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

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

Metagenomic next-generation sequencing (mNGS) of samples from 15 patients with documented Zika virus (ZIKV) infection in Bahia, Brazil, from April 2015 to January 2016 identified coinfections with chikungunya virus (CHIKV) in 2 of 15 ZIKV-positive cases by PCR (13.3%). While generally nonspecific, the clinical presentation corresponding to these two CHIKV/ZIKV coinfections reflected infection by the virus present at a higher titer. Aside from CHIKV and ZIKV, coinfections of other viral pathogens were not detected. The mNGS approach is promising for differential diagnosis of acute febrile illness and identification of coinfections, although targeted arbovirus screening may be sufficient in the current ZIKV outbreak setting

PCR testing for Human herpesvirus 3 (HHV) and Suid herpesvirus 1 (SuHV1).

<p>(A) PCR amplification was performed using a primer set designed from a single HHV3 read found in the dVIN sample 1 next-generation sequencing (NGS) data. The expected 163-bp product, identified as HHV3 by confirmatory Sanger sequencing, is only seen in an HHV3-positive cerebrospinal fluid (CSF) sample processed in parallel on the same NGS run, indicating that the single HHV3 read in the dVIN sample 1 NGS dataset was most likely due to cross-contamination. (B) PCR amplification was performed using a primer set designed from two SuHV1 ViroChip probes. Note that the dVIN sample 1 is negative for SuHV1. Abbreviations: L, DNA ladder; d1-d10, dVIN samples 1 through 10; N, normal skin vulvar biopsy; H, HHV3-positive CSF sample;; ntC, no template control.</p

Histology and p16^ink4a immunostaining patterns of high-grade dVIN and uVIN samples.

<p>(A) dVIN, showing characteristic elongation and anastomosis of rete ridges. The epithelium shows enlarged keratinoocytes with abundant eosinophilic cytoplasm. The inset displays prominent cytologic atypia localized to the lower 1/3 of the epithelium. (B) uVIN, warty subtype. A spiked surface epithelium with hyperkeratosis and hypergranulosis is visualized. The inset displays full-thickness cytologic atypia. (C) p16^ink4a immunostaining is negative in dVIN. (D) Full-thickness p16^ink4a immunopositivity is seen in high-grade uVIN. Abbreviations: dVIN, differentiated vulvar intraepithelial neoplasia; uVIN, usual-type vulvar intraepithelial neoplasia.</p

Schematic flowchart showing testing of FFPE vulvar tissues from 28 cases of high-grade differentiated VIN (dVIN) and usual-type VIN (uVIN).

<p>Histological (yellow boxes), p16^ink4a immunostaining (yellow boxes), and genomic (pink boxes) analyses were performed. Abbreviations: FFPE, formalin-fixed, paraffin-embedded; VIN, vulvar intraepithelial neoplasia; VSCC, vulvar squamous cell carcinoma; PCR, polymerase chain reaction.</p

Viruses and bacteria identified in tissue samples by next-generation sequencing.

<p>Viruses and bacteria identified in tissue samples by next-generation sequencing.</p

Number of next-generation sequencing (NGS) reads at each step of the SURPI bioinformatics pipeline for pathogen identification.

<p>Number of next-generation sequencing (NGS) reads at each step of the SURPI bioinformatics pipeline for pathogen identification.</p

Ranked Z-score analysis of ViroChip microarrays corresponding to dVIN samples and controls for virus identification.

<p>*Criteria: ≥5 hits out of top 50 probes (10%) and probe hits mapped to ≥3 distinct locations on the viral genome.</p><p>**Distinct locations: mapped probe locations on the viral genome separated by at least 5% of the total genome length.</p><p>Ranked Z-score analysis of ViroChip microarrays corresponding to dVIN samples and controls for virus identification.</p