Use Of Molecular Methods Research Articles

The first record of Culicoides deltus as a putative vector of Onchocerca flexuosa in Slovak red deer (Cervus elaphus)

Within the research conducted in the years 2016–2022 in the area of Volovsky Mountains in Slovakia, 63,950 biting midges were collected during 74 trapping sessions. The aim of the study was to identify species composition of biting midges, their host preference and potential transmission of parasites by these insects under natural conditions. The collected biting midges fell into 29 species and the most common were the Culicoides (C. obsoletus/C. scoticus/C. montanus) that accounted for 47.9% of the collected biting midges. Identification of species was based on the morphology of biting midges and the use of molecular methods. We confirmed positive suckling results on red deer in three samples namely C. montanus, C. scoticus and C. deltus. We examined these samples for the presence of Onchocerca worm DNA. We confirmed the presence of O. flexuosa DNA in one C. deltus sample. The host preference of biting midges was identified by molecular technique that involved sequencing a 350-bp sequence of the mitochondrial cytochrome b gene (cyt b). The presence of Onchocerca flexuosa DNA in C. deltus was confirmed by sequencing of fragments of mitochondrial genes cox1. The sequences matched the previously published sequences for O. flexuosa. Data on high prevalence of infections caused by Onchocerca worms in red deer in Slovakia have already been published and indicated favourable conditions for the vectors and a suitable environment for parasite circulation in Slovakia. According to the authors’ knowledge, this was the first ever detection of O. flexuosa in C. deltus in wild nature.

Thyroid Lesion with a Novel Concurrent ETV6-NTRK3 Translocation and DICER1 Mutation

Abstract Introduction/Objective Understanding of the behavior of thyroid tumors expand as the basis of their classification gradually move from morphologic to molecular/genetic. The use of molecular methods in detecting mutations in fine needle aspiration (FNA) specimens further helped in tailoring patient management. Here, we describe a case of a thyroid lesion which was diagnosed with atypia of undetermined significance (AUS) on FNA and discovered to have an unreported concurrent mutation pattern. Methods/Case Report The patient is a 26 year-old female with long standing history of prominent anterior neck mass. New onset of neck pain prompted sonographic analysis which revealed a 1.1 cm hypoechoic left thyroid nodule with microcalcification. FNA was done, and was signed out as AUS which initiated molecular genetic testing. The lesion was found to have both ETV6/NTRK3 translocation and DICER1 p.P375R mutation. Existing literature on NTRK3 rearrangements in thyroid tumors confer a >95% risk of malignancy. It is commonly associated with BRAF-negative papillary thyroid carcinomas (PTC) with both in BRAF-like behavior in terms of nodal metastases and extrathyroidal extension. On the other hand, there are no existing literature specifying the malignancy risk of the exact DICER1 mutation identified since it has been linked to follicular neoplasms in general. Furthermore, isolated DICER1 mutations in the thyroid showed RAS-like behavior. Subsequent resection of the thyroid revealed that the tumor is morphologically a PTC, with lymphovascular invasion. Results (if a Case Study enter NA) NA Conclusion Molecular testing in thyroid lesions with morphologically indeterminable features on FNA significantly aided in prognostication of patients and prediction of malignancy risk. In this case with a previously unreported combination of mutations, existing literature helped in describing the probable behavior of the patient’s tumor. Thus, the researchers emphasize the importance of databases of identified mutations in tumors and their associated characteristics such as The Cancer Genomic Atlas (TCGA) and The Catalogue of Somatic Mutations in Cancer (COSMIC).

A low-cost culture- and DNA extraction-free method for the molecular detection of pneumococcal carriage in saliva.

Molecular methods have improved the sensitivity of the detection of pneumococcal carriage in saliva. However, they typically require sample culture enrichment and nucleic acid extraction prior to performing the detection assay and may limit scalability for extensive surveillance of pneumococcus, particularly in low-resource settings. We evaluated the performance of a DNA-extraction-free method for the detection of pneumococcus in saliva. We developed a streamlined qPCR-based protocol for the detection of pneumococcus, omitting culture enrichment and DNA extraction. Using saliva samples collected from children attending childcare centers (New Haven, CT, USA), we evaluated the detection of pneumococcus using saliva lysates as compared to purified DNA extracted from culture-enriched aliquots of the paired samples using qPCR targeting the pneumococcal piaB gene. Of the 759 saliva samples tested from 92 children [median age 3.65 years; IQR (2.46-4.78)], pneumococcus was detected in 358 (47.2%) saliva lysates prepared using the extraction-free protocol and in 369 (48.6%) DNA extracted from culture-enriched samples. We observed near-perfect agreement between the two protocols (Cohen's kappa: 0.92; 95% CI: 0.90-0.95). Despite a high correlation between CT values generated by the two methods (r = 0.93, P < 0.0001), the CT values generated from saliva lysates were higher (lower concentration) than those from culture-enriched samples (ΔCT = 6.69, P < 0.00001). The cost of detecting pneumococcus using saliva lysates was at least fivefold lower (US$2.53) compared to the cost of the culture-enriched method (range: US$13.60-US$19.46). For pneumococcal carriage surveillance in children, our findings suggest that a DNA extraction-free approach may offer a cost-effective alternative to the resource-intensive culture-enrichment method.IMPORTANCESurveillance for carriage of pneumococcus is a key component of evaluating the performance of pneumococcal vaccines and informing new vaccination strategies. To improve the scalability of pneumococcal carriage surveillance, we show that molecular detection of pneumococcus in saliva from children can be performed without culture enrichment and DNA extraction. Our findings show that using the extraction-free method can improve surveillance efforts for pneumococcal carriage in children, overcoming the resource-intensive hurdle that comes with the use of molecular methods, particularly in low-resource settings.

Morphomolecular identification of heavy parasitic typhlitis in layer flocks: tissue response and cell-mediated reaction

BackgroundHeterakis gallinarum (H. gallinarum) is a common poultry parasite that can be found in the ceca of many gallinaceous bird species, causing minor pathology and reduced weight gain. Most infections go unnoticed in commercial flocks due to the dependence on fecal egg counts, which are prone to false-negative diagnoses. Furthermore, there is a lack of research on gastrointestinal nematodes that use molecular identification methods, which could be essential for rapid diagnosis and developing efficient control approaches. As a result, the study aimed to look at the cause of mortality in layer chickens induced by H. gallinarum in Egyptian poultry farms using morphological, ultrastructural, and molecular characterization. Histopathological, immunohistochemical, and cell-mediated immune responses from damaged cecal tissues were also examined.ResultsSeventy bird samples from ten-layer flocks of different breeds (Native, white, and brown layers) suffering from diarrhea, decreased egg output, and emaciation were collected. Cecal samples were collected from affected and non-affected birds and were examined for parasitic diseases using light and a scanning electron microscope. The mitochondrial cytochrome oxidase 1 (COX1) gene was used to characterize H. gallinarum. Our results showed that the collected nematodal worms were identified as H. gallinarum (male and female), further confirmed by COX1 gene amplification and sequence alignment. Gene expression analysis of the inflammatory markers in infected tissues showed a significant up-regulation of IL-2, IFN-γ, TLR-4, and IL-1β and a significant down-regulation of the anti-inflammatory IL-10. The mRNA level of the apoptotic cas-3 revealed apoptotic activity among the H. gallinarum samples compared to the control group.ConclusionsOur results implemented the use of molecular methods for the diagnosis of Heterakis, and this is the first report showing the tissue immune response following infection in layers: upregulation of IL-1β, IFN-γ, Il-2, and TLR-4, while down-regulation of anti-inflammatory IL-10 in cecal tissue, Cas-3 apoptotic activity and Nuclear factor-κB (NF-κB)activity with immunophenotyping of T-cells in Heterakis infected tissue.

Detection of Macrolide and/or Fluoroquinolone Resistance Genes in Mycoplasma genitalium Strains Isolated from Men in the Northwest Region of Croatia in 2018-2023.

Mycoplasma genitalium (M. genitalium) poses a significant public health challenge due to its association with non-gonococcal urethritis (particularly in men) and antimicrobial resistance. However, despite the prevalence of M. genitalium infections and the rise in resistance rates, routine testing and surveillance remain limited. This is the first study from Croatia that aimed to assess the prevalence and trends of resistance in M. genitalium strains isolated from male individuals by detecting macrolide and fluoroquinolone resistance genes. The study also aimed to explore the factors associated with resistance and changes in resistance patterns over time. Urine samples collected from male individuals in the Zagreb County and northwest region of Croatia between 2018 and 2023 were tested for M. genitalium with the use of molecular methods. Positive samples were subjected to DNA extraction and multiplex tandem polymerase chain reaction (MT-PCR) targeting genetic mutations associated with macrolide (23S rRNA gene) and fluoroquinolone (parC gene) resistance. Of the 8073 urine samples tested from 6480 male individuals (and following the exclusion of repeated specimens), we found that the prevalence of M. genitalium infection was 2.2%. Macrolide resistance was observed in 60.4% of strains, while fluoroquinolone resistance was found in 19.2%. Co-resistance to both antibiotics was present in 18.2% of cases. A statistically significant increase in fluoroquinolone resistance was noted over the study period (p = 0.010), but this was not evident for azithromycin resistance (p = 0.165). There were no statistically significant differences in resistance patterns between age groups, whereas re-testing of patients revealed dynamic changes in resistance profiles over time. The high burden of macrolide resistance and increasing fluoroquinolone resistance underscore the urgent need for comprehensive resistance testing and surveillance programs. The implementation of resistance-guided treatment strategies, along with enhanced access to molecular diagnostics, is pivotal for effectively managing M. genitalium infections.

Vaginal microbiota molecular profiling and diagnostic performance of artificial intelligence-assisted multiplex PCR testing in women with bacterial vaginosis: a single-center experience.

Bacterial vaginosis (BV) is a most common microbiological syndrome. The use of molecular methods, such as multiplex real-time PCR (mPCR) and next-generation sequencing, has revolutionized our understanding of microbial communities. Here, we aimed to use a novel multiplex PCR test to evaluate the microbial composition and dominant lactobacilli in non-pregnant women with BV, and combined with machine learning algorithms to determine its diagnostic significance. Residual material of 288 samples of vaginal secretions derived from the vagina from healthy women and BV patients that were sent for routine diagnostics was collected and subjected to the mPCR test. Subsequently, Decision tree (DT), random forest (RF), and support vector machine (SVM) hybrid diagnostic models were constructed and validated in a cohort of 99 women that included 74 BV patients and 25 healthy controls, and a separate cohort of 189 women comprising 75 BV patients, 30 intermediate vaginal microbiota subjects and 84 healthy controls, respectively. The rate or abundance of Lactobacillus crispatus and Lactobacillus jensenii were significantly reduced in BV-affected patients when compared with healthy women, while Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae, BVAB2, Megasphaera type 2, Prevotella bivia, and Mycoplasma hominis were significantly increased. Then the hybrid diagnostic models were constructed and validated by an independent cohort. The model constructed with support vector machine algorithm achieved excellent prediction performance (Area under curve: 0.969, sensitivity: 90.4%, specificity: 96.1%). Moreover, for subjects with a Nugent score of 4 to 6, the SVM-BV model might be more robust and sensitive than the Nugent scoring method. The application of this mPCR test can be effectively used in key vaginal microbiota evaluation in women with BV, intermediate vaginal microbiota, and healthy women. In addition, this test may be used as an alternative to the clinical examination and Nugent scoring method in diagnosing BV.

From Shadows to Spotlight: Enhancing Bacterial DNA Detection in Blood Samples through Cutting-Edge Molecular Pre-Amplification.

One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium. A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae, respectively. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates.

Enteroaggregative Escherichia coli in mid-Norway: A prospective, case control study.

The use of molecular methods has led to increased detection of Enteroaggregative Escherichia coli (EAEC) in faecal samples. Studies have yielded conflicting results regarding the clinical relevance of this finding. The objective of this study was to investigate the prevalence of EAEC in faecal samples from patients with diarrhoea and healthy controls and describe characteristics of EAEC positive persons. From March 1st, 2017 to February 28th, 2019, we investigated all consecutive faecal samples from patients with diarrhoea received at the laboratory and collected faecal samples from randomly invited healthy controls from mid-Norway. Real-time multiplex PCR was used for detection of bacterial, viral, and parasitic pathogens. We registered sex, age, urban versus non-urban residency, and travel history for all participants. Statistical analyses were performed with Pearson chi-squared test, Kruskal-Wallis test, and Mann-Whitney U test. We identified EAEC in 440 of 9487 (4.6%) patients with diarrhoea and 8 of 375 (2.2%) healthy controls. The EAEC prevalence was 19.1% among those with diarrhoea and recent foreign travel and 2.2% in those without travel history independent of diarrhoea. Concomitant pathogens were detected in 64.3% of EAEC-positive patients with diarrhoea. The median age was 28.5 in those with EAEC-positive diarrhoea and 38 in those with EAEC-negative diarrhoea (p <0.01). In patients with diarrhoea, travel was reported in 72% of those with EAEC and concomitant pathogens, and 54% and 12% in those with only EAEC and no EAEC, respectively (p <0.01). EAEC was a common detection, particularly in patients with diarrhoea and recent international travel, and was found together with other intestinal pathogens in the majority of cases. Our results suggest that domestically acquired EAEC is not associated with diarrhoea. Patients with EAEC-positive diarrhoea and concomitant pathogens were young and often reported recent travel history compared to other patients with diarrhoea.

Rational Use of Molecular Methods in the Diagnosis of Meningitis/Encephalitis

Rational Use of Molecular Methods in the Diagnosis of Meningitis/Encephalitis

Use of molecular methods for the identification of yeast species isolated from fermentations of table olives produced traditionally in Kahramanmaraş

In this study, yeast species involved in the naturally fermented green table olive produced in Southern Turkey were investigated. Table olive samples were prepared with regional olive cultivars and traditional methods were employed in the production. Yeasts were isolated from the brines of the samples at the late fermentation stage and identified at the species level by the DNA sequences of the D1/D2 domain of 26S rRNA genes. The D1/D2 domains were amplified by PCR, sequenced and compared to reference sequences deposited in the NCBI database. According to the results, isolated yeasts belonged to the Candida, Debaryomyces, and Rhodotorula genera and salt tolerant species were dominant as the salt content of the brines exceeded 11%. Among the determined species, Candida oleophila was the most dominant one and it was thought that isolated strains of Candida oleophila may be taken into consideration to be used as starter culture in table olive production.

Clinical use of molecular methods for Trypanosoma cruzi infection in endemic and non-endemic countries: Benefits, limitations and challenges

Trypanosoma cruzi infection is diagnosed by parasitological, molecular, and serological tests. Molecular methods based on DNA amplification provide a more sensitive alternative to classical parasitological techniques for detecting evidence of T. cruzi parasitemia, and are the preferred tests for congenital and oral transmission cases and parasite reactivation in chronically infected immunosuppressed individuals. In newborns at risk of vertical transmission, simplified diagnostic algorithms that provide timely results can reduce the high follow-up losses observed with current algorithms. Molecular methods have also proved useful for monitoring T. cruzi infection in solid organ transplantation recipients, regardless of host immune status, allowing parasite detection even before symptom manifestation. Furthermore, in the absence of other biomarkers and a practical test of cure, and given the limitations of serological methods, recent clinical guidelines have included polymerase chain reaction (PCR) to detect therapeutic failure after antiparasitic treatment in chronically infected adults. Increasing evidence supports the use of molecular tests in a clinical context, given the improved sensitivity and specificity of current assays – characteristics which largely depend on epidemiological factors and genetic and antigenic variability among T. cruzi strains. Further development and registration of commercial PCR kits will improve the use of molecular tests. We discuss the attributes of PCR and other molecular tests for clinical management in people with T. cruzi infection.

Genetic variation of sequencing 18srDNA gene of Trichophyton mentagrophytes isolates

Background. Trichophyton species are considered the most frequent causative and considerable agents in infection concerns. Objective. The existing training designed to conduct the sequencing analysis of nucleotides of ITS1-5.8S-ITS2 for Trichophyton mentagrophytes. Methods. The isolation and identification of pathogenic fungi, Trichophyton mentagrophytes from clinical specimens was done based on the standard morphological and molecular methods. The genomic DNA of fungal isolates were extracted and purified to amplify with primers of 18S rRNA gene for detection and sequencing the nitrogenous bases to define the genetic variation among clinical isolates of Trichophyton mentagrophytes in compared with strains recorded in NCBI GenBank. Results. Eight isolates of Trichophyton mentagrophytes were isolated and identified from clinical specimen of dermatophytosis. The DNA sequencing analysis showed the presence of some genetic variation in nitrogenous bases between the local isolates and that recorded in locus of world NCBI GenBank. Multiple Alignment sequence using NCBI BLAST revealed the recoding a new mutant isolate among the local isolates based on DNA homology percent. Conclusion. The present results proved the successful the use of molecular methods in diagnosis the fungal isolates of Trichophyton mentagrophytes especially the sequencing analysis to find the genetically bases of these isolates.

Severe viral lower respiratory tract infections in Brazilian children: Clinical features of a national cohort

BackgroundThe accurate etiological diagnosis of lower respiratory tract infections (LRTI) is essential for their effective clinical management. The extensive use of molecular methods during the COVID-19 pandemic has enabled massive data acquisition on viral lower respiratory tract infections. The current study aims to identify clinical features associated with eight viral agents among children presenting severe LRTI. Methodsretrospective cohort study of data from the Brazilian Influenza Epidemiological Surveillance Information System. Patients under 20 years-old who had severe LRTI with etiological confirmation through RT-PCR between 2020 and 2022 were included. Binary logistic regressions were used to examine associations between pathogens and symptoms. Results60,657 cases were assessed. The main viral agents detected were Sars-CoV-2 (COV2) (41.2%), Respiratory Syncytial Virus (29.1%), Human Rhinovirus (HRV) (12.1%), and Influenza (FLU) (5.5%). A general mortality rate of 4.3% was observed. The multivariate analysis evidenced that COV2 less likely presented with cough (OR: 0.34; 95%CI: 0.32–0.36), respiratory discomfort (Adjusted Odds Ratio (aOR): 0.61; 95%Confidence Interval (CI): 0.59–0.64), and desaturation (aOR: 0.71; 95%CI: 0.69–0.75). RSV strongly associated with cough (aOR: 2.59; 95%CI: 2.45–2.75) and respiratory discomfort (aOR: 1.54; 95%CI: 1.46–1.62), whereas FLU was linked to fever (aOR: 2.27; 95%CI: 2.06–2.50) and sore throat (aOR: 1.48; 95%CI: 1.34–1.64). ConclusionsThe viral agents responsible for severe LRTI have distinct associations with clinical features in children.

Microscopic, Molecular and Antigen Detection and Isolation of Cryptosporidium parvumParasites in Diarrheal Disease of Calves in Iran.

Cryptosporidiosis is a parasitic disease caused by the protozoan Cryptosporidium in vertebrates. In livestock, especially ruminants, infants develop diarrheal syndromes. The infection is common worldwide , including Iran, where it is reported in several species. Morphological diagnosis of Cryptosporidium species is associated with many limitations and has no taxonomic value on its own, so the use of molecular methods can overcome these limitations to some extent. The present aims at microscopic, molecular and antigen detection and isolation ofCryptosporidiumparvum parasites. Firstly, 300 samples were collected from different parts of Iran. Subsequently oocysts from feces were purified by the method of Casemoreet al. using the flotation technique and stained by the modified Ziehl-Neelsen method (Henriksen method) and identified by diagnostic keys. ELISA test was also performed on the samples with results ranging from 1 to 4 positive. The results of our study show that, of the 300 cases tested for Cryptosporidium, 48 cases (16%) and 54 (18%) were positive in ELISA and PCR, respectively. Microscopic evaluation also mainly confirmed the ELISA results. These cases were collected in summer, autumn, and winter, with, more than 50% of the positive cases found among the samples collected in autumn. In addition, 54 positive cases were found by PCR test, which is 6 cases more than ELISA results. Finally, the results of PCR detection and ELISA were subjected to chi-square analysis, where no significant difference was found between the collected data (p=0.0587).

TWO LIFE STRATEGIES IN COPEPOD CRYPTIC SPECIES: COEXISTENCE AND DISPLACEMENT

Among invasive species there is a special group so named cryptic species for which morphological identification is very difficult. This review is devoted to analysis of the dispersal routes of two copepod cryptic species complexes into aquatic ecosystems. Eurytemora carolleeae introduction was revealed in 2007 with bar-code. The species was described as a new taxon; its distribution was also studied using morphology. Biological invasions of two other Copepod species Acanthocyclops americanus and Eurytemora caspica were mainly studied using morphological methods since the species have already been described. At the same time, to confirm their distinctions from local forms, molecular genetic tools were also used. Two scenarios resulting from cryptic species’ invasions and their competitions with native species were the partly ( E. carrolleeae ) or full displacement ( A. americanus ). Example: the invasion of Eurytemora carolleeae into the Baltic Sea and the Atlantic coast of Europe. When assessing the possible negative impact of invasive species on competitors, the most attention should be paid to predator species. Identification of cryptic species significantly complicates the situation with the assessment of bioinvasion and needs the use of molecular methods.

New terrestrial cyanobacteria from the Azores Islands: description of Venetifunis gen. nov. and new species of Albertania, Kovacikia and Pegethrix

ABSTRACT The taxonomy of cyanobacteria has advanced quickly with the use of molecular methods in combination with well-defined morphological and ecological characters. Using this approach, many taxonomical changes have occurred in the Synechococcales and Oscillatoriales, with the description of new families, genera and species. Here we present the study of four cyanobacterial strains from the Bank of Algae and Cyanobacteria of the Azores (BACA) culture collection, all isolated from the Azores Archipelago. The strains were genetically characterized through the amplification of the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer (ITS) region, as well as morphologically by light and transmission electron microscopy. One of the strains presented a high genetic divergence from known genera of Synechococcales and was described as Venetifunis florensis gen. & sp. nov. (Trichocoleusaceae, Cyanobacteria). The remaining three strains were found to be new species of the genera Albertania, Kovacikia and Pegethrix. Venetifunis florensis was isolated from the shores of Lake Rasa in Flores Island, Kovacikia atmophytica sp. nov. is a new species mainly distinguished genetically from other Kovacikia species from Terceira Island. Albertania obscura sp. nov. was isolated from inside a volcanic cave in Terceira Island and is distinct both morphologically and genetically from the already described species. Pegethrix atlantica sp. nov. is a new morphologically and genetically different species isolated from São Miguel Island. The description of these new taxa increases the biogeographic knowledge of the four mentioned cyanobacterial genera to remote oceanic islands and new terrestrial habitats.

Effective Use of microRNA, BRAF and Sonographic Risk Assessment in Bethesda III Thyroid Nodules Requires a Different Approach to Nodules with Features of Nuclear Atypia and Other Types of Atypia.

The aim of the study was to analyze the diagnostic usefulness of the combined assessment of the ultrasound risk category of the nodule (evaluated with EU-TIRADS system), the presence of BRAF V600E mutation and the expression of selected microRNAs (miR-146b, miR-221 and miR-222) in Bethesda category III thyroid nodules, separately for cases with nuclear atypia (AUS-nuclear) and cases with other types of atypia (AUS-other). We evaluated 161 nodules (66 AUS-nuclear and 95 AUS-other) with known results of postoperative histopathological examination. The rate of cancer and the rate of PTC among cancers were nearly three times higher in the AUS-nuclear than the AUS-other group. For AUS-nuclear nodules, the most effective diagnostic panel included, in addition to repeat FNA, the assessment of BRAF V600E mutation and the expression of miR-146b and miR-222 (sensitivity: 93.5%, specificity: 80.0%). For AUS-other nodules, a two-step procedure was most effective: at the first stage, forgoing surgical treatment in subjects with a benign repeat FNA outcome, and, at the second stage, the assessment of miR-222 expression and the EU-TIRADS category (sensitivity: 92.3%, specificity: 76.8%). The optimal use of molecular methods in the diagnostics of category III thyroid nodules requires a separate approach for nodules with nuclear atypia and nodules with other types of atypia.

Molecular tools prove little auks from Svalbard are extremely selective for Calanus glacialis even when exposed to Atlantification

Two Calanus species, C. glacialis and C. finmarchicus, due to different life strategies and environmental preferences act as an ecological indicators of Arctic Atlantification. Their high lipid content makes them important food source for higher trophic levels of Arctic ecosystems including the most abundant Northern Hemisphere's seabird, the little auk (Alle alle). Recent studies indicate a critical need for the use of molecular methods to reliably identify these two sympatric Calanus species. We performed genetic and morphology-based identification of 2600 Calanus individuals collected in little auks foraging grounds and diet in summer seasons 2019–2021 in regions of Svalbard with varying levels of Atlantification. Genetic identification proved that 40% of Calanus individuals were wrongly classified as C. finmarchicus according to morphology-based identification in both types of samples. The diet of little auks consisted almost entirely of C. glacialis even in more Atlantified regions. Due to the substantial bias in morphology-based identification, we expect that the scale of the northern expansion of boreal C. finmarchicus may have been largely overestimated and that higher costs for birds exposed to Atlantification could be mostly driven by a decrease in the size of C. glacialis rather than by shift from C. glacialis to C. finmarchicus.

Cryptic species Hydatigera kamiyai and other taeniid metacestodes in the populations of small mammals in Serbia

BackgroundHydatigera (Cestoda: Taeniidae) is a recently resurrected genus with the description of a new species, Hydatigera kamiyai, a cryptic entity within the Hydatigera taeniaeformis species complex. Rodents are intermediate hosts and correct taxonomic identification of H. taeniaeformis sensu lato (s.l.) species is difficult without the use of molecular methods. The aim of this study was to identify and explore the genetic diversity of Hydatigera and other taeniid species.MethodsTen different small mammals species (856 individuals) (Rattus rattus, three Apodemus, three Arvicolinae and three Soricidae species) were examined from 2013 to 2023. Captured animals were visually examined for cysts and visible lesions. Two markers were used for amplification and sequencing: cox1 and 12S rDNA.ResultsMolecular analysis of cysts and visible lesions revealed four taeniid species: Hydatigera kamiyai, H. taeniaeformis sensu stricto (s.s.), Taenia martis and T. crassiceps. Hydatigera kamiyai was found in Apodemus flavicollis, A. agrarius, Microtus arvalis and Crocidrua leucodon, while H. taeniaeformis s.s. is registered in R. rattus. Hydatigera kamiyai cox1 sequences clustered with European populations and showed at least 25 nucleotid differences compared to Asian, African, Australian and one of our isolates of H. taeniaeformis s.s acquired from a rat, followed by large sequence distances (9.4% to 12.9%), indicating clear molecular distinction of two species.ConclusionsThis is one of the few mitochondrial gene-based studies performed after the description of cryptic entities within the Hydatigera taeniaeformis s.l. complex and represents a valuable contribution to understanding of genetic diversity, host suitability and geographic distribution of these tapeworm species. Also, our study provides an important basis of molecular data from this part of Europe for further studies. We emphasize the importance of additional studies of intermediate hosts, especially rats from Europe and Apodemus spp. and voles from Asia and Africa.Graphical abstract

Advances in Molecular Techniques for Developing Drought-Tolerant Maize (Zea Mays L.)

Drought is one of the major limiting factors in maize (Zea mays L.) production, causing significant yield losses globally. Breeding for drought tolerance has been a key strategy in increasing maize yield in drought-prone areas. The use of molecular methods in maize breeding has facilitated the identification of genes and quantitative trait loci (QTL) associated with drought tolerance. This review summarizes the progress made in using molecular methods to identify and utilize genetic improvements in drought-tolerant maize. It covers various molecular methods used in identifying drought tolerance genes and QTLs, and the application of these findings in marker-assisted selection (MAS) and genomic selection (GS). The review also highlights the potential of CRISPR-Cas9 genome editing to improve drought tolerance in maize.