Candida guilliermondii and Other Species of Candida Misidentified as Candida famata: Assessment by Vitek 2, DNA Sequencing Analysis,and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Two Global Antifungal Surveillance Programs

Candida famata (teleomorph Debaryomyces hansenii) has been described as a medically relevant yeast, and this species has been included in many commercial identification systems that are currently used in clinical laboratories. Among 53 strains collected during the SENTRY and ARTEMIS surveillance programs and previously identified as C. famata (includes all submitted strains with this identification) by a variety of commercial methods (Vitek, MicroScan, API, and AuxaColor), DNA sequencing methods demonstrated that 19 strains were C. guilliermondii, 14 were C. parapsilosis, 5 were C. lusitaniae, 4 were C. albicans, and 3 were C. tropicalis, and five isolates belonged to other Candida species (two C. fermentati and one each C. intermedia, C. pelliculosa, and Pichia fabianni). Additionally, three misidentified C. famata strains were correctly identified as Kodomaea ohmeri, Debaryomyces nepalensis, and Debaryomyces fabryi using intergenic transcribed spacer (ITS) and/or intergenic spacer (IGS) sequencing. The Vitek 2 system identified three isolates with high confidence to be C. famata and another 15 with low confidence between C. famata and C. guilliermondii or C. parapsilosis, displaying only 56.6% agreement with DNA sequencing results. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) results displayed 81.1% agreement with DNA sequencing. One strain each of C. metapsilosis, C. fermentati, and C. intermedia demonstrated a low score for identification (<2.0) in the MALDI Biotyper. K. ohmeri, D. nepalensis, and D. fabryi identified by DNA sequencing in this study were not in the current database for the MALDI Biotyper. These results suggest that the occurrence of C. famata in fungal infections is much lower than previously appreciated and that commercial systems do not produce accurate identifications except for the newly introduced MALDI-TOF instruments.     

Assessment of caspofungin susceptibility of Candida glabrata by the Etest®, CLSI,and EUCAST methods,and detection of FKS1 and FKS2 mutations

Candida glabrata has emerged as a major pathogen in invasive candidiasis in recent years. Currently, guidelines for invasive candidiasis treatment recommend fluconazole or an echinocandin as the first-line therapy. Nevertheless, the resistance of Candida glabrata to echinocandin is an emerging problem and has been partly associated with mutations in the FKS1 and FKS2 genes. The Etest® is an appropriate method for determining antifungal susceptibility in emergency routine diagnosis. In this work, we evaluated the reliability of the Etest® in comparison with the two reference broth microdilution methods, Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST), to assess the caspofungin resistance of 193 isolates of Candida glabrata. The interpretation of minimum inhibitory concentration (MIC) values was also discussed according to different breakpoints. Moreover, FKS1 and FKS2 mutations were investigated for isolates with high MICs. Our results showed that the MIC₅₀ value was similar to the MIC₉₀ value for each method. The Etest® method showed the lowest MIC values, whereas EUCAST presented the highest. Categorical agreement between the Etest® and CLSI methods was 100 % and 36 % using the breakpoints proposed by Arendrup et al. (Antimicrob Agents Chemother 56(7):3965–3968, 2012) and Pfaller et al. (Int J Antimicrob Agents 38(1):65–69, 2011), respectively. Two isolates showed high MIC values with the three methods and both presented FKS2 mutations. A novel FKS2 mutation was also reported for one isolate. Future epidemiological studies should also evaluate the reliability of the Etest® to detect echinocandin resistance, as it remains a routine method.     

Value of (1-3)-β-d-glucan, Candida mannan and Candida DNA detection in the diagnosis of candidaemia

This study determined the value of (1,3)-β-d-glucan (BDG), Candida mannan (MN) and Candida species-specific DNA as surrogates for diagnosis of candidaemia. Thirty-nine patients yielding Candida species in blood cultures were investigated for presence of BDG, MN and Candida species-specific DNA in serum samples. The Candida spp. bloodstream isolates included C. albicans (n = 16), C. tropicalis (n = 10), C. parapsilosis (n = 7), C. glabrata (n = 3) and C. dubliniensis (n = 3). Positivity of the three markers was as follows: Candida DNA for corresponding Candida species, 100%; BDG, 87%; MN, 59%. Despite varying sensitivities of these biomarkers, they provided a useful adjunct to the diagnosis of candidaemia.     

Efficient capture of Candida albicans and zymosan by SIGNR1 augments TLR2-dependent TNF-α production

SIGNR1, a mouse C-type lectin, binds various pathogens, including Candida albicans. In this study, we explore the impact of SIGNR1 in the recognition of C. albicans/zymosan and the subsequent tumor necrosis factor (TNF)-α production using SIGNR1-transfected RAW264.7 (RAW-SIGNR1) cells and resident peritoneal macrophages. Compared with RAW-control cells, RAW-SIGNR1 cells dramatically enhanced TNF-α production upon the stimulation with heat-killed C. albicans and zymosan. Recognition of microbes via carbohydrate recognition domain (CRD) of SIGNR1 was crucial for the enhanced TNF-α production. Consistently, such an enhancement was significantly decreased by anti-SIGNR1 mAb. Laminarin, antagonistic Dectin-1 ligand, cooperated to further diminish the response, although no effect was observed by itself in RAW-SIGNR1 cells. However, it moderately reduced the response of RAW-control cells. Zymosan depleted of toll-like receptor (TLR) ligands decreased the response, even though it was recognized by SIGNR1 and Dectin-1. Moreover, antagonistic anti-TLR2 abolished the response, suggesting that TNF-α production largely relies on TLR2-mediated signaling. Resident peritoneal macrophages expressing SIGNR1 predominantly captured zymosan injected intra-peritoneally and produced TNF-α, which was dependent on TLR2 and partly inhibited by anti-SIGNR1 mAb. Finally, physical association of SIGNR1 with the extracellular portion of TLR2 through CRD was confirmed by immunoprecipitation using various deletion mutants. These results suggest that SIGNR1 recognizing microbes participates in the enhanced TNF-α production by M? in cooperation with TLR2.     

β-lapachone and α-nor-lapachone modulate Candida albicans viability and virulence factors

Background

Candida albicans is the most important fungal pathogen that causes infections in humans, and the search for new therapeutic strategies for its treatment is essential.

Clinical manifestations of candidemia caused by uncommon Candida species and antifungal susceptibility of the isolates in a regional hospital in Taiwan, 2007–2014

ObjectiveThis retrospective study investigated clinical manifestations of candidemia caused by uncommon Candida species and antifungal susceptibility of the isolates in a regional hospital in Taiwan.MethodsThe uncommon Candida species was initially defined as Candida species other than C. albicans, C. tropicalis, C. glabrata complex, C. parapsilosis complex and C. krusei. All uncommon Candida isolates were identified and confirmed by molecular methods. In vitro susceptibility testing of the uncommon Candida species to nine antifungal agents was conducted using the broth microdilution method with the Sensititre YeastOne (SYO) system (Trek Diagnostic Systems, Ltd., East Grimstead, UK).ResultsTwenty-one patients, comprising 11 males and 10 females with a median age of 69 years, were recruited. Cancer (n = 11) was the most common underlying disease, 19 (90.5%) cases had prior antibiotic exposure, and only two patients had prior antifungal use. The overall in-hospital mortality rate was 38.1% (n = 8). C. guilliermondii (n = 11) was the most common pathogen, followed by C. curvata (n = 3). C. guilliermondii isolates exhibited relatively high rates of azole minimum inhibitory concentrations (MICs) above epidemiological cut-off values (ECVs), whereas C. pelliculosa and C. lusitaniae isolates all remained susceptible to azoles. All three C. curvata isolates had high caspofungin (>8 mg/L) and fluconazole MICs (8 mg/L) and could be defined as multidrug-resistant.ConclusionsUncommon Candida species frequently exhibit high rates of non-susceptibility to antifungals. Identification of all Candida isolates at the species level from blood samples is of value for treatment.     

Comparison of β-D-Glucan levels between Candida auris and other Candida species at the time of candidaemia: a retrospective study

ObjectivesTo compare serum β-D-glucan (BDG) levels in candidaemia with different Candida species, especially C. auris.MethodsAga Khan University clinical laboratory database was retrospectively reviewed from January 2015 to December 2019. Blood culture positive cases with any Candida species and concomitant BDG level were included.ResultsAmong the 192 cases included in our study, 48 were C. albicans, 54 C. auris, eight C. glabrata, 32 C. parapsilosis, 43 C. tropicalis and seven other Candida species. The level of BDG was significantly lower in C. auris (median 62.43, interquartile range (IQR) 12.80–182.94 pg/mL) compared to C. albicans (median 266.83, IQR 66.29–523.43 pg/mL) and C. tropicalis (median 324.41, IQR 105.20–523.44 pg/mL). The sensitivity of serum BDG was significantly lower for C. auris (43.75%, 95% CI 29.5–58.8%) than C. tropicalis (79.07%, 95% CI 64.0–90.0%).DiscussionSerum BDG has lower sensitivity in patients with suspected C. auris candidaemia in our setting. Considering that C. auris has higher morbidity and mortality than other species, a more sensitive test is required.     

Rosiglitazone,a PPAR-γ agonist,inhibits VEGF secretion by peripheral blood mononuclear cells and ROS production by human leukocytes

Objective  

To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, on the secretion of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMCs) and on the generation of reactive oxygen species (ROS) by leukocytes.     

Recognition of emotional‐prosodic meanings in speech by autistic,schizophrenic, and normal children

The abilities of autistic, schizophrenic, and normal‐control children to label four emotional intonations (the emotional task) in speech were tested, along with a linguistic task. All stimuli were pretested on normal adults. Older (≥ 8 years of age) normal children performed as well as adults on both tasks; younger normal children and both younger and older autistic children performed poorly on the emotional task; children (all older) diagnosed as schizophrenic were not significantly impaired in either task. Mental age was not correlated with performance in autistic children. The relevance of these results to other findings regarding emotional and linguistic behaviors in normal and disabled children is considered.     

β-adrenoreceptor activation in brain, lung and adipose tissue, measured by microdialysis in pig

PurposeThe aim of this study is to investigate the effect of local activation of β-adrenoreceptor by Isoprenaline on metabolism in brain, fat and lung measured by microdialysis.MethodsWe used 8 healthy pigs under general anaesthesia and placed microdialysis catheters in brain, fat, lung and artery. We performed a direct measurement of glucose, lactate, pyruvate and glycerol. The stimulation was performed by one-hour infusion of Isoprenaline, a β-adrenoreceptor agonist.ResultsThe infusion of isoprenaline did not affect the glucose in any tissue. The levels of lactate (p=0.008) and pyruvate (p=0.011) decreased significantly in lung after isoprenaline infusion. There was a significant increase in L/P ratio in fat tissue (p=0.001) while no significant changes could be found in brain (p=0.086) and lung (p=0.679).The most pronounced and significant change was observed in glycerol in fat (p<0.001) that increased by 95%.ConclusionThe prominent increase in glycerol in fat proved to be a good measure of β-adrenoreceptor activation and a measure of lipolysis. This can be used to online monitor β-adrenoreceptor activation by glycerol measurement in patients.     

Interferon-�� and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells

Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio‐defence systems through the production of cytokines such as interferon‐α (IFN‐α) and interleukin‐12 (IL‐12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti‐microbial innate immune responses but also in the pathogenesis of IFN‐related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA‐mediated innate immunity by possessing DNA‐sensing toll‐like receptor 9 (TLR9). We here found that double‐stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte‐derived DCs (moDCs), leading to the preferential production of IFN‐α but not IL‐12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA‐mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co‐localization of endosomal LAMP1 staining, and the dsDNA‐mediated IFN‐α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single‐stranded RNA (ssRNA) stimulated moDCs to secrete IL‐12 but not IFN‐α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA‐mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN‐α production and acquired immunity via IL‐12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA‐mediated activation of protective or pathogenic immunity.     

Ethyl-p-methoxycinnamate isolated from kaempferia galanga inhibits inflammation by suppressing interleukin-1, tumor necrosis factor-α, and angiogenesis by blocking endothelial functions

OBJECTIVE:

The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga.

METHODS:

The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals'' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells.

RESULTS:

Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor.

CONCLUSION:

Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent for the treatment of inflammatory and angiogenesis-related diseases.     

Comparison of (1→3)-β-d-Glucan,Mannan/Anti-Mannan Antibodies,and Cand-Tec Candida Antigen as Serum Biomarkers for Candidemia

We conducted a case-control study using the Fungitell assay, the novel Platelia Candida Antigen (Ag) Plus and Candida Antibody (Ab) Plus assays, and the Cand-Tec latex agglutination test to evaluate the usefulness of (1→3)-β-d-glucan (BDG), mannan antigen with/without anti-mannan antibody, and Cand-Tec Candida antigen measurement for the diagnosis of candidemia. A total of 56 patients fulfilled the inclusion criteria and were enrolled. One hundred patients with bacteremia and 100 patients with sterile blood cultures served as negative controls. In the candidemia group, median (1→3)-β-d-glucan, mannan antigen, and anti-mannan antibody levels were 427 pg/ml, 190 pg/ml, and 18.6 antibody units (AU)/ml, respectively. All three parameters were significantly elevated in patients with candidemia. The sensitivity and specificity were, respectively, 87.5% and 85.5% for (1→3)-β-d-glucan, 58.9% and 97.5% for mannan antigen, 62.5% and 65.0% for anti-mannan antibody, 89.3% and 63.0% for mannan antigen plus anti-mannan antibody, 89.3% and 85.0% for BDG plus mannan antigen, and 13.0% and 93.9% for Cand-Tec Candida antigen. The low mannan antigen sensitivity was in part caused by Candida parapsilosis and Candida guilliermondii fungemias, which were not detected by the Platelia Candida Ag Plus assay. When the cutoff was lowered from 125 pg/ml to 50 pg/ml, mannan antigen sensitivity increased to 69.6% without severely affecting the specificity (93.5%). Contrary to recently published data, superficial candidiasis was not associated with elevated mannan antigen levels, not even after the cutoff was lowered. Combining procalcitonin (PCT) with (1→3)-β-d-glucan to increase specificity provided a limited advantage because the benefit of the combination did not outweigh the loss of sensitivity. Our results demonstrate that the Cand-Tec Candida antigen and the mannan antigen plus anti-mannan antibody measurements have unacceptably low sensitivity or specificity. Of the four tests compared, (1→3)-β-d-glucan and mannan antigen are the superior biomarkers, depending on whether a sensitivity-driven or specificity-driven approach is used.     

Structural,cellular, and molecular evaluation of bone erosion in experimental models of rheumatoid arthritis: Assessment by μCT,histology, and serum biomarkers

Bone erosion is a clinical endpoint for various diseases including rheumatoid arthritis. In this paper, we used rodent arthritis models with severe bone erosion to examine the structural, cellular, and molecular aspects of the inflammation-driven bone resorption process. Our data show that bone loss is observed only in chronically, severely inflamed joints. The most severely affected anatomic sites were the metatarsal phalangeal joint and tarsal bones of the paw. The magnitude of the inflammation-driven bone erosion was dependent on both the duration of inflammatory response and the severity of the joint swelling response. The application of micro-computed tomography well demonstrated the therapeutic benefit of anti-IL-17A in protection of bones from erosion. Alterations in the cellular profile of the joint occurred prior to any major structural deterioration of the bone. Receptor activator for nuclear factor κB ligand, a potent inducer of osteoclast differentiation and bone resorption, was elevated in animals coincident with severe arthritis initiation. The experimental approaches and concepts outlined in this paper provide a valuable process to evaluate and quantify therapies that modulate rodent arthritis-associated bone-erosion models.