Monitoring of sterigmatocystin biosynthesis using RT-qPCR in airborne Aspergillus species of the series Versicolores.

Aspergilli series Versicolores have been shown to be explanatory variables for different symptoms like coughing and dizziness experienced by residents of mold-damaged homes. Among these species, eight are particularly recurrent in bioaerosols: Aspergillus amoenus, A. creber, A. fructus, A. jensenii, A. protuberus, A. puulaauensis, A. sydowii and A. tabacinus. In order to monitor the biosynthesis of sterigmatocystin (a mycotoxin associated with a risk of cancer development) and the development of these molds, we developed an RT-qPCR tool by targeting the aflR and rho1 genes. A total of 30 fungal isolates representing these eight species were included. For each of them, sterigmatocystin was quantified by UPLC-HRMS and (1 â†’ 3)-ß-D-glucan by visible spectrophotometry using Endosafe®-PTS™-Glucan Cartridges. After validation of our method by RT-qPCR, the direct assay was compared to the amount of aflR and rho1 cDNA. The sterigmatocystin and aflR assays showed a significant correlation between these two approaches (p < 0.0001), demonstrated for the first time the production of sterigmatocystin by A. tabacinus and suggested the ability of A. sydowii to synthesize sterigmatocystin. Assays conducted on (1 â†’ 3)-ß-D-glucan and rho1 did not show a correlation, supporting the multiplicity of functions performed in fungal cells by the RHO1 GTPase. The proposed tool could allow monitoring of sterigmatocystin biosynthesis by Aspergillus of the series Versicolores under different culture and climatic conditions.

A novel qPCR based-method for detection and quantification of three recurrent species of Penicillium isolated from bioaerosols in mold-damaged homes.

Fungal contamination of indoor environments can cause respiratory diseases and induce damages to building materials. Among the fungal species found in mold-damaged homes, Penicillium brevicompactum, P. chrysogenum and P. crustosum can be considered as recurrent strains. In this study, we therefore propose a rapid and novel qPCR-based method in order to allow the monitoring of these three fungal species. The method developed allows the quantification of the target DNA of these three Penicillium species with a limit of quantification of 0.01 ng/µL without significant difference with spectrophotometry quantification assay for DNA concentrations between 5 and 100 ng/µL. This technique also enables the rapid detection of these three species in complex mixtures of DNA extracted from 15 bioaerosols collected in mold-damaged homes and previously cultured on agar plate. This new sensitive and specific qPCR technique can thus be easily integrated into bioaerosol studies.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

Appropriate use of antifungals: impact of an antifungal stewardship program on the clinical outcome of candidaemia in a French University Hospital.

OBJECTIVES: The objective of this study was to compare clinical outcomes of patients with candidaemia before and after implementation of an antifungal stewardship program (AFSP). METHODS: This study included all consecutive cases of candidaemia identified from January 2012 to December 2015 in a French University Hospital. Data were collected retrospectively for a period of 2 years before implementation of the AFSP, and prospectively for 2 years after. All cases were reviewed by a multidisciplinary panel of experts including infectiologists, a microbiologist and pharmacists to have a complete follow-up of patients. RESULTS: 33 and 37 patients were finally included in the first and second period, respectively. The sites of entry of the candidaemia cases studied were as follows: intraabdominal in 29 cases (41.4%), central venous catheter 21 (30.0%), other or unknown: 20 (28.6%). Infectiologist consultations increased from 36.4 to 86.5% between the two periods with a significative impact on daily blood cultures which were more frequently performed in the second period (p = 0.04), and the use of echinocandins which was more frequent in the second period (97.1% of cases vs 78.8%, p = 0.03). The 3-month mortality rate declined from 36.4% in the first period to 27.0% in the second period (p = 0.4). CONCLUSIONS: Despite the insufficient number of candidaemia cases and the presence of other unmodifiable risk factors of mortality which did not allow us to show a significant effect on the 3-month mortality, AFSP had a significant effect on daily blood cultures and echinocandin use as first-line therapy.

Laser-assisted Fluorescent Angiography to Assess Tissue Perfusion in the Setting of Traumatic Elbow Dislocation.

INTRODUCTION: In the surgical setting, the most accepted technique for measuring tissue perfusion includes subjective identification, with visual and tactile inspections of the tissue, performed by a surgeon. Recently, fields such as ophthalmology, plastic surgery, and other surgical specialties, such as cardiac, vascular, and transplant surgery, have seen the emergence of laser-assisted fluorescent angiography (LAFA) to visualize real-time tissue perfusion during procedures. CASE REPORT: The case of an 80-year-old woman, who presented with a wound over the left medial elbow following a subacute recurrent elbow dislocation, illustrates the use of this technology in the field of orthopedics. Initially, the patient was treated at an outside facility where the dislocation was reduced, and she was placed in a long arm splint. With concern of recurrent dislocation and wound development, she presented to the authors with a complex wound measuring about 9 cm x 5 cm with variable thickness ranging from 0 mm to 5 mm in depth. Her initial emergent irrigation and debridement and wound management was assisted by LAFA and the administration of indocyanine green to guide debridement and determine viable wound margins. After staging with external fixation and a negative pressure wound dressing, she later returned for skin grafting and healed uneventfully. CONCLUSIONS: In conjunction with plastic surgeons, the orthopaedic team utilized LAFA for debridement that led to both a successful wound repair and skin grafting procedure.

Neurotoxocariasis: a systematic literature review.

PURPOSE: Toxocariasis is a widespread zoonosis, which may result in central nervous system injury. METHODS: We conducted a systematic literature review in MEDLINE, SciELO, ScienceDirect and Google Scholar up to April 2015 using a combination of the following search terms: "neurotoxocariasis" or "neurotoxocarosis", "toxocariasis" or "toxocarosis" and "cerebral" or "neurologic". RESULTS: One hundred cases of neurotoxocariasis were identified in literature. The majority of patients were male (58 %), with a median age of 42 years. The predominant clinical pictures were myelitis (60 %), encephalitis (47 %) and/or meningitis (29 %). Fever was inconstant (23 %). The suspected mode of transmission, mentioned in only 49 % of cases, was mainly contact with dogs and/or cats (67 %) and ingestion of contaminated food (31 %). Diagnostic imaging examinations found hypodense lesions in cerebral scanner sequences and hyperintense lesions in cerebral MRI T2-weighted sequences in 65 and 57 % of encephalitis cases respectively, and in 92 % of myelitis cases in medullary MRI T2-weighted sequences. The detection of antibodies against Toxocara spp. was almost constant in blood and cerebrospinal fluid (CSF), 99 and 93 %, respectively. The two most commonly used drugs were corticosteroids (72 %) and/or albendazole (68 %) for a period of at least 3 weeks, which often needed to be repeated. Despite a low mortality rate (6 %), complete remission was observed in only 40 % of cases. CONCLUSIONS: Neurotoxocariasis, a completely preventable zoonosis, could lead to severe sequelae failing prompt diagnosis. A compatible clinical picture, presence of risk factors, blood eosinophilia and high titers of antibodies against Toxocara spp. in CSF should alert physicians.

Severe cryptococcal-associated neurological immune reconstitution inflammatory syndrome in a renal transplant recipient treated with adalimumab.

Cryptococcosis is a major concern in organ transplant recipients. A decrease in immunosuppressants following the initiation of antifungal therapy is currently recommended, but can occasionally be complicated by the onset of immune reconstitution inflammatory syndrome (IRIS). We report on a case of cryptococcosis in a kidney transplant recipient, compounded by severe neurological IRIS, the outcome of which was unfavorable despite the use of anti-tumor necrosis factor-alpha monoclonal antibodies.

Aspergillus vertebral osteomyelitis in immunocompetent subjects: case report and review of the literature.

Aspergillus spondylodiscitis (AS) is rare in immunocompetent (IC) patients. A 65-year-old diabetic IC male subject presented with cervical AS 18 months after otomycosis. Two serological tests, mastoidectomy and biopsy of the sphenoid bone, were negative. A prevertebral biopsy identified A. flavus. The patient was successfully treated with voriconazole. Forty-three cases of AS in IC patients have been published. A predisposition was found in 84 % of cases. Fever was reported in 20 % of cases, whereas neurological defects were present in 41 %. Serology was inconsistently positive (5/7) and diagnosis was confirmed by biopsy or surgery. A. fumigatus was the most frequently isolated species (74 %). All episodes were medically treated, associated with surgery in 57 % of cases, and 73 % of patients fully recovered. AS must be discussed in IC patients presenting with risk factors, including diabetes mellitus. Biopsy is necessary to confirm diagnosis, since serology offers low sensitivity. Nevertheless, the prognosis is good.

[Human dirofilariasis: a new French case of Dirofilaria repens]. / Dirofilariose humaine: un nouveau cas français à Dirofilaria repens.

Dirofilariosis is an endemic filarial parasitic disease in the Mediterranean basin, unfamiliar in France. Its incidence and geographic area are increasing due to global warming. Dogs and cats are the usual hosts, but humans may be accidentally infected. We reported the 91st case of French dirofilariosis, contracted in Camargue (South France) which appeared as a subcutaneous abdominal nodule. Ultrasound strongly guided the diagnosis by showing a linear structure moving in a fibrocystic structure. Surgical excision confirmed the diagnosis of species (Dirofilaria repens) and this remains the only curative treatment.

Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisum.

Aphids respond to environmental changes by developing alternative phenotypes with differing reproductive modes. Parthenogenetic reproduction occurs in spring and summer, whereas decreasing day lengths in autumn provoke the production of sexual forms. Changing environmental signals are relayed by brain neuroendocrine signals to the ovarioles. We combined bioinformatic analyses with brain peptidomics and cDNA analyses to establish a catalogue of pea aphid neuropeptides and neurohormones. 42 genes encoding neuropeptides and neurohormones were identified, of which several were supported by expressed sequence tags and/or peptide mass analyses. Interesting features of the pea aphid peptidome are the absence of genes coding for corazonin, vasopressin and sulfakinin and the presence of 10 different genes coding insulin related peptides, one of which appears to be very abundantly expressed.

Transcriptomic and proteomic analyses of seasonal photoperiodism in the pea aphid.

BACKGROUND: Aphid adaptation to harsh winter conditions is illustrated by an alternation of their reproductive mode. Aphids detect photoperiod shortening by sensing the length of the night and switch from viviparous parthenogenesis in spring and summer, to oviparous sexual reproduction in autumn. The photoperiodic signal is transduced from the head to the reproductive tract to change the fate of the future oocytes from mitotic diploid embryogenesis to haploid formation of gametes. This process takes place in three consecutive generations due to viviparous parthenogenesis. To understand the molecular basis of the switch in the reproductive mode, transcriptomic and proteomic approaches were used to detect significantly regulated transcripts and polypeptides in the heads of the pea aphid Acyrthosiphon pisum. RESULTS: The transcriptomic profiles of the heads of the first generation were slightly affected by photoperiod shortening. This suggests that trans-generation signalling between the grand-mothers and the viviparous embryos they contain is not essential. By analogy, many of the genes and some of the proteins regulated in the heads of the second generation are implicated in visual functions, photoreception and cuticle structure. The modification of the cuticle could be accompanied by a down-regulation of the N-beta-alanyldopamine pathway and desclerotization. In Drosophila, modification of the insulin pathway could cause a decrease of juvenile hormones in short-day reared aphids. CONCLUSION: This work led to the construction of hypotheses for photoperiodic regulation of the switch of the reproductive mode in aphids.

Gene flow between sexual and facultatively asexual lineages of an aphid species and the maintenance of reproductive mode variation.

Many organisms considered as strictly clonal may in fact experience some rare events of sexual reproduction with their sexual relatives. However, the rate of sexual-asexual gene flow has rarely been assessed mainly because its evaluation is difficult to achieve in the field. In the cyclically parthenogenetic aphid Rhopalosiphum padi, two main sets of lineages, differing in their investment in sexual reproduction and in their genetic attributes, co-exist even at a very fine scale: the 'sexual' lineages which have a full commitment to the sexual reproduction, and the 'facultatively asexual' lineages, which allocate investment in the sexual and parthenogenetic reproduction. This system offers a unique opportunity to tackle the genetic interactions between two contrasting reproductive modes. Here, we provide evidence that gene flow occurred between sexual and facultatively asexual lineages of R. padi. We carefully examined the shuffling in phenotypic and genotypic variation following a sexual reproduction event that took place in the field. Combining genotypic data and phenotypic measurements showed that this gene mixing led to the production of a wide array of reproductive modes, including strictly asexual lineages. Finally, we discuss the central role played by facultatively asexual lineages on the maintenance of reproductive mode variation.

Host range expansion of an introduced insect pest through multiple colonizations of specialized clones.

Asexuality confers demographic advantages to invasive taxa, but generally limits adaptive potential for colonizing of new habitats. Therefore, pre-existing adaptations and habitat tolerance are essential in the success of asexual invaders. We investigated these key factors of invasiveness by assessing reproductive modes and host-plant adaptations in the pea aphid, Acyrthosiphon pisum, a pest recently introduced into Chile. The pea aphid encompasses lineages differing in their reproductive mode, ranging from obligatory cyclical parthenogenesis to fully asexual reproduction. This species also shows variation in host use, with distinct biotypes specialized on different species of legumes as well as more polyphagous populations. In central Chile, microsatellite genotyping of pea aphids sampled on five crops and wild legumes revealed three main clonal genotypes, which showed striking associations with particular host plants rather than sampling locations. Phenotypic analyses confirmed their strong host specialization and demonstrated parthenogenesis as their sole reproductive mode. The genetic relatedness of these clonal genotypes with corresponding host-specialized populations from the Old World indicated that each clone descended from a particular Eurasian biotype, which involved at least three successful introduction events followed by spread on different crops. This study illustrates that multiple introductions of highly specialized clones, rather than local evolution in resource use and/or selection of generalist genotypes, can explain the demographic success of a strictly asexual invader.

Seasonal photoperiodism regulates the expression of cuticular and signalling protein genes in the pea aphid.

Seasonal photoperiodism in aphids is responsible for the spectacular switch from asexual to sexual reproduction. However, little is known on the molecular and physiological mechanisms involved in reproductive mode shift through the action of day length. Earlier works showed that aphid head, but not eyes, directly perceives the photoperiodic signal through the cuticle. In order to identify genes regulating the photoperiodic response, a 3321 cDNA microarray developed for the pea aphid, Acyrthosiphon pisum was used to compare RNA populations extracted from heads of short- and long-day reared aphids. Microarray analyses revealed that 59 different transcripts were significantly regulated, among which a majority encoded cuticular proteins and several encoded proteins involved in cellular signalling or signal transduction. These results were confirmed by quantitative RT-PCR experiments on two cuticular and three signalling protein genes. Complementary experiments eliminated moulting and circadian rhythms as putative confounding effects. Quantitative RT-PCR performed at additional developmental stages demonstrated the regulation of expression of cuticular and signalling protein genes during the whole process of photoperiod shortening. This suggests that photoperiodic changes could affect cuticle structure and cell to cell communication in the head of aphids in relation with the switch of reproductive modes.

Annotated expressed sequence tags for studies of the regulation of reproductive modes in aphids.

The damaging effect of aphids to crops is largely determined by the spectacular rate of increase of populational expansion due to their parthenogenetic generations. Despite this, the molecular processes triggering the transition between the parthenogenetic and sexual phases between their annual life cycle have received little attention. Here, we describe a collection of genes from the cereal aphid Rhopalosiphum padi expressed during the switch from parthenogenetic to sexual reproduction. After cDNA cloning and sequencing, 726 expressed sequence tags (EST) were annotated. The R. padi EST collection contained a substantial number (139) of bacterial endosymbiont sequences. The majority of R. padi cDNAs encoded either unknown proteins (56%) or housekeeping polypeptides (38%). The large proportion of sequences without similarities in the databases is related to both their small size and their high GC content, corresponding probably to the presence of 5'-unstranslated regions. Fifteen genes involved in developmental and differentiation events were identified by similarity to known genes. Some of these may be useful candidates for markers of the early steps of sexual differentiation.

Multiple routes to asexuality in an aphid species.

Cyclical parthenogens, including aphids, are important models for studying the evolution of sex. However, little is known about transitions to asexuality in aphids, although the mode of origin of asexual lineages has important consequences for their level of genetic diversity, ecological adaptability and the outcome of competition with their sexual relatives. Thus, we surveyed nuclear, mitochondrial and biological data obtained on cyclical and obligate parthenogens of the bird cherry-oat aphid, Rhopalosiphum padi (L), to investigate the frequency of transitions from sexuality to permanent asexuality. Many instances of asexual lineages retaining the ability to produce males are known in aphids, so particular attention was paid to the existence of occasional matings between females from sexual lineages and males produced by asexual lineages, which have the potential to produce new asexual lineages. Phylogenetic inference based on microsatellite and mitochondrial data indicates at least three independent origins of asexuality in R. padi, yielding the strongest evidence to date for multiple origins of asexuality in an aphid. Moreover, several lines of evidence demonstrate that transitions to asexuality result from two mechanisms: a complete spontaneous loss of sex and repeated gene flow from essentially asexual lineages into sexual ones.

Uncontrolled-rate freezing and storage at -80 degrees C, with only 3.5-percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations.

BACKGROUND: Although controlled-rate freezing and storage in liquid nitrogen are the standard procedure for peripheral blood progenitor cell (PBPC) cryopreservation, uncontrolled-rate freezing and storage at -80 degrees C have been reported. STUDY DESIGN AND METHODS: The prospective evaluation of 109 autologous PBPC transplantations after uncontrolled-rate freezing and storage at -80 degrees C of apheresis products is reported. The cryoprotectant solution contained final concentrations of 1-percent human serum albumin, 2.5-percent hydroxyethyl starch, and 3.5-percent DMSO. RESULTS: With in vitro assays, the median recoveries of nucleated cells (NCs), CD34+ cells, CFU-GM, and BFU-E were 60.8 percent (range, 11.2-107.1%), 79.6 percent (6.3-158.1%), 35.6 percent (0.3-149.5%), and 32.6 percent (1.7-151.1%), respectively. The median length of storage was 7 weeks (range, 1-98). The median cell dose, per kg of body weight, given to patients after the preparative regimen was 6.34 x 10(8) NCs (range, 0.02-38.3), 3.77 x 10(6) CD34+ cells (0.23-58.5), and 66.04 x 10(4) CFU-GM (1.38-405.7). The median time to reach 0.5 x 10(9) granulocytes per L, 20 x 10(9) platelets per L, and 50 x 10(9) reticulocytes per L was 11 (range, 0-37), 11 (0-129), and 17 (0-200) days, respectively. Hematopoietic reconstitution did not differ in patients undergoing myeloablative or nonmyeloablative conditioning regimens before transplantation. CONCLUSION: This simple and less expensive cryopreservation procedure can produce successful engraftment, comparable to that obtained with the standard storage procedure.

Granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells from children contain the same levels of long-term culture-initiating cells producing the same numbers of colony-forming cells as those from adults, but display greater in vitro monocyte/macrophage potential.

Autologous peripheral blood progenitor cell (PBPC) transplantation is now commonly used in children. The ontogenic differences in haematopoiesis published in recent years suggest differences in the categories of mobilized PBPCs between children and adults. We investigated the frequency and distribution of mature progenitor cells (colony-forming cells, CFCs) and primitive progenitor cells [CD34+ CD38- and CD34+ Thy-1+ cells, long-term culture-initiating cells (LTC-ICs)] in children and adults mobilized using granulocyte colony-stimulating factor alone. We found similar proportions of granulocyte colony-forming units (CFU-G) and/or macrophage CFUs (CFU-M), mixed lineage CFUs (CFU-Mix) and megakarocyte CFUs (CFU-Mk), CD34+ CD38- and CD34+ Thy-1+ cells, and LTC-ICs (16.5 +/- 3.5 vs. 10.65 +/- 5 per 104 CD34+ cells), which produced the same number of CFCs (5 +/- 1 vs. 6 +/- 1 CFCs/LTC-ICs) in PB CD34+ cells from children and adults. However, we noted a higher proportion of erythroid blast-forming units (BFU-E) in PB CD34+ cells from adults (x 1.5, P = 0.003). Using cord blood as a third ageing point, we observed an inverse age-related propensity for commitment to the monocyte/macrophage lineage that was still found after normalizing the data per body weight and processed blood mass. This ontogeny-related programming was detected from the LTC-IC level, which produced 1.7 times more CFU-M in children than in adults (P = 0.048). These subtle differences in commitment between children and adults, shown here for the first time, are of interest for the in vitro manipulation of PBPCs and, in particular, for application in adoptive immunotherapy in children.