International External Quality Assessment Study for Molecular Detection of Lassa Virus

Abstract

Lassa virus (LASV) is a causative agent of hemorrhagic fever in West Africa. In recent years, it has been imported several times to Europe and North America. The method of choice for early detection of LASV in blood is RT-PCR. Therefore, the European Network for Diagnostics of ‘Imported’ Viral Diseases (ENIVD) performed an external quality assessment (EQA) study for molecular detection of LASV. A proficiency panel of 13 samples containing various concentrations of inactivated LASV strains Josiah, Lib-1580/121, CSF, or AV was prepared. Samples containing the LASV-related lymphocytic choriomeningitis virus (LCMV) and negative sera were included as specificity controls. Twenty-four laboratories from 17 countries (13 European, one African, one Asian, two American countries) participated in the study. Thirteen laboratories (54%) reported correct results, 4 (17%) laboratories reported 1 to 2 false-negative results, and 7 (29%) laboratories reported 3 to 5 false-negative results. This EQA study indicates that most participating laboratories have a good or acceptable performance in molecular detection of LASV. However, several laboratories need to review and improve their diagnostic procedures.

Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

“…Reactions were performed on a LightCycler 96 machine (Roche). For detection of LASV clade II, the primers Nikisins_F and Nikisins_R were used 26 . For detection of LASV clade IV, the in-house assay including primers Broad_F and Broad_R and probe Broad_P, was also used with the TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems).…”

“…We evaluated the sensitivity on a panel of 10 RNA and cDNA samples per clade, derived from suspected LF patients (Fig 2E,G). We compared SHERLOCK results using both fluorescent and lateral flow readouts head-to-head with the ‘Nikisin’ RT-qPCR assay 26 and benchmarked both results against sequencing data (Fig 2F,H). The LASV-II fluorescent readout was positive for all seven sequencing-positive samples, and negative for all three sequencing-negative samples (100% sensitivity, 100% concordance), as was the gold-standard Nikisin RT-qPCR.…”

Laboratory Diagnosis of Lassa Fever

“…However, with highly diverse pathogens such as Lassa virus, genetic diversity can be problematic for nucleic acid-based assays, as even a single nucleotide variant in one of the primers can have a significant negative impact on assay sensitivity depending on the location of the nucleotide variant (43). Multiple real-time RT-PCR assays are published in the literature for Lassa virus (41,(44)(45)(46). For example, Safronetz and colleagues initially detected Lassa virus circulating in Mali using a SYBR green real-time RT-PCR assay targeting the Lassa virus S RNA segment (45), and Trombley and colleagues developed multiple probe-based Lassa virus assays due to strain diversity (44).…”

“…For example, Safronetz and colleagues initially detected Lassa virus circulating in Mali using a SYBR green real-time RT-PCR assay targeting the Lassa virus S RNA segment (45), and Trombley and colleagues developed multiple probe-based Lassa virus assays due to strain diversity (44). However, standard RT-PCR assays are commonly used (28,29,46) due to their ease of use and the decreased specificity for probe-based real-time RT-PCR. Probe-based real-time RT-PCR (two primers and a probe) introduces the possibility of probe mismatches due to the high degree of Lassa virus diversity, potentially increasing the false-negative rate compared with that from RT-PCR using only two primers.…”

“…For example, Ölschläger and colleagues redesigned a commonly used standard RT-PCR assay for Lassa virus after identifying decreased assay sensitivity due to sequence variants for the reverse primer (29). This new RT-PCR assay is widely used for screening samples for Lassa virus and performed well in an external quality assessment study conducted by the European Network for Diagnostics of Imported Viral Diseases (46). Multiplex panels to simultaneously detect a multitude of viruses that can produce hemorrhagic fever syndromes, including Lassa and Ebola viruses, using RT-PCR alone or in combination with either enzyme hybridization or ligase detection reactions have also been developed (47)(48)(49).…”