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|Title: ||Loop-mediated isothermal amplification (LAMP) for the diagnosis of human sleeping sickness: towards a point-of-care diagnostic test.|
|Authors: ||Wastling, Sally Louise|
|Supervisor(s): ||Welburn, Sue|
|Issue Date: ||25-Nov-2011|
|Publisher: ||The University of Edinburgh|
|Abstract: ||Acute and chronic sleeping sickness are fatal neglected tropical diseases caused by
Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense respectively
(members of the sub-genus Trypanozoon). Accurate diagnostics are needed to guide
treatment since the symptoms of disease are non-specific and the drugs that are used
for treatment are too toxic to be administered to unconfirmed cases. Tests need to be
simple enough to confirm clinical diagnosis of sleeping sickness in poorly-resourced,
peripheral health centres and for use as epidemiological tools to detect T. b.
rhodesiense in the zoonotic reservoirs of infection.
This study focuses upon LAMP (loop-mediated isothermal amplification) as a novel
diagnostic for sleeping sickness that may serve to bridge the gap between the need
for sensitive, specific molecular diagnostics on the one hand and ‘field-friendly’
diagnostics on the other.
Here, two previously published LAMP assays for Trypanozoons were compared to
classic PCR based methods for the diagnosis of Trypanozoon infection status in 428
cattle blood samples. The results did not support the use of LAMP as an improved
system for surveillance of T. b. rhodesiense in the zoonotic cattle reservoir.
T. b. rhodesiense and T. b. gambiense subspecies specific LAMP assays were
evaluated against traditional reference subspecies specific PCR tests, using DNA
purified from 86 cryopreserved trypanosome isolates. Novel LAMP assays for these
subspecies were also designed and evaluated. Both the published and novel assays
for T. b. rhodesiense (targeting different regions of the SRA gene) were sensitive,
specific and reliable when applied to purified DNAs, but were less consistent on field
samples. The novel T. b. gambiense LAMP (targeting TgsGP) was sensitive and
specific but this was not the case for the published LAMP assay (targeting the 5.8S
rRNA gene). However reliability may be less than optimal for LAMP TgsGP. Finally, simple endpoint readout methods for LAMP were evaluated. The colour
change reagent hydroxynaphthol blue was identified as the best currently available
method taking cost, ease of use and reliability into consideration.
In 2009 the number of reported sleeping sickness cases fell below 10,000 for the first
time in 50 years. Improved LAMP diagnostics could facilitate the diagnosis of
sleeping sickness and support the continued fight against this neglected, but deadly
|Sponsor(s): ||Medical Research Council (MRC)|
loop-mediated isothermal amplification
|Appears in Collections:||School of Biomedical Sciences thesis and dissertation collection|
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