances hypoxia-driven T-cell immunosuppression via upregulation of adenosine A(2A) receptors.

Hypoxia affects inflammation by modulating T-cell activation via the adenosinergic system. We supposed that, in turn, inflammation influences cell hypoxic behavior and that stimulation of T-cells in inflammatory conditions involves the concerted action of the nuclear factor κB (NF-κB) and the related hypoxia-inducible factor 1α (HIF-1α) on the adenosinergic system. We addressed this hypothesis by monitoring both transcription factors and four adenosinergic signaling parameters - namely adenosine, adenosine deaminase (ADA), adenosine A2A receptor (A2AR) and cAMP - in T-cells stimulated using phorbol myristate acetate and phytohemagglutinin and submitted to hypoxic conditions which were mimicked using CoCl2 treatment. We found that cell viability was more altered in stimulated than in resting cells under hypoxia. Detailed analysis showed that: i) NF-κB activation remained at basal level in resting hypoxic cells but greatly increased following stimulation, stimulated hypoxic cells exhibiting the higher level; ii) HIF-1α production induced by hypoxia was boosted via NF-κB activation in stimulated cells whereas hypoxia increased HIF-1α production in resting cells without further activating NF-κB; iii) A2AR expression and cAMP production increased in stimulated hypoxic cells whereas adenosine level remained unchanged due to ADA regulation; and iv) the presence of H2S, an endogenous signaling molecule in inflammation, reversed the effect of stimulation on cell viability by down-regulating the activity of transcription factors and adenosinergic immunosuppression. We also found that: i) the specific A2AR agonist CGS-21680 increased the suppressive effect of hypoxia on stimulated T-cells, the antagonist ZM-241385 exhibiting the opposite effect; and ii) Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase 4, and 8-Br-cAMP, a cAMP analog which preferentially activates cAMP-dependent protein kinase A (PKA), increased T-cell immunosuppression whereas H-89, a potent and selective inhibitor of cAMP-dependent PKA, restored cell viability. Together, these data indicate that inflammation enhances T-cell sensitivity to hypoxia via NF-κB activation. This process upregulates A2AR expression and enhances cAMP production and PKA activation, resulting in adenosinergic T-cell immunosuppression that can be modulated via H2S.

Search for adenosine A2A spare receptors on peripheral human lymphocytes.

Some ligand-receptor couples involve spare receptors, which are apparent when a maximal response is achieved with only a small fraction of the receptor population occupied. This situation favours cross-reactions with low-affinity ligands, which may be detrimental for cell signaling. In the case of the adenosine A2A receptors (A2AR), which have an immunosuppressive effect on lymphocytes through cAMP production, the presence of spare A2AR remains to be established. We examined the situation using patients over-expressing lymphocyte A2AR and an agonist-like mAb to A2AR. We found that maximal mAb binding and functional response varied among the patients whereas the dissociation constant and half-maximal effective concentration had similar mean values (0.19 and 0.18 µM, respectively). Lymphocyte A2AR expression was correlated to plasma adenosine level and A2AR occupation but not to A2AR response. These results are consistent with a lack of a reserve of functional A2AR on human lymphocytes as a general rule and suggest that the amount and functional state of the expressed A2AR determine the maximal level of the lymphocyte response to adenosine.

SKCa Channels Blockage Increases the Expression of Adenosine A2A Receptor in Jurkat Human T Cells.

Adenosine is a nucleoside displaying various biological effects via stimulation of four G-protein-coupled receptors, A1, A2A, A2B, and A3. Adenosine also modulates voltage-gated (Kv) and small conductance calcium-activated (SKCa) potassium channels. The effect of these potassium channels on the expression of adenosine receptors is poorly understood. We evaluated the action of BgK (a natural Kv channel blocker) and Lei-Dab7 (a synthetic SKCa channel blocker) on the expression of adenosine A2A receptors (A2AR) in Jurkat human T cells. We found that Lei-Dab7, but not BgK, increased the maximal binding value of the tritiated ligand ZM241385 to A2AR in a dose-dependent manner (+45% at 5 nM; +70% at 50 nM as compared to control). These results were further confirmed by Western blotting using a specific monoclonal antibody to human A2AR. The ligand affinity-related dissociation constant and A2AR mRNA amount were not significantly modified by either drug. We suggest that modulation of SKCa channels can influence membrane expression of A2AR and thus has a therapeutic potential.

Mononuclear cell adenosine deaminase and CD26/dipeptidylpeptidase-IV activities are sensitive markers of reperfusion during percutaneous transluminal angioplasty.

UNLABELLED: During ischaemia, the extracellular level of adenosine increases, which has cytotoxic effects. In endothelium, cell surface adenosine deaminase (ADA) complexing CD26 is coordinately induced during ischaemia as part of an adaptative response by eliminating adenosine. We examined whether a similar mechanism exists for mononuclear cells. We studied mononuclear cell surface ADA (MCADA) and dipeptidyl-peptidase IV activity (DPPIV) of membrane CD26 during percutaneous transluminal coronary angioplasty (PTCA) as a model of ischaemia-reperfusion. Enzymatic activities were compared with levels of ischaemia-modified albumin (IMA), a marker of ischaemia-reperfusion. METHODS AND RESULTS: Patients (15 men and 5 women) with non-ST segment elevation acute coronary syndrome related to a stenosis of proximal left anterior descending artery were prospectively included before revascularization. MCADA, DPPIV and IMA were measured before PTCA (T0) then 15 (T15) and 120 (T120) minutes after reperfusion. Fifteen healthy control subjects were enrolled. At T0, MCADA and IMA levels were higher in patients than in controls. MCADA decreased at T15 (median, IQR: 8.2 [7.6-9.8] IU) relative to T0 (11.25 [10-13.5] IU, p<0.01) and remained low at T120. DPPIV decreased at T15 (0.9 [0.7-1.1] AU) relative to T0 (1.05 [0.99-1.48] AU; p<0.01) and remained low at T120. IMA level increased only at T120. MCADA and DPPIV were correlated. Our findings are that MCADA and DPPIV decreased rapidly after angioplasty, suggesting that both catalysts are early markers of reperfusion. CONCLUSION: MCADA and DPPIV are sensitive and early markers of ischaemia-reperfusion process during PTCA.

Adenosine plasma level and A2A adenosine receptor expression: correlation with laboratory tests in patients with neurally mediated syncope.

OBJECTIVES: The purpose of this study was to investigate the hypothesis that responses to the ATP test and head-up tilt test (HUT) may be correlated with different purinergic profiles. DESIGN AND SETTING: The ATP and HUT identify distinct subsets of patients with neurally mediated syncope (NMS). Adenosine and its A(2A) receptors (A(2A)R) may be implicated in the pathophysiology of NMS in patients with positive HUT. Nothing is known about the purinergic profile of patients with positive ATP. PATIENTS AND MEASURES: This prospective study includes a consecutive series of patients with suspected NMS. All patients underwent both HUT and ATP. Before testing, samples were collected for measurement of baseline adenosine plasma level (APL) and expression. RESULTS: A total of 46 patients (25 men and 21 women) with a mean age of 57±18 years were enrolled. The HUT test was positive in 27 patients and the ATP test in 20. Both tests were positive in 9 and negative in 8. High APL was associated with high probability of positive HUT while low APL was associated with high probability of positive ATP. Expression of A(2A)R was lower in patients with positive ATP than in those with positive HUT. CONCLUSION: These findings indicate that patients with NMS present different purinergic profiles and that responses to HUT and ATP are correlated with these profiles.

Intracerebroventricular injection of an agonist-like monoclonal antibody to adenosine A(2A) receptor has antinociceptive effects in mice.

Adenosine is a modulator of nociceptive pathways, both at the spinal and supraspinal levels. Adenosine A(1) and A(2A) receptors (A(1)R, A(2A)R) are expressed in the basal ganglia where they are the target of caffeine, the most widely use psychoactive drug which acts as an antagonist to both types of receptors. Given the controversial role of A(2A)R versus A(1)R in modulating pain in brain areas, mice received intracerebroventricular injection of Adonis, an agonist-like monoclonal antibody with high specificity for the A(2A)R and were subjected to behavioral tests investigating nociceptive thresholds. We report that Adonis led to a significant dose-dependent increase in hot-plate and tail-flick latencies in mice and that such increase was prevented by caffeine and ZM 241385, a specific A(2A)R antagonist. The Adonis antinociceptive effects were also inhibited by naloxone, a non selective antagonist for opioid receptors, suggesting that Adonis acts, at least in part, through the stimulation of the endogenous opioid system. These results confirm the A(2A)R as a target for pain control and Adonis as a potential drug with therapeutic interest.