Dried Cerebrospinal Fluid Spots Detect Anti-Japanese Encephalitis IgM

The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, “Dried Blood Spots” or Guthrie cards, for diagnostic assays is well-established, but in contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid spots.

Japanese encephalitis virus (JEV) infection is widespread in Asia, and primarily affects children in poor, rural areas and the virus spreads to the brain and spinal cord resulting in significant death and disability. Diagnosis requires testing for the immune response “antibody” specific to JEV in the cerebrospinal fluid (CSF) that surrounds the brain and spinal cord.


An international team of scientists led by those at the London School of Hygiene and Tropical Medicine (UK) developed a dried CSF spot protocol to evaluate its diagnostic performance using the World Health Organization (Geneva, Switzerland) recommended anti-JEV immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). Patient CSF and serum samples were collected at three hospitals in Laos from 2009 to 2015.

The investigators employed a novel technique using a pre-cut circle of cellulose-cotton paper. Samples were dried on the pre-cut filter paper and then left for 30 days at room temperature. Two types of filter paper were chosen, and utilized in all the studies: the Whatman 903 Protein Saver Card (903) and the 3MM Chr Blotting paper (3MM) (GE Healthcare Life Sciences; UK). The commercial JEV MAC-ELISA assay used was is the JE Detect IgM Capture ELISA (Inbios; Seattle, WA, USA). This assay measures the Optical Density (OD) of each sample with JEV Recombinant Antigen, JERA, compared to Normal Control Antigen (NCA) to adjust for background nonspecific reactivity.

There were a total of 132 samples containing sufficient fluid volume for testing. When tested for the presence of viral antibodies 34 dried samples and 38 neat samples tested positive, with overall agreement of 92.4%. Compared with neat non-dried samples the dried spots showed 81.6% positive agreement and 96.8% negative agreement. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids. The authors concluded that the novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies.

Tehmina Bharucha, PhD, the lead author of the study, said, “This novel method for saturating dried cerebrospinal fluid spots has the potential to enhance our knowledge of Japanese encephalitis virus epidemiology, and inform health policies where they are most needed. It could also be transferred for use in diagnosing other infectious diseases, including using other body fluid samples.” The study was published on March 17, 2016, in the journal Public Library of Science Neglected Tropical Diseases.

Related Links:
London School of Hygiene and Tropical Medicine
World Health Organization
GE Healthcare Life Sciences