Search Results (102)

This study aimed to identify genetic variants using a customized next-generation sequencing (NGS) panel for pediatric myelodysplastic syndrome (pMDS) and to explore their associations with cytogenetic and clinical characteristics. Cytogenetic analyses were conducted using G-banding and fluorescence in situ hybridization. NGS was performed with the Ion Torrent Personal Genome Machine for the following genes: GATA2, RUNX1, CEBPA, ANKRD26, ETV6, SAMD9, SAMD9L, PTPN11, NRAS, SETBP1, DDX41, TP53, FLT3, SRP72, and JAK3. Analyses were performed with Ion Reporter 5.20.8.0 software. Genetic variants were classified using the dbSNP, 1000 Genomes, COSMIC, and Varsome databases. We analyzed 25 cases of pMDS; 15 presented abnormal karyotypes, and 19 showed genetic variants. Among the 29 variants identified across 12/15 genes, 27% were pathogenic and 14% were likely pathogenic, with NRAS and GATA2 most frequently associated with disease progression. A new somatic variant of uncertain significance in SETBP1 was detected in seven patients showing heterogeneous clinical outcomes. Genetic variants were found in 7/10 patients with normal karyotypes, indicating that submicroscopic alterations can shed light on disease biology. Our results highlight the critical role of a targeted NGS panel in identifying molecular alterations associated with pMDS pathogenesis, thereby enhancing diagnostic precision, prognosis, and aiding in treatment selection. Full article

A backyard cat with symptoms of otitis was transferred to a veterinary clinic in Central Greece. A sample was obtained and P. aeruginosa was isolated. The strain exhibited an extensively drug-resistant (XDR) profile, as it was non-susceptible to all tested agents except colistin. DNA extraction and whole-genome sequencing (WGS) were performed using a robotic extractor and Ion Torrent technology, respectively. The genome was assembled and screened for resistance and virulence determinants. The isolate belonged to the high-risk clone ST308 with a total of 67 antibiotic resistance genes (ARGs) and 221 virulence factor-related genes being identified. No plasmids were detected. The metallo-beta-lactamase (MBL) bla_NDM-1 gene and 46 efflux pumps were included in the strain’s resistome. Both ARGs conferring tolerance to disinfecting agents and biofilm-related genes were identified, associated with the ability of this clone to adapt and persist in healthcare facilities. This case highlights the risk of relevant bacterial clones spreading in the community and even being transmitted to companion animals, causing challenging opportunistic infections to susceptible individuals, while others may become carriers, further spreading the clones to their owners, other animals and the environment. Full article

Fungal communities can be used as indicators of various environmental processes in forest ecosystems. The diversity of these communities is linked to aboveground plants and soil properties. We assessed fungal diversity at four Norway spruce sampling sites that were growing on fertile mineral soils (Oxalidosa) in northwestern Latvia. Three sites were managed—a three-year-old clear-cut and fifty- and eighty-five-year-old stands; one site was unmanaged—a naturally regenerated site after wind damage in 1969. For metabarcoding, we used a fungal internal transcribed spacer (ITS2) and high throughput sequencing with the Ion Torrent platform. Our results showed high operational taxonomic unit richness in the samples, with notable variation in community composition between individual plots both within and among sites, with the highest being in managed, middle-aged stands and the lowest in unmanaged. Significant differences in the diversity of soil fungal communities were not detected between the sites. Redundancy analysis indicated that pH, soil organic matter, organic carbon, and nitrogen were the most important soil variables that explained the variation in fungal communities. The unmanaged stand differed notably by community composition. This study highlights the importance of monitoring forest soil environmental parameters and fungal communities to gain a more comprehensive assessment of forestry management regimes. Full article

Background/Objectives: Acinetobacter baumannii is an increasingly significant nosocomial pathogen causing severe infections globally. The emergence of multidrug-resistant A. baumannii strains has raised concerns about the efficacy of current treatment options. This study aimed to investigate the molecular epidemiology and antimicrobial resistance patterns of A. baumannii isolates from Kazakhstan. Methods: We collected nine A. baumannii isolates in 2022–2023 in Karaganda, Kazakhstan, which were then subjected to whole-genome sequencing (WGS) using the IonTorrent platform for genome characterization. Multilocus sequence typing (MLST) was used to classify the isolates into distinct clonal complexes. In addition, antibiotic susceptibility testing was conducted using the standard methods for a range of antibiotics commonly used against A. baumannii. Results: Our results revealed a high degree of genomic diversity among isolates from Kazakhstan, with multiple distinct classes identified: ST78 (n = 4, 44.4%), ST15 (n = 2, 22.2%), ST2 (n = 2, 22.2%), and ST193 (n = 1, 11%). MLST analysis showed that ST78^Pas/1104^Oxf (harboring blaOXA-72 and blaOXA-90 genes) were prevalent among the multidrug-resistant isolates. Based on the results of MLST, KL, and OCL, the analyzed isolates were assigned to specific international clones: IC2—ST2(Pas)-KL2/168-OCL1, IC4—ST15(Pas)-KL9-OCL7, and IC6—ST78(Pas)-KL49-OCL1. Notably, these isolates exhibited resistance to multiple antibiotics including meropenem, imipenem, gentamicin, amikacin, and ciprofloxacin. Conclusions: This study highlighted the complex molecular epidemiology of A. baumannii in Kazakhstan over a two-year period, underscoring the need for targeted surveillance strategies to monitor antimicrobial resistance patterns. The emergence and dissemination of multidrug-resistant strains within this timeframe emphasizes the importance of whole-genome sequencing as a diagnostic tool and underscores the challenges posed by these infections. Full article

Diagnosis of biliopancreatic cancers by the available serum tumor markers, imaging, and histopathological tissue specimen examination remains a challenge. Circulating cell-free DNA derived from matched pairs of secretin-stimulated duodenal fluid (DF) and plasma from 10 patients with biliopancreatic diseases and 8 control subjects was analyzed using AmpliSeq™ HD technology for Ion Torrent Next-Generation Sequencing to evaluate the potential of liquid biopsy with DF in biliopancreatic cancers. The median cfDNA concentration was greater in DF-derived than in plasma-derived samples. A total of 13 variants were detected: 11 vs. 1 were exclusive for DF relative to the plasma source, and 1 was shared between the two body fluids. According to the four-tier systems, 10 clinical tier-I–II (76.9%), 1 tier–III (7.7%), and 2 tier–IV (15.4%) variants were identified. Notably, the 11 tier-I-III variants were exclusively found in DF-derived cfDNA from five patients with biliopancreatic cancers, and were detected in seven genes (KRAS, TP53, BRAF, CDKN2A, RNF43, GNAS, and PIK3CA); 82% of the tier-I-III variants had a low abundance, with a VAF < 6%. The mutational profiling of DF seems to be a reliable and promising tool for identifying cancer-associated alterations in malignant cancers of the biliopancreatic tract. Full article

We sequenced and analyzed the complete mitogenome of a Norwegian isolate of the octocoral Alcyonium digitatum using the Ion Torrent sequencing technology. The 18,790 bp circular mitochondrial genome was found to harbor the same set of 17 genes, which encode 14 protein subunits, two structural ribosomal RNAs and one tRNA, as reported in other octocorals. In addition, we detected a new tRNA^Pro-like gene sequence nested within the MutS protein coding region. This putative tRNA gene feature appears to be conserved among the octocorals but has not been reported previously. The A. digitatum mitogenome was also shown to harbor an optional gene (ORFA) that encodes a putative protein of 191 amino acids with unknown function. A mitogenome-based phylogenetic analysis, presented as a maximum likelihood tree, showed that A. digitatum clustered with high statistical confidence with two other Alcyonium species endemic to the Mediterranean Sea and the Southeast Pacific Ocean. Full article

Background: Irritable bowel syndrome (IBS) is a common condition that affects the lifestyle of patients. It is associated with significant changes in the composition of the gut microbiome, but the underlying microbial mechanisms remain to be fully understood. We study the fecal microbiome of patients with constipation-predominant IBS (IBS-C) and mixed-type IBS (IBS-M). Methods: We sequenced the V3 region of the 16S rRNA on the Ion Torrent PGM sequencing platform to study the microbiome. Results: In the patients with IBS-C and IBS-M, an increase in alpha diversity was found, compared to the healthy group, and differences in beta diversity were also noted. At the phylum level, both IBS subtypes showed an increase in the Firmicutes/Bacteroidetes ratio, as well as an increase in the abundance of Actinobacteria and Verrucomicrobiota. Changes in some types of bacteria were characteristic of only one of the IBS subtypes, while no statistically significant differences in the composition of the microbiome were detected between IBS-C and IBS-M. Conclusions: This study was the first to demonstrate the association of Turicibacter sanguinis, Mitsuokella jalaludinii, Erysipelotrichaceae UCG-003, Senegalimassilia anaerobia, Corynebacterium jeikeium, Bacteroides faecichinchillae, Leuconostoc carnosum, and Parabacteroides merdae with IBS subtypes. Full article

We conducted a pilot study to analyze the microbiome in cervical samples of women living with HIV with various profiles of HPV infections. The participants had an average age of 41.5 years. Sequence analysis of 16S rRNA V3 gene amplicons was performed using next-generation sequencing technology (Ion Torrent PGM^TM). The bioinformatics pipeline was analyzed using the Find, Rapidly, OTUs with Galaxy Solution system (FROGS). Common genera were determined to identify Community State Types (CSTs). The cervical microbiome profiles showed a dominance of lactobacilli in 56% (five out of nine) of samples. All three women with normal cervical cells and high-risk HPV infection were classified as CST IV, characterized by anaerobic bacteria associated with bacterial vaginitis, such as Gardnerella, Prevotella, Atopobium, and Sneathia. Among the two women with abnormal cervical cells and high-risk HPV infection, one was classified as CST III, and the other had an unclassified profile dominated by L. helveticus. Four women with normal cervical cells and no HPV infection exhibited various CSTs. Our study demonstrated the feasibility of the protocol in analyzing the cervical microbiome. However, further analysis with a larger number of longitudinal samples is necessary to determine the role of cervical microbiota in HPV persistence, clearance, or the development of precancerous lesions. Full article

Colorectal cancer (CRC) outcomes in terms of incidence and mortality are significantly worse in African Americans than other Americans. While differences in primary preventions for neoplasia (diet, obesity remediation, aspirin prophylaxis) are being elucidated, genetic mutations affecting premalignant lesions and immune response mechanisms may possibly also explain the increased incidence and mortality, particularly from right-sided disease. Objective: Our team therefore examined colonic segments seeking to test the hypothesis that the immune response and somatic genetic profiles of the colonic anatomic segments may vary and thus account for variations in neoplasia risk among the various colonic segments revealing an antigenic relationship with precancerous lesions. The p87 antigenic field effect is recognized via Adnab-9 antibody immunohistochemistry to be significantly less in the right colon in African Americans, particularly in the cecum. Method: Since small high-grade dysplastic adenomas (SHiGDA) likely missed by CRC screening may progress to cancer, we used Ion Torrent™ sequencing of DNA extracted from four normal colonic segments (two left-sided and two right) of patients with SHiGDAs. We also contrasted unique mutational fields in one patient with a large HiGDA (APC with unique mutations) and one patient who prospectively developed a SHiGDA (JAK3). Result: The SHiGDA (small high-grade dysplastic polyp) patient was p87 negative for any extracted stool, saliva, or colonic effluent via ELISA (enzyme linked immunoadsorbant assay). Furthermore, mean values of expression in segments from the right colon were reduced with respect to the means obtained from the left segments in 233 patients evaluated for a p87 field effect. This has recently been shown to be the case in a large cohort of AA and Caucasian 2294 patients, possibly explaining the right-sided CRC disparity in African Americans and the subsequent increase in mortality. This field effect disparity is also true for two cancers contracted by the SHiGDa patient (lung and prostate). Conclusion: Thus, this pilot study suggests that the reduction in p87 in the right colon is possibly correlated with JAK3 mutations. If confirmed, JAK3 mutations, known to be associated with immune aberrations, may provide a mechanistic explanation for the lack of a p87 (protein 87 kilodaltons) field in some patients with HGD polyps who might benefit from possible intervention such as more intensive screening. Limited microbiome studies were also performed on two patients with familial cancer syndromes and these compared favorably with controls available from the literature. Full article

Multiple sclerosis (MS) arises from a complex interplay between host genetic factors and environmental components, with the gut microbiota emerging as a key area of investigation. In the current study, we used ion torrent sequencing to delve into the bacteriome (bacterial microbiota) and mycobiome (fungal microbiota) of people with MS (pwMS), and compared them to healthy controls (HC). Through principal coordinate, diversity, and abundance analyses, as well as clustering and cross-kingdom microbial correlation assessments, we uncovered significant differences in the microbial profiles between pwMS and HC. Elevated levels of the fungus Torulaspora and the bacterial family Enterobacteriaceae were observed in pwMS, whereas beneficial bacterial taxa, such as Prevotelladaceae and Dialister, were reduced. Notably, clustering analysis revealed overlapping patterns in the bacteriome and mycobiome data for 74% of the participants, with weakened cross-kingdom interactions evident in the altered microbiota of pwMS. Our findings highlight the dysbiosis of both bacterial and fungal microbiota in MS, characterized by shifts in biodiversity and composition. Furthermore, the distinct disease-associated pattern of fungi–bacteria interactions suggests that fungi, in addition to bacteria, contribute to the pathogenesis of MS. Overall, our study sheds light on the intricate microbial dynamics underlying MS, paving the way for further investigation into the potential therapeutic targeting of the gut microbiota in MS management. Full article

Sex-specific discrepancies in bladder cancer (BCa) are reported, and new studies imply that microbiome may partially explain the diversity. We aim to provide characterization of the bladder microbiome in both sexes diagnosed with non-muscle-invasive BCa with specific insight into cancer grade. In our study, 16S rRNA next-generation sequencing was performed on midstream urine, bladder tumor sample, and healthy-appearing bladder mucosa. Bacterial DNA was isolated using QIAamp Viral RNA Mini Kit. Metagenomic analysis was performed using hypervariable fragments of the 16S rRNA gene on Ion Torrent Personal Genome Machine platform. Of 41 sample triplets, 2153 taxa were discovered: 1739 in tumor samples, 1801 in healthy-appearing bladder mucosa and 1370 in midstream urine. Women were found to have smaller taxa richness in Chao1 index than men (p = 0.03). In comparison to low-grade tumors, patients with high-grade lesions had lower bacterial diversity and richness in urine. Significant differences between sexes in relative abundance of communities at family level were only observed in high-grade tumors. Full article

Acute respiratory diseases in felines can be attributed to a diverse range of pathogens. The recent emergence of novel viruses, particularly SARS-CoV-2 and its variants, has also been associated with respiratory ailments in cats and other pets, underscoring the need for a highly sensitive diagnostic assay capable of concurrently detecting multiple respiratory pathogens. In this study, we developed a targeted next generation sequencing panel using Ion Torrent Ampliseq technology to detect multiple respiratory pathogens, including recent SARS-CoV-2 variants and Feline herpesvirus-1, Feline calicivirus, Bordetella bronchiseptica, Mycoplasmopsis (previously Mycoplasma) felis, and Chlamydia felis. A PCR amplification-based library preparation, employing primers designed for pathogen target regions, was synthesized and divided into two pools, followed by sequencing and assembly to a repertoire of target pathogen genomes. Analytical sensitivity was assessed based on Ct values from real-time PCR for the corresponding pathogens, indicating an equivalent detection limit. Most of the pathogens under study were positively identified to a limit of approximately Ct 36, whereas for Feline herpesvirus-1 and SARS-CoV-2, positive reads were observed in samples with a Ct of 37. Based on a limited number of samples, the diagnostic sensitivity values for the SARS-CoV-2, Feline herpesvirus-1, and M. felis samples were 100% with no false negative results. The diagnostic specificity of SARS-CoV-2, Feline herpesvirus-1, Feline calicivirus, and C. felis were 100%. Importantly, none of the target primers exhibited non-specific amplification, ensuring the absence of false positive results for other pathogens within the study. Additionally, the assay’s specificity was validated by cross-referencing the raw sequencing data with established databases like BLAST, affirming the high specificity of the targeted Next-Generation Sequencing (tNGS) assay. Variations in the sequencing reads of different pathogens were observed when subjected to diverse extraction methods. Rigorous assessment of the assay’s reliability involved reproducibility across testing personnel and repeated runs. The developed assay’s clinical applicability was tested using samples submitted to the diagnostic laboratory from cat shelters and suspected cases. The developed targeted next-generation sequencing methodology empowers the detection of multiple respiratory pathogens manifesting similar clinical symptoms while offering confirmation of results through genome sequencing. Full article

The application of bacterial metagenomic analysis as a biomarker for cancer detection is emerging. Our aim was to discover gut microbiota signatures with potential utility in the diagnosis of colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). A prospective study was performed on a total of 77 fecal samples from CRC and NSCLC patients and controls. DNA from stool was analyzed for bacterial genomic sequencing using the Ion Torrent™ technology. Bioinformatic analysis was performed using the QIIME2 pipeline. We applied logistic regression to adjust for differences attributable to sex, age, and body mass index, and the diagnostic accuracy of our gut signatures was compared with other previously published results. The feces of patients affected by different tumor types, such as CRC and NSCLC, showed a differential intestinal microbiota profile. After adjusting for confounders, Parvimonas (OR = 53.3), Gemella (OR = 6.01), Eisenbergiella (OR = 5.35), Peptostreptococcus (OR = 9.42), Lactobacillus (OR = 6.72), Salmonella (OR = 5.44), and Fusobacterium (OR = 78.9) remained significantly associated with the risk of CRC. Two genera from the Ruminococcaceae family, DTU089 (OR = 20.1) and an uncharacterized genus (OR = 160.1), were associated with the risk of NSCLC. Our two panels had better diagnostic capacity for CRC (AUC = 0.840) and NSLC (AUC = 0.747) compared to the application of two other published panels to our population. Thus, we propose a gut bacteria panel for each cancer type and show its potential application in cancer diagnosis. Full article

Next-generation sequencing is a vital tool for personalized diagnostics and therapies in cancer. Despite numerous advantages, the method depends on multiple parameters regarding the sample material, e.g., sample fixation. A panel’s ability to ensure balanced pre-amplification of the regions of interest is challenging, especially in targeted sequencing approaches, but of significant importance to its applicability across hematological malignancies and solid tumors. This study comparatively evaluated the technical performance of the commercially available Oncomine^TM Myeloid Panel in fresh and Formalin-fixed paraffin-embedded (FFPE) material by using an Ion Torrent™ Personal Genome Machine™ System and Ion GeneStudio S5 System platform. In total, 114 samples were analyzed, including 55 fresh materials and 59 FFPE samples. Samples were sequenced with a minimum of one million reads. Amplicons with coverage below 400 reads were classified as underperforming. In fresh material, 49/526 amplicons were identified as performing insufficiently, corresponding with 18 genes. Using FFPE material, 103/526 amplicons underperformed. Independent of input material, regions in 27 genes, including ASXL1, BCOR and BRAF, did not match quality parameters. Subsequently, exemplary mutations were extracted from the Catalogue of Somatic Mutations in Cancer database. This technical evaluation of the Oncomine^TM Myeloid Panel identified amplicons that do not achieve adequate coverage levels and which need to be considered when interpreting sequencing. Full article

Genotypic testing is often recommended to improve the management of patients infected with human immunodeficiency virus (HIV). To help combat this major pandemic, next-generation sequencing (NGS) techniques are widely used to analyse resistance to antiretroviral drugs. In this study, we used a Vela Sentosa kit (Vela Diagnostics, Kendall, Singapore), which is usually used for the Ion Torrent personal genome machine (PGM) platform, to sequence HIV using the Illumina Miseq platform. After RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR), minor modifications were applied to the Vela Sentosa kit to adapt it to the Illumina Miseq platform. Analysis of the results showed the same mutations present in the samples using both sequencing platforms. The total number of reads varied from 185,069 to 752,343 and from 642,162 to 2,074,028 in the Ion Torrent PGM platform and the Illumina Miseq platform, respectively. The average depth was 21,955 and 46,856 for Ion Torrent PGM and Illumina Miseq platforms, respectively. The cost of sequencing a run of eight samples was quite similar between the two platforms (about USD 1790 for Illumina Miseq and about USD 1833 for Ion Torrent PGM platform). We have shown for the first time that it is possible to adapt and use the Vela Sentosa kit for the Illumina Miseq platform to obtain high-quality results with a similar cost. Full article