title: Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots.
abstract: BACKGROUND
  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. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA).
  
  
  RESULTS
  Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200 μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009-15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25-30 °C showed 81.6% (65.7-92.3 95%CI) positive agreement, 96.8% (91.0-99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70-0.92 95%CI).
  
  
  CONCLUSIONS
  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. 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.
authors.affiliation: London School of Hygiene and Tropical Medicine, London, United Kingdom.|Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.
authors.forename: Tehmina
authors.initials: T
authors.lastname: Bharucha
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.
authors.forename: Anisone
authors.initials: A
authors.lastname: Chanthongthip
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.
authors.forename: Soumphou
authors.initials: S
authors.lastname: Phuangpanom
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.
authors.forename: Ooyanong
authors.initials: O
authors.lastname: Phonemixay
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.
authors.forename: Onanong
authors.initials: O
authors.lastname: Sengvilaipaseuth
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.
authors.forename: Manivanh
authors.initials: M
authors.lastname: Vongsouvath
authors.affiliation: Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom.|Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
authors.forename: Sue
authors.initials: S
authors.lastname: Lee
authors.affiliation: London School of Hygiene and Tropical Medicine, London, United Kingdom.|Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.|Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Paul N
authors.initials: PN
authors.lastname: Newton
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR.|Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom.|UMR_D 190 "Emergence des Pathologies Virales" (Aix-Marseille Univ, IRD French Institute of Research for Development, EHESP French School of Public Health), Marseille, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Dubot-Pérès
mesh_terms: D000914:Antibodies, Viral / Q000134:cerebrospinal fluid*; D002555:Cerebrospinal Fluid / Q000737:chemistry*; D003890:Desiccation; D004664:Encephalitis Virus, Japanese / Q000276:immunology*; D004672:Encephalitis, Japanese / Q000175:diagnosis*; D006801:Humans; D007075:Immunoglobulin M / Q000134:cerebrospinal fluid*; D007812:Laos; D009161:Mycobacterium; D010209:Paper; D010865:Pilot Projects; D012189:Retrospective Studies; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature
chemical_list: D000914:Antibodies, Viral; D007075:Immunoglobulin M