Use of a diagnostic Puumala virus real-time RT-PCR in an orthohantavirus endemic region in the Netherlands

Abstract

Laboratory diagnosis of orthohantavirus infection is primarily based on serology. However, for a confirmed serological diagnosis, evaluation of a follow-up serum sample is essential, which is time consuming and causes delay. Real-time reverse transcription polymerase chain reaction (RT-PCR) tests, if positive, provide an immediate and definitive diagnosis, and accurately identify the causative agent, where the discriminative nature of serology is suboptimal. We re-evaluated sera from orthohantavirus-suspected clinical cases in the Dutch regions of Twente and Achterhoek from July 2014 to April 2016 for the presence of Puumala orthohantavirus (PUUV), Tula orthohantavirus (TULV), and Seoul orthohantavirus (SEOV) RNA. PUUV RNA was detected in 11% of the total number (n = 85) of sera tested, in 50% of sera positive for anti-PUUV/TULV IgM (n = 16), and in 1.4% of sera negative or indeterminate for anti-PUUV/TULV IgM (n = 69). No evidence was found for the presence of TULV or SEOV viral RNA. Based on these findings, we propose two algorithms to implement real-time RT-PCR testing in routine orthohantavirus diagnostics, which optimally provide clinicians with early confirmed diagnoses and could prevent possible further invasive testing and treatment.

Importance: The addition of a real-time reverse transcription polymerase chain reaction test to routine orthohantavirus diagnostics may better aid clinical decision making than the use of standard serology tests alone. Awareness by clinicians and clinical microbiologists of this advantage may ultimately lead to a reduction in over-hospitalization and unnecessary invasive diagnostic procedures.

Keywords: Puumala virus; diagnostics; hantavirus; molecular methods; nephrology; nucleic acid amplification test; serology; zoonotic infections.

Fig 1

Algorithm 1: If suspected for orthohantavirus infection and if symptoms exist for 7 days or less, perform real-time RT-PCR and serology simultaneously. If real-time RT-PCR is negative, perform serology on a second sample after 2 weeks for a confirmed diagnosis. Data from real-time RT-PCR-tested sera from patients who had been routinely tested by serology previously (n = 85) were run through the algorithm as a model (on the right); the resulting numbers of confirmed cases and presumptive cases at a given time are depicted in the light gray boxes and dark gray boxes, respectively, and may be compared with those resulting from the strategy of serology alone (on the left). Indeterminate (indet.) or IgG-positive cases generally require further evaluation, like the presumptive cases, and are depicted in the same dark gray box.

Fig 2

Algorithm 2: If suspected for orthohantavirus infection, perform serology on day 1. Perform real-time RT-PCR on day 2 on the IgM-positive and indeterminate sera of patients who have symptoms for 7 days or less. If real-time RT-PCR is negative, perform serology on a second sample after 2 weeks for a confirmed diagnosis. Data from real-time RT-PCR-tested sera from patients who had been routinely tested by serology previously were run through the algorithm as a model; for explanation of the given numbers of cases and abbreviations, see Fig. 1.