Detection of Human Bocavirus in the Cerebrospinal Fluid of Children With Encephalitis

We report 4 children with encephalitis associated with human bocavirus (HBoV) 1 or 2. All children were severely underweight, and 2 died; 1 of them had a matching HBoV2 nucleotide sequence isolated from serum and bocavirus like particles in the cerebrospinal fluid that were observed with electron microscopy.No further pathogens were detected in the cerebrospinal fluid of these patients.Encephalitis is an important cause of morbidity and mortality worldwide.In the industrialized countries of Europe and America, the annual incidence of acute encephalitis is 7.3 cases per 100 000 persons [1].Up to 71% of these patients experience lasting sequelae, and up to 7.4% die [1,2].A myriad of infectious agents is associated with encephalitis, such as herpes simplex virus, which is common in adults, and the varicella zoster virus and enterovirus, which are common in children [2].The cause of 70%-85% of cases of encephalitis worldwide remains unknown, despite the use of a wide range of diagnostic methods [3,4].This indicates the presence of unknown agents or known pathogens that normally infect other organs but are not expected to cause encephalitis.Because hematogenous spread is the most common route that viruses take to enter the central nervous system and cause encephalitis, we focused our attention on the human bocavirus (HBoV), which is able to cause viremia [5] and, therefore, has the potential to disseminate to other parts of the body to initiate infection.In children, HBoV1 is prevalent in respiratory and enteric infections.Three more related human bocavirus species, named HBoV2-4, have been found preferentially in stool; HBoV2 has been associated with gastrointestinal disease [6].Encephalitis-causing viruses such as enteroviruses use the gastrointestinal tract as a route for infection.We therefore hypothesize that HBoV1-4 may cause encephalitis in children.In this study, we did not limit testing to the detection of HBoV but also evaluated for other diarrheagenic viruses, including rotavirus, norovirus, adenovirus, and astrovirus, as possible causes of encephalitis. METHODS PatientsThe study was conducted at the hospital of the Institute of Child and Mother Health (ICMH) situated in Matuail, Dhaka, Bangladesh.The study occurred from April 2009 through March 2010 and was approved by the ethics committee of the ICMH.A total of 79 children were enrolled during the study period, and 69 gave consent to participate in the study.The male-female ratio was 2:1.3, and the mean age was 40.4 months (range, 4-144 months).The parents gave their consent for participation in this study.Encephalitis was defined as the presence of fever, convulsion, and unconsciousness, with or without signs of meningeal irritation [7].Cerebrospinal fluid (CSF) and serum samples were collected from each patient.The nutritional status of the patients was determined using the World Health Organization child growth standards. Routine Laboratory InvestigationCSF samples were subjected to macroscopic and microscopic examination, and were cultured for bacteria.Total and differential white blood cell count, erythrocyte sedimentation rate, hemoglobin and C-reactive protein (CRP) levels were determined. Pathogen Detection and IdentificationEncephalitis-causing pathogens and diarrheagenic viruses (rotaviruses, astroviruses, noroviruses and adenoviruses) were determined in the CSF by polymerase chain reaction (PCR).Employed primers and their corresponding references are detailed in Supplementary Table 1.HBoV was detected by nested touchdown PCR that targeted the VP1/2 region of HBoV1-4