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

Abstract

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.

Figure 1

Signal development of the ReLASV Pan-Lassa antigen rapid diagnostic test. The ReLASV Pan-Lassa Antigen RDT is designed as a dipstick style lateral flow immunoassay. It can be visually scored on a scale of 0 to 5.

Figure 2

Characteristics of subjects from 2018 outbreak of Lassa fever presenting to Irrua Specialist Teaching Hospital in Nigeria. Panel A: Demographics of subject cohort classified by Lassa immunoassay results. Subjects were classified as Acute Lassa fever, Post-Acute Lassa fever, and Non-Lassa Illness based on ReLASV immunoassay screening. Panel B: Case fatality rates of subjects classified by Lassa immunoassay results. Panel C: Aspartate Aminotransferase levels in samples from subjects classified by Lassa immunoassay results. Panel D: Detection of LASV genomes (>400 reads) by Next Generation Sequencing from samples of subjects classified by Lassa immunoassay results.

Figure 3

Reported clinical signs and symptoms in the 2018 cohort of suspected Lassa fever cases presenting to Irrua Specialist Teaching Hospital. Subjects were classified as Acute Lassa fever, Post-Acute Lassa fever, and Other Illness based on Lassa immunoassay screening. Asterisks represent P > ChiSq for difference between three patient groups (*p < 0.05; ***p < 0.001).

Figure 4

Correlations of quantitative polymerase chain reaction assay results with Next Generation sequencing of Lassa virus genomes. Panel A: Correlation between Altona 1.0 qPCR and Nikisins qPCR cycle threshold (Ct) results and log₁₀ of viral genome equivalents per mL (n = 178, R² = 0.64, Linear Regression = 1.80 + 0.79). Panel B: LASV genome assembly length correlation to post-filtering mapped reads (cubic fit of Log-Log transformation of data, R² = 0.97). Panel C: Log – Linear transformed linear fit of Altona 1.0 qPCR Ct and log₁₀ of viral genome equivalents per mL to mapped reads (n = 199, R² = 0.60). Panel D: Log – Linear transformed linear fit of Nikisins qPCR Ct and log₁₀ of viral genome equivalents per mL to mapped reads (n = 201, R² = 0.45). Panel E: Receiver operator curve determination of Altona 1.0 qPCR Ct cut-off based on LASV genomic sequencing. Panel F: Receiver operator curve determination of Nikisins qPCR Ct cut-off based on LASV genomic sequencing.

Figure 5

Demographics of the 2018 cohort of suspected Lassa fever cases presenting to Irrua Specialist Teaching Hospital classified by Lassa quantitative polymerase chain reaction results. Samples from subjects were classified as positive, equivocal or negative on the Altona 1.0 qPCR (Panel A) or the Nikisons qPCR (Panel B). Case fatality rates of subjects by qPCR results are compared. Aspartate aminotransferase levels and presence of LASV sequences in samples from subjects are also compared.

Figure 6

Relationship of Pan-Lassa rapid diagnostic test and antibody capture immunoassay results to quantitative polymerase chain assay results. Samples from subjects presenting to Irrua Specialist Teaching Hospital with suspected Lassa fever in 2018 were used to compare results of the recombinant Lassa immunoassays to results of the Altona 1.0 qPCR assay (Panel A) and the Nikisins qPCR assay (Panel B).

Figure 7

Correlation of quantitative polymerase chain reaction cycle threshold values and Pan-Lassa rapid diagnostic test signal intensity. Signal intensity based on a visual score aid for the Pan-Lassa RDT was compared to cycle threshold (Ct) values for the Altona 1.0 (R² = 0.65; Panel A) and Nikisins qPCR (R² = 0.62; Panel B) for samples from subjects with acute Lassa fever (IgG seronegative).