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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). Methodology and Principal Findings 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 200μl 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/Significance 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., Author Summary Japanese encephalitis virus infection is widespread in Asia, and primarily affects children in poor, rural areas. 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 Japanese encephalitis virus in the cerebrospinal fluid that surrounds the brain and spinal cord. However, in most areas where the infection occurs there are no laboratory facilities. In the absence of laboratory diagnosis, we have poor knowledge of the extent of the problem in these areas. Dried blood spots on filter paper have been used widely for many years as simple, cost-effective tools for transporting samples for testing. However, there have been few attempts at using dried cerebrospinal fluid spots. We developed a technique for using dried spots to store and test cerebrospinal fluid for antibodies to Japanese encephalitis virus. We compared the results of dried spots with routine neat cerebrospinal fluid in 132 patient samples, and demonstrated excellent agreement between the two tests. 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.

Three types of paper-covered and one type of plastic-covered Kodak dental film were used to determine if bacteria could penetrate the coverings and contaminate the inner film. Films from each group were immersed for 30 or 120 seconds in high concentrations of known bacterial suspensions with or without 10% sterilized calf serum added to the incubation media. The plastic-covered film effectively excluded all bacteria, whereas the paper-covered film showed contamination with all organisms even at 30 seconds. Increasing the viscosity of the incubation medium with calf serum decreased the level of contamination.

The suitability of phage-impregnated paper discs for the phage typing of mycobacteria was studied. The relevance of the routine test dilution, the volume of the phage used, the mode of incubation, and the effect of prolonged storage of phage-impregnated paper discs were considered. By using paper discs, each impregnated with one of 5 different mycobacteriophages (BG1, BK1, G37, CRI-3 and LG) that lyse Mycobacterium smegmatis 607B, it was determined that 100 x the routine test dilution in a volume of at least 20 microliter was required for phage lysis. Soaked and dried paper discs produced larger areas of lysis than those with 20-microliter volumes. Soaked discs were found to be stable even after storage for 8 weeks at 4 degrees C.

Biologically active 14 C-labeled purified protein derivative ( 14 C-PPD) has been prepared from the culture filtrates of seven species of mycobacteria, namely Mycobacterium tuberculosis Johnston strain (PPD), M. bovis BCG (PPD-BCG), M. avium (PPD-A), M. kansasii (PPD-Y), M. intracellulare (PPD-B), M. scrofulaceum (PPD-G), and M. fortuitum (PPD-F). These mycobacteria were grown in a culture medium containing a mixture of 14 C-labeled amino acids. The yield and specific radioactivity of the PPD, of the nucleic acid, of the bacterial cells, and of the CO 2 developed during growth have been determined for each of the seven species of mycobacteria. Although the yields of 14 C-PPD antigens differed greatly for the different species of mycobacteria tested, their specific radioactivities were similar. The 14 C-PPD antigens have been used as a means to measure their adsorption to glass. When glass ampoules containing dilute solutions (0.001 mg of PPD per ml) of these PPD antigens (PPD, PPD-BCG, PPD-A, PPD-Y, PPD-G, PPD-B, and PPD-F) were stored for 12 months at 5 C, it was found that they all adsorbed equally well to glass surfaces. In fact, regardless of the origin of the PPD, a loss due to adsorption of about 90% occurred during the first month of storage, and thereafter the PPD content remained practically constant for the rest of the duration of the storage period. The addition of 0.0005% Tween 80 to the PPD solutions effectively reduced the adsorption to glass of most PPD antigens. However, adsorption of PPD-BCG was not quite so effectively prevented, even when the Tween 80 concentration was increased from 0.0005 to 0.0005%.

Paper strips impregnated with stains and chemicals for bacterial staining (Gugol Scientific Corporation, distributed by Wampole Laboratories) were evaluated. The quality of staining 100 Gram-positive and 80 Gram-negative microorganisms was compared with Hucker's modification. Over-decolorization of strip-stained Gram-positive organisms was the most frequent cause of inconsistency. Paper strip “acid-fast” stain was compared with the Ziehl-Neelsen technic for 90 mycobacteria and nine Nocardia strains. Mycobacteria and Nocardia were consistently well stained by the paper strip technic; however, Nocardia were not stained by the true acid-fast method. Ninety-five clinical smears, stripstained by physicians on the wards, were evaluated. Ease of performance and stability of reagents make the paper strip technics practical for use in clinics, wards, and small bncteriologic laboratories.

A commercial reagent-impregnated paper strip test for the determination of production of niacin by mycobacteria has been evaluated. Safety, simplicity, uniformity, and stability were found to be inherent features of the test strips. Results compare favorably with those obtained using the Konno test as modified by Runyon and associates. Precautions which must be observed in performing this determination are listed.

An ultraviolet chamber for decontaminating single sheets of paper seeded with mycobacteria was investigated. The ultraviolet killing effect ranged from 50% for Mycobacterium kansasii to more than 99% for M. tuberculosis.