We tested 332 consecutively submitted CSF samples from 319 patients <4 years of age from Denmark that had been submitted to Statens Serum Institut, Copenhagen, from January 2006 through December 2009 for viral diagnostic testing. We tested for the following viruses: herpes simplex virus types 1 and 2 (228 samples), varicella zoster virus (228 samples), human enterovirus (261 samples), and human parechovirus (88 samples since November 2008). For all samples, initial test results were negative.

We selected fecal samples from 479 children <5 years of age with gastroenteritis that had been submitted for viral diagnostic testing from September 2009 through February 2010 and tested them for SAFV. Nucleic acid extracted from these samples was combined into 48 pools, with 9 or 10 samples per pool. Samples from pools with positive results were identified, and new extractions from these pools were tested individually. However, enough sample material for new extractions was available for only about half of the samples.

Nucleic acids were extracted from 200 µL of CSF or blood (from SAFV-positive patients) by using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) and semiautomatic extraction on the QIAcube instrument (QIAGEN). Nucleic acid was extracted from 200-µL fecal suspension (10% in phosphate-buffered saline) by using the Total Nucleic Acid Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany) on the MagnaPure LC instrument (Roche Diagnostics GmbH).

Five microliters of extracted material was used per reverse transcription PCR (total volume 25 µL) by using the OneStep RT-PCR Kit (QIAGEN). The reaction mixtures contained 1 µmol/L of each primer and 0.2 µmol/L of probe.

Design of the primers and probe was based on an alignment of all available SAFV sequences in GenBank (www.ncbi.nlm.nih.gov/genbank) in July 2010 by using ClustalW (www.clustal.org) and Primer3 (http://frodo.wi.mit.edu/primer3) software. The primers and probe are selective for a highly conserved part of the 5′ untranslated region (Table). The Strategene Mx3005P real-time thermocycler instrument (Agilent Technologies A/S, Horsholm, Denmark) was used for amplification and detection with the following settings: 50°C for 20 min, 95°C for 15 min, followed by 45 cycles of 95°C for 15 s and 55°C for 1 min.

Genotyping was conducted by nested PCR and sequencing of parts of the viral protein (VP) 1 and VP2 regions of the capsid gene by using primers listed in the Table. The inner VP1 and VP2 primers amplified DNA fragments of ≈599 and 577 bp, respectively. PCR products were purified by using the High Pure PCR Purification Kit (Roche Diagnostics GmbH) before sequencing, which was performed by using the inner PCR primers on an ABI automated sequencer and BigDye version 1.1 chemistry (both from Applied Biosystems, Darmstadt, Germany). Sequences were aligned, and phylogenetic analysis with known reference sequences was performed by using MEGA4 software (www.megasoftware.net). The sequences have been submitted to GenBank under accession nos. JF693612–23.

Child 1 was a 16-month-old, previously healthy boy who became ill in May 2009. The boy had a fever 6 days before hospital admission, followed 1 day later by sudden onset of monosymptomatic ataxia, with no fever. The ataxia fluctuated from causing an insecure gait to walking into things and falling. The patient also had intermittent difficulty controlling his arms when trying to eat. No history of recent travel was reported. The boy was in otherwise good health; he had no abnormal psychological symptoms and retained a normal degree of consciousness throughout the acute phase of the disease. Differential diagnoses at hospital admission were intracranial tumor or viral cerebellitis. The boy’s 4-year-old sister remained healthy.

Laboratory test results are listed in Table A1. At hospital admission, CSF values (leukocyte count, protein level, and glucose level) were within reference ranges, and no microorganisms were detected. A magnetic resonance imaging scan of the brain showed a small venous anomaly in the left frontal lobe but no tumor, hemorrhage, or inflammation. A fecal sample collected 2 weeks later yielded positive test results for parechovirus type 3 and negative results for enterovirus and adenovirus. Parechovirus was not found in the CSF or blood.

During the next 2 months, the ataxia remitted completely without sequelae. The diagnosis at this time was viral encephalitis, possibly caused by parechovirus type 3.

Child 2 was a 27-month-old, previously healthy girl who was found dead in her bed in August 2009; she had no known history of disease or symptoms. During necropsy, signs of cerebral herniation were detected. A small vascular malformation surrounded by edema was found in the brain. No signs of encephalitis or hemolytic uremic syndrome were found.

The following sample materials were collected: CSF, blood, feces, myocardium, pericardial aspirate, lung tissue, and respiratory secretions (Table A1). In the CSF, mononuclear pleocytosis was noted. Results of routine bacteriologic culture found coagulase-negative staphylococci in the CSF, a few nonhemolytic streptococci in the lung tissue, and a few nonhemolytic streptococci and a few Staphylococcus aureus organisms in the pericardial aspirate. Verotoxin-producing Escherichia coli was cultured from the fecal sample. The conclusion of the autopsy and laboratory findings was that cerebral herniation was the immediate cause of death.

Virus from each of the 2 children was characterized by sequencing part of the VP1 region of the capsid gene, and the sequences were compared with those of other SAFVs detected in fecal samples from the patients with gastroenteritis (Figure 2). Of the 48 fecal sample pools, 10 were positive for SAFV by PCR. From these pools, 6 individual samples were available for further testing; the VP2 capsid region was successfully sequenced for 4 of these 6 samples, and they were all SAFV type 2. Later, an SAFV type 2–specific VP1 PCR was designed, which provided VP1 sequences from all 6 fecal samples and samples from the 2 children reported here: the fecal sample from child 1 and the blood sample from child 2. The phylogenetic analysis (Figure 2) showed that all viruses were SAFV type 2 and that the sequences arranged in 2 clusters with 8% nt differences and 5% aa differences between the clusters.