Unbiased metagenomic next-generation sequencing of blood from hospitalized febrile children in Gabon.
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ABSTRACT:
SUBMITTER: Fernandes JF
PROVIDER: S-EPMC7448917 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:IntroductionMetagenomic next-generation sequencing (mNGS) assay for detecting infectious agents is now in the stage of being translated into clinical practice. With no approved approaches or guidelines available, laboratories adopt customized mNGS assays to detect clinical samples. However, the accuracy, reliability, and problems of these routinely implemented assays are not clear.ObjectivesTo evaluate the performance of 90 mNGS laboratories under routine testing conditions through analyzing identical samples.MethodsEleven microbial communities were generated using 15 quantitative microbial suspensions. They were used as reference materials to evaluate the false negatives and false positives of participating mNGS protocols, as well as the ability to distinguish genetically similar organisms and to identify true pathogens from other microbes based on fictitious case reports.ResultsHigh interlaboratory variability was found in the identification and the quantitative reads per million reads (RPM) values of each microbe in the samples, especially when testing microbes present at low concentrations (1 × 103 cell/ml or less). 42.2% (38/90) of the laboratories reported unexpected microbes (i.e. false positive problem). Only 56.7% (51/90) to 83.3% (75/90) of the laboratories showed a sufficient ability to obtain clear etiological diagnoses for three simulated cases combined with patient information. The analysis of the performance of mNGS in distinguishing genetically similar organisms in three samples revealed that only 56.6% to 63.0% of the laboratories recovered RPM ratios (RPM S. aureus /RPM S. epidermidis ) within the range of a 2-fold change of the initial input ratios (indicating a relatively low level of bias).ConclusionThe high interlaboratory variability found in both identifying microbes and distinguishing true pathogens emphasizes the urgent need for improving the accuracy and comparability of the results generated across different mNGS laboratories, especially in the detection of low-microbial-biomass samples.
| S-EPMC9091723 | biostudies-literature
Project description:Febrile illness is a major burden in African children, and non-malarial causes of fever are uncertain. In this retrospective exploratory study, we used metagenomic next-generation sequencing (mNGS) to evaluate serum, nasopharyngeal, and stool specimens from 94 children (aged 2-54 months) with febrile illness admitted to Tororo District Hospital, Uganda. The most common microbes identified were Plasmodium falciparum (51.1% of samples) and parvovirus B19 (4.4%) from serum; human rhinoviruses A and C (40%), respiratory syncytial virus (10%), and human herpesvirus 5 (10%) from nasopharyngeal swabs; and rotavirus A (50% of those with diarrhea) from stool. We also report the near complete genome of a highly divergent orthobunyavirus, tentatively named Nyangole virus, identified from the serum of a child diagnosed with malaria and pneumonia, a Bwamba orthobunyavirus in the nasopharynx of a child with rash and sepsis, and the genomes of two novel human rhinovirus C species. In this retrospective exploratory study, mNGS identified multiple potential pathogens, including 3 new viral species, associated with fever in Ugandan children.
| S-EPMC6586300 | biostudies-literature
Project description:ObjectivesTravel-associated infections are challenging to diagnose because of the broad spectrum of potential aetiologies. As a proof-of-principle study, we used MNGS to identify viral pathogens in clinical samples from returning travellers in a single center to explore its suitability as a diagnostic tool.MethodsPlasma samples from 40 returning travellers presenting with a fever of ≥38°C were sequenced using MNGS on the Illumina MiSeq platform and compared with standard-of-care diagnostic assays.ResultsIn total, 11/40 patients were diagnosed with a viral infection. Standard of care diagnostics revealed 5 viral infections using plasma samples; dengue virus 1 (n = 2), hepatitis E (n = 1), Ebola virus (n = 1) and hepatitis A (n = 1), all of which were detected by MNGS. Three additional patients with Chikungunya virus (n = 2) and mumps virus were diagnosed by MNGS only. Respiratory infections detected by nasal/throat swabs only were not detected by MNGS of plasma. One patient had infection with malaria and mumps virus during the same admission.ConclusionsMNGS analysis of plasma samples improves the sensitivity of diagnosis of viral infections and has potential as an all-in-one diagnostic test. It can be used to identify infections that have not been considered by the treating physician, co-infections and new or emerging pathogens.SummaryNext generation sequencing (NGS) has potential as an all-in-one diagnostic test. In this study we used NGS to diagnose returning travellers with acute febrile illness in the UK, highlighting cases where the diagnosis was missed using standard methods.
| S-EPMC6859916 | biostudies-literature
Project description:BackgroundEarly availability of pathogen identification in urinary tract infections (UTIs) has critical importance in disease management. Metagenomic next-generation sequencing (mNGS) has the potential to transform how acute and serious infections are diagnosed by offering unbiased and culture-free pathogen detection. However, clinical experience with application of the mNGS test is relatively limited.MethodsWe therefore established a MinION-based mNGS pathogens diagnostic platform and evaluated its potential for clinical implementation in UTIs with clinical samples. 213 urine samples from patients with suspected UTIs were included and subjected to mNGS testing using the MinION platform. mNGS results were compared to the gold standard of clinical culture and composite standard of combining clinical testing, confirmatory qPCR testing, and clinical adjudication by doctors.ResultsThe mNGS exhibited a sensitivity of 81.4% and a specificity of 92.3%, along with a positive predictive value of 96.6%, a negative predictive value of 64.9%, and an overall accuracy of 84.4%, all of which were determined based on the gold standard of routine culture results. When assessed against the composite standard, the sensitivity and specificity both increased to 89.9% and 100%, respectively, while the accuracy rose to 92.4%. Notably, the positive predictive value and negative predictive value also saw improvements, reaching 100% and 76.8%, respectively. Moreover, this diagnostic platform successfully identified dsDNA viruses. Among the 65 culture-negative samples, the viral detection rate reached 33.8% (22/65) and was subsequently validated through qPCR. Furthermore, the automatic bioinformatics pipeline we developed enabled one-click analysis from data to results, leading to a significant reduction in diagnosis time.ConclusionThese results demonstrate that the pathogen detection performance of mNGS is sufficient for diagnostic testing in clinical settings. As the method is generally unbiased, it can improve diagnostic testing of UTIs and other microbial infections.
| S-EPMC10612365 | biostudies-literature
Project description:BackgroundMetagenomic next-generation sequencing (mNGS) has made a revolution in the mode of pathogen identification. We decided to explore the diagnostic value of blood and bronchoalveolar lavage fluid (BALF) as mNGS samples in pneumonia.MethodsWe retrospectively reviewed 467 mNGS results and assessed the diagnostic performance of paired blood and BALF mNGS in 39 patients with pneumonia.ResultsFor bacteria and fungi, 16 patients had culture-confirmed pathogen diagnosis, while 13 patients were culture-negative. BALF mNGS was more sensitive than blood mNGS (81.3% vs. 25.0%, p=0.003), and the specificity in BALF and blood mNGS was not statistically significant different (76.9% vs. 84.6%, p=0.317). For 10 patients without culture test, treatments were changed in 2 patients. For viruses, Epstein-Barr virus was positive in blood mNGS in 9 patients. Human adenovirus was detected in both BALF and blood mNGS in 3 patients.ConclusionOur study suggests that BALF mNGS is more sensitive than blood mNGS in detecting bacteria and fungi, but blood also has advantages to identify the pathogens of pneumonia, especially for some viruses.
| S-EPMC7448216 | biostudies-literature
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.
| S-EPMC7538539 | biostudies-literature
Project description:BACKGROUND: The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown. RESULTS: In the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis. CONCLUSION: The analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.
| S-EPMC3922542 | biostudies-literature
Project description:BackgroundMetagenomic next-generation sequencing (mNGS) technology has been widely used to diagnose various infections. Based on the most common pathogen profiles, targeted mNGS (tNGS) using multiplex PCR has been developed to detect pathogens with predesigned primers in the panel, significantly improving sensitivity and reducing economic burden on patients. However, there are few studies on summarizing pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China on large scale.MethodsFrom January 2021 to December 2023, bronchoalveolar lavage fluid (BALF) or sputum samples from 546 immunocompetent and immunocompromised patients with suspected community-acquired pneumonia were collected. Pathogen profiles in those patients on whom mNGS was performed were summarized. Additionally, we also evaluated the performance of tNGS in diagnosing pulmonary infections.ResultsCombined with results of mNGS and culture, we found that the most common bacterial pathogens were Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii in both immunocompromised and immunocompetent patients with high detection rates of Staphylococcus aureus and Enterococcus faecium, respectively. For fungal pathogens, Pneumocystis jirovecii was commonly detected in patients, while fungal infections in immunocompetent patients were mainly caused by Candida albicans. Most of viral infections in patients were caused by Human betaherpesvirus 5 and Human gammaherpesvirus 4. It is worth noting that, compared with immunocompetent patients (34.9%, 76/218), more mixed infections were found in immunocompromised patients (37.8%, 14/37). Additionally, taking final comprehensive clinical diagnoses as reference standard, total coincidence rate of BALF tNGS (81.4%, 48/59) was much higher than that of BALF mNGS (40.0%, 112/280).ConclusionsOur findings supplemented and classified the pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China. Most importantly, our findings can accelerate the development and design of tNGS specifically used for regional pulmonary infections.
| S-EPMC11333343 | biostudies-literature
Project description:ObjectivesThis study aimed to evaluate the clinical performance of plasma cell-free DNA (cfDNA) next-generation sequencing (NGS) for pathogen detection in patients with sepsis.MethodsA total of 43 pairs of blood and plasma samples form 33 blood culture-positive patients were used as testing samples in metagenomic NGS (mNGS) and NGS of 16S ribosomal RNA gene amplicons (16S rRNA NGS). The results of routine tests, including microbial culture, complete blood count, and biochemical tests, were collected from electronic medical records.ResultsUsing blood as an mNGS testing sample, the proportion of host DNA was 99.9%, with only three bacteria and no fungi detected. When using plasma in mNGS, the proportion of host DNA was approximately 97%, with 84 bacteria and two fungi detected. Notably, 16S rRNA NGS detected 15 and 16 bacteria in 43 pairs of blood and plasma samples, respectively. Blood culture detected 49 bacteria (23 gram-negative bacilli and 26 gram-positive cocci) and four fungi, with 14 bacteria considered contaminants by clinical microbiologists. For all blood cultures, plasma cfDNA mNGS detected 78.26% (19/23) gram-negative rods, 17% (2/12) gram-positive cocci, and no fungi. Compared to blood cultures, the sensitivity and specificity of plasma cfDNA mNGS for detecting bacteria and fungi were 62.07% and 57.14%, respectively.ConclusionCompared to blood, plasma is more suitable for the detection of bloodstream infections using mNGS and is less affected by host DNA. The positive detection rate of plasma cfDNA mNGS for bloodstream infections caused by gram-negative bacteria was higher than that caused by gram-positive cocci.
| S-EPMC10847245 | biostudies-literature
Project description:A diagnosis of brucellosis can be difficult because routine culture and serological methods exhibit variable sensitivity and specificity. We present the use of a metagenomic next- generation sequencing assay to diagnose a case of neurobrucellosis from cerebrospinal fluid, resulting in the institution of appropriate antibiotic treatment and a favorable clinical outcome.
| S-EPMC6251619 | biostudies-literature