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.

Focussed Assessment with Sonography in acute Lassa Fever (FASLa): Development of a point-of-care protocol and description of common ultrasound findings.

BACKGROUND: Managing Lassa fever (LF) patients is challenging because of the complexity of this life-threatening infectious disease, the necessary isolation measures, and the limited resources in countries where it is endemic. Point-of-care ultrasonography (POCUS) is a promising low-cost imaging technique that may help in guiding the management of patients. METHODS: We conducted this observational study at the Irrua Specialist Teaching Hospital in Nigeria. We developed a POCUS protocol, trained local physicians who applied the protocol to LF patients and recorded and interpreted the clips. These were then independently re-evaluated by an external expert, and associations with clinical, laboratory and virological data were analyzed. FINDINGS: We developed the POCUS protocol based on existing literature and expert opinion and trained two clinicians, who then used POCUS to examine 46 patients. We observed at least one pathological finding in 29 (63%) patients. Ascites was found in 14 (30%), pericardial effusion in 10 (22%), pleural effusion in 5 (11%), and polyserositis in 7 (15%) patients, respectively. Eight patients (17%) showed hyperechoic kidneys. Seven patients succumbed to the disease while 39 patients survived, resulting in a fatality rate of 15%. Pleural effusions and hyper-echoic kidneys were associated with increased mortality. INTERPRETATION: In acute LF, a newly established POCUS protocol readily identified a high prevalence of clinically relevant pathological findings. The assessment by POCUS required minimal resources and training; the detected pathologies such as pleural effusions and kidney injury may help to guide the clinical management of the most at-risk LF patients.

Factors associated with progression to death in patients with Lassa fever in Nigeria: an observational study.

BACKGROUND: Lassa fever is endemic in several west African countries. Case-fatality rates ranging from 21% to 69% have been reported. The pathophysiology of the disease in humans and determinants of mortality remain poorly understood. We aimed to determine host protein biomarkers capable of determining disease outcome. METHODS: In this observational study, we analysed left-over blood samples from patients who tested positive for Lassa fever at Irrua Specialist Teaching Hospital, Nigeria, between January, 2014, and April, 2017. We measured viral load, concentrations of clinical chemistry parameters, and levels of 62 circulating proteins involved in inflammation, immune response, and haemostasis. Patients with a known outcome (survival or death) and at least 200 µL of good-quality diagnostic sample were included in logistic regression modelling to assess the correlation of parameters with Lassa fever outcome. Individuals who gave consent could further be enrolled into a longitudinal analysis to assess the association of parameters with Lassa fever outcome over time. Participants were divided into two datasets for the statistical analysis: a primary dataset (samples taken between Jan 1, 2014, and April 1, 2016), and a secondary dataset (samples taken between April 1, 2016, and April 1, 2017). Biomarkers were ranked by area under the receiver operating characteristic curve (AUC) from highest (most predictive) to lowest (least predictive). FINDINGS: Of 554 patients who tested positive for Lassa fever during the study period, 201 (131 in the primary dataset and 70 in the secondary dataset) were included in the biomarker analysis, of whom 74 (49 in the primary dataset and 25 in the secondary dataset) had died and 127 (82 in the primary dataset and 45 in the secondary dataset) had survived. Cycle threshold values (indicating viral load) and levels of 18 host proteins at the time of admission to hospital were significantly correlated with fatal outcome. The best predictors of outcome in both datasets were plasminogen activator inhibitor-1 (PAI-1; AUC 0·878 in the primary dataset and 0·876 in the secondary dataset), soluble thrombomodulin (TM; 0·839 in the primary dataset and 0·875 in the secondary dataset), and soluble tumour necrosis factor receptor superfamily member 1A (TNF-R1; 0·807 in the primary dataset and 0·851 in the secondary dataset), all of which had higher prediction accuracy than viral load (0·774 in the primary dataset and 0·837 in the secondary dataset). Longitudinal analysis (150 patients, of whom 36 died) showed that of the biomarkers that were predictive at admission, PAI-1 levels consistently decreased to normal levels in survivors but not in those who died. INTERPRETATION: The identification of PAI-1 and soluble TM as markers of fatal Lassa fever at admission, and of PAI-1 as a marker of fatal Lassa fever over time, suggests that dysregulated coagulation and fibrinolysis and endothelial damage have roles in the pathophysiology of Lassa fever, providing a mechanistic explanation for the association of Lassa fever with oedema and bleeding. These novel markers might aid in clinical risk stratification and disease monitoring. FUNDING: German Research Foundation, Leibniz Association, and US National Institutes of Health.

Acute kidney injury and mortality in pediatric Lassa fever versus question of access to dialysis.

OBJECTIVES: To assess the prevalence of acute kidney injury (AKI), and its impact on outcome in hospitalized pediatric patients with Lassa fever (LF). METHODS: We reviewed the presenting clinical and laboratory features and outcomes of 40 successive hospitalized children with PCR-confirmed LF. The diagnosis and staging of AKI was based on KDIGO criteria. We compared groups of patients using t- or χ2 tests as necessary, and took p-values <0.05 as indicative of the presence of significant differences. RESULTS: Sixteen (40%) children had AKI. Case fatality rate (CFR) was 9/16 (56%) in children with and 1/24 (4%) in those without AKI (OR [95% CI] of CFR associated with AKI = 29.57 [3.17, 275.7]). Presentation with abnormal bleeding (p = 0.008), encephalopathy (p = 0.004), hematuria plus proteinuria (p = 0.013), and elevated serum transaminase levels (p <0.02) were significantly associated with an increased prevalence of AKI. CONCLUSION: AKI prevalence in hospitalized pediatric patients with Lassa fever is high, and correlated with illness severity/CFR. The high prevalence underscores the need for access to hemodialysis, and clinical presentation and/or presence of hematuria plus proteinuria could serve as a ready prompt for referral for such specialized care.

Field evaluation of a Pan-Lassa rapid diagnostic test during the 2018 Nigerian Lassa fever outbreak.

Lassa virus (LASV) is the causative agent of Lassa fever (LF), an often-fatal hemorrhagic disease. LF is endemic in Nigeria, Sierra Leone and other West African countries. Diagnosis of LASV infection is challenged by the genetic diversity of the virus, which is greatest in Nigeria. The ReLASV Pan-Lassa Antigen Rapid Test (Pan-Lassa RDT) is a point-of-care, in vitro diagnostic test that utilizes a mixture of polyclonal antibodies raised against recombinant nucleoproteins of representative strains from the three most prevalent LASV lineages (II, III and IV). We compared the performance of the Pan-LASV RDT to available quantitative PCR (qPCR) assays during the 2018 LF outbreak in Nigeria. For patients with acute LF (RDT positive, IgG/IgM negative) during initial screening, RDT performance was 83.3% sensitivity and 92.8% specificity when compared to composite results of two qPCR assays. 100% of samples that gave Ct values below 22 on both qPCR assays were positive on the Pan-Lassa RDT. There were significantly elevated case fatality rates and elevated liver transaminase levels in subjects whose samples were RDT positive compared to RDT negative.

Acute Lassa Virus Encephalitis with Lassa Virus in the Cerebrospinal Fluid but Absent in the Blood: A Case Report with a Positive Outcome.

It is rare both to have the central nervous system (CNS) as the main focus in the acute phase of Lassa fever infection without associated bleeding, and to find Lassa virus (LAV) in the cerebrospinal fluid (CSF) but not in the serum. We report the case of a 38-year-old Nigerian woman with mainly CNS manifestation of Lassa fever. She was admitted twice within 11 days because of persistent fever. A clinical diagnosis of acute LAV encephalitis was made because of a high index of suspicion and CNS involvement confirmed by positive reverse transcriptase polymerase chain reaction (RT-PCR) for LAV in the CSF, while her blood was repeatedly negative for LAV by RT-PCR test. She recovered fully following supportive care coupled with treatment with an 18-day course of ribavirin, and suffered no long-term neurological complication or relapse. Post-treatment CSF examination by RT-PCR did not detect LAV.

Clinical and laboratory predictors of Lassa fever outcome in a dedicated treatment facility in Nigeria: a retrospective, observational cohort study.

BACKGROUND: Lassa fever is a viral haemorrhagic disease endemic to west Africa. No large-scale studies exist from Nigeria, where the Lassa virus (LASV) is most diverse. LASV diversity, coupled with host genetic and environmental factors, might cause differences in disease pathophysiology. Small-scale studies in Nigeria suggest that acute kidney injury is an important clinical feature and might be a determinant of survival. We aimed to establish the demographic, clinical, and laboratory factors associated with mortality in Nigerian patients with Lassa fever, and hypothesised that LASV was the direct cause of intrinsic renal damage for a subset of the patients with Lassa fever. METHODS: We did a retrospective, observational cohort study of consecutive patients in Nigeria with Lassa fever, who tested positive for LASV with RT-PCR, and were treated in Irrua Specialist Teaching Hospital. We did univariate and multivariate statistical analyses, including logistic regression, of all demographic, clinical, and laboratory variables available at presentation to identify the factors associated with patient mortality. FINDINGS: Of 291 patients treated in Irrua Specialist Teaching Hospital between Jan 3, 2011, and Dec 11, 2015, 284 (98%) had known outcomes (died or survived) and seven (2%) were discharged against medical advice. Overall case-fatality rate was 24% (68 of 284 patients), with a 1·4 times increase in mortality risk for each 10 years of age (p=0·00017), reaching 39% (22 of 57) for patients older than 50 years. Of 284 patients, 81 (28%) had acute kidney injury and 104 (37%) had CNS manifestations and thus both were considered important complications of acute Lassa fever in Nigeria. Acute kidney injury was strongly associated with poor outcome (case-fatality rate of 60% [49 of 81 patients]; odds ratio [OR] 15, p<0·00001). Compared with patients without acute kidney injury, those with acute kidney injury had higher incidence of proteinuria (32 [82%] of 39 patients) and haematuria (29 [76%] of 38) and higher mean serum potassium (4·63 [SD 1·04] mmol/L) and lower blood urea nitrogen to creatinine ratio (8·6 for patients without clinical history of fluid loss), suggesting intrinsic renal damage. Normalisation of creatinine concentration was associated with recovery. Elevated serum creatinine (OR 1·3; p=0·046), aspartate aminotransferase (OR 1·5; p=0·075), and potassium (OR 3·6; p=0·0024) were independent predictors of death. INTERPRETATION: Our study presents detailed clinical and laboratory data for Nigerian patients with Lassa fever and provides strong evidence for intrinsic renal dysfunction in acute Lassa fever. Early recognition and treatment of acute kidney injury might significantly reduce mortality. FUNDING: German Research Foundation, German Center for Infection Research, Howard Hughes Medical Institute, US National Institutes of Health, and World Bank.

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.