Working towards novel diagnostics
Improving parasite detection
Assembly of mini Anion
Exchange Chromatography Technique (mAECT)
minicolumns in the new manufacturing facility at
INRB, Kinshasa
The most widely used method for diagnosis
of HAT is demonstration of parasites in
body fluids by microscopy. For patients infected
with T.b. rhodesiense, the number of parasites
in the blood and CSF is usually high
enough for them to be seen directly under a
light microscope. However, T.b. gambiense
exists in such low numbers that they have to
be concentrated before viewing.
The most sensitive method for detection of
trypanosomes in blood is the mini Anion
Exchange Centrifugation Technique (mAECT).
This involves separating trypanosomes from
blood by anion exchange chromatography and
concentrating them at the bottom of a sealed
glass tube by low speed centrifugation (3000
rpm). After centrifugation, the tip of the glass
tube is examined for the presence of motile
trypanosomes under low magnification
microscopy. The technique enables detection
of parasites in samples containing less than 100
parasites/ml, improving sensitivity significantly.
Until recently, attempts to produce mAECT
kits in Africa have been faced with problems
of sustainability. FIND
has been working with
the Institute of Tropical
Medicine (ITM) in Antwerp
to improve and
standardize the mAECT
test, and to establish a
mAECT production unit
at the Institut National
de Recherche Biomédicale
(INRB) in Kinshasa,
DRC. A quality control
system has been put in place at the institute,
while lot release quality control is carried out
at the ITM. The kits are made available to
control programmes at costs that will ensure
sustainable production. The target of these
activities is an initial production of 30,000 kits
by April 2008.
Stored minicolumns produced at INRB, ready for packaging
and shipping
FIND is also working with a number of
partners to develop an alternative parasite
detection technique, which should be more
sensitive and simpler to use than mAECT.
One such method, which is being explored
with the Flanders Interuniversity Institute
for Biotechnology (VIB) at the Free
University of Brussels, involves separating
parasites in a magnetic field. In this
approach, camel heavy-chain VHH antibodies
(nanobodies) that target the carbohydrate
region of the variable surface glycoprotein
(VSG) of trypanosomes are bound to
magnetic beads. They are then mixed with
test samples, and “fished” using a magnet,
dragging bound parasites with them. A similar
approach is being investigated in collaboration
with the Darmstadt University of
Technology in Germany by using aptamers
that target the protein moiety of the VSG.
Other options that are being considered
include separation in an electric field, density
and filter separation, separation using
other gels, biosensors, fluorimetry and fluorescence
microscopy.
In search of serological tests
with improved sensitivity
and specificity
Trypanosomes have a densely packed antigenic
coat that changes frequently in a
process called antigenic variation, posing a
major obstacle in the development of diagnostic
tests for HAT based on antigen detection.
The fast growing field of nanotechnology is
giving rise to a new generation of engineered
antibody fragments that have advantages
over traditional approaches (such as classic
immunoglobulins), including small size, high affinity and specificity,
and ease in tailoring into suitable probes.
Accessing new epitopes using novel probes such as single chain
variable fragment (scFv) Ab, VHH Ab and aptamer
FIND is working with its academic partners
to develop novel tools for antigen detection. For
example, scientists from the Seattle Biomedical
Research Institute (SBRI), USA, are generating
specific 32kDa anti-trypanosome single chain
variable fragment (scFv) antibodies that target
epitopes on the invariant surface glycoproteins
(ISG’s) and oligopeptidase B. The antibody
probes contain both the light and the heavy
chain variable domains (VL and VH) connected
via a flexible linker. Another group at the Free
University of Brussels, Belgium, has generated a
trypanosome-specific 15kDa nanobody that
contains only a single domain (VHH) of
a heavy chain, recognizing a carbohydrate
epitope that is present on both T.b. gambiense
and T.b. rhodesiense. Another approach by
scientists from Darmstadt University in
Germany, has been based on the interaction
between nucleic acid-derived RNA
aptamers and trypanosome antigens. The aptamers bind antigens from T.b. gambiense
and T.b. rhodesiense with high affinity
and specificity. These efforts have increased
the prospects of developing a simple and rapid
antigen detection test.
The Card Agglutination Trypanosomiasis
Test (CATT) is still the reference serological
screening assay for T.b. gambiense. There is no
equivalent test for screening T.b. rhodesiense,
which relies on clinical symptoms and signs;
indicators that are far from satisfactory. In
intensified efforts to develop an antibody
detection test for HAT, FIND is for the first
time coordinating the collection and screening
of a broad spectrum of recombinant and
native trypanosome antigens from universities
and institutes from around the world. At
present, these antigens are being screened
for specificity and sensitivity, using a panel
of well-defined sera from HAT patients
and non-HAT controls. The screening,
performed by an independent company
(Microcoat, Germany), has yielded very
promising results. The best antigen candidates
will be used to develop a diagnostic test,
preferably targeting both forms of the disease.
Moving molecular diagnostics from
the lab to the treatment center
An illustration of the essential primers in a LAMP reaction, and their target sites on double-stranded DNA. They include the forward 3
primer (F3), forward inner primer (FIP), backward 3 primer (B3), backward inner primer (BIP), loop primer F, and loop primer B.
Loop-mediated isothermal amplification
(LAMP) of DNA is a simple and rapid nucleic acid
amplification method with high sensitivity and specificity,
and is performed under isothermal conditions
using specific primers. This novel method
relies on autocycling due to a strand-displacement
activity of Bst DNA polymerase, in
which dumbbell-like DNA structures are
formed and quickly converted into stem-loop
DNA by self-primed DNA synthesis. Unlike
polymerase chain reaction (PCR), the LAMP
assay does not require purified DNA and thermal
cyclers to obtain an efficient amplification
product. It can be performed using heat-treated
blood as starting material. The assay requires
only basic instruments such as a heating block
that can maintain a constant temperature during
DNA amplification. Moreover, the large
amount of amplified DNA produced during the
LAMP reaction can be detected by visual
inspection based on turbidity or fluorescence.
For these reasons, LAMP represents a promising
new molecular technique with both
high sensitivity and specificity. LAMP can also
be used for the simultaneous analysis of large
numbers of samples, and can be performed by staff with only minimal experience in molecular
biology. The test could also be useful for
confirming cure after treatment.
FIND has been working with both Murdoch
(Australia) and Obihiro (Japan) Universities to
confirm the feasibility of using the LAMP technology
in the diagnosis of HAT. Highly sensitive
and specific LAMP tests have been reproduced
successfully in laboratories in endemic
countries, including Tanzania, Uganda and
Kenya. This work has given sufficiently promising
results to support adaptation of the LAMP
technique for diagnosis of sleeping sickness, and
has greatly increased the prospects for the development
of a commercial test.
Tools for staging HAT and
follow-up after treatment
At present, a painful and invasive lumbar puncture
has to be performed to determine whether trypanosomes
have penetrated into the central nervous system. Photo courtesy of WHO, 2003
Staging and follow-up of HAT patients are
important to guide treatment and to confirm cure after treatment. Patients require a lumbar
puncture and examination of the CSF for
presence of parasites and number of white blood
cells. This procedure is invasive and painful, and
the examinations performed currently have significant
shortcomings. FIND is investing in the
development of more accurate methods for staging
of HAT and for follow-up after treatment.
Recently, FIND and other partners, including the ITM and Universities of Geneva, Makerere
and Aberdeen, initiated discovery
research projects that will lead to the
identification of novel brain biomarkers that
are characteristic for HAT. Two-dimensional
gel eletrophoresis, TMT isobaric labeling and
different types of mass spectrometry will be used
to identify previously unknown markers.
Potential biomarkers, some of which are already
under evaluation, include components generated
through the cellular host immune response to
brain involvement, such as different cytokines
and chemokines, including IL-1ra, IL9, G-CSF,
IP-10, MIP-1beta and VEGF. Proteins indicating
brain destruction or immune activation, including
GSTP1, H-FABP, glial fibrillary acidic protein,
neuron-specific enolase and neurofilament,
are also being studied. Detection of some of
these proteins in CSF and plasma may lead to
development of less invasive staging tests.
Another staging project supported by FIND
involves the transformation of LATEX/IgM,
an existing test for IgM quantification in CSF,
into a single format test with improved stability
through collaboration with the Tropical
Institutes of Antwerp and Amsterdam.
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