Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda)

<p>Abstract</p> <p>Background</p> <p>We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (<it>coxI </it>and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms.</p> <p>Results</p> <p>DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both <it>coxI </it>and 12S rDNA allow to reach high-quality performances, only <it>coxI </it>revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using <it>coxI </it>and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species.</p> <p>Conclusion</p> <p>An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.</p

Litomosa chiropterorum Ortlepp, 1932 (Nematoda: Filarioidea) from a South African miniopterid: redescription, Wolbachia screening and phylogenetic relationships with Litomosoides.

69 Miniopterus notalensis, type host of the onchocercid Litomosa chiropterorum, were collected in caves in the Western Province and Gauteng Province, South Africa. The prevalence of these filariae was about 50 %. The microfilaria is folded, as in other Litomosa and an area rugosa composed of cuticular bosses is present in the male posterior region. L. chiropterorum is close to the species parasitic in other Miniopterus spp. and some Rhinolophus spp. from Africa, Madagascar and Europe; it is unique with the expanded anterior extremity and the four cephalic submedian bosses. The molecular analysis of L. chiropterorum, the first done with Litomosa species from a bat, supports the hypothesis that Litomosa and Litomosoides, which have an exceptionally large buccal capsule in common, form a group in which Litomosa has a basal position. Interestingly, L. chiropterorum does not harbour Wolbachia, as proved with immunohistological staining and PCR screening using the 16S rDNA gene as target. This is contrary to L. westi from rodents and the majority of the Litomosoides species parasitic in bats or rodents. The absence of Wolbachia in a filarioid group considered ancient based on traditional and molecular approaches opens interesting scenarios on the evolution of the endosymbionts spread through filarial lineages

Breast carcinoma detection in ex vivo fresh human breast surgical specimens using a fast slide-free confocal microscopy scanner: HIBISCUSS project

Background: New generation ultra-fast fluorescence confocal microscopy allows the ex vivo intraoperative analysis of fresh tissue. The High resolution Imaging for Breast carcInoma detection in ex vivo Specimens after breast Conserving sUrgery by hiStolog Scanner (HIBISCUSS) project aimed to develop an online learning program to recognize the main breast tissue features on ultra-fast fluorescence confocal microscopy images and to evaluate the performance of surgeons and pathologists in diagnosing cancerous and non-cancerous breast tissue in ultra-fast fluorescence confocal microscopy images. Methods: Patients who underwent conservative surgery or mastectomy for breast carcinoma (invasive or in situ lesions) were included. The fresh specimens were stained with a fluorescent dye and imaged using a large field-of-view (20 cm2) ultra-fast fluorescence confocal microscope. Results: One hundred and eighty-one patients were included. The images from 55 patients were annotated to generate learning sheets and images from 126 patients were blindly interpreted by seven surgeons and two pathologists. The time for tissue processing and ultra-fast fluorescence confocal microscopy imaging was between 8 and 10 min. The training program was composed of 110 images divided into nine learning sessions. The final database for blind performance assessment comprised 300 images. The mean duration for one training session and one performance round was 17 and 27 min respectively. The performance of pathologists was almost perfect with 99.6 per cent (standard deviation (s.d.) 5.4 per cent) accuracy. Surgeons' accuracy significantly increased (P = 0.001) from 83 per cent (s.d. 8.4 per cent) in round 1 to 98 per cent (s.d. 4.1 per cent) in round 7 as well as the sensitivity (P = 0.004). Specificity increased without significance from 84 per cent (s.d. 16.7 per cent) in round 1 to 87 per cent (s.d. 16.4 per cent) in round 7 (P = 0.060). Conclusion: Pathologists and surgeons showed a short learning curve in differentiating breast cancer from non-cancerous tissue in ultra-fast fluorescence confocal microscopy images. Performance assessment for both specialties supports ultra-fast fluorescence confocal microscopy evaluation for intraoperative management. Registration number: NCT04976556 (http://www.clinicaltrials.gov)

New Insights into the Evolution of Wolbachia Infections in Filarial Nematodes Inferred from a Large Range of Screened Species

Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals.We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected.The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions

Mapping the Presence of \u3ci\u3eWolbachia pipientis\u3c/i\u3e on the Phylogeny of Filarial Nematodes: Evidence for Symbiont Loss During Evolution

Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbor these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbor these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes. The mapping of the presence/absence of W. pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes

Thyroid cartilage invasion in early-stage squamous cell carcinoma involving the anterior commissure

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Litomosa chiropterorum

69 Miniopterus notalensis, type host of the onchocercid Litomosa chiropterorum, were collected in caves in the Western Province and Gauteng Province, South Africa. The prevalence of these filariae was about 50 %. The microfilaria is folded, as in other Litomosa and an area rugosa composed of cuticular bosses is present in the male posterior region. L. chiropterorum is close to the species parasitic in other Miniopterus spp. and some Rhinolophus spp. from Africa, Madagascar and Europe; it is unique with the expanded anterior extremity and the four cephalic submedian bosses. The molecular analysis of L. chiropterorum, the first done with Litomosa species from a bat, supports the hypothesis that Litomosa and Litomosoides, which have an exceptionally large buccal capsule in common, form a group in which Litomosa has a basal position. Interestingly, L. chiropterorum does not harbour Wolbachia, as proved with immunohistological staining and PCR screening using the 16S rDNA gene as target. This is contrary to L. westi from rodents and the majority of the Litomosoides species parasitic in bats or rodents. The absence of Wolbachia in a filarioid group considered ancient based on traditional and molecular approaches opens interesting scenarios on the evolution of the endosymbionts spread through filarial lineages