Rapid detection of Trypanosoma cruzi in human serum by use of an immunochromatographic dipstick test.
ABSTRACT We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador, Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%).
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ABSTRACT: The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)3 (2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)3 (2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimethoxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when "dipstick" format was used.Biomedical Microdevices 03/2013; · 2.72 Impact Factor
- PLoS Neglected Tropical Diseases 10/2011; 5(10):e1250. · 4.57 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2010, p. 3003–3007
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 8
Rapid Detection of Trypanosoma cruzi in Human Serum by Use of an
Immunochromatographic Dipstick Test?
Richard Reithinger,1,2* Mario J. Grijalva,3,4Rosa F. Chiriboga,4Belkisyole ´ Alarco ´n de Noya,5
Jaime R. Torres,6Norma Pavia-Ruz,7Pablo Manrique-Saide,8
Marta V. Cardinal,8and Ricardo E. Gu ¨rtler9
Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom1;
School of Medical Services and Health Science, George Washington University, Washington, DC2; Tropical Disease Institute,
Biomedical Sciences Department, College of Osteopathic Medicine, Ohio University, Athens, Ohio 457013; Centre for
Infectious Disease Research, Catholic University of Ecuador, Quito, Ecuador4; Immunology Section, Instituto de
Medicina Tropical, Universidad Central de Venezuela, Caracas, Venezuela5; Infectology Section, Instituto de
Medicina Tropical, Universidad Central de Venezuela, Caracas, Venezuela6; A´rea de Apoyo al Diagno ´stico Clínico,
Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad de Yucata ´n, Me ´rida, Mexico7;
Department of Zoology, Campus de Ciencias Biolo ´gicas y Agropecuarias, Universidad de
Yucata ´n, Me ´rida, Mexico8; and Department of Ecology, Genetics and
Evolution, Universidad de Buenos Aires, Buenos Aires, Argentina9
Received 21 December 2009/Returned for modification 26 January 2010/Accepted 27 May 2010
We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma
cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador,
Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested
positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range
between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%).
Chagas disease is caused by Trypanosoma cruzi and is
found in wildlife, domestic animals, and humans in rural as
well as peri-urban areas of Mexico, Central America, and
South America; in the United States, T. cruzi is found in
wildlife, but human cases are rare (29). Although transmis-
sion of T. cruzi can occur orally, congenitally, or transfu-
sionally, most transmission to mammalian hosts is through
the feces of blood-feeding triatomine bugs when T. cruzi
trypomastigotes in the feces contaminate the bite wound or
enter the host through mucosal surfaces (22). By causing the
loss of an estimated 670,000 disability-adjusted life years
(i.e., a measure that sums years of potential life lost due to
premature mortality and years of productive life lost due to
disability), Chagas disease is the most important parasitic
disease in the Americas; 8 to 10 million people are currently
infected with T. cruzi, with up to 100 million at risk of
contracting the disease (32).
There are several methods to diagnose T. cruzi infection
(11), but none are ideal when mass screening of samples is
required (e.g., epidemiological surveys, blood unit screen-
ing). While comparatively easy to use and sensitive, sero-
logical tests (i.e., enzyme-linked immunosorbent assay
[ELISA], immunofluorescence antibody test [IFAT], indi-
rect hemagglutination test [IHAT], or radioimmunosorbent
assay [RIA]) are of varied specificities (i.e., 60 to 100%) (12,
16, 26). Molecular tests, including PCR-based approaches,
are very specific but lack sensitivity (i.e., 30 to 95%) and
require technological expertise and specialized, expensive
laboratory equipment (11, 21, 23). Hemoculture and xeno-
diagnosis are the current gold standard for T. cruzi parasi-
tological diagnosis (6, 11, 21). Though these techniques are
specific, their sensitivity in the chronic phase of infection is
quite variable (e.g., 0 to 50% ); they also are labor-
intensive and time-consuming (e.g., because of the necessity
of mass-rearing bugs for xenodiagnosis and examination of
them). Thus, a rapid, sensitive, and specific diagnostic test to
detect T. cruzi infection would be extremely valuable for
mass-screening surveys and intervention campaigns as well
as during the onset of outbreaks; results could be read
immediately, and control measures could be implemented in
Immunochromatographic dipstick tests have been devel-
oped for a range of tropical diseases, including malaria (31),
leishmaniasis (7), and schistosomiasis (3); until recently (4,
5, 8, 14, 17, 20, 25, 28, 30), none was available for Chagas
Recently, the World Health Organization announced re-
newed efforts to eliminate Chagas disease (27). For such efforts
to succeed, an easy-to-use, sensitive, and specific diagnostic
test will be crucial for both detecting and treating cases early as
well as monitoring the implementation of elimination efforts
and evaluating their impact (18, 24).
We evaluated the sensitivity and specificity of a commer-
cially available immunochromatographic dipstick test to de-
tect antibodies to T. cruzi infection in human serum samples
with known serological results collected in areas of both
Chagas disease endemicity and nonendemicity in four dif-
ferent Latin American countries.
* Corresponding author. Present address: 2030 Addis Ababa Place,
Dulles, VA 20189. Phone: (202) 216-6203. E-mail: rreithinger@yahoo
?Published ahead of print on 9 June 2010.
Test samples. Study samples were collected between 2000
and 2007 from the general population, cases suspected of hav-
ing the disease, or blood donors during a range of epidemio-
logical studies in Argentina, Ecuador, Mexico, and Venezuela
(Table 1). Samples had been stored at ?20°C and had previ-
ously been tested by at least one of the following methods
according to standard in-country protocols: ELISA (n ? 366),
IHAT (n ? 166), and IFAT (n ? 101) (Table 1). Samples were
considered negative and positive as per in-country Chagas dis-
ease diagnostic guidelines (Table 1).
Dipstick. The dipstick test (Trypanosoma Detect MRA
rapid test; Inbios, Seattle, WA) was carried out according to
the manufacturer’s instructions, where 20 ?l of serum fol-
lowed by 3 to 4 drops of buffer (150 to 200 ?l) were added
to the dipstick’s sample pad. After 10 min, a red control line
and, if positive, a second line appeared on the test field of
the dipstick cassette. The test is based on a proprietary gold
mix containing multiepitope recombinant antigen derived
from different T. cruzi antigens. All dipsticks originated
from the same lot and were distributed to the various study
locations in January 2007; all dipsticks were used on study
samples between May and October 2007, within the speci-
fied date of expiration of the dipstick.
Figure 1A summarizes the dipstick and serology results of
samples evaluated. One hundred forty-nine of 366 (40.7%)
and 171/366 (46.7%) samples tested positive by the dipstick
test and serology, respectively. A total of 30 (8.2%) samples
had discordant results when tested by conventional serolog-
ical tests and with dipsticks (Fig. 1A). Samples that were
negative by standard serology but positive by the dipstick
method included 2 of the 47 (4.3%) negative-control sam-
ples and 2 of the 61 (3.3%) samples from individuals with
other infections (i.e., HIV, syphilis, hepatitis B virus [HBV],
hepatitis C virus [HCV], brucellosis, and tuberculosis). The
remaining 26 samples yielding discordant results were from
individuals living in areas of T. cruzi endemicity and who had
tested positive by conventional serology but negative with
the dipstick. For samples that were tested by ELISA and for
which exact optical density values were available, an asso-
ciation between ELISA optical density and dipstick positiv-
ity was observed (Fig. 1B).
Based on the total number of samples testing positive or
negative by serology, the sensitivity and specificity of the
rapid diagnostic test was calculated to be 84.8% (range
between countries, 77.5 to 95.0%) and 97.9% (95.9 to
100%); negative and positive predictive values were 88.0%
(85.0 to 92.6%) and 97.3% (94.3 to 100%), respectively (Fig.
1A). The kappa value, which is an index comparing the
observed agreement between tests against the agreement
which might have been expected by chance, was calculated
to be 0.83 (range between countries, 0.80 to 0.94).
To our knowledge, this is the first multicenter study to
evaluate the Inbios Trypanosoma Detect test to detect T.
cruzi infection in blood samples from people in the general
population living in areas of T. cruzi endemicity or nonen-
demicity, cases suspected of having the disease, or blood
donors. Our study indicates that the tested dipstick test has
high specificity (97.9%) and moderate to high sensitivity
(84.8%). Agreement between the results of standard sero-
logical tests and the tested dipstick was good. This comple-
ments the results from other studies testing the same dip-
stick (5, 14, 30) or dipsticks from other manufacturers
(i.e., Chagas Stat-Pak, Chembio Diagnostics Inc.; SD Bio-
line Chagas Ab rapid test (Standard Diagnostics); and On-
Site Chagas Ab rapid test cassette, CTK Biotech) (4, 8, 14,
20, 25, 28).
Four samples that were negative by standard serological
tests were positive with the dipstick, and we are currently
assessing whether these samples are true or false positives;
clearly, the tested dipstick yielded false negatives (n ? 26).
As similar studies evaluating dipsticks for malaria (31) and
leishmaniasis (7) diagnosis have shown, variability in dip-
stick performance will depend on factors such as the type of
diagnostic antigen and conjugate used. Commercially avail-
able dipstick tests can be highly variable in terms of sensi-
tivity and specificity (7). Rates of false positivity for malaria
dipsticks can be as high as 28%, which may, for example, be
due to cross-reactivity to rheumatoid factor (15). For sam-
ples from individuals suspected of T. cruzi infections, false-
positive results may be caused by other protozoan organ-
isms, including Leishmania spp. (2).
There are many potential advantages of using dipsticks
over other diagnostic methods. First, when using dipsticks, a
large number of samples can be processed quickly and with
minimum effort. Second, compared to the technological ex-
pertise needed for serology, molecular methods, or xenodi-
agnosis, the expertise (i.e., training of personnel) necessary
to perform the dipstick tests is minimal, as is the require-
ment for specialized laboratory equipment. Another advan-
tage of dipstick tests is that patients can see the results for
themselves, which will contribute to a better working rela-
tionship between local communities and people carrying out
the testing (e.g., during surveys). Third, from an epidemio-
logical point of view, a dipstick test allows intervention
strategies to be implemented in situ, such as for serologic
surveillance, vaccine or clinical trials, and rapid initiation of
treatment of infected individuals during outbreaks of acute
Chagas disease. Compared to what is required following
currently used serological tests, the need for follow-up visits
to surveyed individuals is reduced and hence operational
costs are reduced. Fourth, in settings normally considered
nonsupportive of T. cruzi and Chagas disease (22, 29), dip-
stick tests could easily be included in laboratory testing
algorithms. These could apply to people potentially exposed
to T. cruzi when traveling (e.g., tourists, military personnel)
as well as blood and organ donors from countries where
Chagas disease is endemic.
Immunochromatographic dipstick tests are comparatively
expensive (the tested dipstick has a retail price of $1.40), but
considering the above, a sensitive and specific dipstick test
such as the one tested here could prove very cost-effective
relative to currently available diagnostic tests, especially
when used in mass-screening surveys, investigations of acute
outbreaks of Chagas disease, and even tests in blood donor
A more comprehensive multicenter study is now planned
to evaluate dipsticks using a standardized protocol on
batches of sera from known negative and positive controls
and a random selection of samples from areas of endemicity
and nonendemicity, as well as to assess the relationship
3004NOTESJ. CLIN. MICROBIOL.
TABLE 1. Characteristics of samples and serological assays useda
No. of samples that were
Definition (per in-country guidelines) of:
(cutoff, 0.2 ?
T. cruzi epimastigotes,
A sample was considered
seropositive if at least two serological tests were positive followingtesting by ELISA, IHAT, and IFAT
negative followingtesting by threedifferenttests (ELISA,IFAT, andIHA)
Readings were discordant
following testing bythree different tests (ELISA, IFAT, andIHAT)
(BiosChileIngenierı ´aGenética S.A., Santiago,Chile) (cutoff,?CPos ?
CNeg? ? 0.35)
(bioMérieux,Buenos Aires, Argentina) (cutoff,CNeg ? 0.100)
Readings were positive
following testing bytwo different ELISAs
negative followingtesting by two differentELISAs
Readings were discordant
following testing by two different ELISAs, orone reading was closeto the test’s cutoff (?10%); the third
reading by either test was considereddefinitive
Kit Chagas III
(BiosChileIngenieria Genetica S.A.,Santiago,Chile) (cutoff,?CPos ?
CNeg? ? 0.35)
Readings were positive
following testing bytwo different ELISAs
negativefollowing testing byone ELISA
Readings were discordant
following testing by two different ELISAs, or one reading was closeto the test’s cutoff; the third reading by eithertest was considereddefinitive
(cutoff, 0.2 ? 2
T. cruzi epimastigotes,
PM strain locally isolated from apatient in 1985
Readings were positive
by serological testingof two patient samples taken 1 month apart
negativefollowing testing by one ELISA
Readings were discordant
following testing by two different ELISAs, orone reading was close to the test’s cutoff; thethird reading by either test was considereddefinitive
aPositive and negative samples included samples that had previously tested positive and negative by serological methods, as per in-country Chagas disease diagnostic guidelines. Abbreviations: CPos and CNeg, optical
density value of positive and negative controls, respectively; ND, not done.
VOL. 48, 2010NOTES 3005
between dipstick positivity and standard serology and the
heat stability and ease of use of dipsticks.
We thank Otilia Rodríguez Carvajal from the Instituto Mexicano del
Seguro Social in Me ´rida, Mexico, as well as Ana María de Rissio from
the Instituto Nacional de Parasitología Mario Fatala Chabe ´n in Bue-
nos Aires, Argentina, for testing and providing the serum samples used
in this study.
R.E.G. is supported by awards from the NIH/NSF Ecology of Infectious
Diseases program, which is funded by the Fogarty International Center and
the National Institute of Environmental Health Sciences (grant R01
TW05836 to Uriel Kitron and R.E.G.); the Agencia Nacional de Promocio ´n
Científica y Te ´cnica (Argentina); and the University of Buenos Aires.
M.V.C. and R.E.G. are members of CONICET’s Researcher’s Career.
The opinions expressed are those of the authors and may not reflect
the position of their employing organization or of their work’s sources
FIG. 1. (A) Comparative diagnosis of T. cruzi in 366 human blood samples. Samples were evaluated by different serological tests and considered
positive or negative as per in-country Chagas disease diagnostic guidelines. The number of samples that were tested in each country is specified,
as is whether they were serologically positive or negative, whether they were samples from patients with other infections (and also serologically
negative), and how they tested when the dipsticks were used. Sensitivities, specificities, positive and negative predictive values, kappa values, and
95% confidence intervals are also given. Shown below the standard-serology results are results for samples that had serologically concordant and
discordant results and how they tested with the dipstick. One sample from Ecuador from a case with another infection (indicated with*) had
discordant ELISA results, testing negative and testing borderline positive; it retested negative. Abbreviations: PS, samples testing positive as per
country guidelines; NS, samples testing negative as per country guidelines; OI, samples from individuals with other infections (i.e., brucellosis,
HBV, HCV, HIV, syphilis, tuberculosis); Dipstick, Trypanosoma cruzi Detect dipstick; N/A, not available; 95% CI, 95% confidence interval; PPV,
positive predictive value; NPV, negative predictive value. (B) Relationship between ELISA optical density values and rapid diagnostic test
positivity. Samples represented are samples for which exact optical density values were available, i.e., samples from Ecuador and Mexico (n ? 200).
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