PCR and other NAAT methods for TB have great advantages of speed (compared to culture) and sensitivity (compared to microscopy). Furthermore, recently developed real-time assays may generate results in less than an hour, a key requirement for point-of-care testing. The utility of current NAAT methods is limited, however, by their cost and complexity, particularly in countries where TB is endemic. The required precision instruments are beyond the capacity of most diagnostic sites to purchase, maintain, or operate, and the complexity of testing obviates the possibility of point-of-care use.
Beyond amplification and detection itself, preparatory steps for NAAT make current formats cumbersome. Existing TB NAAT methods require separate steps for sputum liquefaction, DNA extraction, target amplification, and amplicon detection. This translates into multiple steps, separate workstations, and greater opportunity for cross-contamination with either bacterial DNA or amplicon. Automation of this process may be feasible, but is likely to further drive up the cost of equipment and reagents.
LAMP has inherent properties that make amplification and detection possible in one uninterrupted process, with no need to open the amplification vessel or any need for a luminometer or other detection device. The assay utilizes a single polymerase that is active at relatively high isothermal amplification temperatures, diminishing the likelihood of nonspecific priming. The use of six primers increases specificity at the same time that it enhances speed. In our hands, TB could usually be detected within 30 min, although a 40-min amplification time was optimal.
This paper reports the development and first use of a prototype LAMP assay for TB intended for use in peripheral laboratories. The study demonstrated that technicians without molecular training could perform the test with high reproducibility in a simple laboratory space without specialized equipment. Reader-to-reader variability was negligible, and there were no examples of absolute discordance (positive versus negative). Reaction results were stable for hours following automated enzyme inactivation with a heat spike at the end of amplification. The need for repeat testing for unexpected control tube results did not arise during the enrollment period, except for a limited number of runs in which an inadequately prepared positive control was used.
Quantitatively, the assay significantly outperformed microscopy, detecting M. tuberculosis
DNA in almost all smear-positive sputum specimens and half of smear-negative, culture-positive specimens. These results are similar to those reported previously for sophisticated commercialized NAATs for M. tuberculosis
). The total hands-on time to process and read results on a batch of sputum samples was similar to that of microscopy, with significantly simpler result readout. Notably, the presence of frank blood in the samples did not appear to inhibit the reaction, reinforcing findings reported previously by Poon et al. (18
), who were able to detect malaria parasites at low density in boiled whole blood.
Further simplification of the LAMP assay was noted as being a priority by study participants. This is clearly possible, potentially leading to the first molecular test system that can be deployed in developing countries outside of reference laboratories. A significant amount of work has already been done to simplify and optimize the processing and extraction steps. Prior development work on NAAT methods for M. tuberculosis has always been held hostage by the N-acetyl-l-cysteine-NaOH processing method that is in standard use prior to culture in industrialized countries. This has been true largely because developers realized that the systems would be used in higher-level laboratories already performing culture and that culture and susceptibility testing would continue. Integration mandated the same initial processing steps. By working toward a method that would replace microscopy, we were able to explore alternative processing methods that could be more easily converted into a few-step process not requiring external equipment.
Eiken and the Foundation for Innovative New Diagnostics are developing an improved version of the test in which the number of steps will be reduced by a third and the number of electrical devices will be reduced to only a single heating block. With these simplifications, the ease of use and the robustness will increase, and the risk of laboratory DNA contamination will drop further. Larger studies are planned for the next version of TB LAMP in which performance, especially in smear-negative TB, will be evaluated and compared to those of other molecular amplification tests in addition to microscopy and culture.