Unbiased Metagenomic Sequencing Research Articles

Nanopore sequencing of infectious fluid is a promising supplement for gold-standard culture in real-world clinical scenario.

Infectious diseases are major causes of morbidity and mortality worldwide, necessitating the rapid identification and accurate diagnosis of pathogens. While unbiased metagenomic next-generation sequencing (mNGS) has been extensively utilized in clinical pathogen identification and scientific microbiome detection, there is limited research about the application of nanopore platform-based mNGS in the diagnostic performance of various infectious fluid samples. In this study, we collected 297 suspected infectious fluids from 10 clinical centers and detected them with conventional microbiology culture and nanopore platform-based mNGS. The objective was to assess detective and diagnostic performance of nanopore-sequencing technology (NST) in real-world scenarios. Combined with gold-standard culture and clinical adjudication, nanopore sequencing demonstrated nearly 100% positive predictive agreements in microbial-colonized sites, such as the respiratory and urinary tracts. For samples collected from initially sterile body sites, the detected microorganisms were highly suspected pathogens, and the negative predictive agreements were relatively higher than those in the microbial-colonized sites, particularly with 100% in abscess and 95.7% in cerebrospinal fluid. Furthermore, consistent performance was also observed in the identification of antimicrobial resistance genes and drug susceptibility testing of pathogenic strains of Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii. Rapid NST is a promising clinical tool to supplement gold-standard culture, and it has the potential improve patient prognosis and facilitate clinical treatment of infectious diseases.

Direct Dengue Virus Genome Sequencing from Antigen NS1 Rapid Diagnostic Tests: A Proof-of-Concept with the Standard Q Dengue Duo Assay.

With nearly half of the world's population being at risk of infection, dengue virus represents a major global health issue. The use of dengue antigen rapid diagnostic tests (Ag-RDTs) represents an alternative to PCR methods for the diagnosis of acute infections since they display excellent sensitivities and specificities and can be performed outside the laboratory. The high genetic diversity of the dengue virus genome represents a challenge for vaccine development, and the progressive expansion of this virus into previously nonendemic regions justifies the implementation of a genomic surveillance program. In this proof-of-concept study, we show the feasibility of sequencing dengue virus genomes directly from positive Ag-RDT (Standard Q Dengue Duo Test assay, n = 7) cassettes stored up to 31 days at room temperature after testing. For 5 of the 7 samples, a high number of reads were obtained allowing phylogenetic analyses to be carried out to determine not only the serotypes (dengue 1, 2, 3 and 4 were detected) but also the genotypes. Furthermore, in one sample, our unbiased metagenomic next-generation sequencing approach made it possible to detect epizootic hemorrhagic disease virus sequences, an arthropod-transmitted virus in ruminants. To conclude, as such an approach requires no cold storage or freezing of samples, dengue Ag-RDTs represent a very pragmatic and robust alternative for the genomic surveillance of dengue virus.

Successful Confirmation of Dual Genital Herpes Co-Infection with Herpes Simplex Virus 1 and Herpes Simplex Virus 2 Using Unbiased Metagenomic Next-Generation Sequencing.

Dual co-infection with both HSV-1 and HSV-2 is rare, with few cases reported in the literature. In this case report, we describe the successful use of unbiased metagenomic next-generation sequencing (mNGS) as a rapid and alternative method for confirming dual genital herpes co-infection. Our case involves a 74-year-old woman who presented with genital lesions and initially tested positive for both HSV-1 and HSV-2 via the Luminex ARIES HSV 1&2 assay. The entire mNGS process, from nucleic acid extraction to result analysis, was completed in less than 48 h. Using mNGS, we identified mapped reads specific to either HSV-1 or HSV-2 and screened the sequences to rule out mis-genotyping by the Luminex ARIES assay. Notably, the generated sequences can reveal sequence variations within multiple gene regions, demonstrating the potential of mNGS for identifying novel HSV-1 and HSV-2 variants. Our findings suggest that mNGS can serve as a rapid and reliable alternative confirmatory method for dual genital herpes infections, providing valuable information to guide appropriate treatment options for patients. By eliminating the need for prior knowledge of causative agents, mNGS offers an unbiased approach for detecting and characterizing viral co-infections.

Metagenomic surveillance uncovers diverse and novel viral taxa in febrile patients from Nigeria

Effective infectious disease surveillance in high-risk regions is critical for clinical care and pandemic preemption; however, few clinical diagnostics are available for the wide range of potential human pathogens. Here, we conduct unbiased metagenomic sequencing of 593 samples from febrile Nigerian patients collected in three settings: i) population-level surveillance of individuals presenting with symptoms consistent with Lassa Fever (LF); ii) real-time investigations of outbreaks with suspected infectious etiologies; and iii) undiagnosed clinically challenging cases. We identify 13 distinct viruses, including the second and third documented cases of human blood-associated dicistrovirus, and a highly divergent, unclassified dicistrovirus that we name human blood-associated dicistrovirus 2. We show that pegivirus C is a common co-infection in individuals with LF and is associated with lower Lassa viral loads and favorable outcomes. We help uncover the causes of three outbreaks as yellow fever virus, monkeypox virus, and a noninfectious cause, the latter ultimately determined to be pesticide poisoning. We demonstrate that a local, Nigerian-driven metagenomics response to complex public health scenarios generates accurate, real-time differential diagnoses, yielding insights that inform policy.

Metagenomic Next-Generation Sequencing for Pathogens in Bronchoalveolar Lavage Fluid Improves the Survival of Patients with Pulmonary Complications After Allogeneic Hematopoietic Stem Cell Transplantation.

Unbiased metagenomic next-generation sequencing (mNGS) has been used for infection diagnosis. In this study, we explored the clinical diagnosis value of mNGS for pulmonary complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). From August 2019 to June 2021, a prospective study was performed to comparatively analyze the pathogenic results of mNGS and conventional tests for bronchoalveolar lavage fluid (BALF) from 134 cases involving 101 patients with pulmonary complications after allo-HSCT. More pathogens were identified by mNGS than with conventional tests (226 vs 120). For bacteria, the diagnostic sensitivity (P = 0.144) and specificity (P = 0.687) were similar between the two methods. For fungus except Pneumocystis jirovecii (PJ), conventional tests had a significantly higher sensitivity (P = 0.013) with a similarly high specificity (P = 0.109). The sensitivities for bacteria and fungi could be increased with the combination of the two methods. As for PJ, both the sensitivity (100%) and specificity (99.12%) of mNGS were very high. For viruses, the sensitivity of mNGS was significantly higher (P = 0.021) and the negative predictive value (NPV) was 95.74% (84.27-99.26%). Pulmonary infection complications accounted for 90.30% and bacterium was the most common pathogen whether in single infection (63.43%) or mixed infection (81.08%). The 6-month overall survival (OS) of 88.89% in the early group (mNGS ≤ 7days) was significantly higher than that of 65.52% (HR 0.287, 95% CI 0.101-0.819, P = 0.006) in the late group (mNGS > 7days). mNGS for BALF could facilitate accurate and fast diagnosis for pulmonary complications. Early mNGS could improve the prognosis of patients with pulmonary complications after allo-HSCT. ClinicalTrials.gov identifier, NCT04051372.

Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms.

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.

Cutaneous ulcerative lesions of unknown etiology affecting lionfish Pterois spp. in the Gulf of Mexico.

Cutaneous ulcerative skin lesions in a complex of invasive Gulf of Mexico lionfish (Red Lionfish Pterois volitans, Devil Firefish P.miles, and the hybrid Red Lionfish × Devil Firefish) became epizootic beginning in mid-August 2017. Herein, we provide the first pathological descriptions of these lesions and summarize our analyses to elucidate the etiology of the disease. We examined ulcerated and normal fishthrough gross pathology and histopathology, bacterial sampling, and unbiased metagenomic next-generation sequencing. We tracked prevalence of the disease, and we used biological health indicators (condition factor, splenosomatic and hepatosomatic index) to evaluate impacts to health, while considering sex and age as potential risk factors. Typical ulcerative lesions were deep, exposing skeletal muscle, and were bordered by pale or reddened areas often with some degree of scale loss. Only incidental parasites were found in our examinations. Most fish (86%; n=50) exhibited wound healing grossly and histologically, confirmed by the presence of granulation tissues. A primary bacterial pathogen was not evident through bacterial culture or histopathology. Metagenomic next-generation sequencing did not reveal a viral pathogen (DNA or RNA) but did provide information about the microbiome of some ulcerated specimens. Compared with clinically healthy fish, ulcerated fish had a significantly lower condition factor and a higher splenosomatic index. Disease prevalence at monitored sites through July 2021 indicated that ulcerated fish were still present but at substantially lower prevalence than observed in 2017. Although some common findings in a number of specimens suggest a potential role for opportunistic bacteria, collectively our suite of diagnostics and analyses did not reveal an intralesional infectious agent, and we must consider the possibility that there was no communicable pathogen.

Detection and Characterization of a Novel Picornavirus in European Badger (Meles meles).

The recent development of unbiased metagenomic next-generation sequencing has provided a richer view of the wild animal virome making it necessary to expand the knowledge about virus diversity in wildlife, as well as to monitor their potential transmission to domestic animals or humans. In the present study, by screening collections of enteric specimens from wild animals, a novel picornavirus was identified in the intestinal content of a badger (Meles meles). By enrichment with a sequence-independent single-primer amplification (SISPA) approach and deep sequencing with Oxford Nanopore Technologies (ONT) platform, the genome sequence of a novel picornavirus strain, Badger/3A-2019/ITA, was reconstructed. On comparison based on the polyprotein sequences, the virus was distantly related (58.7% and 59.7% sequence identity at the nucleotide and amino acid level, respectively) to the feline picornavirus strain FFUP1, identified in 2012 in Portugal and classified into genus Sakobovirus within the species Sakobuvirus A. Upon phylogenetic, pairwise homology, and distance analyses performed on the P1, 2Chel, 3Cpro, and 3Dpol proteins and the complete genomic sequence, the badger picornavirus may be considered a member of a new sakobuvirus species, which we propose as Sakobuvirus B.

Absence of pathogenic viruses in COVID-19 convalescent plasma.

It is important to maintain the safety of blood products by avoiding the transfusion of units with known and novel viral pathogens. It is unknown whether COVID-19 convalescent plasma (CCP) may contain pathogenic viruses (either newly acquired or reactivated) that are not routinely screened for by blood centers. The DNA virome was characterized in potential CCP donors (n=30) using viral genome specific PCR primers to identify DNA plasma virome members of the Herpesviridae [Epstein Barr Virus (EBV), cytomegalovirus (CMV), human herpesvirus 6A/B, human herpesvirus 7] and Anelloviridae [Torque teno viruses (TTV), Torque teno mini viruses (TTMV), and Torque teno midi viruses (TTMDV)] families. In addition, the RNA plasma virome was characterized using unbiased metagenomic sequencing. Sequencing was done on a HiSeq2500 using high output mode with a read length of 2X100 bp. The sequencing reads were taxonomically classified using Kraken2. CMV and EBV seroprevalence were evaluated using a chemiluminescent immunoassay. TTV and TTMDV were detected in 12 (40%) and 4 (13%) of the 30 study participants, respectively; TTMDV was always associated with infection with TTV. We did not observe TTMV DNAemia. Despite CMV and EBV seroprevalences of 33.3% and 93.3%, respectively, we did not detect Herpesviridae DNA among the study participants. Metagenomic sequencing did not reveal any human RNA viruses in CCP, including no evidence of circulating SARS-CoV-2. There was no evidence of pathogenic viruses, whether newly acquired or reactivated, in CCP despite the presence of non-pathogenic Anelloviridae. These results confirm the growing safety data supporting CCP.

Prevalence of Epstein–Barr Virus in Patients with Intraocular Inflammation

ABSTRACT The relationship between Epstein–Barr virus (EBV) infection and uveitis is unclear. We conducted an observational cross-sectional study to determine the prevalence of EBV in uveitis and to describe the clinical features of EBV-positive uveitis cases. This study was carried out at the F.I. Proctor Foundation at the University of California, San Francisco. All patients with suspected infectious uveitis who underwent unbiased metagenomic deep sequencing (MDS) were included. Demographics, testing information, and clinical features were documented. Eleven out of 288 patients with suspected infectious uveitis had EBV detected by RNA-seq in intraocular fluid. The prevalence of EBV in uveitis in our study sample is 4%. Three out of 11 EBV-positive eyes (27%) were found to have biopsy-proven vitreoretinal lymphoma. Future studies are needed to determine if EBV may drive the development of vitreoretinal lymphoma and if its presence should heighten the suspicion of vitreoretinal lymphoma.

Diversity and evolution of the animal virome.

The COVID-19 pandemic has given the study of virus evolution and ecology new relevance. Although viruses were first identified more than a century ago, we likely know less about their diversity than that of any other biological entity. Most documented animal viruses have been sampled from just two phyla - the Chordata and the Arthropoda - with a strong bias towards viruses that infect humans or animals of economic and social importance, often in association with strong disease phenotypes. Fortunately, the recent development of unbiased metagenomic next-generation sequencing is providing a richer view of the animal virome and shedding new light on virus evolution. In this Review, we explore our changing understanding of the diversity, composition and evolution of the animal virome. We outline the factors that determine the phylogenetic diversity and genomic structure of animal viruses on evolutionary timescales and show how this impacts assessment of the risk of disease emergence in the short term. We also describe the ongoing challenges in metagenomic analysis and outline key themes for future research. A central question is how major events in the evolutionary history of animals, such as the origin of the vertebrates and periodic mass extinction events, have shaped the diversity and evolution of the viruses they carry.

Novel Applications of Metagenomics for Detection of Tickborne Pathogens.

Tick populations have expanded in many parts of the globe, bringing with them an enhanced appreciation and discovery of novel tickborne pathogens, as well an increased in reported human cases of tickborne disease. Targeted and unbiased (shotgun) clinical metagenomic sequencing tests are increasingly used for detection of known and emerging infectious agents and have recently been employed for detection of tickborne pathogens. This review describes the types of metagenomic sequencing assays used for detection of emerging tickborne pathogens and reviews the recent literature on this topic. Important diagnostic and interpretative challenges are also covered. Metagenomic analysis has emerged as a powerful tool for detection, discovery, characterization, and classification of tickborne pathogens. Shotgun metagenomics is especially promising because it allows for detection of all tickborne bacteria, viruses, and parasites in a single specimen. Despite the potential advantages, there are several important challenges, including high cost, complexity of testing and interpretation, and slow turnaround time. No doubt, these challenges will diminish with increased use and advances in the field.

Cerebrospinal Fluid from Healthy Pregnant Women Does Not Harbor a Detectable Microbial Community.

ABSTRACTCerebrospinal fluid (CSF) circulating in the human central nervous system has long been considered aseptic in healthy individuals, because normally, the blood-brain barrier can protect against microbial invasions. However, this dogma has been called into question by several reports that microbes were identified in human brains, raising the question of whether there is a microbial community in the CSF of healthy individuals without neurological diseases. Here, we collected CSF samples and other samples, including one-to-one matched oral and skin swab samples (positive controls), from 23 pregnant women aged between 23 and 40 years. Normal saline samples (negative controls), sterile swabs, and extraction buffer samples (contamination controls) were also collected. Twelve of the CSF specimens were also used to evaluate the physiological activities of detected microbes. Metagenomic and metatranscriptomic sequencing was performed in these 116 specimens. A total of 620 nonredundant microbes were detected, which were dominated by bacteria (74.6%) and viruses (24.2%), while in CSF samples, metagenomic sequencing found only 26 nonredundant microbes, including one eukaryote, four bacteria, and 21 viruses (mostly bacteriophages). The beta diversity of microbes compared between CSF metagenomic samples and other types of samples (except negative controls) was significantly different from that of the CSF self-comparison. In addition, there was no active or viable microbe in the matched metagenomic and metatranscriptomic sequencing of CSF specimens after subtracting those also found in normal saline, DNA extraction buffer, and skin swab specimens. In conclusion, our results showed no strong evidence of a colonized microbial community present in the CSF of healthy individuals.IMPORTANCE The microbiome is prevalent throughout human bodies, with profound health implications. However, it remains unclear whether it is present and active in human CSF, which has been long considered aseptic due to the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect the presence of a microbiome in CSF collected from 23 pregnant women with matched controls. Analysis of 116 specimens found no strong evidence to support the presence of a colonized microbiome in CSF. Our findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological infections and disorders, and will help further disease diagnostics, prevention, and therapeutics in clinical settings.

Intrathecal inflammatory responses in the absence of SARS-CoV-2 nucleic acid in the CSF of COVID-19 hospitalized patients

ObjectiveLittle is known about CSF profiles in patients with acute COVID-19 infection and neurological symptoms. Here, CSF was tested for SARS-CoV-2 RNA and inflammatory cytokines and chemokines and compared to controls and patients with known neurotropic pathogens. MethodsCSF from twenty-seven consecutive patients with COVID-19 and neurological symptoms was assayed for SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR) and unbiased metagenomic sequencing. Assays for blood brain barrier (BBB) breakdown (CSF:serum albumin ratio (Q-Alb)), and proinflammatory cytokines and chemokines (IL-6, IL-8, IL-15, IL-16, monocyte chemoattractant protein −1 (MCP-1) and monocyte inhibitory protein – 1β (MIP-1β)) were performed in 23 patients and compared to CSF from patients with HIV-1 (16 virally suppressed, 5 unsuppressed), West Nile virus (WNV) (n = 4) and 16 healthy controls (HC). ResultsMedian CSF cell count for COVID-19 patients was 1 white blood cell/μL; two patients were infected with a second pathogen (Neisseria, Cryptococcus neoformans). No CSF samples had detectable SARS-CoV-2 RNA by either detection method. In patients with COVID-19 only, CSF IL-6, IL-8, IL-15, and MIP-1β levels were higher than HC and suppressed HIV (corrected-p < 0.05). MCP-1 and MIP-1β levels were higher, while IL-6, IL-8, IL-15 were similar in COVID-19 compared to WNV patients. Q-Alb correlated with all proinflammatory markers, with IL-6, IL-8, and MIP-1β (r ≥ 0.6, p < 0.01) demonstrating the strongest associations. ConclusionsLack of SARS-CoV-2 RNA in CSF is consistent with pre-existing literature. Evidence of intrathecal proinflammatory markers in a subset of COVID-19 patients with BBB breakdown despite minimal CSF pleocytosis is atypical for neurotropic pathogens.

No Metagenomic Evidence of Causative Viral Pathogens in Postencephalitic Parkinsonism Following Encephalitis Lethargica.

Postencephalitic parkinsonism (PEP) is a disease of unknown etiology and pathophysiology following encephalitis lethargica (EL), an acute-onset polioencephalitis of cryptic cause in the 1920s. PEP is a tauopathy with multisystem neuronal loss and gliosis, clinically characterized by bradykinesia, rigidity, rest tremor, and oculogyric crises. Though a viral cause of EL is likely, past polymerase chain reaction-based investigations in the etiology of both PEP and EL were negative. PEP might be caused directly by an unknown viral pathogen or the consequence of a post-infectious immunopathology. The development of metagenomic next-generation sequencing in conjunction with bioinformatic techniques has generated a broad-range tool for the detection of unknown pathogens in the recent past. Retrospective identification and characterization of pathogens responsible for past infectious diseases can be successfully performed with formalin-fixed paraffin-embedded (FFPE) tissue samples. In this study, we analyzed 24 FFPE brain samples from six patients with PEP by unbiased metagenomic next-generation sequencing. Our results show that no evidence for the presence of a specific or putative (novel) viral pathogen was found, suggesting a likely post-infectious immune-mediated etiology of PEP.

Genomic investigation of atypical malaria cases in Kanel, northern Senegal

BackgroundThe diagnosis of malaria cases in regions where the malaria burden has decreased significantly and prevalence is very low is more challenging, in part because of reduced clinical presumption of malaria. The appearance of a cluster of malaria cases with atypical symptoms in Mbounguiel, a village in northern Senegal where malaria transmission is low, in September 2018 exemplifies this scenario. The collaboration between the National Malaria Control Programme (NMCP) at the Senegal Ministry of Health and the Laboratory of Parasitology and Mycology at Cheikh Anta Diop University worked together to evaluate this cluster of malaria cases using molecular and serological tools.MethodsMalaria cases were diagnosed primarily by rapid diagnostic test (RDT), and confirmed by photo-induced electron transfer-polymerase chain reaction (PET-PCR). 24 single nucleotide polymorphisms (SNPs) barcoding was used for Plasmodium falciparum genotyping. Unbiased metagenomic sequencing and Luminex-based multi-pathogen antibody and antigen profiling were used to assess exposure to other pathogens.ResultsNine patients, of 15 suspected cases, were evaluated, and all nine samples were found to be positive for P. falciparum only. The 24 SNPs molecular barcode showed the predominance of polygenomic infections, with identifiable strains being different from one another. All patients tested positive for the P. falciparum antigens. No other pathogenic infection was detected by either the serological panel or metagenomic sequencing.ConclusionsThis work, undertaken locally within Senegal as a collaboration between the NMCP and a research laboratory at University of Cheikh Anta Diop (UCAD) revealed that a cluster of malaria cases were caused by different strains of P. falciparum. The public health response in real time demonstrates the value of local molecular and genomics capacity in affected countries for disease control and elimination.

POECIVIRUS IS PRESENT IN INDIVIDUALS WITH BEAK DEFORMITIES IN SEVEN SPECIES OF NORTH AMERICAN BIRDS.

Avian keratin disorder (AKD), a disease of unknown etiology characterized by debilitating beak overgrowth, has increasingly affected wild bird populations since the 1990s. A novel picornavirus, poecivirus, is closely correlated with disease status in Black-capped Chickadees (Poecile atricapillus) in Alaska, US. However, our knowledge of the relationship between poecivirus and beak deformities in other species and other geographic areas remains limited. The growing geographic scope and number of species affected by AKD-like beak deformities require a better understanding of the causative agent to evaluate the population-level impacts of this epizootic. Here, we tested eight individuals from six avian species with AKD-consistent deformities for the presence of poecivirus: Mew Gull (Larus canus), Hairy Woodpecker (Picoides villosus), Black-billed Magpie (Pica hudsonia), American Crow (Corvus brachyrhynchos), Red-breasted Nuthatch (Sitta canadensis), and Blackpoll Warbler (Setophaga striata). The birds were sampled in Alaska and Maine (1999-2016). We used targeted PCR followed by Sanger sequencing to test for the presence of poecivirus in each specimen and to obtain viral genome sequence from virus-positive host individuals. We detected poecivirus in all individuals tested, but not in negative controls (water and tissue samples). Furthermore, we used unbiased metagenomic sequencing to test for the presence of other pathogens in six of these specimens (Hairy Woodpecker, two American Crows, two Red-breasted Nuthatches, Blackpoll Warbler). This analysis yielded additional viral sequences from several specimens, including the complete coding region of poecivirus from one Red-breasted Nuthatch, which we confirmed via targeted PCR followed by Sanger sequencing. This study demonstrates that poecivirus is present in individuals with AKD-consistent deformities from six avian species other than Black-capped Chickadee. While further investigation will be required to explore whether there exists a causal link between this virus and AKD, this study demonstrates that poecivirus is not geographically restricted to Alaska, but rather occurs elsewhere in North America.

64. Metagenomic Sequencing to Identify Alternative Infections and Co-infections in Persons Under Investigation for covid-19

BackgroundBroad testing for respiratory viruses among persons under investigation (PUI) for SARS-CoV-2 is performed inconsistently, limiting our understanding of alternative infections and co-infections in these patients. Here, we used unbiased metagenomic next-generation sequencing (mNGS) to assess the frequencies of 1) alternative viral infections in SARS-CoV-2 RT-PCR negative PUIs and 2) viral co-infections in SARS-CoV-2 RT-PCR positive PUIs.MethodsA convenience sample set was selected from PUIs who were tested for SARS-CoV-2 in the Emory Healthcare system during the first 2 months of the pandemic from 02/26-04/23/20. Laboratory results were extracted by chart review; Flu/RSV and multiplex respiratory pathogen PCRs had been performed at the discretion of treating physicians. Excess nasopharyngeal swab samples were retrieved within 72 hours of collection and underwent RNA extraction and SARS-CoV-2 testing by triplex RT-PCR. mNGS was performed by DNAse treatment, random primer cDNA synthesis, Nextera XT tagmentation, and high-depth Illumina sequencing. Reads underwent taxonomic classification by KrakenUniq, as implemented in viral-ngs.Results53 PUIs were included, 30 negative and 23 positive for SARS-CoV-2 by RT-PCR. Among SARS-CoV-2 negative PUIs, 28 (93%) underwent clinical testing for alternative infections, and 8 (29%) tested positive for another respiratory virus. In all cases, mNGS identified the same virus (Table 1). In another 3 PUIs, mNGS identified two viruses that were not tested for and one that was missed by routine testing. No SARS-CoV-2 was detected by mNGS among RT-PCR negative PUIs. Among SARS-CoV-2 RT-PCR positive PUIs, 18 (69%) underwent clinical testing for co-infections, and none were detected. mNGS did not identify any viral co-infections but did detect SARS-CoV-2 in all 23 PUIs.Table 1: Molecular and Metagenomic Testing of Persons Under Investigation ConclusionUnbiased mNGS offers the powerful opportunity to streamline testing for PUIs by assessing for SARS-CoV-2 and alternative infections simultaneously; this technique can also be used to identify co-infections, but none were observed in our study population. Interestingly, many PUIs had no infection identified on routine testing or mNGS, which may reflect inadequate sampling, rapid virus clearance, or a non-viral process.Disclosures All Authors: No reported disclosures

Identification of co-circulating pathogens that are clinically indistinguishable from Lassa fever during the 2018 Lassa outbreak in Nigeria

Background: The burden of undiagnosed febrile illness in low and middle-income countries is high and infectious diseases remain the leading cause of morbidity and mortality. The aetiology of systemic febrile illness and sepsis are particularly badly described partly because many pathogens cause a similar initial clinical presentation. In early 2018, Nigeria experienced its largest recorded outbreak of Lassa fever (LF). During this period of heightened transmission, there were 1,968 persons who met the clinical case definition for LF. However, only 431 could be laboratory confirmed, while for 1,537 no causative pathogen could be identified. The unprecedented scale of this outbreak prompted the Nigeria Centre for Disease Control (NCDC) and the WHO to reconsider their diagnostic strategy and re-prioritise LF diagnostics. A joint research collaboration between the NCDC and the United Kingdom Public Health Rapid Support Team (UK-PHRST) has worked to address this question. Methods and materials: Archived clinical diagnostic samples collected under the auspices of the Nigerian National Lassa Virus Testing Algorithm taken from patients who met NCDCs case definition for LF during the 2018 LF outbreak in Nigeria were sought and a non-probability sampling technique on consecutive cases who meet the selection criteria (lassa-negative by PCR and malaria-negative by RDT) was employed. RNA extracts were screened TaqMan array card system for 50 pathogens known to occur in West Africa and a subset of TAC+/- samples underwent unbiased metagenomic sequencing to determine which pathogen(s) are co-circulating and potentially mimicking the presentation of LF. Associations between pathogens and selected epidemiological and clinical variables were tested by Chi-square with significance level set at p < 0.05. Results: Results from this study indicate that there was no one specific pathogen responsible for patients developing clinical presentations indistinguishable from LF. Rather, a variety of pathogens (both viral and bacterial) were found to be co-circulating at the time of heightened LF transmission resulting in both single and co-infections (particularly in the immunocompromised). Conclusion: We anticipate that this study will help ensure proper and expedited diagnosis of diseases in the differential diagnosis for LF, helping to target treatment of patients with both LF and non-LF acute febrile illnesses in Nigeria.

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next-Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty-four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialised kits including a hybridisation capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP), and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples (n = 8), with mNGS and RVOP producing the most complete genomes. For mid viral loads (Ct 20–25), amplicon-based enrichment methods led to genome coverage >99 per cent for all samples while 1/8 sample sequenced with RVOP and 2/8 samples sequenced with mNGS had a genome coverage below 99 per cent. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in three samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34nt-deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a loss of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer-annealing or probe-hybridizing regions and would require further validation using unbiased metagenomic sequencing.