Direct Blood Dry LAMP Diagnoses African Trypanosomiasis

Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive tool used for the diagnosis of a variety of infectious diseases, but still requires complicated sample preparation steps and a well-equipped laboratory to produce reliable and reproducible results.

Several substantial modifications to the LAMP technique have been made to carry out on-site diagnosis of Human African Trypanosomiasis (HAT) in remote areas where there are normally resource-poor laboratories in most developing countries.

Scientists at the Hokkaido University (Sapporo, Japan) working with colleagues in Zambia, refined an existing LAMP system for HAT diagnosis that is cost effective and stable at high temperature. In their system, lysed blood can be used directly with high detection sensitivity. This dry LAMP system will be widely applicable in the field or for bedside diagnosis even in areas lacking adequate infrastructure.

The first essential improvement was that LAMP reagents were dried and stabilized in a single tube by incorporating trehalose as a cryoprotectant to prolong shelf life at ambient temperature. The second technical improvement was achieved by simplifying the sample preparation step so that DNA or ribonucleic acid (RNA) could be amplified directly from detergent-lysed blood samples. Primer sets were evaluated by real-time LAMP and also melting curve analysis using the Rotor-Gene 3000 thermal cycler (Corbett Research, Sydney, Australia) to monitor the reaction, and optimal amplification time and temperature for primer sets were determined.

The investigators developed a combination of dyes that they have called a “colori-fluorometric indicator (CFI) for the LAMP reaction. They also developed a battery-driven hand-made light emitting diodes (LED) illuminator that emits 500 nm wavelength light, which was suitable for field or bedside use. Using CFI, the LAMP reactions could be detected in two ways: either by detecting amplified DNA products with GelGreen or by a decreased Magnesium (Mg2+) concentration with hydroxyl-naphtol blue (HNB). The resulting fluorescence was visible under a blue-green LED illuminator. The color of HNB changed from violet to blue during the progression of the LAMP reaction as the Mg2+ ion concentration decreased by forming insoluble magnesium pyrophosphate. These color changes were easily visible by the naked eye. The sensitivity, as determined by a ten-fold detection limit was comparable to that of calcein, a commonly used color-development reagent for LAMP.

The authors concluded that they had refined the HAT LAMP detection system, making the feasibility of LAMP for bedside diagnosis and field surveillance. Their LAMP system can be applied to a wide range of other infectious diseases and therefore paves the way for possible utilization of rapid molecular diagnostic tests at point of care stations in resource poor countries. Human African trypanosomiasis (HAT) is an endemic protozoan disease affecting many African countries, predominantly in rural areas where the tsetse fly vector is present. The study was published on March 13, 2015, in the journal Public Library of Science Neglected Tropical Diseases.

Related Links:
Hokkaido University 
Corbett Research