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Journal of Neurovirology Oct 2023Little is known about concomitant central nervous system (CNS) infections by more than one virus. Current diagnostics are based on molecular tests for particular...
Little is known about concomitant central nervous system (CNS) infections by more than one virus. Current diagnostics are based on molecular tests for particular pathogens making it difficult to identify multi-viral infections. In the present study, we applied DNA- and RNA-based next-generation sequencing metagenomics (mNGS) to detect viruses in cerebrospinal fluids from 20 patients with herpes simplex encephalitis. Coinfection was detected in one patient: sequences in cerebrospinal fluids matched enterovirus A (2.660 reads; 4% of recovered genome) and enterovirus B (1.571 reads; 13% of recovered genome). Subsequent PCR combined with serotyping allowed to identify human echovirus 6, a representative of enterovirus B. Several other mNGS hits (human pegivirus, Merkel cell polyomavirus, human papillomavirus type 5) were not considered to represent a genuine signal as they could not be confirmed by specific RT-PCR/PCR. HSV DNA, while being detectable by PCR in every patient, was detected by mNGS in only one. In conclusion, contaminations and false signals may complicate mNGS interpretation; however, the method can be useful in diagnostics of viral coinfections in CNS, particularly in the case of rare pathogens.
Topics: Humans; Coinfection; Encephalitis, Herpes Simplex; Virus Diseases; Polymerase Chain Reaction; Enterovirus B, Human; Central Nervous System Infections; DNA; High-Throughput Nucleotide Sequencing
PubMed: 37490185
DOI: 10.1007/s13365-023-01157-9 -
Journal of Insect Science (Online) Sep 2023The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from...
The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ± 3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ± 4.57 ng/µl) and a microwave-based method (119.26 ± 3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ± 0.02, 1.79 ± 0.01, and 1.87 ± 0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.
Topics: Animals; Culicidae; Culex; DNA; Polymerase Chain Reaction; DNA Primers
PubMed: 37804500
DOI: 10.1093/jisesa/iead080 -
New Biotechnology Dec 2023Analysis of circulating cell-free DNA (ccfDNA) isolated from liquid biopsies is rapidly being implemented into clinical practice. However, diagnostic accuracy is...
Analysis of circulating cell-free DNA (ccfDNA) isolated from liquid biopsies is rapidly being implemented into clinical practice. However, diagnostic accuracy is significantly impacted by sample quality and standardised approaches for assessing the quality of ccfDNA are not yet established. In this study we evaluated the application of nucleic acid "spike-in" control materials to aid quality control (QC) and standardisation of cfDNA isolation for use in in vitro diagnostic assays. We describe an approach for the design and characterisation of in-process QC materials, illustrating it with a spike-in material containing an exogenous Arabidopsis sequence and DNA fragments approximating to ccfDNA and genomic DNA lengths. Protocols for inclusion of the spike-in material in plasma ccfDNA extraction and quantification of its recovery by digital PCR (dPCR) were assessed for their suitability for process QC in an inter-laboratory study between five expert laboratories, using a range of blood collection devices and ccfDNA extraction methods. The results successfully demonstrated that spiking plasmid-derived material into plasma did not deleteriously interfere with endogenous ccfDNA recovery. The approach performed consistently across a range of commonly-used extraction protocols and was able to highlight differences in efficiency and variability between the methods, with the dPCR quantification assay performing with good repeatability (generally CV <5%). We conclude that initial findings demonstrate that this approach appears "fit for purpose" and spike-in recovery can be combined with other extraction QC metrics for monitoring the performance of a process over time, or in the context of external quality assessment.
Topics: Cell-Free Nucleic Acids; Liquid Biopsy; Quality Control; DNA; Polymerase Chain Reaction
PubMed: 37730172
DOI: 10.1016/j.nbt.2023.09.005 -
Pathogens and Global Health Jul 2023The cycle threshold (Ct) in quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) is inversely correlated to the amount of viral nucleic acid...
The cycle threshold (Ct) in quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) is inversely correlated to the amount of viral nucleic acid or viral load and can be regarded as an indicator of infectivity. We examined the association of socio-demographic and clinical characteristics of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) polymerase chain reaction (PCR) positive cases with PCR cycle threshold (Ct) values at the time of diagnosis. SARS-CoV-2 cases reported between 12 October 2020 and 24 January 2021 in Regensburg were analyzed employing bivariate and multivariable methods. We included 3,029 SARS-CoV-2 cases (31% asymptomatic at diagnosis) and analyzed the association of case characteristics with Ct values in 2,606 cases. Among symptomatic patients, cough (38.0%), rhinitis (32.4%), headache (32.0), and fever/chills (29.9%) were the most frequent complaints. Ct values ≤20 were more frequent in symptomatic cases (20.9% vs. 11.3%), whereas Ct values >30 were more common in asymptomatic cases (32.6% vs. 18.0%). Ct values >20 and ≤30 were most common in symptomatic and asymptomatic cases (48.0% vs 40.7%). We observed lower median Ct values of E and N gene in symptomatic cases. In a random forest model, the total number of symptoms, respiratory symptoms, and age were most strongly associated with low Ct values. In conclusion, certain symptoms and age were associated with lower Ct values. Ct values can be used as a pragmatic approach in estimating infectivity at the first notification of a case and, thus, in guiding containment measures.
Topics: Humans; COVID-19; SARS-CoV-2; Cross-Sectional Studies; Real-Time Polymerase Chain Reaction; Viral Load; COVID-19 Testing
PubMed: 36519354
DOI: 10.1080/20477724.2022.2158003 -
International Journal of Molecular... Jan 2024Real-time quantitative polymerase chain reaction (qRT-PCR) has been widely used in gene expression analyses due to its advantages of sensitivity, accuracy and high... (Review)
Review
Real-time quantitative polymerase chain reaction (qRT-PCR) has been widely used in gene expression analyses due to its advantages of sensitivity, accuracy and high throughput. The stability of internal reference genes has progressively emerged as a major factor affecting the precision of qRT-PCR results. However, the stability of the expression of the reference genes needs to be determined further in different cells or organs, physiological and experimental conditions. Methods for evaluating these candidate internal reference genes have also evolved from simple single software evaluation to more reliable and accurate internal reference gene evaluation by combining different software tools in a comprehensive analysis. This study intends to provide a definitive reference for upcoming research that will be conducted on fruit trees. The primary focus of this review is to summarize the research progress in recent years regarding the selection and stability analysis of candidate reference genes for different fruit trees.
Topics: Fruit; Trees; Gene Expression Profiling; Real-Time Polymerase Chain Reaction; Software
PubMed: 38256212
DOI: 10.3390/ijms25021142 -
Virology Journal Aug 2023Viral infections of the central nervous system (CNS) are common worldwide and result in considerable morbidity and mortality associated with neurologic illness. Until...
Viral infections of the central nervous system (CNS) are common worldwide and result in considerable morbidity and mortality associated with neurologic illness. Until now, there have been no epidemiologic data regarding viruses causing aseptic meningitis, encephalitis, and CNS infections in Egypt. We investigated 1735 archived cerebrospinal fluid samples collected from Egyptian patients between 2016 and 2019 and performed molecular characterization for infection for12 different viruses: herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesviruses 6 and 7 (HHV-6 and HHV-7), human enteroviruses (HEVs), human parechovirus (HPeV), parvovirus B19 (B19V), adenovirus (AdV), and mumps virus (MuV). All included samples were negative for bacterial infection. Our results indicated a relatively high prevalence of viral infection, with HEVs being the most prevalent viruses, followed by HSV-1, EBV, and then HSV-2. The highest prevalence was among male patients, peaking during the summer. Data obtained from this study will contribute to improving the clinical management of viral infections of the CNS in Egypt.
Topics: Humans; Male; Egypt; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Polymerase Chain Reaction; Virus Diseases; Viruses; Central Nervous System Infections; Herpesvirus 3, Human; Herpesvirus 2, Human; Enterovirus; DNA, Viral
PubMed: 37533069
DOI: 10.1186/s12985-023-02079-y -
Stem Cell Research & Therapy Nov 2023The functional impairment of adipose stem cells (ASCs) during aging limits their clinical transformation. Studies have shown that extrachromosomal circular DNAs...
BACKGROUND
The functional impairment of adipose stem cells (ASCs) during aging limits their clinical transformation. Studies have shown that extrachromosomal circular DNAs (eccDNAs) are associated with tumor progression and cell aging, but the roles of eccDNAs in ASCs remain unknown.
METHOD
We conducted Circle sequencing (Circle-seq) to identify eccDNAs in ASCs isolated from young and old donors. The differentially expressed eccDNAs were calculated, annotated and validated via polymerase chain reaction.
RESULTS
Thousands of eccDNAs were identified and comprehensively characterized. Most of them were GC-rich, < 1000 base pairs in size, and were enriched on chromosome 19 and 17 with a high density of Alu elements and genes, 2 kb upstream/downstream of genes and satellites. In total, 3025 eccDNAs were differentially expressed among the two ASC groups. Conjoint analysis of the Circle-seq results and previous RNA-seq results revealed that 73 eccDNAs and 55 genes exhibited the same differential expression between the two groups. KEGG and GO analyses revealed that genes encoding differentially expressed eccDNAs were enriched for cell adhesion, cellular senescence and TGF-β receptor signaling pathway. We also found that aged ASCs exhibited loss of eccDNAs, including CAMK2G , TRABD2B and TRABD2B .
CONCLUSION
In this study, we elucidated the first eccDNA profile relating to ASCs and demonstrated that three eccDNAs are lost in aged ASCs, which may be potential biomarkers of stem cell aging and valuable targets for stem cell rejuvenation.
Topics: DNA, Circular; DNA; Polymerase Chain Reaction; Biomarkers
PubMed: 38017497
DOI: 10.1186/s13287-023-03575-2 -
Gut and Liver Sep 2023Dual priming oligonucleotide-based multiplex polymerase chain reaction (DPO-PCR) has recently been used for both the detection of and the identification of 23S...
Effective Eradication Regimen and Duration According to the Clarithromycin Susceptibility of Determined Using Dual Priming Oligonucleotide-Based Multiplex Polymerase Chain Reaction.
BACKGROUND/AIMS
Dual priming oligonucleotide-based multiplex polymerase chain reaction (DPO-PCR) has recently been used for both the detection of and the identification of 23S ribosomal RNA point mutations that cause clarithromycin resistance. The aim of this study was to investigate the duration of effective standard triple therapy in a clarithromycin susceptible group and of bismuth-based quadruple therapy in a resistant group based on DPO-PCR.
METHODS
We retrospectively analyzed the electronic medical records of 184 patients who, between September 2019 and December 2020, received eradication therapy following detection of , and the subsequent identification of the clarithromycin susceptibility of their using DPO-PCR. Patients were treated with 7- or 14-day standard triple therapy in the clarithromycin susceptible group, whereas 7- or 14-day bismuth-based quadruple therapy in the clarithromycin resistance group.
RESULTS
In the clarithromycin susceptible group, per-protocol analyses showed eradication rates of 87.5% (42/48; 95% confidence interval [CI], 77.1% to 95.8%) for 7-day therapy and 87.2% (41/47; 95% CI, 78.7% to 95.7%) for 14-day therapy (p=0.969). The eradication rates in the clarithromycin resistance group were 91.4% (32/35; 95% CI, 80.0% to 100.0%) for 7-day therapy and 90.3% (28/31; 95% CI, 77.4% to 100.0%) for 14-day therapy (p=0.876). There was no significant difference in the eradication rates, patient compliance, or rate of adverse events between the 7- and 14-day therapies for both groups.
CONCLUSIONS
Compared to the 14-day therapy, 7-day eradication therapy is sufficient after DPO-PCR-based clarithromycin susceptibility testing.
Topics: Humans; Clarithromycin; Anti-Bacterial Agents; Helicobacter pylori; Helicobacter Infections; Bismuth; Oligonucleotides; Multiplex Polymerase Chain Reaction; Retrospective Studies; Drug Therapy, Combination; Amoxicillin
PubMed: 36168964
DOI: 10.5009/gnl220256 -
International Journal of... 2023Mycobacterial infections can manifest in various anatomical sites, necessitating the analysis of nonsputum specimens for accurate diagnosis. The aim of this study was to... (Observational Study)
Observational Study
BACKGROUND
Mycobacterial infections can manifest in various anatomical sites, necessitating the analysis of nonsputum specimens for accurate diagnosis. The aim of this study was to identify the molecular cases of mycobacterial infections in nonsputum specimens using polymerase chain reaction based assays and gene sequencing methods.
METHODS
This observational study examined 161 nonsputum samples that have been stored in the Clinical Microbiology Laboratory at Hasanuddin University Hospital. Samples were analyzed by microscopy and molecular detection methods according to the standard methods at the Clinical Microbiology Laboratory of Hasanuddin University. Descriptive statistics were utilized to summarize patient demographics, infection characteristics, and outcomes.
RESULTS
The samples were collected from patients with an average age of 39.82 years. The anatomical sites of specimen collection varied, with musculoskeletal organs and eyes being the most common. Microbiological analysis revealed a predominance of Gram positive bacteria, with polymicrobial morphology observed. Methicillin susceptible Staphylococcus aureus were the most frequently isolated organisms. Acid fast bacilli were detected in 8.1% of samples. Phylogenetic analysis, based on 16S rRNA gene sequencing, revealed similarities between the samples and known mycobacterial species, including Mycobacterium parmense, Mycobacterium lacus, and Mycobacterium dioxanotrophicus.
CONCLUSIONS
The findings highlight the microbial diversity observed in these infections. The study advocates for comprehensive diagnostic evaluations and targeted testing strategies based on both clinical and laboratory findings. This knowledge can contribute to improved diagnostic accuracy and optimized treatment strategies for mycobacterial infections.
Topics: Humans; Adult; Mycobacterium Infections, Nontuberculous; RNA, Ribosomal, 16S; Phylogeny; Polymerase Chain Reaction; Hospitals, University; Nontuberculous Mycobacteria
PubMed: 37721231
DOI: 10.4103/ijmy.ijmy_121_23 -
Viruses Nov 2023In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP,...
In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAX PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a C cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.
Topics: Swine; Animals; Porcine respiratory and reproductive syndrome virus; Porcine Reproductive and Respiratory Syndrome; Reverse Transcriptase Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Viral Vaccines
PubMed: 38005917
DOI: 10.3390/v15112240