Trypanosoma brucei gambiense and T. b. rhodesiense are pathogens responsible for human African trypanosomiasis (HAT). Death from HAT is inevitable if untreated. In the rural areas where patients are typically seen, failure to microscopically observe trypanosomes in blood smears and/or CSF in the critical early stages of the disease is probably the single most important factor in failed treatment. We propose a new technology that we believe will be important for the detection of parasites in the CSF during Stage 2 disease (CNS involvement), particularly in the field setting. The procedure uses protein-DNA chimeras termed 'Tadpoles'. To create 'Tadpoles', a 'DNA barcode'is added to a single site on a protein. Amplification of the 'Tadpoles'DNA by PCR creates assays with great dynamic range and specificity. However, as an alternative to PCR we propose to amplify the DNA-barcode associated with Tadpoles by loop-mediated isothermal amplification (LAMP). The LAMP reaction amplifies DNA with high specificity, efficiency and speed under isothermal conditions and allows for easy visual positive identification of the target DNA. We propose to combine the technology of LAMP with 'Tadpoles'to create LAMP-based 'LAMPole'assays for the diagnosis of HAT suitable for diagnostic applications in the field. There are two Specific Aims. These are 1) to design oligonucleotides and create """"""""LAMPoles"""""""" for the highly sensitive detection of trypanosome signature proteins, and 2) to define analytical/clinical sensitivity and specificity for LAMPoles in the diagnosis of Stage 2 HAT. Success in the completion of the Specific Aims of this application will provide novel diagnostic tests for the early detection of Stage 2 HAT applicable in African health care centers and in the field.

Public Health Relevance

Human African trypanosomiasis or sleeping sickness is 100% fatal if not treated. At least 40,000 people die every year in Africa because good tests for diagnosis in the more rural areas do not exist. Using a simple water bath, we believe that a new technology we will develop called 'LAMPoles'will provide rapid, accurate, evidence for infection in field conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI079282-02
Application #
7932036
Study Section
Special Emphasis Panel (ZRG1-IDM-M (12))
Program Officer
Wali, Tonu M
Project Start
2009-09-15
Project End
2011-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
2
Fiscal Year
2010
Total Cost
$202,950
Indirect Cost
Name
Johns Hopkins University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Nikolskaia, Olga V; Thekisoe, Oriel M M; Dumler, J Stephen et al. (2017) Loop-Mediated Isothermal Amplification for Detection of the 5.8S Ribosomal Ribonucleic Acid Internal Transcribed Spacer 2 Gene Found in Trypanosoma brucei gambiense. Am J Trop Med Hyg 96:275-279
Burbulis, Ian E; Yamaguchi, Kumiko; Nikolskaia, Olga V et al. (2015) Detection of pathogen-specific antibodies by loop-mediated isothermal amplification. Clin Vaccine Immunol 22:374-80
Grab, Dennis J; Nenortas, Elizabeth; Bakshi, Rahul P et al. (2013) Membrane active chelators as novel anti-African trypanosome and anti-malarial drugs. Parasitol Int 62:461-3
Grab, Dennis J; Nikolskaia, Olga V; Inoue, Noboru et al. (2011) Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP). PLoS Negl Trop Dis 5:e1249
Grab, Dennis J; Garcia-Garcia, Jose C; Nikolskaia, Olga V et al. (2009) Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells. PLoS Negl Trop Dis 3:e479