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Serological diagnosis

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 11

Serological diagnosis
Veerle Lejon, Philippe Büscher

S
erological diagnosis of human African trypanosomiasis (HAT) can
be achieved by demonstrating the inflammatory responses, by specific
antibody detection or demonstration of parasite antigens in the
putative host. Serological diagnosis should be considered as indirect evidence
of trypanosomiasis, and should be followed by parasitological techniques to
confirm infection with Trypanosoma brucei.

Blood alterations related to inflammation

Numerous non-specific alterations of the blood have been observed in

sleeping sickness patients [1-3].
An increased sedimentation rate and decreased haematocrit reflect the
systemic chronic inflammation. Auto-agglutination of red blood cells and
hemolytic anemia are common features of rhodesiense disease.Thrombocytopenia
if occurring, is mild in gambiense infections, but may be severe in rhodesiense
infections. There may be monocytosis with Mott cells.
Liver transaminases are abnormal in rhodesiense disease, while in gambiense
disease gamma glutamyl transferase and amylase activities have been shown
to be slightly decreased [4]. Low serum C3 levels and split C3 products can
be found, that, in combination with raised immune complex levels point to
complement activation [4-6].
Protein measurements in serum show decreased albumin and increased
immunoglobulin concentrations. Total serum IgM is tremendously increased
up to 5 to 16 times the normal concentration. Serum IgG is moderately
increased (1.5x) while IgA is within the normal range or slightly elevated
[4, 6-9]. Although an increase in serum IgM may be due to other diseases
including leprosy, leishmaniasis, malaria or syphilis, it is rarely as marked as in
sleeping sickness. A raised serum IgM level is therefore a serious indication
that further efforts should be made to confirm diagnosis [10].
During the poly-specific immune response, a variety of non-trypanosome
specific antibodies and auto-antibodies are produced e.g. against fibrin,

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 Veerle Lejon, Philippe Büscher

fibrinogen, DNA, red blood cells, thymocyte antigens and central nervous
system components such as myelin, galactocerebrosides and neurofilament
[11-15]. Antibodies specific for other pathogens, such as T. gondii, S. stercoralis,
Epstein Barr Virus, cytomegalovirus, P. fieldi, P. brasiliana and B. burgdorferi
have been observed in patients with T.b. gambiense sleeping sickness [16-18]
and may constitute a considerable risk for misdiagnosis.

Detection of trypanosome specific antibodies

Trypanosomes have a complex antigenic structure and elicit production of

high concentrations of specific IgG and IgM antibodies which can be detected
by antibody detection tests.
The type of antigen(s) employed, greatly determines the sensitivity and
specificity of an antibody detection test. For serodiagnosis of T.b. gambiense,
tests are better than for rhodesiense disease. T.b. gambiense antibody detection
tests use selected variable surface glycoproteins as antigens (see chapter:
introduction to antigenic variation). For T.b. rhodesiense, diagnostic variable
surface glycoproteins have not yet been identified for reasons of much higher
antigenic variability of this subspecies and non-variable antigens are preferred.
Several test formats exist (Table 1). For diagnosis of T.b. gambiense, rapid
agglutination tests are available that are applicable in the field. Unfortunately,
no equivalent is available for screening of T.b. rhodesiense HAT. Presumptive
diagnosis of rhodesiense HAT in the field therefore still relies on clinical signs
and symptoms. It should be noted however that in countries with the chronic
form of T.b. rhodesiense (Zambia, Malawi), tests for T.b. gambiense diagnosis
seem useful (personal observations). Immunofluorescence, ELISA, immune
trypanolysis and plate agglutination tests are more appropriate for remote
laboratory testing.
Current serological tests detect antibodies only after 3-4 weeks of infection
which may be one of the reasons for occurrence of false negative reactions.
Seropositivity in antibody detection tests must be interpreted with caution
since antibodies can persist for up to five years after cure [19, 20]. Also some
cross-reactivity with other parasitoses can occur, particularly at low serum or
blood dilutions and when IgM is participating in the reaction.
Hereafter, an exhaustive list of antibody detection techniques is described.
Several reviews deal with the techniques in detail [21-24].

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Body fluid
Test HAT type plain filter
serum plasma saliva CSF
blood paper
CATT gambiense x x x x
LATEX gambiense x x x x x
MOPATT rhodesiense x x x
Immuno-
gambiense + rhodesiense x x x x
fluorescence
ELISA gambiense + rhodesiense x x x x x
trypanolysis gambiense x x x

Table I - Overview of antibody detection test types and sample types suited for diagnosis of HAT.

CATT/T.b. gambiense (CATT)

The Card Agglutination Test for Trypanosomiasis (CATT) is a fast and

simple point-of-care card agglutination assay developed for detection of specific
antibodies in T.b. gambiense sleeping sickness patients [25]. The introduction
of the CATT for mass population screening has been a major breakthrough
in the diagnosis of T.b. gambiense HAT, limiting the number of parasitological
examinations on persons found with a positive serology. The CATT antigen
consists of complete bloodstream forms of T.b. gambiense variable antigen type
LiTat 1.3. The antigen production is a fastidious process. Trypanosomes are
purified from infected rat blood, fixed, stained with Coomassie blue giving
the reagent its characteristic color, and freeze-dried. The Institute of Tropical
Medicine, Antwerp, is currently the only producer. Field kits containing
the reagent, control sera and small equipment to perform the test on whole
blood (microlancets, capillary tubes, test cards, stirring rods, suction bulbs,
syringe, droppers) are available at a price of 0.5 €/test (price October 2012,
production@itg.be, TT&P, Institute of Tropical Medicine, Nationalestraat
155, B-2000 Antwerpen, Belgium, +32 3 247 6368).
For screening purposes the test is performed on undiluted whole blood.
Up to 10 persons can be tested at the same time and hundreds of individuals
can be screened daily. A heparinized capillary tube is filled with fingerprick
blood for about ¾ of its length. For the agglutination reaction, one drop (about
45 µl) of the well homogenised CATT antigen is put on a test area of the card.
1 drop of undiluted blood is added in each test area. The reaction mixture is
spread out over the test area using a stirring rod. The test card is rocked at 60
rpm. After 5 minutes of agitation, the results are read. Presence of weak to
very strong agglutination is considered positive, absence of agglutination is
considered negative (Fig. 1).

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 Veerle Lejon, Philippe Büscher

1a - A negative (zone 1) and positive (zone 2) agglutination reaction occurring in CATT on whole blood.

1b - Negative (zones 3, 5, 6, 7, 8, 9), doubtful (zone 2) and positive reaction (zone 1, 4, 10)
in CATT on serum dilution 1:4.

Figure 1 - Agglutination reaction in CATT/T.b. gambiense.

In case of CATT whole blood positivity, plasma or serum dilutions are often
tested prior to parasitological examinations. Twofold dilutions of 1/4, 1/8, 1/16,
1/32 and 1/64 are prepared in CATT buffer. The CATT test is performed
in the same way, using 25 µl of dilution as sample. Only those subjects that
are CATT positive on serum dilution 1/4 or 1/8, depending on the national
control programme, will undergo parasitological examination, decreasing the
number of parasitological examinations and allowing a significant gain in time
and financial resources.
For screening individuals that cannot be reached by full mobile teams
during active case finding, the CATT can be performed on blood impregnated
filter papers [26, 27]. Constraints for widespread use of this “micro-CATT”

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are its difficult reading and interpretation of the agglutination, due to the small
amounts of antigen and test sample used, and the rapid decrease in sensitivity
when filter papers are stored at ambient temperature [28]. An alternative
method, the macro-CATT, developed for testing blood impregnated filter
paper using the standard amount of antigen and a higher volume of eluate,
seems to perform better [29].
Performance of CATT may differ according to the geographical origin of
the patients (Table II).

Test version % sens % spec Authors Location

CATT-WB ND 96 [30] South-Africa
CATT-WB 99-100 78-88 [31] DR Congo
CATT-WB ND 90 [32] Liberia
CATT-WB 83 97 [33] Congo
CATT-WB 92 93 [34] Côte d’Ivoire
CATT-WB 98 94 [28] Central African Republic
CATT-WB 100 97 [28] Côte d’Ivoire
CATT-WB 100 90 [35] Côte d’Ivoire
Uganda, Equatorial Guinea, DR
CATT-WB 90 97 [35]
Congo
CATT-WB 100 94 [36] Cameroon
CATT-WB 86-98 ND [37] DR Congo
CATT-WB 100 90-98 [38] Angola
Europa, DR Congo, Upper Volta, Côte
CATT-s 1:4 92 100 [39]
d’Ivoire
CATT-s 1:5 ND 95 [32] Liberia
CATT-s 1:5 97 94 [33] Congo
CATT-s 1:8 100 95-100 [38] Angola
CATT-s 1:10 92 99 [33] Congo
CATT-s 1:16 97-100 97-100 [38] Angola
CATT-s 1:32 60-62 99-100 [38] Angola
CATT-FP 96 97 [28] Côte d’Ivoire, Central African Republic
CATT-FP 91 ND [29] South Sudan

Table II - Performance expressed as percent sensitivity (sens) and specificity (spec)

of different CATT test versions according to different authors.
(CATT-WB: CATT on whole blood, CATT-s: CATT on serum dilution, CATT-FP: CATT on filter paper eluate)
ND: not done.

The sensitivity of CATT on whole blood (CATT-WB) is around 95%,

varying from 83% to 98%, and the negative predictive value is excellent
during mass population screening [28, 31, 33-39]. Up to serum dilution 1:16,
sensitivity remains high. False negative CATT results may occur in patients
infected with strains of trypanosomes that lack or do no express the LiTat
1.3 gene, explaining the lower sensitivity of CATT in some endemic areas
Sleeping sickness lectures 203
 Veerle Lejon, Philippe Büscher

[40-42]. False negative CATT results may also occur when testing undiluted
blood, or serum dilution <1/4. In such cases, the agglutination may be inhibited
by complement factors, a phenomenon called prozone. This problem can be
resolved by addition of EDTA in the dilution buffer [35, 43, 44].
Despite a specificity of around 95%, the positive predictive value of the
CATT-wb remains limited because the test is used for mass screening in
populations where the prevalence of HAT is low (<2%) [28, 30-36, 38, 39]. False
positive results can occur in patients with malaria and other parasitic diseases
such as transient infection by non-human trypanosomes. The specificity of the
CATT is further improved when performed on serum diluted 1/4 or higher
[32, 33, 38, 39].
The occurrence of CATT seropositives (serum dilution 1/4 or higher) that
cannot be confirmed parasitologically remains a matter of concern. One option
is increasing the sensitivity of parasite detection by examining serological
suspects at regular intervals [45]. Unfortunately, the adherence to follow-up
visits is usually low and the efficiency of this strategy is poor [46]. Another
option is, based on CATT end-titer, to determine a subgroup of serologically
suspected individuals at high risk of being infected and to treat them. This
approach was successfully tested in Angola and Sudan [45, 46]. Based on these
findings, treatment for all serological suspect individuals with a CATT end
titer ³ 1:16 has been proposed when the prevalence of HAT is sufficiently high
(>1%), when access to care is poor, and when availability of parasitological
diagnosis is limited.
Besides the occurrence of non-confirmed seropositives, CATT has some
minor practical inconveniences. The reagents should be kept at 4°C for long
term storage, requiring a cold chain. Vials contain 50 test doses, but once opened
and reconstituted, the reagents can be used only during 1 week when stored
between +2°C and +8°C, or up to 8 hours at 37°C. This limits the use of CATT
in health centers visited by very low number of HAT patients. Development of
an alternative test format containing less doses/vial is ongoing. However, due
to its simplicity, reliability, and low cost, CATT is used by almost all control
programs for screening for T.b. gambiense infection in the population at risk.

LATEX/T.b. gambiense

The latex agglutination test for T.b. gambiense, LATEX/T.b. gambiense is a

point-of-care card agglutination test that has been developed as an alternative
to the CATT [47]. The test is based on the combination of three purified
variable surface antigens, LiTat 1.3, 1.5 and 1.6, coupled onto white latex
particles. The test procedure is similar to the CATT, including the use of a

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similar rotator (Fig. 2). Again field kits containing the reagent, control sera
and small equipment to perform the test are available from the Institute of
Tropical Medicine at a price of 0.5 €/test (price October 2012, production@
itg.be, TT&P, Institute of Tropical Medicine, Nationalestraat 155, B-2000
Antwerpen, Belgium, +32 3 247 6368).
LATEX/T.b. gambiense can be performed on undiluted blood, diluted
blood, serum, filter paper eluates and cerebrospinal fluid. When compared to
the CATT, the LATEX/T. b. gambiense showed a higher specificity (96-100%)
but a lower or similar sensitivity (71-100%) in recent field studies (Table III)
[28, 34, 36, 44]. The combination of three purified variable surface antigens
however results in high sensitivity e.g. in regions (for example Nigeria) where
CATT/T.b. gambiense fails [47].
Inconveniences are similar to the CATT. The reagents should be kept at
4°C for long term storage and vials also contain 50 test doses. Once opened
and reconstituted, the reagents can be used only during a limited time period,
or should be frozen for prolonged storage. Since LATEX/T.b. gambiense is
slightly more complicate to perform in the field than CATT and has similar
performance, its use is not especially promoted, unless in areas with low CATT
sensitivity.
Research to replace the purified native antigens by recombinant antigens or
synthetic peptides and to simplify the test format is ongoing.

Figure 2 - Material used to perform LATEX/T.b. gambiense.

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 Veerle Lejon, Philippe Büscher

Test version % sens % spec Authors Location

LATEX-s 1:8 97 91 [47] DR Congo, Congo, Côte d’Ivoire, Uganda,
LATEX-s 1:16 93 99 [47] Equatorial Guinea, Sudan, Gabon, Nigeria
LATEX-b 1:4 100 99 [44] Equatorial Guinea
LATEX-b 1:4 100 98 [34] Côte d’Ivoire
LATEX-b 1:4 100 98 [36] Cameroon
LATEX-b 1:4 100 96 [36] Central African Republic
LATEX-b 1:8 100 100 [44] Equatorial Guinea
LATEX-b 1:8 92 98 [34] Côte d’Ivoire
LATEX-b 1:8 100 99 [36] Cameroon
LATEX-b 1:8 90 98 [36] Central African Republic
LATEX-b 1:16 100 100 [44] Equatorial Guinea
LATEX-b 1:16 67 100 [34] Côte d’Ivoire
LATEX-WB 68 99 [28] Central African Republic
LATEX-WB 90 99 [28] Côte d’Ivoire
Lejon,
LATEX-FP 89 98 Central African Republic, Côte d’Ivoire
unpublished

Table III - Performance expressed as percent sensitivity (sens) and specificy (spec) of different
LATEX/T.b. gambiense test versions (LATEX-WB: LATEX on whole blood, LATEX-b: LATEX on
blood dilution, LATEX-s: LATEX on serum dilution, LATEX-FP: LATEX on filter paper eluate)
according to different authors.

PATT and MOPATT

The Procyclic Agglutination Test for Trypanosomiasis has been introduced

for detection of T.b. rhodesiense antibodies in blood [48, 49]. The original test,
using live procyclic trypanosomes, has limitations for field application but
the modified test with fixed trypanosomes (MOPATT) has been used with
success in Kenya [50] and deserves further evaluation. The test was available
from KARI (former KETRI in Kenya). The sensitivity of the test for T.b.
gambiense sleeping sickness is insufficient.

Immunofluorescence assays

Immunofluorescence assays have been used with success for T.b. gambiense
sleeping sickness control in Equatorial Guinea [51] and in Gabon [52].
Immune fluorescence can be applied on serum, on filter paper eluates and on
cerebrospinal fluid [3, 20, 53, 54].
For performing the test, slides are coated with whole trypanosomes. A
drop of diluted test sample (serum dilution 1:100-200) is allowed to react
for 30 minutes, remnants are washed off with PBS. Following a 30 minutes
incubation with FITC conjugated anti-human IgG antibodies, slides are

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washed again and mounted with glycerol-PBS and a cover slip. Reading is
done under a fluorescence microscope (40x10).
The availability of standardized and stabilized antigen for T.b. gambiense at low
cost has greatly improved the reliability of the test [39]. This antigen is available
from the Institute of Tropical Medicine, Antwerp at a price of 20 €/250 doses
(price October 2012, production@itg.be, TT&P, Institute of Tropical Medicine,
Nationalestraat 155, B-2000 Antwerpen, Belgium, +32 3 247 6368).
For testing of serum, it is important to use strictly IgG specific fluorescent
conjugates thus avoiding cross-reactive IgM. Research is still needed to
identify better antigens for T.b. rhodesiense sleeping sickness.
Depending on the antigen used, test sensitivity on serum has been described
to be 75-95% for T.b. gambiense [44, 55, 56] and 71-92% for T.b. rhodesiense [3,
51, 57]. Specificity is >90% [55].
Immunofluorescence is cheap and reagents are relatively stable at 4°C,
but an immunofluorescence microscope and electricity are necessary. It is
therefore especially suited for surveillance and remote diagnosis. At present
immunofluorescence is mainly applied for serodiagnosis of T.b. rhodesiense.

ELISA

Numerous ELISA tests for sleeping sickness have been described [58-64].

Figure 3 - ELISA for detection of trypanosome specific antibodies (photo I. El-Rayah).

For indirect ELISA (Fig. 3), the trypanosomal antigen is applied in a

microplate, which is blocked with a protein solution (mainly BSA or milk

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 Veerle Lejon, Philippe Büscher

powder) before incubation with the sample dilution. The plate is washed, and
an anti-human IgG enzyme conjugate is added. After a second wash, the
substrate solution is applied. A color reaction is observed in case of presence of
trypanosome specific antibodies.
Serum, filter paper eluates, saliva and CSF can be tested with strict
standardization and quantification. Furthermore, it is possible to study
different immunoglobulin classes and isotypes [63]. There is a tendency to
use purified antigens instead of crude trypanosome lysates but investigations
should continue into the use of recombinant or synthetic peptides. Important
also is the incorporation of an antigen negative control well for each individual
sample, as trypanosomiasis sera tend to stick to the plates.
Although different test protocols have been used, the sensitivity of ELISA
is described to be 95-100% [58, 60-62, 64] in combination with a specificity
of 97-100% [58, 60-62, 64].
As for immunofluorescence, the need for sophisticated equipment and large
volumes of pure water, remain a serious drawback for widespread application
of the test. Due to its high sensitivity and specificity, ELISA is especially suited
to perform large scale surveys and for remote diagnosis.

Immune Trypanolysis

This test for antibody detection makes use of live bloodstream trypanosomes
and is restricted to laboratories which have facilities to maintain cloned
populations. The test is based on recognition of the variable epitopes on the
surface of the trypanosomes by the corresponding antibodies resulting in
complement mediated lysis. The test is highly specific and therefore mainly
used as the reference test for evaluation of other antibody detection systems
and for research purposes [22].

Detection of trypanosome antigens

The antigen detection tests developed so far [65-68] have not been
validated and cannot be recommended for diagnostic purposes. Reliable
antigen detection tests remain to be developed.

ELISA

ELISA tests for trypanosome antigen detection make use of polyclonal

and monoclonal antibodies [66-68]. The tests can be performed on serum and
CSF samples from T.b. gambiense as well as from T.b. rhodesiense patients. Apart

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from the need of sophisticated equipment, the contradictory results obtained

with similar test systems for animal trypanosomiases have diminished the
interest of implementation in sleeping sickness control programmes.

CIATT

The Card Indirect Agglutination Trypanosomiasis Test is the result of

the combination of latex agglutination technology with monoclonal ELISA
experience [69]. The CIATT is intended to be applied as a pen-side assay
generating immediate results. Preliminary results indicate specificities tested
in non-endemic areas ranging from only 61% in Côte d’Ivoire to 98% in
Tanzania [70]. Due to the occurrence of contradicting results, the use of
CIATT has largely been abandoned.

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[67] OLAHO-MUKANI W, NYANG’AO JMN, NGAIRA JM, OMUSE JK,

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sickness in non-endemic areas. 1999.

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