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.
Lassa fever was first described in 1969 as the cause of a nosocomial outbreak of hemorrhagic fever in Nigeria [1]. Lassa fever is an acute viral infection associated with a wide spectrum of disease manifestations, which range from mild courses to multiorgan failure [1–3]. The etiologic agent of Lassa fever is Lassa virus (LASV, family Arenaviridae, genus Arenavirus) [4]. The natural host of LASV is the small rodent Mastomys natalensis, which lives close to human settlements [5]. The rodents can become chronically infected at birth and excrete infectious virus in urine and other body fluids, with subsequent transmission to humans [6]. There is evidence of human-to-human transmission in both hospital and community settings [7]. The fact that LASV may be transmitted from human to human gives rise to nosocomial or community-based outbreaks. LASV is endemic in the countries of Nigeria, Liberia, Sierra Leone, and Guinea [8,9] and was detected in Mali [10,11]. Seroepidemiological studies and imported cases of Lassa fever indicate that arenaviruses circulate somewhere in the region comprising Côte d’Ivoire and Burkina Faso [12]. The annual incidence is estimated at 300,000 cases, with 5,000 fatalities per year [13,14]. Additionally, LASV has been introduced several times into Europe, Japan, and North America. Among the hemorrhagic fever viruses of risk group 4 (such as Crimean-Congo hemorrhagic fever, Ebola, and Marburg virus), LASV has been most frequently imported [15]. The virus usually is imported by returning travelers [16,17]. Within Europe, LASV infections have been imported to Germany [18,19], The Netherlands [20] and the United Kingdom [21]. Laboratory testing is required to establish a diagnosis, as Lassa fever can hardly be distinguished from other febrile diseases based on clinical symptoms [14,22]. A suspected case must be rapidly ruled out or verified to facilitate appropriate case management, including treatment, the implementation of isolation measures, or the tracking of contact persons [18]. The method of choice for early detection of LASV in blood is reverse transcription (RT) -PCR [23–29]. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays for the reliable detection of all virus strains [30]. The performance of the different techniques applied for molecular diagnosis of LASV may vary between laboratories. External quality assessment (EQA) studies for LASV molecular diagnostics have not been performed since 2004 [31]. An EQA study allows the participating laboratories to monitor the quality of their diagnostics and to identify problems with particular diagnostic assays. For these reasons, an EQA study for the molecular diagnosis of LASV was conducted by the European Network for Diagnostics of ‘Imported’ Viral Diseases (ENIVD) (http: //www. enivd. org) in 2013. ENIVD is concerned with the development of laboratory diagnostic capacities for imported virus infections, quality control, standardization of laboratory procedures, and training of laboratory staff [32]. Based on the results of this study, the quality of LASV diagnostics may be improved.
Twenty-eight laboratories involved in diagnostics of viral hemorrhagic fevers were invited to participate in this study. Invitees were selected from the register of ENIVD network members and from the list of national and regional reference laboratories for rare, emerging, and dangerous viruses. The participation in the study was free of charge. Participants permitted publication of the results in a comparative and anonymous manner. This EQA was coordinated by ENIVD according to the established procedures of the network [33–35]. The proficiency test panel included 13 LASV preparations derived from culture supernatants of Vero E6 cells (ATCC—American Type Culture Collection) infected with 4 different LASV strains. Virus in cell culture supernatant was inactivated by heat (1 h at 60°C) followed by gamma irradiation (25 kilo gray). The test panel consisted of six samples of LASV strain Josiah from Sierra Leone, obtained by serial 10-fold dilution of cell culture supernatant (1: 10 to 1: 106), three samples of LASV strain Lib-1580/121 from Liberia (dilutions 1: 103 to 1: 105), LASV strain CSF from Nigeria (dilution 1: 103), and LASV strain AV from Cote d’Ivoire or Burkina Faso (dilution 1: 103). The samples were freeze-dried in 3% mannitol based formulation using an EPSILON 2-6D Pilot Freeze Dryer (Martin Christ GmbH, Osterode am Harz, Germany). In addition, we included one sample containing LASV strain Josiah at a dilution of 1: 104 (sample #14) that was prepared with a new dry stabilizer method (Biomatrica, Inc. , San Diego, CA, USA), and one sample containing LASV strain Lib-1580/121 at dilution of 1: 104 (sample #6) that was prepared using a liquid stabilizer (Biomatrica). A sample containing lymphocytic choriomeningitis virus (LCMV), the prototype member of the family Arenaviridae, as well as two negative control sera were included in the test panel as specificity controls. After lyophilized sample preparation, the samples were tested and quantified by an in-house real-time PCR assay for quality control purpose. The assay was performed by employing 12. 5 pmol of forward primer LaV F2 (CCACCATYTTRTgCATRTgCCA), 13 pmol of reverse primer LaV R (gCACATgTNTCHTAYAgYATggAYCA) and 5 pmol of probe LaV TM (FAM-AARTggggYCCDATgATgTgYCCWTT-BBQ). The real-time PCR assay was carried out in one-step format on ABI 7500 real-time PCR system using the AgPath-ID One-Step RT-PCR Kit according to manufacturer´s instruction. Plasmid standards were used for the quantification of the genome copies of Lassa virus RNA. The EQA was performed according to National Ethical regulations. Before dispatching the panel, samples were sent to the reference laboratory for testing the quality and obtaining the reference results. Reference laboratory used RT-PCR protocol described by Ölschläger et al. , 2010 [27]. Samples were resuspended in 100 μl of water and the RNA was extracted using the QIAamp viral RNA kit (Qiagen, Hilden, Germany). The presence of LASV or LCMV RNA in the samples was ascertained by RT-PCR and sequencing. The number of LASV genome copies present in these samples was determined by qRT-PCR. Samples were shipped by regular mail at ambient temperature. Participating laboratories were instructed to resuspend the samples in 100 μl of water and to analyze the material like serum samples potentially containing LASV using their routine nucleic acid detection assays. The EQA panel was accompanied by documentation including instructions and an evaluation form for results. Participants were asked to report the assay protocol, the result for each sample, the LASV strain identified, the number of genome copies as well as any problem encountered. To guarantee anonymous data evaluation and reporting, each participating laboratory was coded with an identifier. The results were scored according to detection rate and specificity as in previous EQA studies of ENIVD [33–35]. We assigned one point for correct results; false-negative, false-positive, and indeterminate results did not count. Results were classified as “good”—when all results were correct; “acceptable”—when 1 to 2 results were incorrect; and “need for improvement”—when more than 2 results were incorrect. Results for the sample containing LCMV (sample #3) were not included in the score, as verification of the sequence was optional. In addition, we excluded from scoring the sample containing LASV strain Josiah at a dilution of 1: 106 (sample #15) as this concentration is likely to be below the 95%-detection limit of most assays. Thus, obtaining a positive or negative result becomes a matter of chance. Each laboratory received the complete summary of the results in an anonymous way, by which only the own laboratory was recognizable.
This EQA study indicates that most participating laboratories, located in various countries around the world, have a good or acceptable performance in molecular detection of LASV. However, several laboratories need to improve their performance, in particular with respect to detection of the Liberian strain. The data allow the participating laboratories to identify the weakness in their diagnostic procedures and to review and improve their protocols. One published protocol has achieved 100% detection rate reported by single participant. However, the reference laboratory recommends Ölschläger et al. , 2010 published protocol for LASV detection as most commonly used with good detection rate and ability to detect all described Lassa virus strains. The main aim of this EQA study was not to compare published protocols, rather to give chance to participating laboratories to evaluate their testing performance and provide practical exercise for molecular detection of LASV. There should be a follow-up EQA for molecular detection of LASV to evaluate a possible improvement.
A proficiency test panel for molecular diagnostic of Lassa virus provides objective evidence of testing quality of International diagnostic laboratories. Since there are no commercial assays available, it is very important to assess the quality of diagnostic test used as well as evaluate detection sensitivity and specificity performance. Participating laboratories have received samples containing different inactivated Lassa virus strains as well as two negative controls. Participants were asked to provide information on diagnostic test procedure and protocols used for analysis of samples of Lassa virus External Quality Assessment (EQA). Based on received information we were able to compare and evaluate the quality of diagnostic profile and facilitate further improvement. Participating laboratories may use results of Lassa virus EQA to become accredited for Lassa virus molecular diagnostic. Since different Lassa virus strains are not available for most of the laboratories, participants achieved very advanced training for diagnostic of rare and imported viruses.
International External Quality Assessment Study for Molecular Detection of Lassa Virus