Increased Likelihood of Detecting Ebola Virus RNA in Semen by Using Sample Pelleting.

Ebola virus RNA can reside for months or years in semen of survivors of Ebola virus disease and is probably associated with increased risk for cryptic sexual transmission of the virus. A modified protocol resulted in increased detection of Ebola virus RNA in semen and improved disease surveillance.

Lassa virus circulating in Liberia: a retrospective genomic characterisation.

BACKGROUND: An alarming rise in reported Lassa fever cases continues in west Africa. Liberia has the largest reported per capita incidence of Lassa fever cases in the region, but genomic information on the circulating strains is scarce. The aim of this study was to substantially increase the available pool of data to help foster the generation of targeted diagnostics and therapeutics. METHODS: Clinical serum samples collected from 17 positive Lassa fever cases originating from Liberia (16 cases) and Guinea (one case) within the past decade were processed at the Liberian Institute for Biomedical Research using a targeted-enrichment sequencing approach, producing 17 near-complete genomes. An additional 17 Lassa virus sequences (two from Guinea, seven from Liberia, four from Nigeria, and four from Sierra Leone) were generated from viral stocks at the US Centers for Disease Control and Prevention (Atlanta, GA) from samples originating from the Mano River Union (Guinea, Liberia, and Sierra Leone) region and Nigeria. Sequences were compared with existing Lassa virus genomes and published Lassa virus assays. FINDINGS: The 23 new Liberian Lassa virus genomes grouped within two clades (IV.A and IV.B) and were genetically divergent from those circulating elsewhere in west Africa. A time-calibrated phylogeographic analysis incorporating the new genomes suggests Liberia was the entry point of Lassa virus into the Mano River Union region and estimates the introduction to have occurred between 300-350 years ago. A high level of diversity exists between the Liberian Lassa virus genomes. Nucleotide percent difference between Liberian Lassa virus genomes ranged up to 27% in the L segment and 18% in the S segment. The commonly used Lassa Josiah-MGB assay was up to 25% divergent across the target sites when aligned to the Liberian Lassa virus genomes. INTERPRETATION: The large amount of novel genomic diversity of Lassa virus observed in the Liberian cases emphasises the need to match deployed diagnostic capabilities with locally circulating strains and underscores the importance of evaluating cross-lineage protection in the development of vaccines and therapeutics. FUNDING: Defense Biological Product Assurance Office of the US Department of Defense and the Armed Forces Health Surveillance Branch and its Global Emerging Infections Surveillance and Response Section.

Persistence of Ebola virus after the end of widespread transmission in Liberia: an outbreak report.

BACKGROUND: Outbreak response efforts for the 2014-15 Ebola virus disease epidemic in west Africa brought widespread transmission to an end. However, subsequent clusters of infection have occurred in the region. An Ebola virus disease cluster in Liberia in November, 2015, that was identified after a 15-year-old boy tested positive for Ebola virus infection in Monrovia, raised the possibility of transmission from a persistently infected individual. METHODS: Case investigations were done to ascertain previous contact with cases of Ebola virus disease or infection with Ebola virus. Molecular investigations on blood samples explored a potential linkage between Ebola virus isolated from cases in this November, 2015, cluster and epidemiologically linked cases from the 2014-15 west African outbreak, according to the national case database. FINDINGS: The cluster investigated was the family of the index case (mother, father, three siblings). Ebola virus genomes assembled from two cases in the November, 2015, cluster, and an epidemiologically linked Ebola virus disease case in July, 2014, were phylogenetically related within the LB5 sublineage that circulated in Liberia starting around August, 2014. Partial genomes from two additional individuals, one from each cluster, were also consistent with placement in the LB5 sublineage. Sequencing data indicate infection with a lineage of the virus from a former transmission chain in the country. Based on serology and epidemiological and genomic data, the most plausible scenario is that a female case in the November, 2015, cluster survived Ebola virus disease in 2014, had viral persistence or recurrent disease, and transmitted the virus to three family members a year later. INTERPRETATION: Investigation of the source of infection for the November, 2015, cluster provides evidence of Ebola virus persistence and highlights the risk for outbreaks after interruption of active transmission. These findings underscore the need for focused prevention efforts among survivors and sustained capacity to rapidly detect and respond to new Ebola virus disease cases to prevent recurrence of a widespread outbreak. FUNDING: US Centers for Disease Control and Prevention, Defense Threat Reduction Agency, and WHO.

Xenosurveillance reflects traditional sampling techniques for the identification of human pathogens: A comparative study in West Africa.

BACKGROUND: Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. METHODOLOGY/PRINCIPAL FINDINGS: We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. CONCLUSIONS/SIGNIFICANCE: This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens.

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

West African Anopheles gambiae mosquitoes harbor a taxonomically diverse virome including new insect-specific flaviviruses, mononegaviruses, and totiviruses.

Anopheles gambiae are a major vector of malaria in sub-Saharan Africa. Viruses that naturally infect these mosquitoes may impact their physiology and ability to transmit pathogens. We therefore used metagenomics sequencing to search for viruses in adult Anopheles mosquitoes collected from Liberia, Senegal, and Burkina Faso. We identified a number of virus and virus-like sequences from mosquito midgut contents, including 14 coding-complete genome segments and 26 partial sequences. The coding-complete sequences define new viruses in the order Mononegavirales, and the families Flaviviridae, and Totiviridae. The identification of a flavivirus infecting Anopheles mosquitoes broadens our understanding of the evolution and host range of this virus family. This study increases our understanding of virus diversity in general, begins to define the virome of a medically important vector in its natural setting, and lays groundwork for future studies examining the potential impact of these viruses on anopheles biology and disease transmission.

Reduced evolutionary rate in reemerged Ebola virus transmission chains.

On 29 June 2015, Liberia's respite from Ebola virus disease (EVD) was interrupted for the second time by a renewed outbreak ("flare-up") of seven confirmed cases. We demonstrate that, similar to the March 2015 flare-up associated with sexual transmission, this new flare-up was a reemergence of a Liberian transmission chain originating from a persistently infected source rather than a reintroduction from a reservoir or a neighboring country with active transmission. Although distinct, Ebola virus (EBOV) genomes from both flare-ups exhibit significantly low genetic divergence, indicating a reduced rate of EBOV evolution during persistent infection. Using this rate of change as a signature, we identified two additional EVD clusters that possibly arose from persistently infected sources. These findings highlight the risk of EVD flare-ups even after an outbreak is declared over.

Lateral Flow Immunoassays for Ebola Virus Disease Detection in Liberia.

BACKGROUND: Lateral flow immunoassays (LFIs) are point-of-care diagnostic assays that are designed for single use outside a formal laboratory, with in-home pregnancy tests the best-known example of these tests. Although the LFI has some limitations over more-complex immunoassay procedures, such as reduced sensitivity and the potential for false-positive results when using complex sample matrices, the assay has the benefits of a rapid time to result and ease of use. These benefits make it an attractive option for obtaining rapid results in an austere environment. In an outbreak of any magnitude, a field-based rapid diagnostic assay would allow proper patient transport and for safe burials to be conducted without the delay caused by transport of samples between remote villages and testing facilities. Use of such point-of-care instruments in the ongoing Ebola virus disease (EVD) outbreak in West Africa would have distinct advantages in control and prevention of local outbreaks, but proper understanding of the technology and interpretation of results are important. METHODS: In this study, a LFI, originally developed by the Naval Medical Research Center for Ebola virus environmental testing, was evaluated for its ability to detect the virus in clinical samples in Liberia. Clinical blood and plasma samples and post mortem oral swabs submitted to the Liberian Institute for Biomedical Research, the National Public Health Reference Laboratory for EVD testing, were tested and compared to results of real-time reverse transcription-polymerase chain reaction (rRT-PCR), using assays targeting Ebola virus glycoprotein and nucleoprotein. RESULTS: The LFI findings correlated well with those of the real-time RT-PCR assays used as benchmarks. CONCLUSIONS: Rapid antigen-detection tests such as LFIs are attractive alternatives to traditional immunoassays but have reduced sensitivity and specificity, resulting in increases in false-positive and false-negative results. An understanding of the strengths, weaknesses, and limitations of a particular assay lets the diagnostician choose the correct situation to use the correct assay and properly interpret the results.

Evolution and Spread of Ebola Virus in Liberia, 2014-2015.

The 2013-present Western African Ebola virus disease (EVD) outbreak is the largest ever recorded with >28,000 reported cases. Ebola virus (EBOV) genome sequencing has played an important role throughout this outbreak; however, relatively few sequences have been determined from patients in Liberia, the second worst-affected country. Here, we report 140 EBOV genome sequences from the second wave of the Liberian outbreak and analyze them in combination with 782 previously published sequences from throughout the Western African outbreak. While multiple early introductions of EBOV to Liberia are evident, the majority of Liberian EVD cases are consistent with a single introduction, followed by spread and diversification within the country. Movement of the virus within Liberia was widespread, and reintroductions from Liberia served as an important source for the continuation of the already ongoing EVD outbreak in Guinea. Overall, little evidence was found for incremental adaptation of EBOV to the human host.

Molecular Evidence of Sexual Transmission of Ebola Virus.

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).

Monitoring of Ebola Virus Makona Evolution through Establishment of Advanced Genomic Capability in Liberia.

To support Liberia's response to the ongoing Ebola virus (EBOV) disease epidemic in Western Africa, we established in-country advanced genomic capabilities to monitor EBOV evolution. Twenty-five EBOV genomes were sequenced at the Liberian Institute for Biomedical Research, which provided an in-depth view of EBOV diversity in Liberia during September 2014-February 2015. These sequences were consistent with a single virus introduction to Liberia; however, shared ancestry with isolates from Mali indicated at least 1 additional instance of movement into or out of Liberia. The pace of change is generally consistent with previous estimates of mutation rate. We observed 23 nonsynonymous mutations and 1 nonsense mutation. Six of these changes are within known binding sites for sequence-based EBOV medical countermeasures; however, the diagnostic and therapeutic impact of EBOV evolution within Liberia appears to be low.

Xenosurveillance: a novel mosquito-based approach for examining the human-pathogen landscape.

BACKGROUND: Globally, regions at the highest risk for emerging infectious diseases are often the ones with the fewest resources. As a result, implementing sustainable infectious disease surveillance systems in these regions is challenging. The cost of these programs and difficulties associated with collecting, storing and transporting relevant samples have hindered them in the regions where they are most needed. Therefore, we tested the sensitivity and feasibility of a novel surveillance technique called xenosurveillance. This approach utilizes the host feeding preferences and behaviors of Anopheles gambiae, which are highly anthropophilic and rest indoors after feeding, to sample viruses in human beings. We hypothesized that mosquito bloodmeals could be used to detect vertebrate viral pathogens within realistic field collection timeframes and clinically relevant concentrations. METHODOLOGY/PRINCIPAL FINDINGS: To validate this approach, we examined variables influencing virus detection such as the duration between mosquito blood feeding and mosquito processing, the pathogen nucleic acid stability in the mosquito gut and the pathogen load present in the host's blood at the time of bloodmeal ingestion using our laboratory model. Our findings revealed that viral nucleic acids, at clinically relevant concentrations, could be detected from engorged mosquitoes for up to 24 hours post feeding by qRT-PCR. Subsequently, we tested this approach in the field by examining blood from engorged mosquitoes from two field sites in Liberia. Using next-generation sequencing and PCR we were able to detect the genetic signatures of multiple viral pathogens including Epstein-Barr virus and canine distemper virus. CONCLUSIONS/SIGNIFICANCE: Together, these data demonstrate the feasibility of xenosurveillance and in doing so validated a simple and non-invasive surveillance tool that could be used to complement current biosurveillance efforts.

Sampling host-seeking anthropophilic mosquito vectors in west Africa: comparisons of an active human-baited tent-trap against gold standard methods.

In this study, we characterize the ability of the previously described Infoscitex tent (IST) to capture mosquitoes in comparison to either the Centers for Disease Control Light Trap hung next to individuals under a bed net (LTC) or to human landing catches (HLC). In Senegal, the IST caught 6.14 times the number of Anopheles gambiae sensu lato (s.l.), and 8.78 times the Culex group V mosquitoes as LTC. In one of two locations in Burkina Faso, the IST caught An. gambiae at a rate not significantly different than HLC. Of importance, 9.1-36.1% of HLC caught An. gambiae were blood fed, mostly with fresh blood, suggesting they fed upon the collector, whereas only 0.5-5.0% from the IST had partial or old blood. The IST also caught outdoor biting species in proportions comparable to HLC. The results show this tent provides a safer and effective alternative to the skill-dependent, risky, and laborious HLC method.

Evaluation of ivermectin mass drug administration for malaria transmission control across different West African environments.

BACKGROUND: Mass drug administration (MDA) of ivermectin to humans for control and elimination of filarial parasites can kill biting malaria vectors and lead to Plasmodium transmission reduction. This study examines the degree and duration of mosquitocidal effects resulting from single MDAs conducted in three different West African countries, and the subsequent reductions in parity and Plasmodium sporozoite rates. METHODS: Indoor-resting, blood-fed and outdoor host-seeking Anopheles spp. were captured on days surrounding MDAs from 2008-2013 in Senegalese, Liberian and Burkinabé villages. Mortality was assessed on a portion of the indoor collection, and parity status was determined on host-seeking mosquitoes. The effect of MDA was then analysed against the time relative to the MDA, the distributed drugs and environmental variables. RESULTS: Anopheles gambiae survivorship was reduced by 33.9% for one week following MDA and parity rates were significantly reduced for more than two weeks after the MDAs. Sporozoite rates were significantly reduced by >77% for two weeks following the MDAs in treatment villages despite occurring in the middle of intense transmission seasons. These observed effects were consistent across three different West African transmission dynamics. CONCLUSIONS: These data provide a comprehensive and crucial evidence base for the significant reduction in malaria transmission following single ivermectin MDAs across diverse field sites. Despite the limited duration of transmission reduction, these results support the hypothesis that repeated MDAs with optimal timing could help sustainably control malaria as well as filarial transmission.

Laboratory and field evaluation of a new rapid test for detecting Wuchereria bancrofti antigen in human blood.

Global Program to Eliminate Lymphatic Filariasis (GPELF) guidelines call for using filarial antigen testing to identify endemic areas that require mass drug administration (MDA) and for post-MDA surveillance. We compared a new filarial antigen test (the Alere Filariasis Test Strip) with the reference BinaxNOW Filariasis card test that has been used by the GPELF for more than 10 years. Laboratory testing of 227 archived serum or plasma samples showed that the two tests had similar high rates of sensitivity and specificity and > 99% agreement. However, the test strip detected 26.5% more people with filarial antigenemia (124/503 versus 98/503) and had better test result stability than the card test in a field study conducted in a filariasis-endemic area in Liberia. Based on its increased sensitivity and other practical advantages, we believe that the test strip represents a major step forward that will be welcomed by the GPELF and the filariasis research community.