scholarly journals Review of 16S and ITS Direct Sequencing Results for Clinical Specimens Submitted to a Reference Laboratory

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Michael Payne ◽  
Robert Azana ◽  
Linda M. N. Hoang
Keyword(s):  
Fungal Pathogens ◽  
Direct Sequencing ◽  
Infectious Agent ◽  
Its Region ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Bacterial Dna ◽  
Clinical Specimens ◽  
Detection And Identification ◽  
Rdna Amplification

We evaluated the performance of 16S and internal transcribed spacer (ITS) region amplification and sequencing of rDNA from clinical specimens, for the respective detection and identification of bacterial and fungal pathogens. Direct rDNA amplification of 16S and ITS targets from clinical samples was performed over a 4-year period and reviewed. All specimens were from sterile sites and submitted to a reference laboratory for evaluation. Results of 16S and ITS were compared to histopathology, Gram and/or calcofluor stain microscopy results. A total of 277 16S tests were performed, with 64 (23%) positive for the presence of bacterial DNA. Identification of an organism was more likely in microscopy positive 16S samples 14/21 (67%), compared to 35/175 (20%) of microscopy negative samples. A total of 110 ITS tests were performed, with 14 (13%) positive. The yield of microscopy positive ITS samples, 9/44 (21%), was higher than microscopy negative samples 3/50 (6%). Given these findings, 16S and ITS are valuable options for culture negative specimens from sterile sites, particularly in the setting of positive microscopy findings. Where microscopy results are negative, the limited sensitivity of 16S and ITS in detecting and identifying an infectious agent needs to be considered.

Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics

2019 ◽  
Vol 58 (5) ◽  
pp. 650-660 ◽  
Author(s):  
Laszlo Irinyi ◽  
Yiheng Hu ◽  
Minh Thuy Vi Hoang ◽  
Lana Pasic ◽  
Catriona Halliday ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Real Time ◽  
Direct Sequencing ◽  
Quantitative Polymerase Chain Reaction ◽  
Pneumocystis Jirovecii ◽  
Quality Data ◽  
Clinical Samples ◽  
Human Pathogens ◽  
Clinical Specimens ◽  
Long Read

Abstract The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

scholarly journals DNA Microarray-Based Detection and Identification of Fungal Pathogens in Clinical Samples from Neutropenic Patients

2007 ◽  
Vol 45 (11) ◽  
pp. 3743-3753 ◽  
Author(s):  
B. Spiess ◽  
W. Seifarth ◽  
M. Hummel ◽  
O. Frank ◽  
A. Fabarius ◽  
...  
Keyword(s):  
Dna Microarray ◽  
Fungal Pathogens ◽  
Clinical Samples ◽  
Detection And Identification ◽  
Neutropenic Patients

scholarly journals Distribution of enterovirus genotypes detected in clinical samples in Hungary, 2010–2018

2020 ◽  
Author(s):  
Erika Bujaki ◽  
Ágnes Farkas ◽  
Zita Rigó ◽  
Mária Takács
Keyword(s):  
Clinical Symptoms ◽  
Age Groups ◽  
Foot And Mouth Disease ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Clinical Specimens ◽  
Echovirus 30 ◽  
Occasional Occurrence ◽  
Severe Infections ◽  
Sporadic Cases

AbstractThis report provides the findings of a retrospective surveillance study on the emergence and circulation of enteroviruses with their associated clinical symptoms over a nine-year period detected at the National Enterovirus Reference Laboratory in Hungary between 2010–2018.Enterovirus (EV) detection and genotyping were performed directly from clinical samples. From 4,080 clinical specimens 25 EV types were identified with a median age of patients of 5 years and 68% of all cases affected children aged 10 years or younger, although infections occurred in all age-groups. In 130 cases neurological symptoms were recorded, in 123 cases the infection presented in skin related signs including hand, foot, and mouth disease (HFMD), herpangina and rash. In 2010 EV-A71 was found to cause the majority of diagnosed EV infections while in 2011 and from 2014–2018, Coxsackievirus (CV)-A6 was identified most often. Echovirus E6 accounted for the most cases in 2012 and Echovirus 30 dominated in 2013. EV-D68 was identified only in 2010 and 2013.Widespread circulation of several EV-A and EV-B viruses with occasional occurrence of EV-C and EV-D was detected. The ability of EVs to cause severe infections in sporadic cases and regular outbreaks highlight the importance of continued monitoring of circulating EV types.

115: Detection and Identification of Bacterial DNA and HIV Gag in the Semen of HIV Infected Men

2004 ◽  
Vol 171 (4S) ◽  
pp. 30-30
Author(s):  
Robert C. Eyre ◽  
Ann A. Kiessling ◽  
Thomas E. Mullen ◽  
Rachel L. Kiessling
Keyword(s):  
Bacterial Dna ◽  
Detection And Identification

scholarly journals Molecular Characterisation and Phylogenetic Analysis of Dermatophytic Fungi Isolated from Tinea Capitis in Northwest Nigeria Using Sequence of the 28S rRNA

2021 ◽  
Vol 12 (3) ◽  
pp. 646-655
Author(s):  
Hussain Yahaya Ungo-kore ◽  
Joseph Olorunmola Ehinmidu ◽  
Josiah Ademola Onaolapo ◽  
Olayeni Stephen Olonitola
Keyword(s):  
Phylogenetic Analysis ◽  
Tinea Capitis ◽  
28S Rdna ◽  
Clinical Samples ◽  
28S Rrna ◽  
Trichophyton Tonsurans ◽  
Detection And Identification ◽  
Microsporum Audouinii ◽  
Trichophyton Simii ◽  
Dermatophyte Species

The detection and identification of fungal DNA from clinical samples is one of the fundamental approaches in biomedicine. The incidence, distribution, and control of dermatophytes has progress significantly and the use of phylogenetic species concepts based on rRNA regions have enhanced the taxonomy of dermatophyte species; however, the use of 28S rDNA genes has certain limitations. This gene has been used in dermatophyte taxonomy with limited enumeration; we appraised the sequence disparity within and among groups of the species, the gene ranking in identification, phylogenetic analysis, and taxonomy of 32 strains of eight dermatophyte species. In this study, a set of primers was adopted to amplify the target followed by a partial sequencing of the rDNA. The utilization of a pairwise nucleotide differentiation, an affinity was observed among eight dermatophyte species, with disparity among species ranging from 0 to 197 base pair (bp). Intra-species bp differences were found within strains of Trichophyton eriotrephon, Trichophyton bullosum, Trichophyton simii (Trichophyton genus), Microsporum audouinii, and Trichophyton tonsurans (Microsporum and Trichophyton genus, respectively); however, only some strains of Trichophyton eriotrephon were found to be invariant having three genotypes. Trichophyton tonsurans exhibited most intra-species variability. The characterization and construction of a phylogenetic tree of 28S rDNA gene on dermatophyte species provide a bedrock of an additional finding of connections between species. However, 28S rRNA capture provides a novel method of effective and sensitive detection of dermatophytes lodged in human skin scale. We report for the first time the emergence of T. eriotrephon, T. bullosum, T. simii, T. benhamiae, and Ctenomyces serratus dermatophytes from Tinea capitis in Nigeria.

Detection and identification of bacterial DNA in semen

2008 ◽  
Vol 90 (5) ◽  
pp. 1744-1756 ◽  
Author(s):  
Ann A. Kiessling ◽  
Bryan M. Desmarais ◽  
Hui-Zhong Yin ◽  
Joseph Loverde ◽  
Robert C. Eyre
Keyword(s):  
Bacterial Dna ◽  
Detection And Identification

Fungal composition of lichen thalli assessed by single strand conformation polymorphism

2010 ◽  
Vol 42 (4) ◽  
pp. 461-473 ◽  
Author(s):  
Lucia MUGGIA ◽  
Martin GRUBE
Keyword(s):  
Direct Sequencing ◽  
Its Region ◽  
Single Strand Conformation Polymorphism ◽  
Morphological Characters ◽  
Single Strand ◽  
Specific Primers ◽  
Lichenicolous Fungi ◽  
Large Numbers ◽  
Total Dna ◽  
Conformation Polymorphism

AbstractFungi that are unrelated to the mycobiont species frequently colonize lichens. Some of these fungal colonists are described lichenicolous fungi, lichen parasites and pathogens that produce recognizable morphological characters, while others apparently produce no noticeable structures. Here we apply the single strand conformation polymorphism (SSCP) technique to directly assess the abundance of different fungi in lichens. Twenty-eight lichen thalli were chosen, some with and some without externally visible symptoms of parasite infection, and these were subjected to total DNA extraction. PCR was conducted with fungal-specific primers for the ITS region of ribosomal DNA. Single strands of the products were separated on native acrylamide gels. The majority of lichen specimens, both infected and those without symptoms, displayed more than one band in the stained gels. In one case, 14 bands were detected using SSCP. Some of these bands apparently represent other neighbouring lichens in the habitat, but many are apparently non-lichen-forming. Since few lichen-associated fungi have been cultured and sequenced, it is difficult to know if SSCP bands represent obligate lichenicolous fungi, other asymptomatic lichen parasites, or fungi not obligately associated with lichens, but our results indicate that large numbers of non-lichen-forming fungi commonly co-occur with lichens in nature. For specimens of the filamentous lichens Cystocoleus ebeneus and Racodium rupestre we used cloned sequences to compare the number of sequences obtained by the SSCP method to the number obtained by direct sequencing of thallus extracts, and we generally found that more sequences could be detected by SSCP than could be seen by direct sequencing.

scholarly journals Validation and Implementation of a Diagnostic Algorithm for DNA Detection ofBordetella pertussis,B. parapertussis, andB. holmesiiin a Pediatric Referral Hospital in Barcelona, Spain

2018 ◽  
Vol 57 (1) ◽  
Author(s):  
Ana Valero-Rello ◽  
Desiree Henares ◽  
Lesly Acosta ◽  
Mireia Jane ◽  
Iolanda Jordan ◽  
...  
Keyword(s):  
Internal Control ◽  
Whooping Cough ◽  
Diagnostic Algorithm ◽  
Clinical Samples ◽  
Analytical Validation ◽  
Content Type ◽  
Detection And Identification ◽  
Ofbordetella Pertussis ◽  
Lower Limits

ABSTRACTThis study aimed to validate a comprehensive diagnostic protocol based on real-time PCR for the rapid detection and identification ofBordetella pertussis,Bordetella parapertussis, andBordetella holmesii, as well as its implementation in the diagnostic routine of a reference children’s hospital. The new algorithm included a triplex quantitative PCR (qPCR) targeting IS481gene (inB. pertussis,B. holmesii, and someBordetella bronchisepticastrains), pIS1001(B. parapertussis-specific) andrnaseP as the human internal control. Two confirmatory singleplex tests forB. pertussis(ptxA-Pr) andB. holmesii(hIS1001) were performed if IS481was positive. Analytical validation included determination of linear range, linearity, efficiency, precision, sensitivity, and a reference panel with clinical samples. Once validated, the new algorithm was prospectively implemented in children with clinical suspicion of whooping cough presenting to Hospital Sant Joan de Deu (Barcelona, Spain) over 12 months. Lower limits of detection obtained were 4.4, 13.9, and 27.3 genomic equivalents/ml of sample for IS481(onB. pertussis), pIS1001and hIS1001, and 777.9 forptxA-Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay variabilities were <3% and <5%, respectively. Among 566 samples analyzed,B. pertussis,B. holmesii, andB. parapertussiswere detected in 11.1%, 0.9% (only in females >4 years old), and 0.2% of samples, respectively. The new algorithm proved to be a useful microbiological diagnostic tool for whooping cough, demonstrating a low rate of other non-pertussisBordetellaspecies in our surveilled area.

scholarly journals Comparison of diagnostic clinical samples and environmental sampling for enterovirus and parechovirus surveillance in Scotland, 2010 to 2012

2014 ◽  
Vol 19 (15) ◽  
Author(s):  
H Harvala ◽  
J Calvert ◽  
D Van Nguyen ◽  
L Clasper ◽  
N Gadsby ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Cerebrospinal Fluid ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Environmental Sampling ◽  
Clinical Specimens ◽  
Young Infants ◽  
Coxsackievirus A6 ◽  
The Family ◽  
Common Causes

Human enteroviruses (EV) and parechoviruses (HPeV) within the family Picornaviridae are the most common causes of viral central nervous system (CNS)-associated infections including meningitis and neonatal sepsis-like disease. The frequencies of EV and HPeV types identified in clinical specimens collected in Scotland over an eight-year period were compared to those identified in sewage surveillance established in Edinburgh. Of the 35 different EV types belonging to four EV species (A to D) and the four HPeV types detected in this study, HPeV3 was identified as the most prevalent picornavirus in cerebrospinal fluid samples, followed by species B EV. Interestingly, over half of EV and all HPeV CNS-associated infections were observed in young infants (younger than three months). Detection of species A EV including coxsackievirus A6 and EV71 in clinical samples and sewage indicates that these viruses are already widely circulating in Scotland. Furthermore, species C EV were frequently identified EV in sewage screening but they were not present in any of 606 EV-positive clinical samples studied, indicating their likely lower pathogenicity. Picornavirus surveillance is important not only for monitoring the changing epidemiology of these infections but also for the rapid identification of spread of emerging EV and/or HPeV types.

scholarly journals Control of artefactual variation in reported inter-sample relatedness during clinical use of a Mycobacterium tuberculosis sequencing pipeline

2018 ◽  
Author(s):  
David H Wyllie ◽  
Nicholas Sanderson ◽  
Richard Myers ◽  
Tim Peto ◽  
Esther Robinson ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Read Depth ◽  
Pairwise Distance ◽  
Contact Tracing ◽  
Clinical Samples ◽  
Bacterial Dna ◽  
Consensus Sequences ◽  
Minor Variant ◽  
Validation Set ◽  
Genomic Regions

ABSTRACTContact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artefactual variation between M. tuberculosis isolates during routine next generation sequencing of Mycobacterium spp, we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted, sequenced, reads mapped, and consensus sequences determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of non-Mycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of non-Mycobacterial bacterial DNA, we found significant increases in minor variant frequencies of more than 1.5 fold in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high variation regions strongly influenced by the amount of non-Mycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we have demonstrated an approach identifying critical genomic regions contributing to clinically relevant artefactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multi-step laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

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