Susceptibility profile of Candida rugosa (Diutina rugosa) against antifungals and compounds of essential oils

Candida rugosa (recently reclassified Diutina rugosa) is an emerging pathogen affecting humans and animals. Candida resistance to existing drugs is an important factor to be monitored, as well as the need of researching alternatives to conventional antifungals. Here, we evaluated the in vitro effects of some antifungals and major components of essential oils by the broth microdilution method (CLSI M27-A3) against fifteen C. rugosa strains from animals isolated and molecular identificated. The results showed MIC₉₀ of: 0.125 μg/mL to ketoconazole and voriconazole, 0.25 μg/mL to micafungin, 0.5 μg/mL to anidulafungin, 1 μg/mL to caspofungin, 2 μg/mL to amphotericin B, itraconazole and flucytosin, 8 μg/mL to fluconazole, 16 μg/mL to nystatin and >128 μg/mL to terbinafine. The compounds carvacrol (MIC₉₀ 320 μg/mL), thimol (MIC₉₀ 320 μg/mL) and cinnamaldehyde (MIC₉₀ 160 μg/mL) demonstrated antifungal activity against the samples tested.     

Pathway analysis of genome-wide association study on asthma

Objective

The aims of this study were to identify the candidate causal single nucleotide polymorphisms (SNPs) and candidate causal mechanisms of asthma and to generate SNP to gene to pathway hypotheses.

Methods

SNPs that met a threshold of p ? 0.001 in a genome-wide association study (GWAS) dataset of asthma, which included 292,443 SNPs in 473 asthma cases and 1892 controls, were used in the present study. Identify candidate causal SNPs and pathway (ICSNPathway) analysis was applied to this dataset.

Results

ICSNPathway analysis identified four candidate causal SNPs, four genes, and 21 candidate causal pathways, which in total provided four hypothetical biologic mechanisms: (1) rs7192 (nonsynonymous coding) to HLA-DRA to 21 pathways, such as, the role of eosinophils in the chemokine network of allergy, Th1/Th2 differentiation, and asthma (nominal p ? 0.001, FDR p ? 0.01); (2) rs20541 (nonsynonymous coding) to IL13 to asthma and cytokines and inflammatory response (nominal p < 0.001, FDR p ? 0.008); (3) rs1058808 (frameshift coding) to ERBB2 to transmembrane receptor activity (nominal p = 0.001, FDR p = 0.01); (4) rs17350764 (nonsynonymous coding (deleterious)) to OR52J3 to transmembrane receptor activity (nominal p = 0.001, FDR p = 0.01).

Conclusion

By applying ICSNPathway analysis to asthma GWAS data, we found four candidate causal SNPs, four genes involving HLA-DRA and IL-13, and four hypotheses, which may contribute to asthma susceptibility.     

Evaluation of the inoculum effect of new antibiotics against carbapenem-resistant enterobacterales

ObjectivesNew antibiotics have been developed to treat multidrug-resistant Enterobacterales. We evaluated the impact of the inoculum size on minimal inhibitory concentrations (MICs) of recently commercialized antibiotics.MethodsWe focused on 40 clinical carbapenemase-producing Enterobacterales and evaluated the impact of the inoculum size on the MICs to cefiderocol and to new β-lactams/β-lactamase inhibitors (ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam) at usual and high inocula (10⁵ and 10⁷ CFU/mL, respectively).ResultsAt usual inoculum, 15% were resistant to cefiderocol (n = 6), 30% to meropenem-vaborbactam (n = 12), 42.5% to ceftazidime-avibactam (n = 17), 55% to imipenem-relebactam (n = 22), and 90% to ceftolozane-tazobactam (n = 36). At higher inoculum, a switch from susceptible to resistant category was observed for 88% (n = 30/34; CI, 71.6–96.2), 75% (n = 3/4; CI, 21.9–98.7), 72% (n = 13/18; CI, 46.4–89.3), 50% (n = 14/28; CI, 31.1–68.9), and 8.7% (n = 2/23; CI, 1.5–29.5) isolates regarding cefiderocol, ceftolozane-tazobactam, imipenem-relebactam, meropenem-vaborbactam, and ceftazidime-avibactam, respectively.DiscussionCefiderocol and meropenem-vaborbactam were the most efficient against carbapenemase-producing Enterobacterales at usual inoculum. When increasing inoculum to 10⁷ CFU/mL, all of the molecules were impacted, particularly cefiderocol and imipenem-relebactam, while others, such as ceftazidime-avibactam, remain mildly affected. Our in vitro results deserved to be confirmed in vivo.     

Erythropoietin acute reaction and haematological adaptations to short, intermittent hypobaric hypoxia

This study aimed to determine whether brief hypoxic stimuli in a hypobaric chamber are able to elicit erythropoietin (EPO) secretion, and to effectively stimulate erythropoiesis in the short term. In two different experiments, a set of haematological, biochemical, haemorheological, aerobic performance, and medical tests were performed in two groups of healthy subjects. In the first experiment, the mean plasma concentration of EPO ([EPO]) increased from 8.7 to 13.5 mU · ml⁻¹ (55.2%; P < 0.01) after 90 min of acute exposure at 540 hPa, and continued to rise until a peak was attained 3 h after the termination of hypoxia. In the second experiment, in which subjects were exposed to a simulated altitude of up to 5500 m (504 hPa) for 90 min, three times a week for 3 weeks, all haematological indicators of red cell mass increased significantly, reaching the highest mean values at the end of the programme or during the subsequent 2 weeks, including packed cell volume (from 42.5 to 45.1%; P < 0.01), red blood cell count (from 4.55 × 10⁶ to 4.86 × 10⁶ · l⁻¹; P < 0.01), reticulocytes (from 0.5 to 1.4%; P < 0.01), and haemoglobin concentration (from 14.3 to 16.2 g · dl⁻¹; P < 0.01), without an increase in blood viscosity. Arterial blood oxygen saturation during hypoxia was improved (from 60% to 78%; P < 0.05). Our most relevant finding is the ability to effectively stimulate erythropoiesis through brief intermittent hypoxic stimuli (90 min), in a short period of time (3 weeks), leading to a lower arterial blood desaturation in hypoxia. The proposed mechanism for these haematological and functional adaptations is the repeated triggering effect of EPO production caused by the intermittent hypoxic stimuli. Accepted: 15 December 1999     

In vitro antimicrobial susceptibility of Prototheca spp. isolated from bovine mastitis in a Portugal dairy herd

ObjectiveTo test the in vitro susceptibility of Prototheca spp. associated with bovine mastitis in a dairy farm of Leiria, Portugal to antibacterial and antifungal drugs, in the aim of evaluating their antimicrobial resistance patterns and determining their susceptibility to disinfectants used in the dairy herd, in order to select the appropriate disinfectants, concentrations and exposure times.Material and methodsIn vitro antimicrobial susceptibility of 38 Prototheca spp. isolates was measured using the disk diffusion method. Fifteen antifungal and antibacterial antibiotics were evaluated. Isolates were tested to determine their sensitivity to five disinfectants used in this dairy herd with the membrane filtration method in accordance with the European norms NF EN 1040 and NF EN 1275.ResultsBased on morphological and biochemical properties, all Prototheca strains isolated in this study were assigned to the species Prototheca zopfii. All strains were shown to be in vitro resistant to penicillin, ampicillin, amoxicillin, neomycin, streptomycin, fluconazole, itraconazole, clotrimazole, econazole, and miconazole. All strains were susceptible to kanamicin and gentamicin. In addition, 77.8 and 44.4% were susceptible to ketoconazole and nystatin, respectively. Only 11.1% of the strains were sensitive to amphotericin B. Biocitro^® and Eco Plus^® proved to be efficient for all Prototheca strains. Combicid^® and Prodip G^® were not able to inhibit the growth of all Prototheca strains.ConclusionThis study recommends the use of a germicide, whose action against Prototheca spp. has also been demonstrated and at the recommended concentration and exposure time.     

A model for simulation and patient-specific visualization of the tissue volume of influence during brain microdialysis

Microdialysis can be used in parallel to deep brain stimulation (DBS) to relate biochemical changes to the clinical outcome. The aim of the study was to use the finite element method to predict the tissue volume of influence (TVI_max) and its cross-sectional radius (r _TVImax) when using brain microdialysis, and visualize the TVI_max in relation to patient anatomy. An equation based on Fick’s law was used to simulate the TVI_max. Factorial design and regression analysis were used to investigate the impact of the diffusion coefficient, tortuosity and loss rate on the r _TVImax. A calf brain tissue experiment was performed to further evaluate these parameters. The model was implemented with pre-(MRI) and post-(CT) operative patient images for simulation of the TVI_max for four patients undergoing microdialysis in parallel to DBS. Using physiologically relevant parameter values, the r _TVImax for analytes with a diffusion coefficient D = 7.5 × 10⁻⁶ cm²/s was estimated to 0.85 ± 0.25 mm. The simulations showed agreement with experimental data. Due to an implanted gold thread, the catheter positions were visible in the post-operative images. The TVI_max was visualized for each catheter. The biochemical changes could thereby be related to their anatomical origin, facilitating interpretation of results.     

Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells

 The properties of the calcium stores coupled to a depletion-operated cation current (I _DOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 μM) activated an initial, large (≈ 200 pA), transient, calcium-dependent chloride current (I _ClCa) followed by a smaller (≈ 10 pA) sustained, non-selective cation current (I _DOC). Intracellular application of heparin (5 mg ml^–1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 μM) activated I _DOC but not I _ClCa; ryanodine (30 μM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of I _ClCa by carbachol. In the presence of 30 μM, but not 3 μM, ryanodine, carbachol was able to activate I _DOC. Cyclopiazonic acid (CPA; 10 μM) abolished the response to carbachol; however, caffeine was still able to activate I _ClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 μM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4,5-trisphosphate-(IP₃-) sensitive or ryanodine-sensitive calcium stores is able to activate I _DOC, and, by extension, capacitative calcium entry in this tissue. Received: 21 July 1997 / Received after revision: 29 August 1997 / Accepted: 1 September 1997     

Structural integrity of the substantia nigra and subthalamic nucleus predicts flexibility of instrumental learning in older-age individuals

Flexible instrumental learning is required to harness the appropriate behaviors to obtain rewards and to avoid punishments. The precise contribution of dopaminergic midbrain regions (substantia nigra/ventral tegmental area [SN/VTA]) to this form of behavioral adaptation remains unclear. Normal aging is associated with a variable loss of dopamine neurons in the SN/VTA. We therefore tested the relationship between flexible instrumental learning and midbrain structural integrity. We compared task performance on a probabilistic monetary go/no-go task, involving trial and error learning of: “go to win,” “no-go to win,” “go to avoid losing,” and “no-go to avoid losing” in 42 healthy older adults to previous behavioral data from 47 younger adults. Quantitative structural magnetization transfer images were obtained to index regional structural integrity. On average, both some younger and some older participants demonstrated a behavioral asymmetry whereby they were better at learning to act for reward (“go to win” > “no-go to win”), but better at learning not to act to avoid punishment (“no-go to avoid losing” > “go to avoid losing”). Older, but not younger, participants with greater structural integrity of the SN/VTA and the adjacent subthalamic nucleus could overcome this asymmetry. We show that interindividual variability among healthy older adults of the structural integrity within the SN/VTA and subthalamic nucleus relates to effective acquisition of competing instrumental responses.     

Thirteen years of antibiotic susceptibility surveillance of Pseudomonas aeruginosa from intensive care units and urology services in the Netherlands

The purpose of this study was the evaluation of trends in the antimicrobial resistance of Pseudomonas aeruginosa from intensive care unit (ICU) patients and urology patients in the Netherlands. From 1998 to 2010, 1,927 consecutive P. aeruginosa isolates from ICU (n?=?1,393) and urology service patients (n?=?534) of 14 university and referral hospitals all over the Netherlands were collected and their susceptibility to relevant antibiotics was determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Over time, a significant upward trend in the resistance of P. aeruginosa strains collected from ICUs to piperacillin (1.2 % to 10.6 %, p?=?0.0175), piperacillin–tazobactam (1.2 % to 12.1 %, p?=?0.0008), ceftazidime (1.2 % to 7.8 %, p?=?0.0064), cefepime (4.8 % to 6.4 %, p?=?0.0166), imipenem (6 % to 19.1 %, p?<?0.0001), meropenem (8.3 % to 17 %, p?=?0.0022) and ciprofloxacin (13.1 % to 31.2 %, p?=?0.0024) was observed, as was the prevalence of multi-resistance (1.2 % to 8.5 %, p?=?0.0002). For P. aeruginosa isolates from the urology services, the resistance to imipenem increased (4.1 % to 7.8 %, p?=?0.0006) and to ciprofloxacin it decreased (22.4 % to 18.8 %, p?=?0.025). Like in other countries, in the Netherlands, an increase in multi-resistant Gram-negatives is observed, suggesting the presence and dissemination of several mechanisms of resistance. Our findings emphasise the importance of local surveillance for the setting up of local antibiotic guidelines and to support optimal empiric therapy. With the observed increase in multi-resistance, the direct testing of alternative antibiotics like polymyxins and fosfomycin is essential. Our data also illustrate the importance of adequate outbreak control measures.     

Diagnostic approach to encephalitis and meningoencephalitis in adult returning travellers

Background

Encephalitis and meningoencephalitis are severe, sometime life-threatening infections of the central nervous system. Travellers may be exposed to a variety of neurotropic pathogens.

Permeability properties of cell membranes and tight junctions of normal and cystic fibrosis sweat ducts

The transepithelial permeability properties to Na, K, and Cl in microperfused segments of human eccrine sweat ducts from normal (N) subjects and patients with cystic fibrosis (CF) were examined. Amiloride administered on the luminal surface caused the transepithelial potential (V _t ) of normal ducts to depolarize to 0 mV, but in the absence of Cl in the medium or in CF ducts, amiloride caused theV _t to significantly reverse electrical polarity from lumen negative to lumen positive with respect to the serosal bath. TheV _t responses to changes in Na concentration in the lumen and K concentration in the bath were similar in CF and N ducts and showed that the basolateral membrane of the duct is K permeable and the apical membrane (in the absence of an anion shunt) is an almost ideal Na electrode. TheV _t of N ducts was insensitive to 10-fold changes in luminal K and contraluminal Na solution concentrations. These responses show that in normal ducts, the apical membrane and tight junctions are relatively impermeable to K, and the basal membrane and tight junctions are relatively impermeable to Na. TheV _t was highly sensitive to Cl^– changes on either surface before or after ouabain inhibition in N ducts, but in every case were insensitive to Cl^– changes in CF ducts. By comparison to control ducts the cation selective properties of the CF duct are probably normal, but both cell membranes as well as the tight junctions of the CF duct are relatively impermeable to Cl. The present data are inconclusive as to whether the route of Cl movement across the N duct epithelium is trans- or paracellular.     

Nitric oxide in responses of regional kidney perfusion to renal nerve stimulation and renal ischaemia

The mechanisms underlying the relative insensitivity of medullary blood flow (MBF) to sympathetic drive remain unknown. We tested the effects of nitric oxide synthase blockade on regional kidney perfusion responses to electrical renal nerve stimulation (RNS) in pentobarbitone-anaesthetized rabbits. Under control conditions, RNS reduced renal blood flow (RBF), cortical blood flow (CBF) and MBF in a frequency-dependent manner. MBF was always reduced less than CBF or RBF. N^G-nitro-l-arginine increased mean arterial pressure (31±3 mmHg), reduced RBF (–8±1 ml/min) and MBF (–33±6 units), enhanced responses to RNS of RBF (from –48±6% to –58±6% at 2 Hz), CBF (from –38±6% to –43±4% at 2 Hz) and, particularly at low frequencies, MBF (from +1±18% to –32±11% at 2 Hz) and potentiated the RBF hyperaemic response following RNS (by 27±6% at 4 Hz). When glyceryl trinitrate was co-infused with N^G-nitro-l-arginine to restore basal nitrergic tone, responses to RNS and the subsequent hyperaemia were indistinguishable from control. Since resting renovascular tone or perfusion pressure has little impact on MBF responses to RNS, these present observations suggest that NO contributes to the blunted MBF response to RNS. Paradoxically, NO seems to blunt renal hyperaemia following acute RNS-induced ischaemia.     

How can Dermanyssus gallinae (De Geer 1778) (Acari: Anactinotrichida: Dermanyssidae) walk upwards on slippery surfaces?

ABSTRACT

Scanning electron microscopy observations of the distal leg region of the poultry red mite Dermanyssus gallinae (De Geer 1778) identified the presence of a compound ambulacrum, the part of the leg that contacts the substratum when the mite walks. The ambulacrum is comprised of a praetarsus (the ambulacrum stalk), a pulvillus and two claws. The pulvillus is a weakly sclerotized structure that can be partly expanded or retracted in the praetarsus. When expanded, the pulvillus shows a cushion-like shape which can, as a result of its soft surface, function as a sucker, thus allowing D. gallinae to adhere to a smooth surface. When traversing an irregular surface, or clinging to a soft surface, the mite retracts the pulvillus and uses only its strongly sclerotized movable claws. These observed morphological adaptations explain the ability of D. gallinae to climb upwards on a slippery surface, resist an air flux, walk on smooth and rigid feathers of its avian hosts, and cling to the bird’s or human's soft skin. This knowledge is important to better understand the attachment mechanism of this species to its host and to the substratum on which it moves, and also to provide insight into the circumstances under which it is able to move.

RESEARCH HIGHLIGHTS

• The ambulacrum is the distal part of the leg touching the substratum

• D. gallinae’s ambulacrum consists of a praetarsus, a pulvillus and two claws

• The weakly sclerotized pulvillus can be part expanded/retracted in the praetarsus

• The expanded pulvillus functions as a sucker to adhere to smooth surfaces

• The claws are used to walk on an irregular surface or cling to a soft surface

     

Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms

The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog of csrA. Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.     

Influence of gastric acid on susceptibility to infection with ingested bacterial pathogens

Despite the widely held belief that gastric acid serves as a barrier to bacterial pathogens, there are almost no experimental data to support this hypothesis. We have developed a mouse model to quantify the effectiveness of gastric acid in mediating resistance to infection with ingested bacteria. Mice that were constitutively hypochlorhydric due to a mutation in a gastric H⁺/K⁺-ATPase (proton pump) gene were infected with Yersinia enterocolitica, Salmonella enterica serovar Typhimurium, Citrobacter rodentium, or Clostridium perfringens cells or spores. Significantly greater numbers of Yersinia, Salmonella, and Citrobacter cells (P ≤ 0.006) and Clostridium spores (P = 0.02) survived in hypochlorhydric mice, resulting in reduced median infectious doses. Experiments involving intraperitoneal infection or infection of mice treated with antacids indicated that the increased sensitivity of hypochlorhydric mice to infection was entirely due to the absence of stomach acid. Apart from establishing the role of gastric acid in nonspecific immunity to ingested bacterial pathogens, our model provides an excellent system with which to investigate the effects of hypochlorhydria on susceptibility to infection and to evaluate the in vivo susceptibility to gastric acid of orally administered therapies, such as vaccines and probiotics.     

Short-term effects of oral enoximone on hemodynamics,exercise capacity,anaerobic threshold,and arrhythmias in congestive heart failure

Summary Enoximone, a phosphodiesterase-inhibitor, is a potent inotropic vasodilator agent that causes a marked improvement in hemodynamics in patients with congestive heart failure. The acute effects of oral enoximone on rest and exercise hemodynamics, ejection fraction, aerobic metabolism, exercise capacity, and arrhythmias were studied in 11 patients with moderate to moderately severe dilative cardiomyopathy after 8 days of enoximone (100 mg tid) in addition to baseline therapy (diuretics and digitalis).The cardiac index increased from 2.44±0.45 to 2.72±0.50 l/min/m² (p<0.01) at rest and from 4.00±0.96 to 4.75±0.95 l/min/m² (p<0.005) during exercise. Pulmonary wedge pressure decreased from 16.8±7.3 to 12.5±6.5 mmHg (p<0.005) at rest and from 28.2±8.0 to 24.5±10.3 mmHg (p< 0.05) during exercise. Systemic vascular resistance decreased from 1608±243 to 1495±300 dynes*sec*cm^–5 (p<0.05) at rest and from 1152±155 to 1027±236 dynes*sec*cm^–5 (ns) during exercise. The anaerobic threshold, which was recorded simultaneously, increased from 13.2±2.7 to 15.5± 2.5ml/kg/min VO₂ (p<0.02). The radionuclide ventriculography ejection fraction improved from 21.7±5.0 to 28.1±9.1% (p<0.01) during exercise; the changes at rest were not significant (20.8±6.2 vs 25.8±8.4%). Exercise tolerance showed an increase of 16% (492±133 to 573±135 sec, p< 0.005). The resting heart rate remained unchanged (81.8±13.4 vs 81.8±11.9). Interestingly, 24-h Holter monitoring revealed more or new repetitive arrhythmias in 9/11 patients.Short-term therapy with oral enoximone enhances ventricular performance by increasing cardiac contractility and lowering vascular resistance, both of which extend exercise tolerance and improve aerobic metabolism. Potential proarrhythmic effects need further evaluation, however.Abbreviations AMP adenosine monophosphate - PDE phosphodiesterase - VCO₂ carbon dioxide production - VPB ventricular premature beat - VE minute ventilation - VO₂ oxygen uptake Dedicated to Professor Jahrmärker on the occasion of his seventieth birthday     

Mucosal immunology

In this review, we shall highlight some recent advances in mucosal immunology and also those concepts which seem to us to merit more attention than they normally receive. Since we cannot hope to be all inclusive, we recommend the following articles and books to the reader (Tomasi & Bienenstock, 1968; Tomasi & Grey, 1972; Bienenstock, 1974; Heremans, 1974; Mestecky & Lawton, 1974; Lamm, 1976; Tomasi, 1976; Waksman & Ozer, 1976; Porter & Knight, 1977; McGhee, Mestecky & Babb, 1978; Ogra & Dayton, 1979; Befus & Bienenstock, 1980).     

Combining Parasite Lactate Dehydrogenase-Based and Histidine-Rich Protein 2-Based Rapid Tests To Improve Specificity for Diagnosis of Malaria Due to Plasmodium knowlesi and Other Plasmodium Species in Sabah,Malaysia

Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with “species-specific” parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P. falciparum because of P. knowlesi cross-reactivity and cautions against their use alone in areas where P. knowlesi malaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesi malaria is endemic.     

The contribution of refractoriness to arrhythmic substrate in hypokalemic Langendorff-perfused murine hearts

The clinical effects of hypokalemia including action potential prolongation and arrhythmogenicity suppressible by lidocaine were reproduced in hypokalemic (3.0 mM K⁺) Langendorff-perfused murine hearts before and after exposure to lidocaine (10 μM). Novel limiting criteria for local and transmural, epicardial, and endocardial re-excitation involving action potential duration (at 90% repolarization, APD₉₀), ventricular effective refractory period (VERP), and transmural conduction time (Δlatency), where appropriate, were applied to normokalemic (5.2 mM K⁺) and hypokalemic hearts. Hypokalemia increased epicardial APD₉₀ from 46.6 ± 1.2 to 53.1 ± 0.7 ms yet decreased epicardial VERP from 41 ± 4 to 29 ± 1 ms, left endocardial APD₉₀ unchanged (58.2 ± 3.7 to 56.9 ± 4.0 ms) yet decreased endocardial VERP from 48 ± 4 to 29 ± 2 ms, and left Δlatency unchanged (1.6 ± 1.4 to 1.1 ± 1.1 ms; eight normokalemic and five hypokalemic hearts). These findings precisely matched computational predictions based on previous reports of altered ion channel gating and membrane hyperpolarization. Hypokalemia thus shifted all re-excitation criteria in the positive direction. In contrast, hypokalemia spared epicardial APD₉₀ (54.8 ± 2.7 to 60.6 ± 2.7 ms), epicardial VERP (84 ± 5 to 81 ± 7 ms), endocardial APD₉₀ (56.6 ± 4.2 to 63.7 ± 6.4 ms), endocardial VERP (80 ± 2 to 84 ± 4 ms), and Δlatency (12.5 ± 6.2 to 7.6 ± 3.4 ms; five hearts in each case) in lidocaine-treated hearts. Exposure to lidocaine thus consistently shifted all re-excitation criteria in the negative direction, again precisely agreeing with the arrhythmogenic findings. In contrast, established analyses invoking transmural dispersion of repolarization failed to account for any of these findings. We thus establish novel, more general, criteria predictive of arrhythmogenicity that may be particularly useful where APD₉₀ might diverge sharply from VERP.