International External Quality Assessment Study for Molecular Detection of Lassa Virus

<div><p>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.</p></div

A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.

<p>A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.</p

300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.</p

Vacuolization induced by TBEV infection.

<p>Caco-2 cells were infected with TBEV K23 virus. Cellular morphological changes and vacuolization were monitored by light microscopy. Caco-2 cells were infected with TBEV strain K23 and fixed at 24 h, 48 h and 72 h. Cells were observed with the 40× objective (400× total magnification). Details of cytoplasmic vacuolization are visualized by immunofluorescence (IF) microscopy. 3 representative vacuoles are indicated by white asterisks in each sub-image. Samples were incubated with anti-TBEV E antibody and then stained with secondary anti-mouse antibody conjugated to FITC (green). Nuclei were stained with DAPI (blue).</p

Translocation of TBEV through Caco-2 monolayers without affecting transepithelial electrical resistance (TER).

<p>(<b>A</b>) Virus amount in basal medium. Polarized Caco-2 monolayers grown on permeable supports were infected with TBEV strain K23 from the apical surface. Basal medium was harvest at different time points and viral RNA in each sample was detected by RT-qPCR. The data were displayed as mean with standard deviation. (<b>B</b>) Transepithelial electric resistance (TER) measurements during TBEV infection. Polarized Caco-2 monolayers, grown on permeable supports, infected with TBEV K23 (circles) from the apical surface. Non-infected cells served as controls (triangles). TER values were measured from 0 h to 120 h post infection. n = 5, *<i>P</i><0.05, **<i>P</i><0.01 to control in Student's t test. (<b>C</b>) Cell viability during TBEV infection. Caco-2 cells were infected with TBEV strain K23 at an MOI of 1 and cell viability was analyzed by MTT assay. Cell viability was measured and calculated as a percentage of non-infected control cells. Data were expressed as mean ± standard error of the mean. (<b>D</b>) Analysis of TUNEL-positive cells. Confluent Caco-2 cells were infected with TBEV strain K23 and apoptosis was detected by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) at 48 h and 120 h post infection. The ratios of TUNEL-positive cells to all cells were analyzed in 4 low-power fields from 3 independent samples of each group. **<i>P</i><0.01.</p

Summary of the EQA study for molecular detection of LASV.

<p>^a with stabilizer;</p><p>^b not included in score;</p><p>+, virus correctly detected;—negative result; +/–, indeterminate result</p><p>Summary of the EQA study for molecular detection of LASV.</p

Antigen titration using the indicated amounts of DENV-1 (A), -2 (B), -3 (C) and -4 (D) Equad and DENV-2 Ewt (E) proteins with three different DENV, WNV and negative (NEG) human sera.

<p>Measurements were performed in duplicates in at least two independent experiments.</p

Results of the EQA study for laboratories performing NT for the serological detection of West Nile virus.

<p>WNV: West Nile virus; DENV: dengue virus; JEV; Japanese encephalitis virus; TBEV: tick-borne encephalitis virus; USUV: Usutu virus; Neg: negative control; Sc: Score; IgM: Immunoglobulin M; IgG: Immunoglobulin G; nd: not done;</p>H<p>: NT in-house test;</p>H2<p>NT in-house test (with WNV lineage II strain Austria).</p

LASV detection rate by sample.

<p>^a with stabilizer;</p><p>^b not included in score</p><p>LASV detection rate by sample.</p

List of participating laboratories.

<p>List of participating laboratories.</p