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

Abstract

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 (S_R) 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 (+/- 1.96S_R) 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.

Keywords: RNA virus; accuracy; honeybee; quantitative PCR; ring test.