Abstract
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.
Highlights
Tuberculosis (TB) is an infectious disease that is very often difficult to diagnose
Design of Recombinase Polymerase Amplification (RPA) assays for IS6110 and IS1081 An RPA assay was designed to target the insertion sequence, IS6110 [29,30], a region that has been shown to have high sensitivity for diagnosing tuberculosis using polymerase chain reaction (PCR) [31,32]
IS6110 is often present in M. tuberculosis in multiple copies of up to 25 per genome [33] but Mycobacterium tuberculosis complex (MTBC) isolates with no IS6110 target have been described [34,35]
Summary
Tuberculosis (TB) is an infectious disease that is very often difficult to diagnose. Pulmonary TB, the most infectious form of the disease, is diagnosed by detecting Mycobacterium tuberculosis complex (MTBC) bacilli in samples of sputum expectorated by the patient. Smear microscopy, which is the primary test used for the diagnosis of pulmonary TB endemic countries [1], is a laborious and relatively insensitive test with a case detection rate of only 56 to 68% [2]. The WHO recently noted that in 2012 approximately three million cases of active TB went undiagnosed by country programs [1] and there is a pressing need for improved diagnostic tools to supplant smear microscopy to facilitate rapid detection [3,4]
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