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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 97, Num. 7, 2002, pp. 1015-1018
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Mem Inst Oswaldo Cruz, Rio de
Janeiro, Vol. 97(7), October
2002, pp. 1015-1018
Evaluation of the
Direct Agglutination Test and the rK39 Dipstick Test for the Sero-diagnosis
of Visceral Leishmaniasis
Henk DFH Schallig/+,
Marilene Canto-Cavalheiro*, Eduardo S da Silva**
KIT (Koninklijk Instituut voor de
Tropen/Royal Tropical Institute), Biomedical Research, Meibergdreef 39, 1105
AZ Amsterdam, The Netherlands *Laboratório de Bioquímica de Tripanosomatídeos,
Departamento de Imunologia, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro,
RJ, Brasil **Laboratório de Leishmanioses, Centro de Pesquisas René
Rachou-Fiocruz, Belo Horizonte, MG, Brasil and Centro de Pós-graduação
e Pesquisas/Fundação Educacional de Divinópolis, Universidade
do Estado de Minas Gerais, Divinópolis, MG, Brasil
+Corresponding author. Fax:
+31-20-697.1841. E-mail: H.Schallig@kit.nl
Received 14 December 2001
Acceped 19 June 2002
Code Number: oc02227
The direct agglutination test
(DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated
for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity
of both tests were determined using sera from confirmed VL patients (n = 21),
healthy controls (n = 19) and from patients with other confirmed infectious
diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The
rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also
used to screen blood samples of confirmed VL patients (n = 15) and serum samples
of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients
positive and tested 98.4% of the serum samples of the VL suspects positive.
In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania
chagasi and found 85.3% of the serum samples of the suspected patients positive.
Although the rK39 dipstick is more rapid and user friendlier than the DAT, the
latter has a superior sensitivity and specificity. Furthermore, the reagents
used for DAT do not require cold storage, whereas the buffer of the rK39 must
be stored at 4oC. Therefore, the DAT is the most suitable test for
the sero-diagnosis of VL under field conditions.
Key words: visceral leishmaniasis
- direct agglutination test - rK39 dipstick test - Leishmania chagasi -
diagnosis - human - Brazil
Visceral leishmaniasis (VL or kala-azar)
is the most severe form of leishmaniasis. Approximately 500,000 new cases of
human VL occur annually and the disease is mainly found in Brazil, East Africa
and on the Indian sub-continent. American visceral leishmaniasis (AVL), caused
by Leishmania chagasi, is a major health problem in many parts of Brazil.
Traditionally, the disease was confined to rural and peri-urban areas, but in
the last few years the number of human cases of AVL in metropolitan regions
is dramatically increasing (Silva et al. 2001). It is important to diagnose
AVL as early as possible, because the disease is in most cases fatal if left
untreated. The principal clinical symptoms of AVL are an enlarged spleen and
a prolonged irregular fever (WHO 1996). Other signs and symptoms are loss of
weight, pallor, enlarged liver, enlarged lymph nodes, anaemia, cough and diarrhoea.
These signs and symptoms may mimic those of malaria, typhoid, tuberculosis,
schistosomiasis and other diseases. The clinical suspicion may be confirmed
directly by the detection of parasites in patient material or by culture. However,
sample retrieval is inconvenient for the patient and parasite isolation by culture
is time-consuming, expensive and difficult.
Because of the above-mentioned limitations
of direct diagnostic methods, a number of indirect immunological tests have
been developed. These tests should meet several prerequisites in order to be
of practical use. They should be sensitive and specific, cheap (in particular
for use in developing countries) and easy to perform under harsh field conditions
(Veeken 1999). The direct agglutination test (DAT) remains the first line diagnostic
tool in many developing countries as it is a simple test with a high sensitivity,
specificity and reproducibility, easy-to-perform and not requiring specialised
equipment (Zijlstra et al. 1991, Boelaert et al. 1999a,b, Schallig et al. 2001).
The introduction of a freeze-dried antigen makes the DAT very suitable for use
under remote field conditions as a cold chain for storage of antigen is not
required (Meredith et al. 1995, Zijlstra et al. 1997, Schallig et al. 2001).
A limitation of the DAT is the relatively long incubation time (18 h) and the
fact that serial dilutions of blood or serum must be made. A dipstick, rK39
dipstick, based on the cloned antigen of a 39 amino acid repeat that is part
of a 230 kDa protein encoded by a kinesin-like gene of L. chagasi (Burns
et al. 1993), may circumvent these limitations (Sundar et al. 1998).
In the present study, the performance
of the DAT based on freeze-dried antigen and commercially available rK39 dipstick
in detecting anti-Leishmania antibodies in serum and blood samples from
Brazilian individuals is evaluated. Serum samples from patients suffering from
a variety of other diseases and healthy controls were also included in the study
in order to determine sensitivity and specificity of both tests.
MATERIALS AND METHODS
Serum and blood samples -
Serum samples stored at the serum collection at KIT Biomedical Research (Amsterdam,
The Netherlands) were used to determine the sensitivity and specificity of DAT
and rK39 under our laboratory conditions. The following panel was used: (1)
sera from Ethiopian patients (n = 10; a gift from Institute of Pathobiology,
University of Addis Ababa, Ethiopia) and Brazilian (n = 11; a gift from the
Leishmaniasis Laboratory of the Centro de Pesquisas Rene Rachou-Fiocruz, Belo
Horizonte, Minas Gerais, Brazil) with active microscopically proven VL (n =
21; positive control sera); (2) healthy blood donors from Manaus (Amazonas,
Brazil) who had no history of VL and no clinical symptoms of leishmaniasis (n
= 19; negative control sera); (3) sera from patients with other confirmed diseases:
cutaneous leishmaniasis (n = 5 from Belo Horizonte, Minas Gerais); Chagas disease
(n = 12 from Recife, Pernambuco, Brazil); African trypanosomiasis (n = 5 from
Uganda); leprosy (n = 5 from Indonesia); malaria (n = 5 from Kenya); tuberculosis
(n = 5 from Kenya); toxoplasmosis (n = 5 from the Netherlands).
Furthermore, the study used clinical
samples (serum and blood) of Brazilian individuals. The samples examined are
part of the collection at the Leishmaniasis Laboratory of the Centro de Pesquisas
Rene Rachou-Fiocruz. All samples were sent to the laboratory under the existing
health care system of Metropolitan Region of Belo Horizonte, Minas Gerais, Brazil
and stored at -20oC. The following two groups of samples were included
in the present study: (1) whole blood samples of patients with active VL, microscopically
proved (n = 15); (2) serum samples of patients clinically suspected of VL, all
patients had an enlarged spleen, a prolonged irregular fever and anemia and
were living in endemic area (n = 61).
DAT - The DAT was performed
essentially as described previously (Meredith et al. 1995, Oskam et al. 1999).
In brief, the serum or blood samples were diluted in physiological saline (0.9%
NaCl) containing 0.8% b-mercaptoethanol. Two-fold
dilution series of the sera were made, starting at a dilution of 1:100 and going
up to a maximum serum dilution of 1:102.400. Freeze-dried DAT antigen (L.
donovani promastigotes) produced by KIT Biomedical Research was reconstituted
with physiological saline according to the manufacturer's instructions. Fifty
µl DAT antigen solution (concentration of 5 x 107 parasites
per ml) was added to each well containing 50 µl diluted serum and the results
were read after 18 h of incubation at room temperature. Appropriate control
samples with known DAT titres were included as controls. A sample is considered
positive if it has a titre ³ 1:1600, the cut-off
value of the DAT (ES da Silva, unpublished results).
RK39 dipstick test - Dipsticks
were purchased from InBios International (Seattle, USA) and the test was performed
according to the manufacturer's instruction. The reading of the test was done
after exactly 10 min. A dipstick was considered positive as both the internal
control and the test band were stained (irrespective of the intensity of the
staining). A dipstick was considered negative as only the internal control was
visible. The result of a dipstick was considered not valid if the internal control
was not stained.
Reproducibility - Two observers
independently read the results of DAT and dipstick assays and their results
were compared afterwards. In the case that the interpretation of the dipstick
results was different between the two observers or that the reading of the DAT
differed more than one serum dilution step the sample was re-tested in the appropriate
test. In the case that the result of the dipstick and the DAT differed from
each other, the sample was re-analysed in both tests to confirm the test results.
Determining sensitivity and specificity
of the tests - The sensitivity (i.e. the probability that the assay will
be positive when the infection is present) and the specificity (i.e. the probability
that the assay will be negative when the infection is absent) were calculated
using the formulas: Sensitivity = TP/(TP+FN) x 100% and Specificity = TN/(TN+FP)
x 100%. Where TN represents true negative, TP true positive, FN false negative
and FP false positive. The sensitivity of the two tests was assessed with sera
from confirmed VL patients (n = 21). Sera of healthy controls (n = 19) and sera
of patients with confirmed other diseases (n = 42) were used to determine the
specificity of DAT and rK39 dipstick.
RESULTS
Reproducibility - Neither
the DAT nor rK39 dipstick test had to be repeated due to inconsistencies between
the readings of the observers. A 100% agreement in the reading of the dipstick
test and the DAT (one step difference in titre was allowed) was found between
the two observers. The analysis performed with rK39 dipstick test and DAT were
all valid as the internal control of the dipstick was always positive and the
titres of appropriate control samples determined with DAT correspond to the
pre-determined titre of the control (one step difference in DAT titre was allowed).
Fourteen samples of which a different
result was obtained with DAT and rK39 dipstick were re-tested with both tests
and in all cases re-testing resulted in the same test result as initially obtained.
Sensitivity and specificity of
DAT and rK39 dipstick test - The results of DAT and rK39 testing of positive
and negative controls and of the serum samples of patients with other confirmed
infectious diseases are summarised in Table
I. Calculation of the sensitivity of the assays revealed that the DAT had
a sensitivity of 100% (no false negative results) and the rK39 had a sensitivity
of 85.7% (three false negative results). None of the negative control samples
and samples from patients with other confirmed infectious diseases tested positive
with DAT (all titres < 1:1600; 100% specificity). In contrast, a positive
(albeit sometimes faint) reaction was observed with the rk39 dipstick with some
of the serum samples of patients with other confirmed infectious diseases (8/42)
or healthy endemic controls (3/19). The calculated specificity of the rK39 dipstick
on the basis of the results obtained in the present study is 82%.
The DAT found 14/15 blood samples
of confirmed VL patients positive (93.3%). In contrast, rK39 detected in only
9/15 blood samples (60%) antibodies against L. chagasi.
Evaluation of serum samples of
Brazilian VL suspects - The results of DAT and rK39 testing of serum samples
of clinical suspects (n = 61) are summarised in Table
II. The DAT tested 98.4% of the serum samples of the VL suspects positive,
whereas rK39 found 85.3% of the serum samples of the suspected patients positive.
Fifty-two samples were positive for both DAT and rK39. Furthermore, DAT was
positive for another eight suspects, but these were negative with rK39. Only
one sample tested negative with both tests.
DISCUSSION
In the present study, the rK39 test
had a lower sensitivity than the DAT based on the freeze-dried antigen. It failed
to detect antibodies in three serum samples (one from Ethiopian origin and two
from Brazilian origin) of parasitologically confirmed VL patients. This is in
line with observations of Zijlstra et al. (2001) who also found a lower sensitivity
(67%) of the rK39 compared to DAT when testing serum samples from African VL
patients in Sudan. In addition, Veeken (2001) has observed a low specificity
(59-70%) of the rK39 test. In contrast, Sundar et al. (1998) reported a 100%
sensitivity for the test using peripheral blood samples from Indian patients.
This observation was confirmed by Bern et al. (2000) in a study in Nepal. These
observations suggest a regional limitation of the use of the rK39 test.
Although not the intended use of
the test, according to the manufacturer's instructions, the sensitivity of the
rK39 test was also evaluated in the present study with blood samples of confirmed
VL patients from the collection at the Leishmaniasis Laboratory of the Centro
de Pesquisas Rene Rachou-Fiocruz (n = 15). The sensitivity of rK39 was in this
case low, 60%, whereas DAT found 14 out of the 15 tested samples positive (93.3%
positive).
The specificity of both tests was
assessed by using sera from healthy controls and from patients with other confirmed
infectious diseases. Several samples of healthy endemic controls tested positive
with the dipstick, but not with DAT. This is in line with observations of Zijlstra
et al. (2001) who also found healthy Sudanese controls without any history of
VL positive with the rK39 dipstick (sensitivity of 96.8%). More importantly,
the rK39 dipstick showed in several cases cross reactions with serum samples
of patients with other infectious diseases, among which is Chagas disease (33%
false positives) an infection which in the acute phase should always be included
in the differential diagnosis of any unknown fever in the Brazilian situation.
The sensitivity and specificity of
the DAT in the present study were determined on a limited number of healthy
subjects, patients with visceral leishmaniasis and patients with various other
diseases. Therefore 100% sensitivity and specificity are not claimed and cross-reactions
can not be fully excluded. It is important to determine the cut-off value of
the test using a large as possible number of appropriate local endemic controls
(Oskam et al. 1999). Because of the promising results obtained in the present
study a large field evaluation of the DAT in different regions of Brazil will
be undertaken.
Although the rK39 dipstick has an
ideal format for use in the field, as it is a rapid and simple test not requiring
extensive training of the operator, its lower sensitivity and specificity limits
its use for the sero-diagnosis of VL in Brazil. Furthermore, the test requires
cold storage of the running buffer, the test strips can not be stored at high
ambient temperatures and the test can not be performed with blood samples. In
contrast, the DAT based on freeze-dried antigen does not require cold storage
of reagents and can be performed with blood samples. A limitation of the DAT
is the relatively long incubation time (18 h) and the fact that serial dilutions
of blood or serum must be made.
Finally, we would like to emphasise
that serological tools are an aid in the diagnosis of VL. The outcome of either
rK39 dipstick test or DAT should always be judged judiciously in connection
with clinical, epidemiological and other diagnostic data.
ACKNOWLEDGEMENTS
To professor Asrat Hailu (Institute
of Pathobiology, University of Addis Ababa, Ethiopia) for providing us with
positive control samples, Dr Birgit van Benthem (KIT Biomedical Research) for
critical reading of the manuscript and Nel Kroon (KIT Biomedical Research) and
Gerard Schoone (KIT biomedical Research) for expert technical assistance.
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Copyright 2002 Instituto Oswaldo
Cruz - Fiocruz
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