Recent Progress and Applications of Thermal Lens Spectrometry and Photothermal Beam Deflection Techniques in Environmental Sensing.

This paper presents recent development and applications of thermal lens microscopy (TLM) and beam deflection spectrometry (BDS) for the analysis of water samples and sea ice. Coupling of TLM detection to a microfluidic system for flow injection analysis (µFIA) enables the detection of microcystin-LR in waters with a four samples/min throughput (in triplicate injections) and provides an LOD of 0.08 µg/L which is 12-times lower than the MCL for microcystin-LR in water. µFIA-TLM was also applied for the determination of total Fe and Fe(II) in 3 µL samples of synthetic cloudwater. The LODs were found to be 100 nM for Fe(II) and 70 nM for total Fe. The application of µFIA-TLM for the determination of ammonium in water resulted in an LOD of 2.3 µM for injection of a 5 µL sample and TLM detection in a 100 µm deep microfluidic channel. For the determination of iron species in sea ice, the BDS was coupled to a diffusive gradient in the thin film technique (DGT). The 2D distribution of Fe(II) and total Fe on DGT gels provided by the BDS (LOD of 50 nM) reflected the distribution of Fe species in sea ice put in contact with DGT gels.

Histological determination of the human origin from dry bone: a cautionary note for subadults.

Anthropologists are frequently required to confirm or exclude the human origin of skeletal remains; DNA and protein radioimmunoassays are useful in confirming the human origin of bone fragments but are not always successful. Histology may be the solution, but the young subadult structure could create misinterpretation. Histological tests were conducted on femur and skull of 31 human subjects. Each sample was observed focusing on presence or absence of fibrous bone, lamellar bone, radial lamellar bone, plexiform bone, reticular pattern, osteon banding, Haversian bone, primary osteons, secondary osteon and osteon fragments. Samples were divided into five age classes; 1 (<1 year), 2 (1-5 years), 3 (6-10 years), 4 (11-15 years) and 5 (16-20 years). Regarding femurs, class 1 presented the following: 87.5% fibrous bone, 37.5% plexiform bone, 12.5% reticular pattern and 12.5% lamellar bone radially oriented. Class 2 showed 37.5% of fibrous bone, 12.5% of reticular pattern and 37.5% of osteon banding. In the higher age classes, the classical human structures, lamellar bone and osteons were frequently visible, except for one case of reticular pattern, generally considered a distinctive non-human structure. The situation appeared different for the skull, where there was a lack of similar information, both in human and non-human. An analysis of the percentage of lamellar bone and osteons was conducted on femur and skull fragments. A trend of increase of primary osteon number and a decrease of the lamellar bone area has been detected in the femur. The present study has therefore shed some light on further pitfalls in species determination of subadult bone.

Muscle attachment site (MAS) patterns for species determination in five species of Sarcophaga (Diptera: Sarcophagidae).

To further establish species determination using the muscle attachment site (MAS) pattern method, third instar larvae of five forensically important species of Sarcophaga Meigen were investigated: Sarcophaga argyrostoma (Robineau-Desvoidy), Sarcophaga caerulescens Zetterstedt, Sarcophaga melanura Meigen, Sarcophaga albiceps Meigen and Sarcophaga similis Meade. As in the previously investigated Calliphoridae, patterns were found to be species specific. The main feature of the Sarcophaga patterns is the divided central horizontal row of segment four. A genus pattern was established to be used as base for comparison in further species determination.

MtDNA Analysis for Genetic Identification of Forensically Important Sarcophagid Flies (Diptera: Sarcophagidae) in China.

DNA-based technologies have been increasingly used in species determination of forensically important sarcophagids, as they are often not morphologically distinct, especially for the immature specimens. The mitochondrial genome has been broadly used for species-level identifications. Although Chinese sarcophagid sequences of short fragments (200-600 bp) had been deposited in GenBank, the barcode region and the complete cytochrome oxidase subunit I (COI) and COII sequences are still unavailable. In this study, 78 sarcophagid fly specimens, representing 17 Chinese sarcophagid species, were collected from 29 locations in 18 Chinese provinces. Sequence data of the mitochondrial COI and COII of the most important Chinese flesh fly taxa associated with cadavers were presented for first time, which serve as reference standards for Chinese species determination. Phylogenetic analysis showed that the COI and COII sequences were useful for identifying most sarcophagid species. The results of this research will be conductive for implementation of the Chinese Sarcophagidae in forensic entomology. However, the application of mitochondrial DNA as species identifier requires great circumspection and additional markers and methods should be studied to ensure accuracy of identification in the future.

Molecular species determination of cyathostomins from horses in Ireland.

Cyathostomins are globally important equine parasites, responsible for both chronic and acute pathogenic effects. The occurrence of mixed infections with numerous cyathostomin species hinders our understanding of parasite epidemiology, host-parasite dynamics, and species pathogenicity. There have been few studies of cyathostomin species occurring in horses in Ireland, where temperate climatic conditions with year-round rainfall provide suitable conditions for infection of grazing animals with bursate nematodes. Here, we amplified and sequenced the ITS-2 region of adult worms harvested at post-mortem from eleven adult horses between August 2018 and June 2020, and recorded species prevalence and abundance of worms recovered from the caecum, right ventral colon and left dorsal colon, using both BLAST and IDTAXA for taxonomic attribution. Phylogenetic relationships and community composition were also recorded and compared with other relevant studies, including a global meta-analysis. Overall, our results agree with previous studies that there does not seem to be a major difference in cyathostomin species occurrence in equids in different geographical regions. We confirmed the results of other workers in relation to the difficulties in discriminating between Cylicostephanus calicatus and Coronocyclus coronatus on the basis of ITS-2 sequences.

Quantification of arsenic in real samples using a spectrophotometric cloud point extraction of the formed ion pair with astrazon orange G.

A novel cloud-point extraction (CPE) procedure for the determination of ultra-trace amounts of arsenic species in real samples, purchased from the local market by spectrophotometer was developed. Inorganic arsenic species analysis in water, beverages, and foods has become increasingly important in recent years, as arsenic species are considered carcinogenic and are assessed at significant levels in samples. The technique is established on a selective ternary complex of As(V) with astrazon orange G (AOG+) in the presence of tartaric acid and polyethylene glycol tertoctylphenyl ether (Triton X-114) at pH 4.0. The calibration curve developed within range 3.0-160 ng/mL with a correlation coefficient of 0.9988 for As(V) provided a preconcentration factor of 200 and a limit of detection (3S blank/m) of 0.88 ng/mL under optimum investigation conditions. The results of molar absorptivity and Sandell sensitivity are calculated and found to be 4.38 × 105 L/mol cm and 0.018 ng cm-2, respectively. The statistical treatment of data obtained from the proposed and GF-AAS procedures are compared in terms of Student's t-tests and variance ratio F-tests has revealed no significant differences. The methodology has been effectively confirmed by assessing real samples and comparing it to the GF-AAS method statistically.

The complete ITS2 barcoding region for Strongylus vulgaris and Strongylus edentatus.

Gastrointestinal nematode parasites are of major concern for horses, where Strongylus vulgaris is considered the most pathogenic among the Strongylus species. Diagnosis of S. vulgaris infections can be determined with next generation sequencing techniques, which are inherently dependent on reference sequences. The best marker for parasitic nematodes is internal transcribed spacer 2 (ITS2) and we provide the first complete ITS2 sequences from five morphologically identified S. vulgaris and additional sequences from two S. edentatus. These sequences have high similarity to already published partial sequences and amplicon sequence variants (ASV) based on next generation sequencing (NGS). The ITS2 sequences from S. vulgaris matched available partial ITS2 sequences and the full ASVs, whereas the S. edentatus sequence matched another complete sequence. We also compare Sanger sequencing and NGS methods and conclude that the ITS2 variation is better represented with NGS methods. Based on this, we recommend that further sequencing of morphologically identified specimens of various species should be performed with NGS cover the intraspecific variation in the ITS2.

Clinical Significance of a 16S-rDNA Analysis of Heart Valves in Patients with Infective Endocarditis: a Retrospective Study.

A substantial proportion of patients with infective endocarditis (IE) are subjected to heart valve surgery. Microbiological findings on valves are important both for diagnostics and for tailored antibiotic therapy, post-operatively. The aims of this study were to describe microbiological findings on surgically removed valves and to examine the diagnostic benefits of 16S-rDNA PCR and sequencing (16S-analysis). Adult patients who were subjected to heart valve surgery for IE between 2012 and 2021 at Skåne University Hospital, Lund, where a 16S-analysis had been performed on the valve, constituted the study population. Data were gathered from medical records, and the results from blood cultures, valve cultures, and 16S-analyses of valves were compared. A diagnostic benefit was defined as providing an agent in blood culture negative endocarditis, providing a new agent in episodes with positive blood cultures, or confirming one of the findings in episodes with a discrepancy between blood and valve cultures. 279 episodes in 272 patients were included in the final analysis. Blood cultures were positive in 259 episodes (94%), valve cultures in 60 episodes (22%), and 16S-analyses in 227 episodes (81%). Concordance between the blood cultures and the 16S-analysis was found in 214 episodes (77%). The 16S-analyses provided a diagnostic benefit in 25 (9.0%) of the episodes. In blood culture negative endocarditis, the 16S-analyses had a diagnostic benefit in 15 (75%) of the episodes. A 16S-analysis should be routinely performed on surgically removed valves in blood culture negative endocarditis. In patients with positive blood cultures, 16S-analysis may also be considered, as a diagnostic benefit was provided in some patients. IMPORTANCE This work demonstrates that it can be of importance to perform both cultures and analysis using 16S-rDNA PCR and sequencing of valves excised from patients undergoing surgery for infective endocarditis. 16S-analysis may help both to establish a microbiological etiology in cases of blood culture negative endocarditis and to provide help in situations where there are discrepancies between valve and blood cultures. In addition, our results show a high degree of concordance between blood cultures and 16S-analyses, indicating that the latter has a high sensitivity and specificity for the etiological diagnosis of endocarditis in patients who were subjected to heart valve surgery.

First Study of Ascaris lumbricoides from the Semiwild Population of the Sumatran Orangutan Pongo abelii in the Context of Morphological Description and Molecular Phylogeny.

There is little evidence that the already described and accepted taxa of ascarids (Ascaris lumbricoides, A. suum, and A. ovis) infecting individuals of taxonomically distant groups (hominids, pigs, sheep, goats, and dogs) can be genetically or morphologically distinguished. However, despite described morphological differences, e.g., due to intraspecific variation, these are insufficient for species determination and may indicate differences amongst ascarids because of cross infections, hybrid production, and specific adaptations to hosts. Herein, the results of a molecular and morphological analysis of ascarids parasitising Sumatran orangutans (Pongo abelii Lesson, 1827) in native populations are presented. The research took place in the Bukit Lawang area, Indonesia, in 2009. Throughout the year, fresh faecal samples were collected regularly from 24 orangutans, and all were examined for the presence of nematode adults. Only five adult worms from two orangutan females were found during regular collection. Using the integrative taxonomic approach, the nematodes found were identified as A. lumbricoides. The significance of the find and its rarity is documented by the fact that this is the first confirmed finding of adult ascarids from an original orangutan site (not from a zoo) in more than 130 years (including the long-term study spanning the last 20 years focusing on orangutan parasites and natural antiparasitic drugs). More accurate morphometric parameters and genetic differences for the identification of ascarids were established. These parameters will be helpful for other findings in great apes and will also be suitable for further and precise determination of this parasite. The details distinguishing between male and female specimens are also stated and well defined. A comprehensive evaluation of the situation of Ascaris species parasitising orangutans, including a comparison with previously described orangutan parasite (i.e., A. satyri-species inquirenda), is discussed.

Comparative genomics of Staphylococcus capitis reveals species determinants.

Staphylococcus capitis is primarily described as a human skin commensal but is now emergent as an opportunistic pathogen isolated from the bloodstream and prosthetic joint infections, and neonatal intensive care unit (NICU)-associated sepsis. We used comparative genomic analyses of S. capitis to provide new insights into commensal scalp isolates from varying skin states (healthy, dandruff lesional, and non-lesional), and to expand our current knowledge of the species populations (scalp isolates, n = 59; other skin isolates, n = 7; publicly available isolates, n = 120). A highly recombinogenic population structure was revealed, with genomes including the presence of a range of previously described staphylococcal virulence factors, cell wall-associated proteins, and two-component systems. Genomic differences between the two described S. capitis subspecies were explored, which revealed the determinants associated exclusively with each subspecies. The subspecies ureolyticus was distinguished from subspecies capitis based on the differences in antimicrobial resistance genes, ß-lactam resistance genes, and ß-class phenol soluble modulins and gene clusters linked to biofilm formation and survival on skin. This study will aid further research into the classification of S. capitis and virulence-linked phylogroups to monitor the spread and evolution of S. capitis.

MALDI-TOF MS as a method for rapid identification of Phytophthora de Bary, 1876.

The number of described species of the oomycete genus Phytophthora is growing rapidly, highlighting the need for low-cost, rapid tools for species identification. Here, a collection of 24 Phytophthora species (42 samples) from natural as well as anthropogenic habitats were genetically identified using the internal transcribed spacer (ITS) and cytochrome c oxidase subunit I (COI) regions. Because genetic identification is time consuming, we have created a complementary method based on by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Both methods were compared and hypothesis that the MALDI-TOF MS method can be a fast and reliable method for the identification of oomycetes was confirmed. Over 3500 mass spectra were acquired, manually reviewed for quality control, and consolidated into a single reference library using the Bruker MALDI Biotyper platform. Finally, a database containing 144 main spectra (MSPs) was created and published in repository. The method presented in this study will facilitate the use of MALDI-TOF MS as a complement to existing approaches for fast, reliable identification of Phytophthora isolates.

Fast, economic and simultaneous identification of clinically relevant Gram-negative species with multiplex real-time PCR.

AIM: A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa). MATERIALS & METHODS: Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay. RESULTS: Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative. CONCLUSION: The genes gad, basC, khe and ecfX can reliably identify these four species via multiplex colony rt-PCR.

A revision of Kerivoula hardwickii and occurrence of K. furva (Chiroptera: Vespertilionidae) in China.

Although increased survey efforts using improved capture methods (particularly harp traps) have greatly expanded the quantity of Kerivoula specimens available in China, the understanding of the genus has been long constrained. After the recently published revision of the hardwickii-complex with the description of K. furva and re-evaluation of occurrence of K. titania in Taiwan, the critical overview of the previous data of Chinese Kerivoula (with the exception of K. picta, a strikingly colored and unmistakable species) is imperative. To clarify the taxonomy and distribution of the hardwickii-complex in China, 40 additional specimens collected from South China were studied through detailed morphological comparisons, multivariate statistical methods and phylogenetic inference. Our results evidently classified these specimens as K. furva instead of K. titania or K. hardwickii sensu stricto and together with the critical review of literature data indicate that all previous Chinese records of the two latter species were based on either misidentifications or outdated taxonomy. K. furva have so far been recorded in the Chinese provinces of Chongqing, Fujian, Guangdong, Guangxi, Hainan, Hunan, Jiangxi, Yunnan and Taiwan, but more field surveys are needed to confirm whether it could be found in other nearby provinces.

Determination of neutral diols and carboxylic acids formed during glycerol electrooxidation by capillary electrophoresis with dual C4D.

Methods for determination of glycerol and its electrooxidation products (neutral diols and carboxylates) by capillary electrophoresis (CE) with dual capacitively coupled contactless conductivity detectors (C4D) are presented. Glycerol, dihydroxyacetone and glyceraldehyde were detected as anionic borate complexes in less than 3min under counter Electroosmotic Flow (EOF) mode (resolution of the critical pair of 1.8). Limits of detection (LODs) of 15, 15 and 10µmolL-1 were obtained for glycerol, dihydroxyacetone and glyceraldehyde, respectively. Two methods of separation were used for the separation of carboxylates. The first one used the same Back Ground Electrolyte (BGE) containing borate, and the second used a BGE (pH 6.1) composed by 2-(N-morpholino)ethanesulfonic acid (MES), L-Histidine and a flow modifier. Better separation and LODs for carboxylates were obtained using Mes/Histidine as BGE. However, along with the non-applicability of this BGE to the determination of neutral diols, observation of the C4D signals at two different points of the capillary (10 and 50cm apart from the injection tip) revealed interaction of the flow modifier with some species (mesoxalate and glyoxylate). The electrooxidation of a glycerol sample in alkaline media on an 8cm2 gold working electrode was evaluated by the developed methods. After 16h of electrolysis, 87% of the glycerol had been oxidized and formate, glycolate, hydroxypyruvate and glycerate were detected as the main products.

Connecting the Dots: From an Easy Method to Computerized Species Determination.

Differences in growth rate of forensically important dipteran larvae make species determination an essential requisite for an accurate estimation of time since colonization of the body. Interspecific morphological similarities, however, complicate species determination. Muscle attachment site (MAS) patterns on the inside of the cuticula of fly larvae are species specific and grow proportionally with the animal. The patterns can therefore be used for species identification, as well as age estimation in forensically important dipteran larvae. Additionally, in species where determination has proven to be difficult-even when employing genetic methods-this easy and cheap method can be successfully applied. The method was validated for a number of Calliphoridae, as well as Sarcophagidae; for Piophilidae species, however, the method proved to be inapt. The aim of this article is to assess the utility of the MAS method for applications in forensic entomology. Furthermore, the authors are currently engineering automation for pattern acquisition in order to expand the scope of the method. Automation is also required for the fast and reasonable application of MAS for species determination. Using filters on digital microscope pictures and cross-correlating them within their frequency range allows for a calculation of the correlation coefficients. Such pattern recognition permits an automatic comparison of one larva with a database of MAS reference patterns in order to find the correct, or at least the most likely, species. This facilitates species determination in immature stages of forensically important flies and economizes time investment, as rearing to adult flies will no longer be required.

Using Frons Width to Differentiate Blow Fly Species (Diptera: Calliphoridae) Phormia regina (Meigen) and Protophormia terraenovae (Robineau-Desvoidy).

Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and Phormia regina (Meigen) (Diptera: Calliphoridae) are morphologically similar blow fly species commonly used for estimating postmortem intervals. Field collection and storage of adults can result in color changes, in particular on calypters and palps; often collected specimens show damage such as wing fray or fungal growth. We measured the frons width: total head width ratio using photographs (ImageJ version 1.49) to differentiate these two species. Both sexes were distinguishable to species, with the greatest difference between males: 12.34% P. terraenovae versus 1.62% P. regina, less so for females: 40.25% P. terraenovae, versus 33.65% P. regina. Incorporating this feature into future blow fly keys would help with distinguishing field-caught specimens when other features are obstructed.

Cone beam computed tomography (CBCT) as a tool for the analysis of nonhuman skeletal remains in a medico-legal setting.

To confirm the nature and forensic significance of questioned skeletal material submitted a medico-legal setting is a relatively common procedure, although not without difficulties when the remains are fragmented or burned. Different methodologies have been described for this purpose, many of them invasive, time and money consuming or dependent on the availability of the analytical instrument. We present a case in which skeletal material with unusual conditions of preservation and curious discovery was sent to a medico-legal setting to determine its human/nonhuman origin. A combined strategy of imagenological procedures (macroscopic, radiographic and cone beam computed tomography - CBCT-technology) was performed as non-invasive and rapid methods to assess the nonhuman nature of the material, specifically of pig (Sus scrofa) origin. This hypothesis was later confirmed by DNA analysis. CBCT data sets provide accurate three-dimensional reconstructions, which demonstrate its reliable use as a forensic tool.

Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems.

Species determination within Staphylococcus genus by extended PCR-restriction fragment length polymorphism of saoC gene.

Genetic methods based on PCR-restriction fragment length polymorphism (RFLP) are widely used for microbial species determination. In this study, we present the application of saoC gene as an effective tool for species determination and within-species diversity analysis for Staphylococcus genus. The unique sequence diversity of saoC allows us to apply four restriction enzymes to obtain RFLP patterns, which appear highly distinctive even among closely related species as well as atypical isolates of environmental origin. Such patterns were successfully obtained for 26 species belonging to Staphylococcus genus. What is more, tracing polymorphisms detected by different restriction enzymes allowed for basic phylogeny analysis for Staphylococcus aureus, which is potentially applicable for other staphylococcal species.