Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota.
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ABSTRACT:
SUBMITTER: Shibata T
PROVIDER: S-EPMC8404996 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cervicitis is a common clinical finding often attributed to sexually transmitted infections (STIs), but no etiologic agent is identified in the majority of cases. In this study, we comparatively assessed inflammation among the common infectious etiologies of cervicitis and assessed the potential value of liquid cytology specimens for predicting STIs. Among 473 Louisiana women at low risk for acquiring STIs, the prevalences of Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis in liquid-based cytology specimens were 1.5, 2.1, 0.6, and 4.4%, respectively. N. gonorrhoeae and human papillomavirus 18 (HPV18) infections were significantly more common among subjects infected with M. genitalium. Using direct microscopy, we observed significant increases in leukocyte infiltrates among subjects with monoinfections with M. genitalium or C. trachomatis compared to women with no detectable STIs. Inflammation was highest among subjects with M. genitalium. Using a threshold of ≥ 2 leukocytes per epithelial cell per high-powered field, the positive predictive values for M. genitalium, C. trachomatis, N. gonorrhoeae, and T. vaginalis were 100, 70, 67, and 20%, respectively. Several novel M. genitalium genotypes were identified, all of which were predicted to be susceptible to macrolide antibiotics, suggesting that different strains may circulate among low-risk women and that macrolide resistance is substantially lower than in high-risk populations. This study highlights the capacity of M. genitalium to elicit cervical inflammation and, considering the strong epidemiologic associations between M. genitalium and human immunodeficiency virus (HIV), provides a potential mechanism for acquisition and shedding of HIV via chronic leukocyte recruitment to the cervical mucosa.
| S-EPMC4097708 | biostudies-literature
Project description:Accurate detection of wildlife pathogens is critical in wildlife disease research. False negatives or positives can have catastrophic consequences for conservation and disease-mitigation decisions. Quantitative polymerase chain reaction is commonly used for molecular detection of wildlife pathogens. The reliability of this method depends on the effective extraction of the pathogen's DNA from host samples. A wildlife disease that has been in the centre of conservationist's attention is the amphibian disease Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Here, we compare the efficiency of a spin column extraction kit (QIAGEN), commonly used in Bd DNA extraction, to an alternative spin column kit (BIOKÈ) used in extractions from other types of samples, which is considerably cheaper but not typically used for Bd DNA extraction. Additionally, we explore the effect of an enzymatic pre-treatment on detection efficiency. Both methods showed similar efficiency when extracting Bd DNA from zoospores from laboratory-created cell-cultures, as well as higher efficiency when combined with the enzymatic pre-treatment. Our results indicate that selecting the optimal method for DNA extraction is essential to ensure minimal false negatives and reduce project costs.
| S-EPMC8529346 | biostudies-literature
Project description:The link between the gut microbiota and the human physiological state has been demonstrated in recent years. High gut microbiota diversity has been linked to many beneficial functions necessary or human health, while dysbiosis has been correlated to different pathological states. In this context, the study of the gut microbiota results of high relevance been necessary the development of different techniques capable of characterizing this complex ecosystem. Metaproteomics has been proved useful in the characterization of complex protein samples becoming a suitable tool for the study of these microbial communities. However, due to the complexity of these samples, protein extraction protocols may affect metaproteomics results. In this context, we evaluated stool sample processing (SSP) and microbial cell disruption, assessing the impact of different protocol modifications in the number of peptides and proteins identified. We compared different stool processing conditions and microbial cell disruption methods in terms of protein and peptide identifications and taxonomic profiles.
2021-07-06 | PXD025659 | Pride
Project description:BackgroundLiquid-based cytology (LBC) is a widely used method for processing specimens obtained by endoscopic biopsy. This study evaluated next-generation sequencing (NGS) analysis of LBC specimens to improve the diagnostic accuracy of pancreatic lesions.MethodsUpon the diagnosis of a suspected pancreatic mass, LBC residues were used retrospectively. The quantity and quality of DNA extracted from residual LBC samples were evaluated, and an NGS analysis targeting 6 genes (KRAS, GNAS, TP53, CDKN2A, SMAD4, and PIK3CA) was performed.ResultsThe library was prepared from LBC specimens taken from 52 cases: 44 were successful, and 8 preparations failed. An analysis of DNA quantity and quality suggested that the success or failure of NGS implementation depended on both properties. The final diagnosis was achieved by a combination of the pathological analysis of the surgical excision or biopsy material with clinical information. Among the 33 cases of pancreatic ductal adenocarcinoma (PDAC), KRAS, TP53, CDKN2A, and SMAD4 mutations were identified in 31 (94%), 16 (48%), 3 (9%), and 2 (6%), respectively. Among the 11 benign cases, only a KRAS mutation was identified in 1 case. On the basis of NGS results, 18 of 33 PDACs (55%) were classified as highly dysplastic or more, and 10 of 11 benign lesions were evaluated as nonmalignant, which was consistent with the final diagnosis.ConclusionsNGS analysis using LBC specimens from which DNA of appropriate quantity and quality has been extracted could contribute to improving the assessment of pancreatic tumor malignancies and the application of molecular-targeted drugs.
| S-EPMC9297882 | biostudies-literature
Project description:BackgroundLiquid-based cytology (LBC) is now a widely used method for cytologic screening and cancer diagnosis. Since the cells are fixed with alcohol-based fixatives, and the specimens are stored in a liquid condition, LBC specimens are suitable for genetic analyses.MethodsHere, we established a small cancer gene panel, including 60 genes and 17 microsatellite markers for next-generation sequencing, and applied to residual LBC specimens obtained by endometrial cancer screening to compare with corresponding formalin-fixed paraffin-embedded (FFPE) tissues.ResultsA total of 49 FFPE and LBC specimens (n = 24) were analyzed, revealing characteristic mutations for endometrial cancer, including PTEN, CTNNB1, PIK3CA, and PIK3R1 mutations. Eight cases had higher scores for both tumor mutation burden (TMB) and microsatellite instability (MSI), which agree with defective mismatch repair (MMR) protein expression. Paired endometrial LBC, and biopsied and/or resected FFPE tissues from 7 cases, presented almost identical mutations, TMB, and MSI profiles in all cases.ConclusionThese findings demonstrate that our ad hoc cancer gene panel enabled the detection of therapeutically actionable gene mutations in endometrial LBC and FFPE specimens. Endometrial cancer LBC specimens offer an alternative and affordable source of molecular testing materials.
| S-EPMC7353725 | biostudies-literature
Project description:BACKGROUND:The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly. METHODS:Fecal samples from healthy adults were stored at -80°C as such or diluted in RNAlater® and subjected to 2 extraction protocols with mechanical lysis: the Powersoil® MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote® (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (http://shaman.pasteur.fr/). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance. RESULTS:We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater® impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon. CONCLUSION:Our results showed that RNAlater® moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater® remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction.
| S-EPMC6075747 | biostudies-literature
Project description:Gastric-type cervical adenocarcinoma (GCA) is a human papillomavirus-unassociated, aggressive, chemorefractory tumor. Well-differentiated examples may exhibit bland morphologic appearances, which could potentially lead to misdiagnosis, particularly in limited material. We sought to characterize the morphologic features of GCA in surgical biopsy and cytology specimens. We identified patients with histologic diagnoses of GCA or minimal-deviation adenocarcinoma between 2004 and 2017. Available slides from biopsy, curettage, and cytology specimens were reviewed. Fifty-nine specimens (37 histology, 22 cytology) were reviewed from 23 patients, including histology specimens alone from 6 patients, cytology specimens alone from 4 patients, and both types of specimen from 13 patients. The median patient age was 52?yr (range, 29-83?yr). Biopsies showed well-to-moderately differentiated adenocarcinomas composed of cells with pale or foamy cytoplasm and well-defined cytoplasmic borders. Nuclei exhibited mild-to-moderate pleomorphism with small nucleoli. The diagnosis was challenging in a minority of biopsies in which neoplastic glandular epithelium was scant, fragmented, and/or well differentiated. Cytology slides showed single and crowded clusters of tumor cells with pale, foamy, and/or vacuolated cytoplasm and well-defined cytoplasmic borders. Nuclei were moderately pleomorphic, round to oval with one or more nucleoli. Of 20 submitted biopsies, GCA was suspected by the submitting pathologist in only 5 (25%) cases. Awareness of the morphologic features and use of confirmatory ancillary studies (eg, immunohistochemistry for markers of gastric differentiation and human papillomavirus testing) will allow accurate diagnosis of these aggressive tumors in biopsy and cytology specimens.
| S-EPMC6230321 | biostudies-literature
Project description:The aim of this study is to evaluate different options for introducing liquid-based cytology (LBC) and human papillomavirus (HPV) testing into the UK cervical cancer screening programme. These include options that incorporate HPV testing either as a triage for mild and borderline smear abnormalities or as a primary screening test. Outcomes include the predicted impact on resource use, total cost, life years and cost-effectiveness. Extensive sensitivity analysis has been carried out to explore the importance of the uncertainty associated with disease natural history and the impact of screening. Under baseline assumptions, the cost-effectiveness of different options for introducing LBC appears favourable, and these results are consistent under a range of assumptions for its impact on the diagnostic effectiveness of cytology. However, if we assume a higher marginal cost of LBC in comparison to conventional methods, primary smear testing options are predicted to be more cost-effective without LBC. Combined LBC primary smear and HPV testing with a 5-year interval is similar in both cost and effectiveness to the other 3-yearly options of primary smear testing or primary HPV testing alone. However, both primary HPV testing and combined options would give rise to a far greater risk of inappropriate colposcopy throughout a woman's lifetime. British Journal of Cancer (2004) 91, 84-91. doi:10.1038/sj.bjc.6601884 www.bjcancer.com Published online 25 May 2004
| S-EPMC2364742 | biostudies-other
Project description:Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material "spiked" with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination.IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.
| S-EPMC6821930 | biostudies-literature
Project description:Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, due to the complexity of soil samples the exhaustive extraction of proteins is a major challenge. We compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types. Four different protein extraction procedures were applied based on (a) SDS extraction without phenol, (b) NaOH and subsequent phenol extraction, (c) SDS-phenol extraction and (d) SDS-phenol extraction with prior washing steps. To assess the suitability of these methods for the functional analysis of the soil metaproteome, they were applied to a potting soil high in organic matter and a forest soil. Proteins were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) and the number of unique spectra as well as the number of assigned proteins for each of the respective protocols was compared. In both soil types, extraction with SDS-phenol (c) resulted in "high" numbers of proteins. Moreover, a spiking experiment was conducted to evaluate protein recovery. To this end sterilized forest soil was amended with proteins from pure cultures of Pectobacterium carotovorum and Aspergillus nidulans. The protein recovery in the spiking experiment was almost 50%. Our study demonstrates that a critical evaluation of the extraction protocol is crucial for the quality of the metaproteomics data, especially in highly complex samples like natural soils.
| S-EPMC3413887 | biostudies-literature