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.

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

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.