Japanese Encephalitis Virus as Cause of Acute Encephalitis, Bhutan.

In 2011, Bhutan's Royal Centre for Disease Control began Japanese encephalitis (JE) surveillance at 5 sentinel hospitals throughout Bhutan. During 2011-2018, a total of 20 JE cases were detected, indicating JE virus causes encephalitis in Bhutan. Maintaining JE surveillance will help improve understanding of JE epidemiology in this country.

Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.

Quantitation of rabbit cytokine mRNA by real-time RT-PCR.

Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.