Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases.

To end the largest known outbreak of Ebola virus disease (EVD) in West Africa and to prevent new transmissions, rapid epidemiological tracing of cases and contacts was required. The ability to quickly identify unknown sources and chains of transmission is key to ending the EVD epidemic and of even greater importance in the context of recent reports of Ebola virus (EBOV) persistence in survivors. Phylogenetic analysis of complete EBOV genomes can provide important information on the source of any new infection. A local deep sequencing facility was established at the Mateneh Ebola Treatment Centre in central Sierra Leone. The facility included all wetlab and computational resources to rapidly process EBOV diagnostic samples into full genome sequences. We produced 554 EBOV genomes from EVD cases across Sierra Leone. These genomes provided a detailed description of EBOV evolution and facilitated phylogenetic tracking of new EVD cases. Importantly, we show that linked genomic and epidemiological data can not only support contact tracing but also identify unconventional transmission chains involving body fluids, including semen. Rapid EBOV genome sequencing, when linked to epidemiological information and a comprehensive database of virus sequences across the outbreak, provided a powerful tool for public health epidemic control efforts

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Development and evaluation of antibody-capture immunoassays for detection of Lassa virus nucleoprotein-specific immunoglobulin M and G

<div><p>Background</p><p>The classical method for detection of Lassa virus-specific antibodies is the immunofluorescence assay (IFA) using virus-infected cells as antigen. However, IFA requires laboratories of biosafety level 4 for assay production and an experienced investigator to interpret the fluorescence signals. Therefore, we aimed to establish and evaluate enzyme-linked immunosorbent assays (ELISA) using recombinant Lassa virus nucleoprotein (NP) as antigen.</p><p>Methodology/Principal findings</p><p>The IgM ELISA is based on capturing IgM antibodies using anti-IgM, and the IgG ELISA is based on capturing IgG antibody–antigen complexes using rheumatoid factor or Fc gamma receptor CD32a. Analytical and clinical evaluation was performed with 880 sera from Lassa fever endemic (Nigeria) and non-endemic (Ghana and Germany) areas. Using the IFA as reference method, we observed 91.5–94.3% analytical accuracy of the ELISAs in detecting Lassa virus-specific antibodies. Evaluation of the ELISAs for diagnosis of Lassa fever on admission to hospital in an endemic area revealed a clinical sensitivity for the stand-alone IgM ELISA of 31% (95% CI 25–37) and for combined IgM/IgG detection of 26% (95% CI 21–32) compared to RT-PCR. The specificity of IgM and IgG ELISA was estimated at 96% (95% CI 93–98) and 100% (95% CI 99–100), respectively, in non-Lassa fever patients from non-endemic areas. In patients who seroconverted during follow-up, Lassa virus-specific IgM and IgG developed simultaneously rather than sequentially. Consistent with this finding, isolated IgM reactivity, i.e. IgM in the absence of IgG, had no diagnostic value.</p><p>Conclusions/Significance</p><p>The ELISAs are not equivalent to RT-PCR for early diagnosis of Lassa fever; however, they are of value in diagnosing patients at later stage. The IgG ELISA may be useful for epidemiological studies and clinical trials due its high specificity, and the higher throughput rate and easier operation compared to IFA.</p></div

Clinical performance characteristics of the IgM ELISA as stand-alone test and in combination with the IgG ELISA for early diagnosis of Lassa fever.

<p>Clinical performance characteristics of the IgM ELISA as stand-alone test and in combination with the IgG ELISA for early diagnosis of Lassa fever.</p

Clinical performance characteristics of the IgM ELISA as stand-alone test and in combination with the IgG ELISA depending on prevalence of Lassa fever and pre-existing IgG among all patients tested.

<p>The calculations are based on the data shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006361#pntd.0006361.t003" target="_blank">Table 3</a>. PPV and NPV of the stand-alone IgM ELISA depend only on the Lassa fever prevalence in diagnostics. However, PPV, NPV, sensitivity, and positive likelihood ratio for combined detection of IgM and IgG (i.e. a positive test result means that both IgM and IgG is positive) depend on the prevalence of Lassa fever as well as the prevalence of pre-existing IgG among all patients tested. To simplify calculation, we assumed a ratio of 1:3 between prevalence of Lassa fever and pre-existing IgG, which roughly corresponds to the setting in Nigeria where the study was performed.</p

Sample-to-cut-off (S/CO) values for 880 sera tested in IgM ELISA, RF-based IgG ELISA, and CD32-based IgG ELISA and comparison with IFA results.

<p>The S/CO values obtained with the ELISA are shown as histograms according to sample origin, type of ELISA, and IFA results. The cut-offs for the ELISAs are indicated by vertical dotted lines. The number of samples with OD < CO and OD > CO is indicated in left and right corner, respectively, of each diagram.</p

Analytical performance of IgM and IgG ELISA in detecting Lassa virus-specific antibodies in 576 serum samples from a Lassa fever endemic area in Nigeria using IFA as a reference method.

<p>Analytical performance of IgM and IgG ELISA in detecting Lassa virus-specific antibodies in 576 serum samples from a Lassa fever endemic area in Nigeria using IFA as a reference method.</p

Comparison of RF- and CD32-based IgG ELISA results obtained from 776 sera.

<p>The S/CO values obtained with the RF ELISA were plotted against the S/CO values of the CD32 ELISA. The cut-offs for both assays are indicated by horizontal and vertical dotted lines. The number of samples per quadrant is also given. The regression curve is shown as straight line. Samples from Lassa fever non-endemic and endemic countries were plotted separately.</p

Phylogeography of Lassa Virus in Nigeria

Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatiotemporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian states. The final data set, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 states. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while a few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi Within lineages II and III, seven and five sublineages, respectively, were distinguishable. Phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal toward the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast toward Taraba and Adamawa and south toward Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sublineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at a higher resolution. In addition, we estimated the direction and time frame of virus dispersal in the country.IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sublineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows us to predict into which geographical areas the virus might spread in the future and prioritize areas for Lassa fever surveillance. Our study not only aimed to generate Lassa virus sequences from across Nigeria but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.status: publishe

IgM and IgG ELISA results in patients grom various settings.

<p>IgM and IgG ELISA results in patients grom various settings.</p