scholarly journals A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification

2020 ◽  
Author(s):  
Xiaofang Jia ◽  
Lvyin Hu ◽  
Min Wu ◽  
Yun Ling ◽  
Wei Wang ◽  
...  
Keyword(s):  
Illumina Sequencing ◽  
Influenza A ◽  
Rna Virus ◽  
Time Frame ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Pathogen Identification ◽  
Influenza A H1n1 ◽  
Hiv 1

Abstract Metagenomic next generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on the assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing based mNGS method was first developed and evaluated using a set of samples with known etiology. Its sensitivity for RNA virus (Influenza A, H1N1) was <6.4×102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then the rapid turnaround time of Nanopore sequencing was testified by sequencing of an Influenza A virus and Adenoviruses. Further, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analyzed and the pathogens identified by illumina sequencing showed 81.8% concordance with qPCR-based results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1 positive patients with meningitis/encephalitis detected HIV-1 RNA and toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol which realized facile metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.

scholarly journals A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaofang Jia ◽  
Lvyin Hu ◽  
Min Wu ◽  
Yun Ling ◽  
Wei Wang ◽  
...  
Keyword(s):  
Illumina Sequencing ◽  
Influenza A ◽  
Time Frame ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Pathogen Identification ◽  
Viral Loads ◽  
Influenza A H1n1 ◽  
Hiv 1

AbstractMetagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.

scholarly journals Detection of human Metapneumovirus infection in children under 18 years old hospitalized in Lima-Peru

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7266 ◽  
Author(s):  
Juana del Valle-Mendoza ◽  
Fiorella Orellana-Peralta ◽  
Luis J. del Valle ◽  
Eduardo Verne ◽  
Claudia Ugarte ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Respiratory Infection ◽  
Acute Respiratory Infection ◽  
Influenza A ◽  
Rna Virus ◽  
Pediatric Population ◽  
Time Frame ◽  
Human Metapneumovirus ◽  
Influenza A H1n1 ◽  
One Year

BackgroundHuman Metapneumovirus (hMPV) is a negative single-stranded RNA virus. Infection by hMPV mainly affects the pediatric population and can cause upper or lower respiratory tract pathologies which can develop life threating complications. This study was carried out between 2009 and 2010 in a high complexity national hospital in Lima, Peru. The time frame corresponds to the pandemic of influenza A H1N1.MethodsA prospective study was performed between September 2009 and September 2010. Patients with a clinical diagnosis suggestive of an acute respiratory infection were included. RT-PCR was utilized to attain the amplification and identification of the hMPV.ResultsA total of 539 samples were analyzed from patients with a clinical context suggestive of an acute respiratory tract infection. Of these samples 73, (13.54%) were positive for hMPV. Out of the positive cases, 63% were under one year old, and increased to nearly 80% when considering children younger than two years old. Cough was the most frequent symptom presented by our population with a number of 62 cases (84.93%). Viral seasonality was also established, noting its predominance during the months of summer in the southern hemisphere. The infection by hMPV has an important prevalence in Peru. It mainly affects children under one year old and should be considered an important differential diagnosis in a patient with an acute respiratory infection.

scholarly journals Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1358
Author(s):  
Leonard Schuele ◽  
Hayley Cassidy ◽  
Erley Lizarazo ◽  
Katrin Strutzberg-Minder ◽  
Sabine Schuetze ◽  
...  
Keyword(s):  
Influenza A ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Genome Coverage ◽  
Sequence Capture ◽  
Shotgun Metagenomic Sequencing ◽  
Oxford Nanopore ◽  
The One ◽  
Targeted Sequence Capture

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

scholarly journals Sequence analysis of the 2009 pandemic influenza A H1N1 virus haemagglutinin gene from 2009-2010 Brazilian clinical samples

2011 ◽  
Vol 106 (5) ◽  
pp. 613-616 ◽  
Author(s):  
João Leandro de Paula Ferreira ◽  
Samanta Etel Treiger Borborema ◽  
Luis Fernando de Macedo Brígido ◽  
Maria Isabel de Oliveira ◽  
Terezinha Maria de Paiva ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Pandemic Influenza ◽  
Influenza A ◽  
H1n1 Virus ◽  
Clinical Samples ◽  
Influenza A H1n1 ◽  
Virus Haemagglutinin

scholarly journals HIV-1 and Its gp120 Inhibits the Influenza A(H1N1)pdm09 Life Cycle in an IFITM3-Dependent Fashion

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e101056 ◽  
Author(s):  
Milene Mesquita ◽  
Natalia Fintelman-Rodrigues ◽  
Carolina Q. Sacramento ◽  
Juliana L. Abrantes ◽  
Eduardo Costa ◽  
...  
Keyword(s):  
Life Cycle ◽  
Influenza A ◽  
Influenza A H1n1 ◽  
Hiv 1

scholarly journals Sirtuins Are Evolutionarily Conserved Viral Restriction Factors

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Emre Koyuncu ◽  
Hanna G. Budayeva ◽  
Yana V. Miteva ◽  
Dante P. Ricci ◽  
Thomas J. Silhavy ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Influenza A ◽  
Rna Virus ◽  
Immune Defense ◽  
Human Pathogens ◽  
Activity Increase ◽  
Virus Growth ◽  
Cell Defense ◽  
Influenza A H1n1 ◽  
Evolutionarily Conserved

ABSTRACTThe seven human sirtuins are a family of ubiquitously expressed and evolutionarily conserved NAD+-dependent deacylases/mono-ADP ribosyltransferases that regulate numerous cellular and organismal functions, including metabolism, cell cycle, and longevity. Here, we report the discovery that all seven sirtuins have broad-range antiviral properties. We demonstrate that small interfering RNA (siRNA)-mediated knockdown of individual sirtuins and drug-mediated inhibition of sirtuin enzymatic activity increase the production of virus progeny in infected human cells. This impact on virus growth is observed for both DNA and RNA viruses. Importantly, sirtuin-activating drugs inhibit the replication of diverse viruses, as we demonstrate for human cytomegalovirus, a slowly replicating DNA virus, and influenza A (H1N1) virus, an RNA virus that multiplies rapidly. Furthermore, sirtuin defense functions are evolutionarily conserved, since CobB, the sirtuin homologue inEscherichia coli, protects against bacteriophages. Altogether, our findings establish sirtuins as broad-spectrum and evolutionarily conserved components of the immune defense system, providing a framework for elucidating a new set of host cell defense mechanisms and developing sirtuin modulators with antiviral activity.IMPORTANCEWe live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens.

scholarly journals A random priming amplification method for whole genome sequencing of SARS-CoV-2 and H1N1 influenza A virus.

2021 ◽  
Author(s):  
Klaudia Chrzastek ◽  
Chandana Tennakoon ◽  
Dagmara Bialy ◽  
Graham L Freimanis ◽  
John Flannery ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Assembly ◽  
Influenza A ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Sequence Information ◽  
Whole Genome ◽  
Random Priming ◽  
Influenza A H1n1

Background: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. Methods: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. Results: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of COVID that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. Conclusions: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.

scholarly journals The use of pharmacological protocols during the Covid-19 crisis - a déjà vu of the 2009 A/H1N1 pandemic experience

2020 ◽  
Vol 3 ◽  
Author(s):  
Wladimir Jimenez Alonso ◽  
Cynthia Schuck-Paim ◽  
Veronique Diez Vicera
Keyword(s):  
Influenza A ◽  
Treatment Options ◽  
Risk Groups ◽  
Time Frame ◽  
Conclusive Evidence ◽  
World Health ◽  
Controlled Trials ◽  
Special Importance ◽  
H1n1 Pandemic ◽  
Influenza A H1n1

Only one decade ago, the World Health Organization declared the outbreak of a novel influenza A (H1N1) virus a worldwide pandemic. Then, just as in today’s Covid-19 crisis, treatment options were the center of debate, one of which is of special importance to the current pandemic: the existence of pharmacological treatments that, although available and potentially effective, are approached with reluctance due to concerns around side effects, the development of resistance and lack of conclusive evidence of effectiveness from randomized-controlled trials. History has proven that reluctance to use antivirals during the 2009 A/H1N1 pandemic was unwise, as those countries that restricted their use to at-risk groups and to patients with advanced disease experienced higher mortality. Those same antiviral are now routinely used for seasonal influenza. We revisit this forgotten lesson as a means of weighing in on the debate over the use of new treatments with promising outcomes as observed in clinical practice, but lacking strong evidence from controlled trials, which require a time frame incompatible with that available in unfolding public health emergencies triggered by novel pathogens.

Influenza

2021 ◽  
Author(s):  
Leong Hoe Nam
Keyword(s):  
Influenza Virus ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Respiratory Infections ◽  
Rna Virus ◽  
Treatment Options ◽  
Antigenic Drift ◽  
New Techniques ◽  
Influenza A H1n1 ◽  
Viral Respiratory Infections

The influenza virus is a segmented RNA virus with different mechanisms for mutations, and hence for minor (antigenic drift) and major (antigenic shift) changes. Influenza virus A was responsible for pandemics on average every 30 years in the past, with the most recent being the 2009 swine-origin influenza A H1N1 (SO-H1N1). The clinical picture is unspecific: seasonal or pandemic influenza cannot be differentiated from other viral respiratory infections on clinical grounds. PCR has become the standard for microbiological confirmation of the diagnosis. Treatment options remain limited with neuraminidase inhibitors (oseltamivir; zanamivir). Resistance may occur under treatment or under prophylaxis; however, it is still rare overall. Vaccination is still the preferred method for prevention. However, the long lead time for production (at least 6 months) poses a challenge. Innovative new techniques like cell culture or recombinant productions are urgently needed. Pandemic influenza vaccines for SO-H1N1 were shown to be effective and safe in children, pregnant women, adults, and also in elderly. Pre-pandemic vaccines (H5N1) are also available.

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