Applications of Machine Learning to Improve the Clinical Viability of Compton Camera Based in vivo Range Verification in Proton Radiotherapy.

We studied the application of a deep, fully connected Neural Network (NN) to process prompt gamma (PG) data measured by a Compton camera (CC) during the delivery of clinical proton radiotherapy beams. The network identifies 1) recorded "bad" PG events arising from background noise during the measurement, and 2) the correct ordering of PG interactions in the CC to help improve the fidelity of "good" data used for image reconstruction. PG emission from a tissue-equivalent target during irradiation with a 150 MeV proton beam delivered at clinical dose rates was measured with a prototype CC. Images were reconstructed from both the raw measured data and the measured data that was further processed with a neural network (NN) trained to identify "good" and "bad" PG events and predict the ordering of individual interactions within the good PG events. We determine if NN processing of the CC data could improve the reconstructed PG images to a level in which they could provide clinically useful information about the in vivo range and range shifts of the proton beams delivered at full clinical dose rates. Results showed that a deep, fully connected NN improved the achievable contrast to noise ratio (CNR) in our images by more than a factor of 8x. This allowed the path, range, and lateral width of the clinical proton beam within a tissue equivalent target to easily be identified from the PG images, even at the highest dose rates of a 150 MeV proton beam used for clinical treatments. On average, shifts in the beam range as small as 3 mm could be identified. However, when limited by the amount of PG data measured with our prototype CC during the delivery of a single proton pencil beam (~1 × 109 protons), the uncertainty in the reconstructed PG images limited the identification of range shift to ~5 mm. Substantial improvements in CC images were obtained during clinical beam delivery through NN pre-processing of the measured PG data. We believe this shows the potential of NNs to help improve and push CC-based PG imaging toward eventual clinical application for proton RT treatment delivery verification.

Measuring prompt gamma-ray emissions from elements found in tissue during passive-beam proton therapy.

Prompt gamma detection during proton radiotherapy for range verification purposes will need to operate in both active and passive treatment beam environments. This paper describes prompt gamma measurements using a high resolution 2″ × 2″ LaBr3detector for a 200 MeV clinical passive-scatter proton beam. These measurements examine the most likely discrete prompt gamma rays emitted from tissue by detecting gammas produced in water, Perspex, carbon and liquid-nitrogen targets. Measurements were carried out at several positions around the depth corresponding to the location of the Bragg peak for water and Perspex targets in order to investigate prompt gamma emission as a function of depth along the beam path. This work also focused on validating the Geant4 Monte Carlo model of the passive-scatter proton beam line and LaBr3detector by making a direct comparison between the simulated and experimental results. The initial prompt gamma measurements were overwhelmed by the high amount of scattered radiation when measuring at isocenter, shifting the target further downstream from the final collimator significantly reduced the background radiation. Prompt gamma peaks were then clearly identified for the water, Perspex and graphite targets. The developed Geant4 Monte Carlo model was able to replicate the measured prompt gamma ray energy spectra, including production for important photopeaks to within 10%, except for the 4.44 MeV peak from the water target, which had more than a 50% overestimation of the number of produced prompt gamma rays. The prompt gamma measurements at various depths correlated well with the proton dose deposition; the 4.44 and 6.13 MeV photopeak profiles peaked within 1 cm of the Bragg peak and the R50%value for the 3-7 MeV energy range predicted the proton range within 8 mm.

Aspergillus incahuasiensis sp. nov., isolated from soil in the semi-arid region of northern Chile.

During a study of the fungi from a semi-arid region of northern Chile, a novel species of Aspergillus was encountered in the soil from an area where pepper trees (Schinusmolle) were growing. Marker genes were sequenced to identify these isolates. The ß-tubulin, calmodulin and DNA-dependent RNA polymerase loci all indicated that this was a novel species in Aspergillus section Nidulantes and in the Aspergillus multicolorclade. The new species was studied morphologically and differences between it and the other members of the A. multicolor clade are described. We provide a name and description for these isolates as Aspergillus incahuasiensis sp. nov.

The Talaromyces pinophilus species complex.

A sample of isolates from Talaromyces pinophilus (55 isolates) and closely related species (76 isolates) was sequenced at four loci, the data were analyzed using maximum likelihood analysis and the GCPSR. The isolates were subjected to growth studies on the recommended media for description of Talaromyces species. On the basis of the combined data, five new species were segregated out of T. pinophilus and placed in newly described species. The T. pinophilus species complex contains ten species. The three other new species, Talaromyces argentinensis, T. californicus and T. louisianensis were not a part of the T. pinophilus species complex but occurred in Talaromyces sect. Talaromyces. T. argentinensis produces a teleomorphic state and is phylogenetically and morphologically distinct from other Talaromyces species.

Aspergillus olivimuriae sp. nov., a halotolerant species isolated from olive brine.

A facultative halo-tolerant Aspergillus strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named Aspergillus olivimuriae. It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 °C, no growth was observed at 40 °C, conidia en masse were avellaneous on all media. The description of the new species Aspergillus olivimuriae brings the total species of Aspergillus sect. Flavipedes to 15. The type strain of A. olivimuriae sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.

New species of Talaromyces isolated from maize, indoor air, and other substrates.

Talaromyces strains isolated from maize seeds and the built environment were examined taxonomically because they could not be identified as previously described species. Using phenotypic analysis, DNA sequencing, and phylogenetic and concordance analyses, the authors discovered and described 10 new species in sect. Islandici and 1 new species in sect. Subinflati. Taxonomic novelties in sect. Islandici are Talaromyces delawarensis, T. herodensis, T. juglandicola, T. kilbournensis, T. novojersensis, T. ricevillensis, T. rogersiae, T. siglerae, T. subtropicalis, and T. tiftonensis, and the species from sect. Subinflata is T. tzapotlensis. The isolate of T. siglerae is unusual in Talaromyces because it produced a Sagenomella-like anamorph, but phylogenetic analysis placed it in Talaromyces. Talaromyces rotundus is known from a few isolates, but searches with internal transcribed spacer (ITS) sequences in GenBank revealed that it is commonly endolichenous with Lasallia hispanica. Talaromyces wortmannii also has a role as an endophyte of the aquatic plant Persicaria amphibia, based on ITS sequence records from GenBank.

Targeting Conserved Genes in Penicillium Species.

Polymerase chain reaction amplification of conserved genes and sequence analysis provides a very powerful tool for the identification of toxigenic as well as non-toxigenic Penicillium species. Sequences are obtained by amplification of the gene fragment, sequencing via capillary electrophoresis of dideoxynucleotide-labeled fragments or NGS. The sequences are compared to a database of validated isolates. Identification of species indicates the potential of the fungus to make particular mycotoxins.

Haenamindole and fumiquinazoline analogs from a fungicolous isolate of Penicillium lanosum.

Three amino acid-derived compounds, haenamindole (1) and 2'-epi-fumiquinazolines C (2) and D (3), were isolated from cultures of a fungicolous isolate of Penicillium lanosum (MYC-1813=NRRL 66231). Compound 1 was also encountered in cultures of P. corylophilum (MYC-418=NRRL 28126). Structure elucidation of these metabolites was based mainly on high resolution mass spectrometry and NMR data analysis. Haenamindole (1) was found to be a recently reported diketopiperazine-type metabolite that incorporates an unusual ß-Phe unit. Analysis of X-ray crystallographic data and the products of acid hydrolysis of 1 enabled a conclusive, slightly modified stereochemical assignment for haenamindole. Fumiquinazoline analog 2 is a new natural product, while related compound 3 has been previously reported only as a product of an in vitro enzymatic step and of a genetically engineered fungal culture. Compounds 1 and 3 showed antiinsectan activity against the fall armyworm Spodoptera frugiperda.

Aspergillus asper sp. nov. and Aspergillus collinsii sp. nov., from Aspergillus section Usti.

In sampling fungi from the built environment, two isolates that could not confidently be placed in described species were encountered. Phenotypic analysis suggested that they belonged in Aspergillus sect. Usti. In order to verify the sectional placement and to assure that they were undescribed rather than phenotypically aberrant isolates, DNA was isolated and sequenced at the beta-tubulin, calmodulin, internal transcribed spacer and RNA polymerase II loci and sequences compared with those from other species in the genus Aspergillus. At each locus, each new isolate was distant from existing species. Phylogenetic trees calculated from these data and GenBank data for species of the section Usti excluded the placement of these isolates in existing species, with statistical support. Because they were excluded from existing taxa, the distinct species Aspergillus asper (type strain NRRL 35910T) and Aspergillus collinsii (type strain NRRL 66196T) in sect. Usti are proposed to accommodate these strains.

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase.

OBJECTIVE: The objective was to phylogenetically classify diverse strains of Aureobasidium pullulans and determine their production of feruloyl esterase. RESULTS: Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were associated with phylogenetic clade 10 and particularly clade 8. Literature strains used for pullulan production all belonged to clade 7. These strains and 36 previously classified strains were tested for feruloyl esterase production, which was found to be associated with phylogenetic clades 4, 11, and particularly clade 8. Clade 8 strains NRRL 58552 and NRRL 62041 produced the highest levels of feruloyl esterase among strains tested. CONCLUSIONS: Production of both xylanase and feruloyl esterase are associated with A. pullulans strains in phylogenetic clade 8, which is thus a promising source of enzymes with potential biotechnological applications.

Evaluation of proton inelastic reaction models in Geant4 for prompt gamma production during proton radiotherapy.

During proton beam radiotherapy, discrete secondary prompt gamma rays are induced by inelastic nuclear reactions between protons and nuclei in the human body. In recent years, the Geant4 Monte Carlo toolkit has played an important role in the development of a device for real time dose range verification purposes using prompt gamma radiation. Unfortunately the default physics models in Geant4 do not reliably replicate the measured prompt gamma emission. Determining a suitable physics model for low energy proton inelastic interactions will boost the accuracy of prompt gamma simulations. Among the built-in physics models, we found that the precompound model with a modified initial exciton state of 2 (1 particle, 1 hole) produced more accurate discrete gamma lines from the most important elements found within the body such as 16O, 12C and 14N when comparing them with the available gamma production cross section data. Using the modified physics model, we investigated the prompt gamma spectra produced in a water phantom by a 200 MeV pencil beam of protons. The spectra were attained using a LaBr3 detector with a time-of-flight (TOF) window and BGO active shield to reduce the secondary neutron and gamma background. The simulations show that a 2 ns TOF window could reduce 99% of the secondary neutron flux hitting the detector. The results show that using both timing and active shielding can remove up to 85% of the background radiation which includes a 33% reduction by BGO subtraction.

Expanding the species and chemical diversity of Penicillium section Cinnamopurpurea.

A set of isolates very similar to or potentially conspecific with an unidentified Penicillium isolate NRRL 735, was assembled using a BLAST search of ITS similarity among described (GenBank) and undescribed Penicillium isolates in our laboratories. DNA was amplified from six loci of the assembled isolates and sequenced. Two species in section Cinnamopurpurea are self-compatible sexual species, but the asexual species had polymorphic loci suggestive of sexual reproduction and variation in conidium size suggestive of ploidy level differences typical of heterothallism. Accordingly we use genealogical concordance analysis, a technique valid only in heterothallic organisms, for putatively asexual species. Seven new species were revealed in the analysis and are described here. Extrolite analysis showed that two of the new species, P. colei and P. monsserratidens produce the mycotoxin citreoviridin that has demonstrated pharmacological activity against human lung tumors. These isolates could provide leads in pharmaceutical research.

Revision of Aspergillus section Flavipedes: seven new species and proposal of section Jani sect. nov.

Aspergillus section Flavipedes contains species found worldwide in soils and rhizospheres, indoor and cave environments, as endophytes, food contaminants and occasionally as human pathogens. They produce many extensively studied bioactive secondary metabolites and biotechnologically relevant enzymes. The taxa were revised based on phylogenetic analysis of sequences from four loci (ß-tubulin, calmodulin, RPB2, ITS rDNA), two PCR fingerprinting methods, micro- and macromorphology and physiology. Section Flavipedes includes three known and seven new species: A. ardalensis, A. frequens, A. luppii, A. mangaliensis, A. movilensis, A. polyporicola and A. spelaeus. The name A. neoflavipes was proposed for Fennellia flavipes a distinct species from its supposed asexual state A. flavipes. Aspergillus iizukae, A. frequens and A. mangaliensis are the most common and widely distributed species, whereas A. flavipes s. str. is rare. A dichotomous key based on the combination of morphology and physiology is provided for all recognized species. Aspergillus section Jani is established to contain A. janus and A. brevijanus, species previously classified as members of sect. Versicolores, Terrei or Flavipedes. This new section is strongly supported by phylogenetic data and morphology. Section Jani species produce three types of conidiophores and conidia, and colonies have green and white sectors making them distinctive. Accessory conidia found in pathogenic A. terreus were found in all members of sects. Flavipedes and Jani. Our data indicated that A. frequens is a clinically relevant and produces accessory conidia during infection.

Acute respiratory distress caused by Neosartorya udagawae.

We describe the first reported case of acute respiratory distress syndrome (ARDS) attributed to Neosartorya udagawae infection. This mold grew rapidly in cultures of multiple respiratory specimens from a previously healthy 43-year-old woman. Neosartorya spp. are a recently recognized cause of invasive disease in immunocompromised patients that can be mistaken for their sexual teleomorph, Aspergillus fumigatus. Because the cultures were sterile, phenotypic identification was not possible. DNA sequencing of ITS, calmodulin and ß-tubulin genes supported identification of Neosartorya udagawae. Our case is the first report of ARDS associated with Neosartorya sp. infection and defines a new clinical entity.

Genetic relatedness versus biological compatibility between Aspergillus fumigatus and related species.

Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management.

Aspergillus pragensis sp. nov. discovered during molecular reidentification of clinical isolates belonging to Aspergillus section Candidi.

The identity of nine clinical isolates recovered from Czech patients and presumptively identified as Aspergillus sp. section Candidi based on colony morphology was revised using sequences of ß-tubulin, calmodulin gene sequence, and internal transcribed spacer rDNA. Six isolates were from suspected and proven onychomycosis, one from otitis externa, and two associated with probable invasive aspergillosis. The results showed that one Aspergillus candidus isolate was the cause of otitis externa, and both isolates obtained from sputa of patients with probable invasive aspergillosis were reidentified as A. carneus (sect. Terrei) and A. flavus (sect. Flavi). Three isolates from nail scrapings were identified as A. tritici, a verified agent of nondermatophyte onychomycosis. One isolate from toenail was determined to be A. candidus and the two isolates belonged to a hitherto undescribed species, Aspergillus pragensis sp. nov. This species is well supported by phylogenetic analysis based on ß-tubulin and calmodulin gene and is distinguishable from other members of sect. Candidi by red-brown reverse on malt extract agar, slow growth on Czapek-Dox agar and inability to grow at 37°C. A secondary metabolite analysis was also provided with comparison of metabolite spectrum to other species. Section Candidi now encompasses five species for which a dichotomous key based on colony characteristics is provided. All clinical isolates were tested for susceptibilities to selected antifungal agents using the Etest and disc diffusion method. Overall sect. Candidi members are highly susceptible to common antifungals.

Clinical, morphological, and molecular characterization of Penicillium canis sp. nov., isolated from a dog with osteomyelitis.

Infections caused by Penicillium species are rare in dogs, and the prognosis in these cases is poor. An unknown species of Penicillium was isolated from a bone lesion in a young dog with osteomyelitis of the right ilium. Extensive diagnostic evaluation did not reveal evidence of dissemination. Resolution of lameness and clinical stability of disease were achieved with intravenous phospholipid-complexed amphotericin B initially, followed by long-term combination therapy with terbinafine and ketoconazole. A detailed morphological and molecular characterization of the mold was undertaken. Sequence analysis of the internal transcribed spacer revealed the isolate to be closely related to Penicillium menonorum and Penicillium pimiteouiense. Additional sequence analysis of ß-tubulin, calmodulin, minichromosome maintenance factor, DNA-dependent RNA polymerase, and pre-rRNA processing protein revealed the isolate to be a novel species; the name Penicillium canis sp. nov. is proposed. Morphologically, smooth, ovoid conidia, a greenish gray colony color, slow growth on all media, and a failure to form ascomata distinguish this species from closely related Penicillium species.

Talaromyces columbinus sp. nov., and genealogical concordance analysis in Talaromyces clade 2a.

During the course of mold surveys, a set of Talaromyces isolates were obtained that did not fit any described species. Phenotypic examination of these isolates showed that they were similar to T. piceus but differed in some growth characteristics. Multilocus DNA sequence data were obtained for the new isolates and some related species in the broader, more inclusive clade, and the data were analyzed using genealogical concordance. The new isolates are described as Talaromyces columbinus. From analysis of the related species, Penicillium rugulosum var. atricolum is given species status in Talaromyces as T. atricola. Penicillium tardum and P. chrysitis were showed to be synonyms of T. rugulosus. Penicillium scorteum and T. phialosporus were showed to be conspecific and under the rule of priority T. scorteus is the proper name for isolates previously known as T. phialosporus. Talaromyces wortmanii was showed to be distinct from Penicillium concavorugulosum and T. variabilis but the relationship of the latter two species remains unresolved. Examination of ITS sequences from GenBank showed that T. columbinus has previously been reported from human lung infections under the name Penicillium piceum.

Sexual reproduction in Aspergillus tubingensis from section Nigri.

A sclerotium-forming member of Aspergillus section Nigri was sampled from a population in a single field in North Carolina, USA, and identified as A. tubingensis based on genealogical concordance analysis. Aspergillus tubingensis was shown to be heterothallic, with individual strains containing either a MAT1-1 or MAT1-2 mating-type gene. Strains of opposite mating type were crossed on mixed cereal agar and incubated for 5-6 months. Stromata typically formed 1-2 indehiscent ascocarps containing asci and ascospores within the pseudo-parenchymatous matrix in a manner similar to the Petromyces sexual stage from section Flavi, which is closely related to section Nigri. Ascospores of A. tubingensis differed from those of section Flavi species in the reticulate ornamentation of ascospores and the presence of two crests that form an equatorial furrow. Sexual reproduction in A. tubingensis may be useful for enhancing enzyme and organic acid production through recombination-mediated genetic engineering of industrial strains.

Taxonomic revision of Eurotium and transfer of species to Aspergillus.

Aspergillus section Aspergillus contains economically important, xerophilic fungi that are widely distributed in nature and the human environment and are known for their ability to grow on substrates with low water activity. The taxa were revised based on sequence data from four loci, PCR fingerprinting, micro- and macromorphology, and physiology. The number of taxa was reduced to 17 species, all of which can be distinguished with sequence data from either the caM or RPB2 locus. The original description of A. proliferans was supplemented by a description of its teleomorph. This species seems to be relatively common and often has been confused with A. glaucus. In addition, green sporulating isolates of A. niveoglaucus isolated from food and several other substrates are indistinguishable in phenotype from A. glaucus. A dichotomous key based on ascospore size and ornamentation and the ability to grow at specific combinations of temperature and water activity is provided for identification of species. In response to recent changes in the botanical code, we transferred the Eurotium species to Aspergillus and selected one name for each species.