Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology.
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ABSTRACT:
SUBMITTER: Adams IP
PROVIDER: S-EPMC6640393 | biostudies-literature | 2009 Jul
REPOSITORIES: biostudies-literature
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Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology.
Adams Ian P IP Glover Rachel H RH Monger Wendy A WA Mumford Rick R Jackeviciene Elena E Navalinskiene Meletele M Samuitiene Marija M Boonham Neil N
Molecular plant pathology 20090701 4
A novel, unbiased approach to plant viral disease diagnosis has been developed which requires no a priori knowledge of the host or pathogen. Next-generation sequencing coupled with metagenomic analysis was used to produce large quantities of cDNA sequence in a model system of tomato infected with Pepino mosaic virus. The method was then applied to a sample of Gomphrena globosa infected with an unknown pathogen originally isolated from the flowering plant Liatris spicata. This plant was found to ...[more]
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Project description:Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I-IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.
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Project description:Agnostic metagenomic next-generation sequencing (mNGS) has emerged as a promising single, universal pathogen detection method for infectious disease diagnostics. This methodology allows for identification and genomic characterization of bacteria, fungi, parasites, and viruses without the need for a priori knowledge of a specific pathogen directly from clinical specimens. Although there are increasing reports of mNGS successes, several hurdles need to be addressed, such as differentiation of colonization from infection, extraneous sources of nucleic acid, method standardization, and data storage, protection, analysis, and interpretation. As more commercial and clinical microbiology laboratories develop mNGS assays, it is important for treating practitioners to understand both the power and limitations of this method as a diagnostic tool for infectious diseases.
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Project description:Many common pathogens are difficult or impossible to detect using conventional microbiological tests. However, the rapid and untargeted nature of metagenomic next-generation sequencing (mNGS) appears to be a promising alternative. To perform a systematic review and meta-analysis of evidence regarding the diagnostic accuracy of mNGS in patients with infectious diseases. An electronic literature search of Embase, PubMed and Scopus databases was performed. Quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Summary receiver operating characteristics (sROC) and the area under the curve (AUC) were calculated; A random-effects model was used in cases of heterogeneity. A total of 20 papers were eligible for inclusion and synthesis. The sensitivity and specificity of diagnostic mNGS were 75% and 68%, respectively. The AUC from the SROC was 85%, corresponding to excellent performance. mNGS demonstrated satisfactory diagnostic performance for infections and yielded an overall detection rate superior to conventional methods.
| S-EPMC9723114 | biostudies-literature
Project description:IntroductionThe diagnosis of pulmonary infection and the identification of pathogens are still clinical challenges in immunocompromised patients. Metagenomic next-generation sequencing (mNGS) has emerged as a promising infection diagnostic technique. However, its diagnostic value in immunocompromised patients needs further exploration.PurposesThis study was to evaluate the diagnostic value of mNGS compared with comprehensive conventional pathogen tests (CTs) in the etiology of pneumonia in immunocompromised patients and immunocompetent patients.MethodsWe retrospectively reviewed 53 patients who were diagnosed with pneumonia from May 2019 to June 2021. There were 32 immunocompromised patients and 21 immunocompetent patients with pneumonia who received both mNGS and CTs. The diagnostic performance was compared between mNGS and CTs in immunocompromised patients, using the composite diagnosis as the reference standard. And, the diagnostic value of mNGS for mixed infections was further analyzed.ResultsCompared to immunocompetent patients, the most commonly pathogens, followed by Cytomegalovirus, Pneumocystis jirovecii and Klebsiella pneumoniae in immunocompromised patients. Furthermore, more mixed infections were diagnosed, and bacterial-fungal-virus coinfection was the most frequent combination (43.8%). mNGS can detect more types of pathogenic microorganisms than CTs in both groups (78.1% vs. 62.5%, P = 0.016and 57.1% vs. 42.9%, P = 0.048). The overall diagnostic positive rate of mNGS for pathogens was higher in immunocompromised patients (P = 0.002). In immunocompromised patients, a comparable diagnostic accuracy of mNGS and CTs was found for bacterial, fungal, and viral infections and coinfection. mNGS had a much higher sensitivity for bacterial infections (92.9% vs. 50%, P < 0.001) and coinfections (68.8% vs. 48.3%, P < 0.05), and it had no significant advantage in the detection of fungal infections, mainly due to the high sensitivity for Pneumocystis jirovecii in both groups.ConclusionmNGS is more valuable in immunocompromised patients and exhibits apparent advantages in detecting bacterial and mixed infections. It may be an alternative or complementary diagnostic method for the diagnosis of complicated infections in immunocompromised patients.
| S-EPMC9731749 | biostudies-literature
Project description:ObjectiveTo evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) in sepsis and bloodstream infection (BSI).MethodsA retrospective analysis of patients diagnosed with sepsis and BSI at the First Affiliated Hospital of Zhengzhou University from January 2020 to February 2022 was conducted. All the patients underwent blood culture and were divided into mNGS group and non-mNGS group according to whether mNGS was performed or not. The mNGS group was further divided into early group (< 1 day), intermediate group (1-3 days), and late group (> 3 days) according to the time of mNGS inspection.ResultsIn 194 patients with sepsis and BSI, the positive rate of mNGS for identifying pathogens was significantly higher than that of blood culture (77.7% vs. 47.9%), and the detection period was shorter (1.41 ± 1.01 days vs. 4.82 ± 0.73 days); the difference was statistically significant (p < 0.05). The 28-day mortality rate of the mNGS group (n = 112) was significantly lower than that of the non-mNGS group (n = 82) (47.32% vs. 62.20%, p = 0.043). The total hospitalization time for the mNGS group was longer than that for the non-mNGS group (18 (9, 33) days vs. 13 (6, 23) days, p = 0.005). There was no significant difference in the ICU hospitalization time, mechanical ventilation time, vasoactive drug use time, and 90-day mortality between the two groups (p > 0.05). Sub-group analysis of patients in the mNGS group showed that the total hospitalization time and the ICU hospitalization time in the late group were longer than those in the early group (30 (18, 43) days vs. 10 (6, 26) days, 17 (6, 31) days vs. 6 (2, 10) days), and the ICU hospitalization time in the intermediate group was longer than that in the early group (6 (3, 15) days vs. 6 (2, 10) days); the differences were statistically significant (p < 0.05). The 28-day mortality rate of the early group was higher than that of the late group (70.21% vs. 30.00%), and the difference was statistically significant (p = 0.001).ConclusionsmNGS has the advantages of a short detection period and a high positive rate in the diagnosis of pathogens causing BSI and, eventually, sepsis. Routine blood culture combined with mNGS can significantly reduce the mortality of septic patients with BSI. Early detection using mNGS can shorten the total hospitalization time and the ICU hospitalization time of patients with sepsis and BSI.
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Project description:Objectives The aim of this study was to evaluate the diagnostic value of bronchoalveolar lavage fluid (BALF) metagenomic next-generation sequencing (mNGS) versus conventional microbiological tests (CMTs) for pediatric pneumonia. Methods This retrospective observational study enrolled 103 children who were diagnosed with pneumonia and hospitalized at Hubei Maternity and Child Health Care Hospital between 15 October 2020 and 15 February 2022. The pneumonia diagnosis was based on clinical manifestations, lung imaging, and microbiological tests. Pathogens in the lower respiratory tract were detected using CMTs and BALF mNGS (of DNA and RNA). The diagnostic performance of BALF mNGS was compared with that of CMTs. Results In 96 patients, pathogens were identified by microbiological tests. The overall pathogen detection rate of mNGS was significantly higher than that of CMTs (91.3% vs. 59.2%, p = 0.000). The diagnostic performance of mNGS varied for different pathogens; however, its sensitivity and accuracy for diagnosing bacterial and viral infections were both higher than those of CMTs (p = 0.000). For the diagnosis of fungi, the sensitivity of mNGS (87.5%) was higher than that of CMTs (25%); however, its specificity and accuracy were lower than those of CMTs (p < 0.01). For the diagnosis of Mycoplasma pneumoniae, the specificity (98.8%) and accuracy (88.3%) of mNGS were high; however, its sensitivity (42.1%) was significantly lower than that of CMTs (100%) (p = 0.001). In 96 patients with definite pathogens, 52 cases (50.5%) were infected with a single pathogen, while 44 cases (42.7%) had polymicrobial infections. Virus–bacteria and virus–virus co-infections were the most common. Staphylococcus aureus, Haemophilus influenzae, rhinovirus, cytomegalovirus, parainfluenza virus, and fungi were more likely to be associated with polymicrobial infections. Conclusions BALF mNGS improved the detection rate of pediatric pneumonia, especially in mixed infections. The diagnostic performance of BALF mNGS varies according to pathogen type. mNGS can be used to supplement CMTs. A combination of mNGS and CMTs may be the best diagnostic strategy.
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Project description:A precise and efficient microbiological diagnosis is essential for sepsis. Metagenomic next-generation sequencing (mNGS) is a novel technique for the diagnosis of infectious diseases, but its current application in multisite sampling and interpretation remains controversial. Therefore, this study was undertaken to evaluate the reliability of multisite mNGS tests and the efficiency of plasma mNGS based on lymphocyte subset counts. A prospective observational study was performed on the intubated patients with sepsis-associated lymphopenia from January 2020 to February 2022. During the study period, data on 71 patients with sepsis-induced lymphopenia were collected. Among the 125 mNGS tests, 95 were positive for pathogens, whereas of the 166 conventional microbiological tests (CMTs), 91 were positive. The comparison showed that 38 patients (53.5%) had at least one matched pair of plasma mNGS and CMT results, while for multisite sampling, 47 patients (66.2%) had at least one. Lymphocyte subset analysis showed that T lymphocyte (577 ± 317 versus 395 ± 207, P = 0.005) and CD4+ T lymphocyte (333 ± 199 versus 230 ± 120, P = 0.009) counts were lower in the matched group. According to receiver operating characteristic (ROC) analysis, a CD4+ T lymphocyte count lower than 266 cells/mm3 was predictive of a match result. For sepsis-associated lymphopenia patients, we found that multisite mNGS tests showed a higher positivity rate. With plasma mNGS, a lower CD4+ T lymphocyte count predicted a better match result with CMT. The lymphocyte subset analysis may promote the clinical interpretation of mNGS results. IMPORTANCE This study was undertaken to evaluate the reliability of pathogenic diagnoses based on multisite mNGS detection at the clinically suspected sites and to analyze the efficiency of plasma mNGS detection based on lymphocyte subset counts in patients with sepsis-associated lymphopenia.
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Project description:Leishmaniasis is a vector-borne disease caused by Leishmania. Although the incidence of leishmaniasis in China is currently low, it has not been completely eradicated. In 2019, visceral leishmaniasis was diagnosed in three patients using bone marrow microscopic examination and metagenomic next-generation sequencing (mNGS). The bone marrow mNGS results from the three patients indicated that 99.9, 99.6, and 30.3% of non-human reads matched the Leishmania genome, and plasma mNGS results from one of the patients revealed that 46.2% of non-human reads matched the Leishmania genome. In the second patient's plasma, no Leishmania sequences were detected by plasma mNGS, and the third patient's plasma was unavailable. The pathogen in all three patients was identified as Leishmania infantum. Leishmania amastigotes were observed by microscopic examination of bone marrow smears in all three patients, but were not found in peripheral blood smears. This indicates that the sensitivity of mNGS is higher than that of smear microscopy and that mNGS can be used to identify Leishmania at the species level. All three patients were elderly male farmers, two from Shanxi and one from Beijing. All three patients had splenomegaly and pancytopenia. Originally, these patients were misdiagnosed and treated for extended periods in other hospitals. Diagnoses of visceral leishmaniasis took place 6, 2, and 2 months after the onset of symptoms in the three patients. In conclusion, this study confirms that bone marrow mNGS can be used to quickly and accurately confirm a diagnosis in patients with suspected leishmaniasis.
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Project description:Pneumonia is one of the most important causes of morbidity and mortality in children. Identification and characterization of pathogens that cause infections are crucial for accurate treatment and accelerated recovery. However, in most cases, the causative agent cannot be identified, which is partly due to the limited spectrum of pathogens covered by current diagnostics based on nucleic acid amplification. Therefore, in this study, we explored the application of metagenomic next-generation sequencing (mNGS) for the diagnosis of children with severe pneumonia. From April to July 2017, 32 hospitalized children with severe nonresponding pneumonia in Shenzhen Children's Hospital were included in this study. Blood tests were conducted immediately after hospitalization to assess cell counts and inflammatory markers, oropharyngeal swabs were collected to identify common pathogens by qPCR and culture. After bronchoscopy, bronchoalveolar lavage fluid (BALF) samples were collected for further pathogen identification using standardized diagnostic tests and mNGS. Blood tests were normal in 3 of the 32 children. In 9 oropharyngeal swabs, bacterial pathogens were detected, in 5 of these Mycoplasma pneumoniae was detected. Adenovirus was detected in 5 BALF samples, using the Direct Immunofluorescence Assay (DFA). In 15 cases, no common pathogens were found in BALF samples, using the current standard diagnostic tests, while in all 32 BALFs, pathogens were identified using mNGS, including adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, cytomegalovirus and bocavirus. This study shows that, with mNGS, the sensitivity of detection of the causative pathogens in children with severe nonresponding pneumonia is significantly improved. In addition, mNGS gives more strain specific information, helps to identify new pathogens and could potentially help to trace and control outbreaks. In this study, we have shown that it is possible to have the results within 24 hours, making the application of mNGS feasible for clinical diagnostics.
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