Date Published: March 13, 2015
Publisher: Public Library of Science
Author(s): Kyoko Hayashida, Kiichi Kajino, Lottie Hachaambwa, Boniface Namangala, Chihiro Sugimoto, Alain Debrabant. http://doi.org/10.1371/journal.pntd.0003578
Abstract: Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive tool used for the diagnosis of a variety of infectious diseases. One of the advantages of this method over the polymerase chain reaction is that DNA amplification occurs at a constant temperature, usually between 60–65°C; therefore, expensive devices are unnecessary for this step. However, LAMP still requires complicated sample preparation steps and a well-equipped laboratory to produce reliable and reproducible results, which limits its use in resource-poor laboratories in most developing countries. In this study, we made several substantial modifications to the technique to carry out on-site diagnosis of Human African Trypanosomiasis (HAT) in remote areas using LAMP. 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 RNA could be amplified directly from detergent-lysed blood samples. With these modifications, diagnosis of HAT in local clinics or villages in endemic areas becomes a reality, which could greatly impact on the application of diagnosis not only for HAT but also for other tropical diseases.
Partial Text: Human African trypanosomiasis (HAT), also known as “sleeping sickness”, is caused by the protozoan parasites Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense. Majority of reported HAT cases are caused by T. b. gambiense in Western and Central Africa, and sporadic HAT cases in East Africa are caused by T. b. rhodesiense. Infection with T. b. gambiense is characterized by chronic disease progression, whereas T. b. rhodesiense causes a more acute infection . In both subspecies, parasites circulating in the blood or lymphatic system often cause general flu-like clinical signs, including fever, general malaise, anemia, lymphadenopathy, and arthralgia, and may therefore be misdiagnosed as other febrile illnesses, such as malaria. In the initial stage, HAT is curable with drugs, such as pentamidine and suramin . In the second stage, trypanosomes eventually cross the blood-brain barrier to invade the central nervous system (CNS), leading to the typical symptoms of sleeping sickness, such as sleep cycle disturbances. In the final stage, patients deteriorate into a coma and the disease is fatal if left untreated. Melarsoprol, and more recently, NECT (nifurtimox–eflornithine combination therapy) for T. b. gambiense , is the drug of choice for the second stage of the disease; however, melarsoprol can be associated with severe side-effects and even death , and NECT is unfortunately less effective to T. b rhodesiense. Therefore, early HAT detection is crucial for the effective treatment and management of this disease.
Currently available diagnostic methods for HAT are based on the microscopic visualization of parasites in the blood, either by Giemsa-staining thin or thick smears or by micro-capillary centrifugation. There are two major problems with these methods: one is low sensitivity and the other is the need for a microscope and an experienced technician. Unless the parasites are concentrated, the sensitivity using microscopy on wet-smears is less than several thousand parasites per ml of blood , which often cause fatal delays in treatment. As such, capillary tube centrifugation (CTC, WOO) , quantitative buffy coat , and mini anion exchange centrifugation technique (mAECT) , and the modified single centrifugation method (MSC)  have been established. These parasite concentration techniques are simple and straightforward, with high sensitivity detecting <2 trypanosomes per ml of CSF in the case of MSC, but still rely on expensive devices like centrifuge and microscope with electricity, and differentiation between T. b. gambiense and T. b. rhodesiense is not possible. Source: http://doi.org/10.1371/journal.pntd.0003578