Molecular method for the detection of Andes hantavirus infection: validation for clinical diagnostics

Abstract

Hantavirus cardiopulmonary syndrome is a severe disease caused by exposure to New World hantaviruses. Early diagnosis is difficult due to the lack of specific initial symptoms. Antihantavirus antibodies are usually negative until late in the febrile prodrome or the beginning of cardiopulmonary phase, while Andes hantavirus (ANDV) RNA genome can be detected before symptoms onset. We analyzed the effectiveness of quantitative reverse transcription polymerase chain reaction (RT-qPCR) as a diagnostic tool detecting ANDV-Sout genome in peripheral blood cells from 78 confirmed hantavirus patients and 166 negative controls. Our results indicate that RT-qPCR had a low detection limit (~10 copies), with a specificity of 100% and a sensitivity of 94.9%. This suggests the potential for establishing RT-qPCR as the assay of choice for early diagnosis, promoting early effective care of patients, and improving other important aspects of ANDV infection management, such as compliance of biosafety recommendations for health personnel in order to avoid nosocomial transmission.

Keywords: Andes virus; Diagnosis; Hantavirus; RT-qPCR.

Figure 1. RT-qPCR Reaction for ANDV

Known amounts of in vitro transcribed S segment RNA was amplified by one-step reaction (circles) or two-step reaction (crosses). A plasmid (P) containing S Segment sequence (triangles) was assayed in a two-step RT-qPCR reaction. All experiments were done in LightCycler 2.0

Figure 2

Comparison of real-time ANDV assays performed in two platforms. Realtime PCR platforms LightCycler 480^® (LC480), and LightCycler 2.0 (LC2.0), were used to assay the same amount of RNA copies. The graphic shows the comparison of the crossing point (Cp) for different amounts of in vitro transcribed RNA, and two technical duplicates for each lightCycler platform.