Trueness and precision of the real-time RT-PCR method for quantifying the chronic bee paralysis virus genome in bee homogenates evaluated by a comparative inter-laboratory study

• Published in: Journal of virological methods 2017-10, Vol.248, p.217-225
• Main Authors: Schurr, Frank, Cougoule, Nicolas, Rivière, Marie-Pierre, Ribière-Chabert, Magali, Achour, Hamid, Ádám, Dán, Castillo, Carlos, de Graaf, Dirk C., Forsgren, Eva, Granato, Anna, Heinikainen, Sirpa, Jurovčíková, Júlia, Kryger, Per, Manson, Christine, Ménard, Marie-Françoise, Perennes, Stéphane, Schäfer, Marc O., Ibañez, Elena San Miguel, Silva, João, Gajger, Ivana Tlak, Tomkies, Victoria, Toplak, Ivan, Viry, Alain, Zdańska, Dagmara, Dubois, Eric
• Format: Article
• Language: English
• Subjects: accuracy; Animal biology; Animals; Bees - virology; Genome, Viral; honeybee; Insect Viruses - genetics; Insect Viruses - physiology; Laboratories; Life Sciences; Pathobiology; Patobiologi; quantitative PCR; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; ring test; RNA virus; RNA Viruses - genetics; RNA Viruses - physiology; RNA, Viral - genetics; Veterinary medicine and animal Health; Viral Load - genetics; Viral Load - methods
• ISSN: 0166-0934; 1879-0984; 1879-0984
• DOI: 10.1016/j.jviromet.2017.07.013

•Chronic bee paralysis is diagnosed by quantifying the causative virus, CBPV, in honeybees by real-time PCR•21 laboratories assayed honeybee homogenates, adjusted to three virus loads (5, 8, and 9 log10 CBPV genome copies per bee)•The accuracy (trueness and precision) of the method implemented in the laboratories was evaluated according to international standards•The inter-laboratory confidence of viral quantification was about+/− 2 log10 for a diagnostic threshold of 8 log10 CBPV genome copies per bee•Taking into account this confidence, viral loads over 6 log10 CBPV RNA copies per bee may indicate probable cases of chronic paralysis The Chronic bee paralysis virus (CBPV) is the aetiological agent of chronic bee paralysis, a contagious disease associated with nervous disorders in adult honeybees leading to massive mortalities in front of the hives. Some of the clinical signs frequently reported, such as trembling, may be confused with intoxication syndromes. Therefore, laboratory diagnosis using real-time PCR to quantify CBPV loads is used to confirm disease. Clinical signs of chronic paralysis are usually associated with viral loads higher than 108 copies of CBPV genome copies per bee (8 log10 CBPV/bee). This threshold is used by the European Union Reference Laboratory for Bee Health to diagnose the disease. In 2015, the accuracy of measurements of three CBPV loads (5, 8 and 9 log10 CBPV/bee) was assessed through an inter-laboratory study. Twenty-one participants, including 16 European National Reference Laboratories, received 13 homogenates of CBPV-infected bees adjusted to the three loads. Participants were requested to use the method usually employed for routine diagnosis. The quantitative results (n=270) were analysed according to international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). The standard deviations of measurement reproducibility (SR) were 0.83, 1.06 and 1.16 at viral loads 5, 8 and 9 log10 CBPV/bee, respectively. The inter-laboratory confidence of viral quantification (+/− 1.96SR) at the diagnostic threshold (8 log10 CBPV/bee) was+/− 2.08 log10 CBPV/bee. These results highlight the need to take into account the confidence of measurements in epidemiological studies using results from different laboratories. Considering this confidence, viral loads over 6 log10 CBPV/bee may be considered to indicate probable cases of chronic paralysis.