Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis.

BACKGROUND: Metagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test. METHODS: In a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review. RESULTS: We enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment. CONCLUSIONS: Routine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).

Introduction, Transmission Dynamics, and Fate of Early Severe Acute Respiratory Syndrome Coronavirus 2 Lineages in Santa Clara County, California.

We combined viral genome sequencing with contact tracing to investigate introduction and evolution of severe acute respiratory syndrome coronavirus 2 lineages in Santa Clara County, California, from 27 January to 21 March 2020. From 558 persons with coronavirus disease 2019, 101 genomes from 143 available clinical samples comprised 17 lineages, including SCC1 (n = 41), WA1 (n = 9; including the first 2 reported deaths in the United States, with postmortem diagnosis), D614G (n = 4), ancestral Wuhan Hu-1 (n = 21), and 13 others (n = 26). Public health intervention may have curtailed the persistence of lineages that appeared transiently during February and March. By August, only D614G lineages introduced after 21 March were circulating in Santa Clara County.

Fatal Case of Chronic Jamestown Canyon Virus Encephalitis Diagnosed by Metagenomic Sequencing in Patient Receiving Rituximab.

A 56-year-old man receiving rituximab who had months of neurologic symptoms was found to have Jamestown Canyon virus in cerebrospinal fluid by clinical metagenomic sequencing. The patient died, and postmortem examination revealed extensive neuropathologic abnormalities. Deep sequencing enabled detailed characterization of viral genomes from the cerebrospinal fluid, cerebellum, and cerebral cortex.

Metagenomic Next-Generation Sequencing of the 2014 Ebola Virus Disease Outbreak in the Democratic Republic of the Congo.

We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient's blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance.

A mouse model of paralytic myelitis caused by enterovirus D68.

In 2014, the United States experienced an epidemic of acute flaccid myelitis (AFM) cases in children coincident with a nationwide outbreak of enterovirus D68 (EV-D68) respiratory disease. Up to half of the 2014 AFM patients had EV-D68 RNA detected by RT-PCR in their respiratory secretions, although EV-D68 was only detected in cerebrospinal fluid (CSF) from one 2014 AFM patient. Given previously described molecular and epidemiologic associations between EV-D68 and AFM, we sought to develop an animal model by screening seven EV-D68 strains for the ability to induce neurological disease in neonatal mice. We found that four EV-D68 strains from the 2014 outbreak (out of five tested) produced a paralytic disease in mice resembling human AFM. The remaining 2014 strain, as well as 1962 prototype EV-D68 strains Fermon and Rhyne, did not produce, or rarely produced, paralysis in mice. In-depth examination of the paralysis caused by a representative 2014 strain, MO/14-18947, revealed infectious virus, virion particles, and viral genome in the spinal cords of paralyzed mice. Paralysis was elicited in mice following intramuscular, intracerebral, intraperitoneal, and intranasal infection, in descending frequency, and was associated with infection and loss of motor neurons in the anterior horns of spinal cord segments corresponding to paralyzed limbs. Virus isolated from spinal cords of infected mice transmitted disease when injected into naïve mice, fulfilling Koch's postulates in this model. Finally, we found that EV-D68 immune sera, but not normal mouse sera, protected mice from development of paralysis and death when administered prior to viral challenge. These studies establish an experimental model to study EV-D68-induced myelitis and to better understand disease pathogenesis and develop potential therapies.

Acute Zika virus infection in an asymptomatic blood donor at the onset of the Puerto Rico epidemic.

BACKGROUND: Zika virus (ZIKV) spread to Puerto Rico likely originated from southeastern Brazil approximately 8.5 months earlier than blood donation screening for ZIKV was initiated, but the time of ZIKV introduction in the blood donor population remains unknown. METHODS: To better understand when arboviral infections first appeared in the blood donor pool in Puerto Rico, we retrospectively screened for ZIKV RNA (as well as chikungunya [CHIKV] and dengue [DENV] viral RNA) a repository of 1186 linked blood donor and recipient samples collected from February 2015 to May 2016 as an endpoint efficacy measure following the introduction of platelet pathogen reduction (PR). Phylogenetic analysis identified relatedness of donor strain to other circulating strains, and molecular clock analysis identified the estimated time of introduction. RESULTS: An asymptomatic donor collected in December 2015 was ZIKV RNA confirmed positive, 4 months BEFORE investigational nucleic acid testing (NAT) implementation in April 2016, coincident and related to the first reported autochthonous cases. No CHIKV RNA or DENV RNA reactives were identified in donors or recipients, and no adverse events were reported from PR use in recipients. Phylogenetic analysis confirmed the molecular relatedness of the donor ZIKV strain to the Puerto Rico lineage likely introduced approximately 4.5 months earlier. CONCLUSION: This study identified an asymptomatic ZIKV infection in a blood donor occurring before those previously recognized by blood donation screening. NAT and PR continue to be used as acceptable strategies to prevent transfusion-transmitted arboviral infections worldwide; however, repeated arboviral outbreaks warrant consideration of PR as a more proactive approach.

Actionable diagnosis of neuroleptospirosis by next-generation sequencing.

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Idiopathic pneumonia syndrome after hematopoietic cell transplantation: evidence of occult infectious etiologies.

Newer diagnostic methods may link more idiopathic pneumonia syndrome (IPS) cases to an infectious agent. Bronchoalveolar lavage (BAL) samples from 69 hematopoietic cell transplant (HCT) recipients with IPS diagnosed between 1992 and 2006 were tested for 28 pathogens (3 bacteria and 25 viruses) by quantitative polymerase chain reaction and for Aspergillus by galactomannan assay. Research BALs from 21 asymptomatic HCT patients served as controls. Among 69 HCT patients with IPS, 39 (56.5%) had a pathogen detected. The most frequent pathogens were human herpesvirus-6 (HHV-6) (N = 20 [29%]) followed by human rhinovirus (HRV), cytomegalovirus (CMV), and Aspergillus (N = 8 [12%] in each). HHV-6 and HRV were rarely detected in controls, whereas CMV and Aspergillus were occasionally detected with low pathogen load. Patients with pathogens had worse day-100 survival than those without (hazard ratio, 1.88; P = .03). Mortality in patients with only pathogens of "uncertain" significance in lung was similar to that in patients with pathogens of "established" significance. Metagenomic next-generation sequencing did not reveal additional significant pathogens. Our study demonstrated that approximately half of patients with IPS had pathogens detected in BAL, and pathogen detection was associated with increased mortality. Thus, an expanded infection detection panel can significantly increase the diagnostic precision for idiopathic pneumonia.

[Clinical effects of different therapeutic regimens for Helicobacter pylori infection in children].

OBJECTIVE: To investigate the clinical effects of sequential therapy, triple therapy, sequential therapy combined with Lactobacillus, and triple therapy combined with Lactobacillus in the eradication of Helicobacter pylori (H.pylori) infection in children. METHODS: A total of 416 children with H.pylori infection were randomly assigned to sequential group (102 children), triple group (100 children), sequential-Lactobacillus group (109 children), and triple-Lactobacillus group (105 children). The clinical outcome, H.pylori eradication rate, cost-effect ratio, and incidence of adverse events were compared between the four groups. RESULTS: The sequential-Lactobacillus and triple-Lactobacillus groups had significantly better clinical outcomes than the sequential group and the triple group (P<0.05). The sequential-Lactobacillus group had the highest marked response rate, followed by the triple-Lactobacillus group. The triple group had the lowest marked response rate. The sequential-Lactobacillus group also had the highest H.pylori eradication rate, followed by the triple-Lactobacillus group. The triple group had the lowest H.pylori eradication rate (P<0.05). The sequential group had the lowest cost-effect ratio, followed by the sequential-Lactobacillus group. The triple group had the highest cost-effect ratio (P<0.01). The sequential-Lactobacillus group had the lowest incidence rate of adverse events, followed by the triple-Lactobacillus group. The triple group had the highest incidence rate. CONCLUSIONS: Sequential therapy combined with Lactobacillus seems to be the best regimen for the eradication of H.pylori infection in children.

Two human immunodeficiency virus Type 2 cases in US blood donors including serologic, molecular, and genomic characterization of an epidemiologically unusual case.

BACKGROUND: Blood donation screening for human immunodeficiency virus Type 2 (HIV-2) has been in place in the United States since 1992. However, only three HIV-2 antibody-positive donors have been reported to date, all detected via HIV-1 cross-reactivity. STUDY DESIGN AND METHODS: Here we identify two additional HIV-2-positive donors by routine anti-HIV-1 and anti-HIV-2 screening, including a first-time male donor living in Georgia having recently immigrated to the United States from West Africa (from a 1998 donation) and a Taiwanese female repeat donor (nurse) living in California with no travel outside of Taiwan or apparent connections to West Africa (from a 2015 donation). Neither donor acknowledged any risk factors, and both remained asymptomatic through follow-up. The second donor was further investigated by serologic, molecular, and genomic assays because of her unusual demographics. She was documented to harbor HIV-2 RNA, albeit sporadically by HIV-2-specific nucleic acid tests (35%-100% of replicates) and at very low levels (<9.6 IU/mL). Metagenomic next-generation sequencing (mNGS) confirmed the identification of a Group B HIV-2 strain, with recovered reads covering 46.9% of the predicted genome. CONCLUSIONS: The estimated frequency of an HIV-2-positive blood donor in the United States is one in 57 million donations. Due to the low frequency and low pathogenicity of HIV-2, public health and blood donation screening efforts must focus on HIV-1 detection and prevention. However, detection of HIV-2 infection in a donor with no apparent link to West Africa suggests that the United States must remain vigilant for HIV-2 virus infections. Ultradeep mNGS may be useful in the future for comprehensive identification of rare transfusion-transmissible agents.

Genomic Assays for Identification of Chikungunya Virus in Blood Donors, Puerto Rico, 2014.

A newly developed transcription-mediated amplification assay was used to detect chikungunya virus infection in 3 of 557 asymptomatic donors (0.54%) from Puerto Rico during the 2014-2015 Caribbean epidemic. Viral detection was confirmed by using PCR, microarray, and next-generation sequencing. Molecular clock analysis dated the emergence of the Puerto Rico strains to early 2013.

Echinococcus multilocularis Calreticulin Interferes with C1q-Mediated Complement Activation.

As a zoonotic disease caused by Echinococcus multilocularis larvae, alveolar echinococcosis (AE) is one of the most severe forms of parasitic infection. Over a long evolutional process E. multilocularis has developed complex strategies to escape host immune attack and survive within a host. However, the mechanisms underlying immune evasion remain unclear. Here we investigated the binding activity of E. multilocularis calreticulin (EmCRT), a highly conserved Ca2+-binding protein, to human complement C1q and its ability to inhibit classical complement activation. ELISA, Far Western blotting and immunoprecipitation results demonstrated that both recombinant and natural EmCRTs bound to human C1q, and the interaction of recombinant EmCRT (rEmCRT) inhibited C1q binding to IgM. Consequently, rEmCRT inhibited classical complement activation manifested as decreasing C4/C3 depositions and antibody-sensitized cell lysis. Moreover, rEmCRT binding to C1q suppressed C1q binding to human mast cell, HMC-1, resulting in reduced C1q-induced mast cell chemotaxis. According to these results, E. multilocularis expresses EmCRT to interfere with C1q-mediated complement activation and C1q-dependent non-complement activation of immune cells, possibly as an immune evasion strategy of the parasite in the host.

Metagenomic Detection of Divergent Insect- and Bat-Associated Viruses in Plasma from Two African Individuals Enrolled in Blood-Borne Surveillance.

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.

Purifying selection decreases the potential for Bangui orthobunyavirus outbreaks in humans.

Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus-positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS.

Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids.

We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S-internal transcribed ribosomal gene spacer (28S-ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respectively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids.

Timely intervention and control of a novel coronavirus (COVID-19) outbreak at a large skilled nursing facility-San Francisco, California, 2020.

OBJECTIVE: To describe epidemiologic and genomic characteristics of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in a large skilled-nursing facility (SNF), and the strategies that controlled transmission. DESIGN, SETTING, AND PARTICIPANTS: This cohort study was conducted during March 22-May 4, 2020, among all staff and residents at a 780-bed SNF in San Francisco, California. METHODS: Contact tracing and symptom screening guided targeted testing of staff and residents; respiratory specimens were also collected through serial point prevalence surveys (PPSs) in units with confirmed cases. Cases were confirmed by real-time reverse transcription-polymerase chain reaction testing for SARS-CoV-2, and whole-genome sequencing (WGS) was used to characterize viral isolate lineages and relatedness. Infection prevention and control (IPC) interventions included restricting from work any staff who had close contact with a confirmed case; restricting movement between units; implementing surgical face masking facility-wide; and the use of recommended PPE (ie, isolation gown, gloves, N95 respirator and eye protection) for clinical interactions in units with confirmed cases. RESULTS: Of 725 staff and residents tested through targeted testing and serial PPSs, 21 (3%) were SARS-CoV-2 positive: 16 (76%) staff and 5 (24%) residents. Fifteen cases (71%) were linked to a single unit. Targeted testing identified 17 cases (81%), and PPSs identified 4 cases (19%). Most cases (71%) were identified before IPC interventions could be implemented. WGS was performed on SARS-CoV-2 isolates from 4 staff and 4 residents: 5 were of Santa Clara County lineage and the 3 others were distinct lineages. CONCLUSIONS: Early implementation of targeted testing, serial PPSs, and multimodal IPC interventions limited SARS-CoV-2 transmission within the SNF.

New Genomes from the Congo Basin Expand History of CRF01_AE Origin and Dissemination.

Although the first HIV circulating recombinant form (CRF01_AE) is the predominant strain in many Asian countries, it is uncommonly found in the Congo Basin from where it first originated. To fill the gap in the evolutionary history of this important strain, we sequenced near complete genomes from HIV samples with subgenomic CRF01_AE regions collected in Cameroon and the Democratic Republic of the Congo from 2001 to 2006. HIV genomes were generated from N = 13 plasma specimens by next-generation sequencing of metagenomic libraries prepared with spiked primers targeting HIV, followed by Sanger gap-filling. Genome sequences were aligned to reference strains, including Asian and African CRF01_AE sequences, and evaluated by phylogenetic and recombinant analysis to identify four CRF01_AE strains from Cameroon. We also identified two CRF02, one CRF27, and six unique recombinant form genomes (01|A1|G, 01|02|F|U, F|G|01, A1|D|01, F|G|01, and A1|G|01). Phylogenetic analysis, including the four new African CRF01_AE genomes, placed these samples as a bridge between basal Central African Republic CRF01_AE strains and all Asian, European, and American CRF01_AE strains. Molecular dating confirmed previous estimates indicating that the most recent common CRF01_AE ancestor emerged in the early 1970s (1968-1970) and spread beyond Africa around 1980 to Asia. The new sequences and analysis presented in this study expand the molecular history of the CRF01_AE clade, and are illustrated in an interactive Next Strain phylogenetic tree, map, and timeline at (https://nextstrain.org/community/EduanWilkinson/hiv-1_crf01).

Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-based DETECTR Lateral Flow Assay.

An outbreak of novel betacoronavirus, SARS-CoV-2 (formerly named 2019-nCoV), began in Wuhan, China in December 2019 and the COVID-19 disease associated with infection has since spread rapidly to multiple countries. Here we report the development of SARS-CoV-2 DETECTR, a rapid (~30 min), low-cost, and accurate CRISPR-Cas12 based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated this method using contrived reference samples and clinical samples from infected US patients and demonstrated comparable performance to the US CDC SARS-CoV-2 real-time RT-PCR assay.

CRISPR-Cas12-based detection of SARS-CoV-2.

An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.