Clonal dispersal of Cryptococcus gattii VGII in an endemic region of cryptococcosis in Colombia

This study characterized the genotype and phenotype of Cryptococcus gattii VGII isolates from Cucuta, an endemic region of cryptococcal disease in Colombia, and compared these traits with those from representative isolates from the Vancouver Island outbreak (VGIIa and VGIIb). Genetic diversity was assessed by multilocus sequence typing (MLST) analysis. Phenotypic characteristics, including growth capacity under different temperature and humidity conditions, macroscopic and microscopic morphology, phenotypic switching, mating type, and activity of extracellular enzymes were studied. Virulence was studied in vivo in a mouse model. MLST analysis showed that the isolates from Cucuta were highly clonal, with ST25 being the most common genotype. Phenotypically, isolates from Cucuta showed large cell and capsular sizes, and shared phenotypic traits and enzymatic activities among them. The mating type a prevailed among the isolates, which were fertile and of considerable virulence in the animal model. This study highlights the need for a continuous surveillance of C. gattii in Colombia, especially in endemic areas like Cucuta, where the highest number of cryptococcosis cases due to this species is reported. This will allow the early detection of potentially highly virulent strains that spread clonally, and can help prevent the occurrence of outbreaks in Colombia and elsewhere. © 2019 by the authors

Cortisol and 17-hydroxyprogesterone levels in saliva of healthy neonates - Normative data and relation to body mass index, arterial cord blood pH and time of sampling after birth

The measurement of cortisol and 17-hydroxyprogesterone (17-OHP) in saliva has become a reliable tool for both the scientist and the clinician for studying adrenal cortical function in the adult and the older child. We have now established in parallel normative data for salivary cortisol and 17-OHP levels in healthy neonates. We have asked whether or not there is a circadian rhythm of cortisol and 17-OHP saliva levels in neonates. Furthermore, we have asked whether salivary hormone levels correlated with auxologic and clinical data and time of sampling. Cortisol and 17-OHP levels in saliva samples from 119 healthy neonates (55 girls, 64 boys) were measured using in-house time-resolved fluorescent immunoassays. Saliva samples were obtained using a saliva collecting tube three times a day on the first or second day of life. Gender and gestational age did not influence salivary cortisol and 17-OHP levels. No significant circadian rhythm of salivary hormone levels was detected in this group of newborns. However, body mass index, arterial cord blood pH and time of saliva sampling significantly influenced salivary hormone levels. In conclusion, measurement of cortisol and 17-OHP in saliva is feasible in healthy neonates. The existence of normative data forms the basis for future studies on pathophysiologic states in the newborn period. Copyright (C) 2000 S. Karger AG, Basel

Coordinate Activation of Activator Protein 1 and Inflammatory Cytokines in Response to Neisseria gonorrhoeae Epithelial Cell Contact Involves Stress Response Kinases

Neisseria gonorrhoeae (Ngo), the etiologic agent of gonorrhea, induce a number of proinflammatory cytokines by contact to epithelial cells. Cytokine genes and a variety of other immune response genes are activated as a result of the regulatory function of immediate early response transcription factors including activator protein 1 (AP-1). Since it is established that phosphorylation of c-Jun, the central component of AP-1, by the stress-activated c-Jun NH2-terminal kinase (JNK) increases the transcriptional activity of AP-1, we studied whether Ngo could induce stress response pathways involving JNK. We found that virulent Ngo strains induce phosphorylation and activation of JNK but not of p38 kinase. Analysis of a nonpathogenic Ngo strain revealed only weak JNK activation. In respect to the molecular components upstream of the JNK signaling cascade, we show that a dominant negative mutant of MAP kinase kinase 4 (MKK4) represses transcription of an AP-1–dependent reporter gene. Regarding upstream stress response factors involved in Ngo-induced MKK4/JNK/AP-1 activation, we identified p21-activated kinase (PAK) but not MAPK/ERK kinase kinase (MEKK1). Inhibition of small GTPases including Rac1 and Cdc42 by Toxin B prevented JNK and AP-1 activation. Our results indicate that Ngo induce the activation of proinflammatory cytokines via a cascade of cellular stress response kinases involving PAK, which directs the signal from the Rho family of small GTPases to JNK/AP-1 activation

Rearing and Maintenance of Galleria mellonella and Its Application to Study Fungal Virulence

Galleria mellonella larvae have been widely used as alternative non-mammalian models for the study of fungal virulence and pathogenesis. The larvae can be acquired in small volumes from worm farms, pet stores, or other independent suppliers commonly found in the United States and parts of Europe. However, in countries with no or limited commercial availability, the process of shipping these larvae can cause them stress, resulting in decreased or altered immunity. Furthermore, the conditions used to rear these larvae including diet, humidity, temperature, and maintenance procedures vary among the suppliers. Variation in these factors can affect the response of G. mellonella larvae to infection, thereby decreasing the reproducibility of fungal virulence experiments. There is a critical need for standardized procedures and incubation conditions for rearing G. mellonella to produce quality, unstressed larvae with the least genetic variability. In order to standardize these procedures, cost-effective protocols for the propagation and maintenance of G. mellonella larvae using an artificial diet, which has been successfully used in our own laboratory, requiring minimal equipment and expertise, are herein described. Examples for the application of this model in fungal pathogenicity and gene knockout studies as feasible alternatives for traditionally used animal models are also provided

Low triiodothyronine syndrome: a prognostic marker for outcome in sepsis?

There is ongoing controversy as to whether hormonal changes of the euthyroid sick syndrome are predictors of poor outcome in sepsis and critical illness. In this prospective study, the prognostic accuracy of thyroid hormone levels in 103 critically ill adult patients on admission and during follow up in a medical intensive care unit (ICU) was assessed and was compared to clinical risk scores, namely, the acute physiology and chronic health evaluation and the simplified acute physiology score. Median T3 levels on admission to the ICU were lower in the 53 septic cases [0.9nmol/l (IQR 0.6-1.1)] as compared with the 50 patients with a systemic inflammatory response syndrome [1.2nmol/l (IQR 0.8-1.4), P<0.001]. The lowest T3 levels were found in patients with severe sepsis [0.8nmol/l (IQR 0.55-0.95)] and septic shock [0.8nmol/l (IQR 0.6-1.0)]. There was no difference in T3 and free thyroxin (fT4) levels on admission in non-survivors compared with survivors overall and in subgroups of patients with SIRS and sepsis. During the follow up, fT4 levels decreased significantly in non-survivors, while they increased in survivors [fT4 difference −1.3 (IQR −2.5 to 0.2) vs. 0.8 (IQR −0.85 to 4.1), P=0.003]. In addition, on the day of death, non-survivors had lower T3 and fT4 levels as compared with survivors (P=0.04 and P=0.02). T3 and fT4 levels on admission were not prognostic in this cohort of critically ill patients. A decrease in fT4 levels in the course of disease, however, may point to adverse outcom

The mycobiome of Australian tree hollows in relation to the Cryptococcus gattii and C. neoformans species complexes

Cryptococcosis is a fungal infection caused by members of the Cryptococcus gattii and C. neoformans species complexes. The C. gattii species complex has a strong environmental association with eucalypt hollows (particularly Eucalyptus camaldulensis), which may present a source of infection. It remains unclear whether a specific mycobiome is required to support its environmental survival and growth. Conventional detection of environmental Cryptococcus spp. involves culture on differential media, such as Guizotia abyssinica seed agar. Next-generation sequencing (NGS)-based culture-independent identification aids in contextualising these species in the environmental mycobiome. Samples from 23 Australian tree hollows were subjected to both culture- and amplicon-based metagenomic analysis to characterize the mycobiome and assess relationships between Cryptococcus spp. and other fungal taxa. The most abundant genera detected were Coniochaeta, Aspergillus, and Penicillium, all being commonly isolated from decaying wood. There was no correlation between the presence of Cryptococcus spp. in a tree hollow and the presence of any other fungal genus. Some differences in the abundance of numerous taxa were noted in a differential heat tree comparing samples with or without Cryptococcus-NGS reads. The study expanded the known environmental niche of the C. gattii and C. neoformans species complexes in Australia with detections from a further five tree species. Discrepancies between the detection of Cryptococcus spp. using culture or NGS suggest that neither is superior per se and that, rather, these methodologies are complementary. The inherent biases of amplicon-based metagenomics require cautious interpretation of data through consideration of its biological relevance

Dating the Cryptococcus gattii Dispersal to the North American Pacific Northwest.

The emergence of Cryptococcus gattii, previously regarded as a predominantly tropical pathogen, in the temperate climate of the North American Pacific Northwest (PNW) in 1999 prompted several questions. The most prevalent among these was the timing of the introduction of this pathogen to this novel environment. Here, we infer tip-dated timing estimates for the three clonal C.&nbsp;gattii populations observed in the PNW, VGIIa, VGIIb, and VGIIc, based on whole-genome sequencing of 134 C.&nbsp;gattii isolates and using Bayesian evolutionary analysis by sampling trees (BEAST). We estimated the nucleotide substitution rate for each lineage (1.59 × 10-8, 1.59 × 10-8, and 2.70 × 10-8, respectively) to be an order of magnitude higher than common neutral fungal mutation rates (2.0 × 10-9), indicating a microevolutionary rate (e.g., successive clonal generations in a laboratory) in comparison to a species' slower, macroevolutionary rate (e.g., when using fossil records). The clonal nature of the PNW C.&nbsp;gattii emergence over a narrow number of years would therefore possibly explain our higher mutation rates. Our results suggest that the mean time to most recent common ancestor for all three sublineages occurred within the last 60 to 100&nbsp;years. While the cause of C.&nbsp;gattii dispersal to the PNW is still unclear, our research estimates that the arrival is neither ancient nor very recent (i.e., &lt;25 years ago), making a strong case for an anthropogenic introduction. IMPORTANCE The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events

Fungal contaminants in drinking water regulation? A tale of ecology, exposure, purification and clinical relevance

Microbiological drinking water safety is traditionally monitored mainly by bacterial parameters that indicate faecal contamination. These parameters correlate with gastro-intestinal illness, despite the fact that viral agents, resulting from faecal contamination, are usually the cause. This leaves behind microbes that can cause illness other than gastro-intestinal and several emerging pathogens, disregarding non-endemic microbial contaminants and those with recent pathogenic activity reported. This white paper focuses on one group of contaminants known to cause allergies, opportunistic infections and intoxications: Fungi. It presents a review on their occurrence, ecology and physiology. Additionally, factors contributing to their presence in water distribution systems, as well as their effect on water quality are discussed. Presence of opportunistic and pathogenic fungi in drinking water can pose a health risk to consumers due to daily contact with water, via several exposure points, such as drinking and showering. The clinical relevance and influence on human health of the most common fungal contaminants in drinking water is discussed. Our goal with this paper is to place fungal contaminants on the roadmap of evidence based and emerging threats for drinking water quality safety regulations.info:eu-repo/semantics/publishedVersio