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Clinical and Diagnostic Laboratory Immunology, March 1998, p. 254-255, Vol. 5, No. 2
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Use of trans-Sialidase Inhibition Assay in a
Population Serologically Negative for Trypanosoma cruzi but
at a High Risk of Infection
María S.
Leguizamón,2
Graciela
Russomando,1
Antonieta
Rojas
de Arias,1
Margarita
Samudio,1
Margarita
Cabral,1
Stella Maris
González-Cappa,2
Alberto
Carlos C.
Frasch,3 and
Oscar
Campetella3,*
Instituto de Investigaciones en Ciencias de
la Salud, Universidad Nacional de Asunción, Asunción,
Paraguay,1 and
Departamento de
Microbiología, Facultad de Medicina, Universidad de Buenos
Aires,2 and
Instituto de Investigaciones
Biotecnológicas, Universidad Nacional de Gral. San
Martín,3 Buenos Aires, Argentina
Received 8 September 1997/Returned for modification 6 October
1997/Accepted 23 December 1997
 |
ABSTRACT |
trans-Sialidase inhibition assay (TIA) was employed in
a population at high risk of Trypanosoma cruzi infection.
From 20 serum samples that were negative by conventional serologic and
parasitologic assays, 18 (90%) were reactive in TIA, providing further
evidence of the higher sensitivity of TIA and suggesting that the
actual prevalence of T. cruzi infection might be
underestimated.
| |
TEXT |
The American trypanosomiasis,
Chagas' disease, is a chronic illness that affects about 16 million
people in the Americas. After the acute phase of the disease, parasites
are rarely found in the blood and diagnosis is mainly based on
serology. Conventional serology employs crude preparations of
parasites, although some relevant antigens have been cloned and tested
as diagnostic tools (9). Serology seems to be equally
sensitive to or even more sensitive than PCR for detecting infection in
patients (2, 13). A new serological test,
trans-sialidase inhibition assay (TIA), is under development
(6-8). The assay is based on the detection of antibodies
able to inhibit the activity of the trans-sialidase, a
virulence factor from Trypanosoma cruzi (12). The
enzyme is absent in other protozoan parasites, such as
Trypanosoma rangeli, Leishmania spp., and
Plasmodium spp., that are frequently found in geographical
regions where T. cruzi is present. These neutralizing antibodies are detected during both the acute and chronic phases of the human infection (7, 8, 10).
Epidemiological surveys of the population at risk of infection are
performed through conventional serology. Very often, when people living
in the same house are tested, only some of them are recorded as
infected even though all were exposed to similar risk factors. Since
TIA seems to be a more sensitive technique (7, 8), it was
employed in a population at very high risk of infection but which was
serologically negative when tested by conventional T. cruzi
assays.
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FIG. 1.
TIA of sera from Paraguayan aborigines. Assayed serum
samples were from persons negative for T. cruzi infection by
conventional serological and parasitological tests. The value of 0%
was assigned to the inhibition obtained with pooled serum samples from
areas of nonendemicity. The positions of the dot spots of recombinant
antigens 1, 2, 30, and SAPA and of the glutathione
S-transferase (GST) from Schistosoma japonicum,
expressed by the pGEX vector without insert (Pharmacia, Uppsala,
Sweden) and used as a negative control, are indicated in the top box.
Open circles denote the samples giving positive dot spot reactions. The
results from two positive and two negative sera in the dot spot assay
are also shown.
|
|
TIA employs a recombinant trans-sialidase (3, 4)
that is preincubated with the serum to be tested, and then the
remnant ability to transfer the sialyl residue from sialyllactose to
[14C]lactose is evaluated. At present we have tested
about 60 normal human sera obtained from the regions of endemicity;
they show variable degrees of trans-sialidase inhibition,
ranging from 
10 to 30%, as reported previously (7, 8). To
perform TIA with experimental samples, normal human pooled serum was
employed as a negative control and the value of inhibition obtained was
taken as 0% (6-8).
Blood samples were obtained from the Sanapa and Angaite Amerindian
communities in western Paraguay (150 samples). The seroprevalence of
T. cruzi infection among this group was 82%. The people of this group live in houses built with palm leaves, where large numbers
of infected vector bugs (70 to 300 triatomines per house) are present.
However, some people in the community are consistently negative by the
normally employed enzyme-linked immunosorbent and immunofluorescence
tests, and also by parasitological tests (xenodiagnosis and
hemoculture). Sera from 20 such serologically negative persons (20 to
40 years old) that had been living in the same place for the last 14 to
30 years were further analyzed by TIA and by a dot spot assay employing
recombinant T. cruzi antigens that are able to detect
chronic (antigens 1, 2, and 30 [5, 9]) or acute (SAPA
antigen [1, 8, 9, 11]) cases of human Chagas'
disease.
The dot spot assay confirmed the negative results obtained by
conventional serology in 16 of the 20 samples. These 16 individuals would be considered not infected with T. cruzi by present
criteria. Sera from the 20 individuals were then analyzed by TIA. As
shown in Fig. 1, 18 of the 20 sera were TIA positive, 15 of them having values ranging from 68 to 98%. The serum components were fractionated by protein A-agarose (Life Technologies, Gaithersburg, Md.), and the
inhibitory activity was found in the immunoglobulin fraction (not
shown).
The TIA results found were similar to those observed with sera obtained
from chagasic patients (7, 8). We have shown that these
antibodies remain for long periods of time (14 years) in acute-stage
patients successfully treated with benznidazole even after T. cruzi infection is no longer detected by conventional serologic
and parasitological tests (8). TIA results for the Amerindians are compatible with those findings and suggest that the
subjects were in contact with the parasite, which might support the
possibility of spontaneous cure or the establishment of a cryptic
infection. In fact, the subjects tested meet the present criteria for
absence of T. cruzi infection by either conventional serology or parasitological tests. However, they should be rejected as
blood or organ donors and should be monitored for the appearance of
chronic Chagas' disease pathologies. The present study also suggests
that the actual seroprevalence of T. cruzi infection might
be underestimated.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from the World Bank/UNDP/WHO
Special Program for Research and Training in Tropical Diseases (TDR),
the Swedish Agency for Research Cooperation with Developing Countries (SAREC), the Universidad de Buenos Aires, the Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), and the Argentina and the Japan International
Cooperation Agency (project on Chagas' and other infectious
diseases). The research of A. C. C. Frasch was supported in
part by an International Research Scholars Grant from the Howard Hughes
Medical Institute and the International Atomic Energy Agency, Vienna,
Austria.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Investigaciones Biotecnológicas, Universidad Nacional de Gral.
San Martín, Casilla de Correo 30, 1650 San
Martín, Buenos Aires, Argentina. Phone: 54 1 752-0021. Fax: 54 1 752-9639. E-mail: oscar{at}inti.gov.ar.
| |
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Clinical and Diagnostic Laboratory Immunology, March 1998, p. 254-255, Vol. 5, No. 2
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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