Capturing sequence diversity in metagenomes with comprehensive and scalable probe design.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. Here we present CATCH, a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs optimal probe sets, with a specified number of oligonucleotides, that achieve full coverage of, and scale well with, known sequence diversity. We focus on applying CATCH to capture viral genomes in complex metagenomic samples. We design, synthesize, and validate multiple probe sets, including one that targets the whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriches unique viral content on average 18-fold, allowing us to assemble genomes that could not be recovered without enrichment, and accurately preserves within-sample diversity. We also use these probe sets to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of uncharacterized viral infections in human and mosquito samples. The results demonstrate that CATCH enables more sensitive and cost-effective metagenomic sequencing

Genome sequencing reveals Zika virus diversity and spread in the Americas

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests

HIV Drug Resistance Surveillance in Honduras after a Decade of Widespread Antiretroviral Therapy.

We assessed HIV drug resistance (DR) in individuals failing ART (acquired DR, ADR) and in ART-naïve individuals (pre-ART DR, PDR) in Honduras, after 10 years of widespread availability of ART.365 HIV-infected, ART-naïve, and 381 ART-experienced Honduran individuals were enrolled in 5 reference centres in Tegucigalpa, San Pedro Sula, La Ceiba, and Choluteca between April 2013 and April 2015. Plasma HIV protease-RT sequences were obtained. HIVDR was assessed using the WHO HIVDR mutation list and the Stanford algorithm. Recently infected (RI) individuals were identified using a multi-assay algorithm.PDR to any ARV drug was 11.5% (95% CI 8.4-15.2%). NNRTI PDR prevalence (8.2%) was higher than NRTI (2.2%) and PI (1.9%, p500 vs. <350 CD4+ T cells/μL. PDR in recently infected individuals was 13.6%, showing no significant difference with PDR in individuals with longstanding infection (10.7%). The most prevalent PDR mutations were M46IL (1.4%), T215 revertants (0.5%), and K103NS (5.5%). The overall ADR prevalence in individuals with <48 months on ART was 87.8% and for the ≥48 months on ART group 81.3%. ADR to three drug families increased in individuals with longer time on ART (p = 0.0343). M184V and K103N were the most frequent ADR mutations. PDR mutation frequency correlated with ADR mutation frequency for PI and NNRTI (p<0.01), but not for NRTI. Clusters of viruses were observed suggesting transmission of HIVDR both from ART-experienced to ART-naïve individuals and between ART-naïve individuals.The global PDR prevalence in Honduras remains at the intermediate level, after 10 years of widespread availability of ART. Evidence of ADR influencing the presence of PDR was observed by phylogenetic analyses and ADR/PDR mutation frequency correlations

Development and validation of a rapid lateral flow E1/E2-antigen test and ELISA in patients infected with emerging Asian strain of Chikungunya virus in the Americas

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.U.S. Public Health Service (award AI100190

Field-deployable viral diagnostics using CRISPR-Cas13

Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015–2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.NIH (Grants AI-100190, 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201

Evaluation of ELISA-Based Multiplex Peptides for the Detection of Human Serum Antibodies Induced by Zika Virus Infection across Various Countries

Zika virus (ZIKV) is a mosquito-borne Flavivirus with a positive-sense RNA genome, which are generally transmitted through the bite of an infected Aedes mosquito. ZIKV infections could be associated with neurological sequelae that, and otherwise produces similar clinical symptoms as other co-circulating pathogens. Past infection with one member of the Flavivirus genus often induces cross-reactive antibodies against other flaviruses. These attributes complicate the ability to differentially diagnose ZIKV infection from other endemic mosquito-borne viruses, making it both a public health issue as well as a diagnostic challenge. We report the results from serological analyses using arbovirus-specific peptides on 339 samples that were previously collected from 6 countries. Overall, we found that our multiplexed peptide-based ELISA was highly efficient for identifying ZIKV antibodies as early as 2 weeks post infection, and that it correlates with microneutralization, plaque reduction neutralization tests (PRNTs) and commercial tests for ZIKV in previously characterized samples. We observed that seropositivity varied by patient cohort, reflecting the sampling period in relation to the 2015–2016 ZIKV outbreak. This work evaluates the accuracy, specificity, and sensitivity of our peptide-based ELISA method for detecting ZIKV antibodies from geographically diverse regions. These findings can contribute to ongoing serological methods development and can be adapted for use in future studies

Clinical evaluation of the BioFire Global Fever Panel for the identification of malaria, leptospirosis, chikungunya, and dengue from whole blood: a prospective, multicentre, cross-sectional diagnostic accuracy study

BACKGROUND: Acute febrile illness is a common presentation for patients at hospitals globally. Assays that can diagnose a variety of common pathogens in blood could help to establish a diagnosis for targeted disease management. We aimed to evaluate the performance of the BioFire Global Fever Panel (GF Panel), a multiplex nucleic acid amplification test performed on whole blood specimens run on the BioFire FilmArray System, in the diagnosis of several pathogens that cause acute febrile illness. METHODS: We did a prospective, multicentre, cross-sectional diagnostic accuracy study to evaluate the GF Panel. Consenting adults and children older than 6 months presenting with fever in the previous 2 days were enrolled consecutively in sub-Saharan Africa (Ghana, Kenya, Tanzania, Uganda), southeast Asia (Cambodia, Thailand), central and South America (Honduras, Peru), and the USA (Washington, DC; St Louis, MO). We assessed the performance of six analytes (chikungunya virus, dengue virus [serotypes 1-4], Leptospira spp, Plasmodium spp, Plasmodium falciparum, and Plasmodium vivax or Plasmodium ovale) on the GF Panel. The performance of the GF Panel was assessed using comparator PCR assays with different primers followed by bidirectional sequencing on nucleic acid extracts from the same specimen. We calculated the positive percent agreement and negative percent agreement of the GF Panel with respect to the comparator assays. This study is registered with ClinicalTrials.gov, NCT02968355. FINDINGS: From March 26, 2018, to Sept 30, 2019, 1965 participants were enrolled at ten sites worldwide. Of the 1875 participants with analysable results, 980 (52·3%) were female and the median age was 22 years (range 0-100). At least one analyte was detected in 657 (35·0%) of 1875 specimens. The GF Panel had a positive percent agreement for the six analytes evaluated as follows: chikungunya virus 100% (95% CI 86·3-100), dengue virus 94·0% (90·6-96·5), Leptospira spp 93·8% (69·8-99·8), Plasmodium spp 98·3% (96·3-99·4), P falciparum 92·7% (88·8-95·6), and P vivax or P ovale 92·7% (86·7-96·6). The GF Panel had a negative percent agreement equal to or greater than 99·2% (98·6-99·6) for all analytes. INTERPRETATION: This 1 h sample-to-answer, molecular device can detect common causative agents of acute febrile illness with excellent positive percent agreement and negative percent agreement directly in whole blood. The targets of the assay are prevalent in tropical and subtropical regions globally, and the assay could help to provide both public health surveillance and individual diagnoses. FUNDING: BioFire Defense, Joint Project Manager for Medical Countermeasure Systems and US Army Medical Materiel Development Activity, and National Institute of Allergy and Infectious Diseases

PDR in a Honduran HIV-1-infected cohort, April 2013-April 2015 (n = 365).

<p>^a Pre-Antiretroviral Treatment Drug Resistance (PDR) estimated using the WHO HIV transmitted drug resistance surveillance mutation list.</p><p>^b PDR estimated with the Stanford algorithm (v7.0), with a threshold of ≥15 for at least one antiretroviral drug of the specified class. ARV, Antiretroviral; NNRTI, Non-Nucleoside Reverse Transcriptase Inhibitors; NRTI, Nucleoside Reverse Transcriptase Inhibitors; PI, Protease Inhibitors.</p><p>PDR in a Honduran HIV-1-infected cohort, April 2013-April 2015 (n = 365).</p

Frequency of pre-ART and acquired HIV drug resistance mutations in Honduras April 2013-April 2015.

<p>^a Frequency in individuals with pre-ART drug resistance (PDR; defined with the WHO list of mutations for HIV drug resistance surveillance) to the corresponding drug class (PI, n = 7; NRTI, n = 7; NNRTI, n = 30). Mutations that contribute with drug resistance penalty scores in the Stanford algorithm are shown. Only mutations found in the cohort are shown. Mutations considered for the analysis are as follows</p><p>NRTIs: M41L, A62V, K65R, D67T, D67H, D67N, D67G, D67E, T69A, T69D, T69ins, T69N, T69C, T69I, T69G, T69S, K70G, K70Q, K70N, K70R, K70E, L74I, L74V, V75L, V75I, V75A, V75T, V75S, V75M, F77L, Y115F, F116Y, V118I, Q151M, M184VI, L210W, T215Y, T215A, T215F, T215CDESIV, K219QEN, K219R.</p><p>NNRTIs: V90I, A98G, L100I, K101E, K101P, K103NS, V106A, V106M, V108I, E138KQ, E138GAR, V179AT, V179D, V179E, V179L, V179F, Y181IV, Y181C, Y188L, Y188H, Y188C, G190S, G190A, G190E, G190C, P225H, F227L, M230L, K238T, Y318F.</p><p>PIs: L10F, K20I, L23I, L24I, D30N, V32I, L33F, E35G, K43T, M46IL, I47A, I47V, G48VM, I50L, I50V, F53L, F53Y, I54VA, I54L, I54M, I54ST, Q58E, G73CSTA, T74S, L76V, V82A, V82F, V82T, V82S, V82M, V82C, V82L, N83D, I84VAC, I85V, N88D, N88S, L90M.</p><p>Frequency of pre-ART and acquired HIV drug resistance mutations in Honduras April 2013-April 2015.</p

Phylogenetic relations between HIV sequences from ART-naïve and ART-experienced Honduran individuals.

<p>A Maximum Likelihood tree including HIV PR-RT sequences from 365 ART-naïve and 381 ART-experienced patients was built, using the General Time Reversible + Γ + I model to estimate genetic distances, with a gamma parameter of 0.4389 estimated for the dataset and 1000 bootstrap repetitions to assess significance. Drug resistance mutation sites as well as positions with less than 95% site coverage were eliminated from the alignment, with a total of 1162 positions included in the final dataset. Branch lengths are measured in number of substitutions per site. All analyses were conducted in MEGA6. Sequences from ART-naïve individuals are shown in grey and sequences from ART-experienced individuals in blue. Sequences with pre-ART drug resistance (PDR) to protease inhibitors (PI, pink), nucleoside RT inhibitors (NRTIs, green), non-nucleoside RT Inhibitors (NNRTIs, red), and more than one ARV family (purple) are coloured. B and non-B reference sequences (shown in black) were obtained from the Los Alamos HIV Database. A-D Clusters of viruses with PDR and bootstrap support >75% are amplified. HIVDR mutations present in the viruses at the tips are shown. Empty triangle, heterosexual male; full-triangle, men who have sex with men; empty circle, female; ART, antiretroviral treatment; USM, Unidad de Salud Metropolitana (La Ceiba); HMCR, Hospital Mario Catarino Rivas (San Pedro Sula); INCP, Instituto Nacional Cardio Pulmonar (Tegucigalpa).</p