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Journal of Clinical Microbiology, August 2007, p. 2491-2497, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.01902-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Puumala Hantavirus Viremia Diagnosed by Real-Time Reverse Transcriptase PCR Using Samples from Patients with Hemorrhagic Fever and Renal Syndrome{triangledown}

Magnus Evander,1* Irene Eriksson,1 Lisa Pettersson,1 Per Juto,1 Clas Ahlm,2 Gert E. Olsson,1,3,4,5 Göran Bucht,3 and Annika Allard1

Department of Virology, Umeå University, Umeå, Sweden,1 Department of Infectious Diseases, Umeå University, Umeå, Sweden,2 Department of Medical Countermeasures, Division of NBC Defense, Swedish Defense Research Agency, Umeå, Sweden,3 Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, Solna, Sweden,4 Wildlife, Fish, and Environmental Studies, Swedish University of Agricultural Sciences, Umeå, Sweden5

Received 13 September 2006/ Returned for modification 15 November 2006/ Accepted 17 May 2007

Puumala virus (PUUV) is the endemic hantavirus in northern Sweden and causes nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome. There is a need for fast and reliable diagnostics to differentiate the disease from other infections. By aligning virus RNA sequences isolated from 11 different bank voles and one human patient, we designed a real-time reverse transcriptase (RT) PCR method for detection of PUUV RNA. The real-time RT-PCR assay showed linearity from 20 to 2 x 106 virus copies with a correlation coefficient above 0.98 to 0.99 for all experiments. The detection threshold for PUUV cDNA was two copies per reaction. A two-step qualitative RT-PCR to detect PUUV RNA showed 100% concordance with the real-time RT-PCR assay. PUUV RNA viremia was detected in 33 of 34 PUUV immunoglobulin M (IgM)-positive patients with typical clinical NE disease from the region of endemicity. One PUUV IgM-negative sample had PUUV RNA, and 4 days later, the patient was IgM positive. Of samples with indeterminate IgM, 43% were PUUV RNA positive. The kinetics of antibody titers and PUUV viremia were studied, and five of six NE patients displayed a decrease in PUUV viremia a few days after disease outbreak coupled with an increase in PUUV IgM and IgG. In one patient with continuously high PUUV RNA levels but low IgM and no IgG response, the infection was lethal. These findings demonstrated that real-time RT-PCR is a useful method for diagnosis of PUUV viremia and for detecting PUUV RNA at early time points, before the appearance of IgM antibodies.

* Corresponding author. Mailing address: Department of Virology, Umeå University, 901 85 Umeå, Sweden. Phone: 46 90 7851790. Fax: 46 90 129905. E-mail: magnus.evander{at}climi.umu.se

{triangledown} Published ahead of print on 30 May 2007.

Journal of Clinical Microbiology, August 2007, p. 2491-2497, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.01902-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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