DETECTION OF ROTAVIRUS RNA IN CEREBROSPINAL FLUID IN A CASE OF ROTAVIRUS GASTROENTERITIS WITH FEBRILE SEIZURES

Abstract

Rotavirus is the most frequent cause of acute gastroenteritis in young children worldwide.1 Rotavirus infection is largely confined to the small intestine,1 but the virus has also been detected in the respiratory tract.2 Rotavirus encephalitis has been suspected on clinical grounds,3-5 and intracerebral rotavirus antibody response has been reported in the cerebrospinal fluid (CSF).6 Recently rotavirus genomic RNA was detected in the CSF of a 2-year-old Japanese girl with encephalitis and gastroenteritis; the detected rotavirus was of unusual serotype G3P9.7 In Japan rotavirus type G1 was also found in the blood and CSF of children with rotavirus gastroenteritis accompanying convulsion.8 We report a case of febrile seizures in the connection of rotavirus gastroenteritis, with rotavirus RNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in the CSF. Case report. A previously healthy 9-month-old girl was admitted to Tampere University Hospital in March, 1995, because of febrile seizures lasting for less than 1 min after a rapid rise of fever to 39°C. On the same day she had had some loose stools and vomiting. On admission the patient was febrile, fully conscious and neurologically normal. The rectal temperature was 39.0°C. She was admitted to the pediatric infectious disease ward where she developed a severe gastroenteritis with vomiting and watery stools. She was febrile and generally ill for 2 days and had vomiting for 4 and diarrhea for 5 days. She was treated with oral rehydration and early full feeding. In laboratory studies the values on admission were: hemoglobin 124 g/l; white blood cell count 8.8 × 109/l with 35% polymorphonuclear leukocytes and 59% mononuclears; C-reactive protein 5 mg/l; sodium 138 mmol/l; potassium 4.6 mmol/l; calcium 2.55 mmol/l; and glucose 4.6 mmol/l. Blood culture was negative. The CSF was unremarkable: erythrocyte count 0; white blood cell count 3/mm3; lactate <0.4 mmol/l; protein 0.9 mg/l; and bacterial culture negative. Rotavirus antigen detection from stools by enzyme-linked immunosorbent assay (Dako Diagnostics Ltd., Glostrup, Denmark) was positive. A significant rise of rotavirus IgA and IgG antibodies was detected in paired sera. For RT-PCR studies of rotavirus stool and CSF specimens were collected on the day of admission, and serum and peripheral blood mononuclear cell specimens were collected 4 days later. Genomic RNA of stool and serum,9 and CSF and peripheral blood mononuclear cell10 specimens were extracted as described previously. RT-PCR was performed as described previously11; in the nested PCR that followed RT-PCR Taq DNA polymerase (Promega, Madison, WI) was used. The presence of rotavirus RNA in the specimens was first studied with the use of primers for VP6 core protein of Subgroup 2, corresponding to nucleotides 519 to 540 and 928 to 909 in the first PCR and nucleotides 616 to 635 and 859 to 838 in the nested PCR of the first PCR products. The nested PCR detecting a 243-base pair (bp) DNA segment of VP6 genome was positive for stool, CSF and peripheral blood mononuclear cell samples and negative for serum (data not shown). The presence of rotavirus RNA in the specimens was further studied with the use of rotavirus VP7-specific primers by RT-PCR followed by nested PCR as described previously.11, 12 Rotavirus VP7 serotype G1-specific RNA was detected in the CSF by demonstration of a 749-bp DNA segment as a nested PCR product on a 1.5% agarose gel stained with ethidium bromide (Fig. 1). The specificity of the finding was confirmed by purifying the PCR product from the gel and hybridizing it with radioactively labeled DNA probe of Wa strain VP7 serotype G1 human rotavirus (Fig. 2). The PCR products were transferred to a nylon membrane by a downward capillary transfer method13 and Southern hybridization was performed according to the protocol recommended by Msi Micron Separation Inc. Discussion. Rotavirus gastroenteritis may be associated with high fever and, as a consequence typical febrile seizures may occur. On the other hand central nervous system invasion by rotavirus and rotavirus encephalitis have been suspected clinically for many years.3-5 These suspicions were recently substantiated by demonstration of rotavirus RNA in the CSF in a Japanese girl with encephalitis and rotavirus gastroenteritis. In our patient we found RT-PCR rotavirus in the CSF on the day of onset of fever in rotavirus gastroenteritis. This suggests extragastrointestinal spread of rotavirus at an early phase of rotavirus infection with systemic symptoms, including high fever and febrile convulsions. Whether rotavirus was directly involved in the induction of convulsions or whether the seizures were induced by high fever cannot be concluded. However, as the course of disease was benign, it would seem possible that systemic spread of rotavirus may take place in the absence of encephalitis. RT-PCR is a sensitive method for the detection of rotavirus very low concentrations,14 as probably is the situation in the CSF or blood in rotavirus disease. On the other hand because RT-PCR is sensitive enough to detect rotavirus even from environmental surfaces,15 contamination from the hospital environment leading to a false positive result is difficult to rule out. Contamination from the laboratory seems unlikely, given that the positive finding was repeated twice from the original CSF sample with both VP6 and VP7 (serotype 1)-specific primers. The finding of rotavirus RNA in the CSF may have few clinical implications, because the clinical picture in this case, as in most cases of seizures in the association with rotavirus gastroenteritis, was typical of febrile seizures. However, our finding suggests that further studies on viral dissemination in rotavirus disease with generalized symptoms are warranted. RT-PCR should provide a powerful tool for such studies. Xiao-Li Pang, M.D.; Jaana Joensuu, M.D.; Timo Vesikari, M.D. Department of Pediatrics University of Tampere Medical School Tampere University Hospital Tampere, FinlandFIG. 1: Visualization of tested PCR products of rotavirus VP7-specific primers. Lanes 1 and 7, a 50-bp ladder PCR marker; Lane 2, Wa strain of serotype G1 reference rotavirus; Lane 3, CSF specimen with a mixture of primers for VP7 serotypes 1, 2, 3, 4, 8 and 9; Lane 4, CSF specimen with a primer for VP7 serotype G1; Lane 5, CSF specimen with a primer for VP7 serotype 3; Lane 6, negative control (water). Arrowhead, 749-bp fragment indicating the nested PCR product of VP7 serotype G1 primer.FIG. 2: Results of Southern hybridization of PCR products from Wa strain of serotype G1 human reference rotavirus and the patient's CSF, using a 32P-labeled DNA probe corresponding to the genomic RNA of the VP7 antigen of the Wa strain. Lane 1, Wa rotavirus; Lane 2, CSF specimen. In Lane 1, there is an excess of first PCR product, in addition to the 749-bp fragment arising from the nested PCR.