Pre-cut filter paper for detecting anti-Japanese encephalitis virus IgM from dried cerebrospinal fluid spots

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 200 mu l 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 degrees 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

Pneumococcal carriage in vaccine-eligible children and unvaccinated infants in Lao PDR two years following the introduction of the 13-valent pneumococcal conjugate vaccine.

Pneumococcal carriage is a prerequisite for disease, and underpins herd protection provided by pneumococcal conjugate vaccines (PCVs). There are few data on the impact of PCVs in lower income settings, particularly in Asia. In 2013, the Lao People's Democratic Republic (Lao PDR) introduced 13-valent PCV (PCV13) as a 3 + 0 schedule (doses at 6, 10 and 14 weeks of age) with limited catch-up vaccination. We conducted two cross-sectional carriage surveys (pre- and two years post-PCV) to assess the impact of PCV13 on nasopharyngeal pneumococcal carriage in 5-8 week old infants (n = 1000) and 12-23 month old children (n = 1010). Pneumococci were detected by quantitative real-time PCR, and molecular serotyping was performed using DNA microarray. Post PCV13, there was a 23% relative reduction in PCV13-type carriage in children aged 12-23 months (adjusted prevalence ratio [aPR] 0.77 [0.61-0.96]), and no significant change in non-PCV13 serotype carriage (aPR 1.11 [0.89-1.38]). In infants too young to be vaccinated, there was no significant change in carriage of PCV13 serotypes (aPR 0.74 [0.43-1.27]) or non-PCV13 serotypes (aPR 1.29 [0.85-1.96]), although trends were suggestive of indirect effects. Over 70% of pneumococcal-positive samples contained at least one antimicrobial resistance gene, which were more common in PCV13 serotypes (p < 0.001). In 12-23 month old children, pneumococcal density of both PCV13 serotypes and non-PCV13 serotypes was higher in PCV13-vaccinated compared with undervaccinated children (p = 0.004 and p < 0.001, respectively). This study provides evidence of PCV13 impact on carriage in a population without prior PCV7 utilisation, and provides important data from a lower-middle income setting in Asia. The reductions in PCV13 serotype carriage in vaccine-eligible children are likely to result in reductions in pneumococcal transmission and disease in Lao PDR

A comparison of two molecular methods for diagnosing leptospirosis from three different sample types in patients presenting with fever in Laos.

OBJECTIVES: To compare two molecular assays (rrs quantitative PCR (qPCR) versus a combined 16SrRNA and LipL32 qPCR) on different sample types for diagnosing leptospirosis in febrile patients presenting to Mahosot Hospital, Vientiane, Laos. METHODS: Serum, buffy coat and urine samples were collected on admission, and follow-up serum ∼10 days later. Leptospira spp. culture and microscopic agglutination tests (MAT) were performed as reference standards. Bayesian latent class modelling was performed to estimate sensitivity and specificity of each diagnostic test. RESULTS: In all, 787 patients were included in the analysis: 4/787 (0.5%) were Leptospira culture positive, 30/787 (3.8%) were MAT positive, 76/787 (9.7%) were rrs qPCR positive and 20/787 (2.5%) were 16SrRNA/LipL32 qPCR positive for pathogenic Leptospira spp. in at least one sample. Estimated sensitivity and specificity (with 95% CI) of 16SrRNA/LipL32 qPCR on serum (53.9% (33.3%-81.8%); 99.6% (99.2%-100%)), buffy coat (58.8% (34.4%-90.9%); 99.9% (99.6%-100%)) and urine samples (45.0% (27.0%-66.7%); 99.6% (99.3%-100%)) were comparable with those of rrs qPCR, except specificity of 16SrRNA/LipL32 qPCR on urine samples was significantly higher (99.6% (99.3%-100%) vs. 92.5% (92.3%-92.8%), p <0.001). Sensitivities of MAT (16% (95% CI 6.3%-29.4%)) and culture (25% (95% CI 13.3%-44.4%)) were low. Mean positive Cq values showed that buffy coat samples were more frequently inhibitory to qPCR than either serum or urine (p <0.001). CONCLUSIONS: Serum and urine are better samples for qPCR than buffy coat, and 16SrRNA/LipL32 qPCR performs better than rrs qPCR on urine. Quantitative PCR on admission is a reliable rapid diagnostic tool, performing better than MAT or culture, with significant implications for clinical and epidemiological investigations of this global neglected disease

Flight investigation of Limited Authority attitude Command Flight control system architectures for Rotorcraft.

NRC publication: Ye

Short report: Comparison of performance of serum and plasma in panbio dengue and Japanese encephalitis virus enzyme-linked immunosorbent assays

We examined the comparative performance of serum and plasma (in dipotassium EDTA) in Panbio Dengue enzyme-linked immunosorbent assays (ELISAs) for detection of non-structural protein 1 (NS1), IgM, and IgG, and a dengue/Japanese encephalitis virus (JEV) combination IgM ELISA in a prospective series of 201 patients with suspected dengue in Laos. Paired comparisons of medians from serum and plasma samples were not significantly different for Dengue IgM, and NS1 which had the highest number of discordant pairs (both 2%; P = 0.13 and P = 0.25, respectively). Comparison of qualitative final diagnostic interpretations for serum and plasma samples were not significantly different: only 1.5% (3 of 201 for Dengue/JEV IgM and Dengue IgG) and 2.0% (4 of 201; IgM and NS1) showed discordant pairs. These results demonstrate that plasma containing EDTA is suitable for use in these ELISAs. Copyright © 2012 by The American Society of Tropical Medicine and Hygiene

Comparison of two commercial ELISA kits for the detection of anti-dengue IgM for routine dengue diagnosis in Laos

The endemicity of Dengue virus (DENV) infection remains a major public health problem in Lao PDR. In this study, we compared two commercial anti-dengue IgM ELISA kits, Panbio® Dengue IgM Capture ELISA (Panbio Kit, Alere, Waltham, MA, USA) and DEN DetectTM MAC-ELISA (InBios kit, InBios International, Inc., Seattle, WA, USA), in the context of diagnosis of patients admitted to hospital with clinical dengue presentation. Two panels of paired blood samples were tested. Panel A was composed of 54 dengue confirmed patients (by DENV real-time RT-PCR) and 11 non-dengue dengue patients (other infections confirmed by corresponding PCR results). Panel B included 74 patients randomly selected from consecutive patients admitted to Mahosot Hospital in 2008 with suspicion of dengue fever according to WHO criteria. Results from panel A showed significantly better sensitivity for Panbio kit (64.8%; 95%CI: 50.6-77.3%) than for InBios kit (18.5%; 95%CI: 9.3-31.4%) when testing admission sera. Sensitivity was increased for both kits when combining results from admission and convalescent sera. Concordant results were obtained from panel B with fair agreement (κ = 0.29) between both kits when testing single admission samples, and moderate agreement (κ = 0.5) when combining results from admission and convalescent sera