The protozoan parasite Trypanosoma cruzi, the agent of Chagas disease (American trypanosomiasis),
remains a major public health concern in Latin America. The insect vectors are haematophagous
triatomine bugs that, during or after their blood meal, pass the infective form of
T. cruzi with feces; the parasite can then enter the host through mucosal membranes,
the conjunctiva or abraded skin. Other routes of infection are congenital, oral or
via blood/organ donation. The acute phase of infection lasts up to a few weeks, with
nonspecific, self-resolving symptoms, although deaths can occur in this phase, particularly
in children or young adults. The subsequent chronic phase of infection is life-long
unless successfully treated, and asymptomatic (indeterminate) in the majority of patients.
However, approximately 30% of infected individuals will develop chronic cardiac and/or
gastrointestinal pathologies, with sudden death due to chagasic cardiomyopathy .
Chronic symptoms may manifest years or decades after the initial infection; currently,
there is no prognostic indicator. A recent WHO report estimates that 5–6 million people
are infected with T. cruzi . Although vector-borne transmission is confined to
the Americas, Chagas disease among migrants from Latin America has become of global
Trypanosoma cruzi displays remarkable intraspecies diversity, with six genetic lineages
(TcI-TcVI) , and a proposed seventh, TcBat . The possible association of different
lineages of T. cruzi with distinct forms of the disease is a long-standing research
interest : the cardiac syndrome is found throughout the endemic area, whereas mega
syndromes of the colon and esophagus have rarely been reported beyond the southern
cone countries of South America.
Trypanosoma cruzi is a zoonosis, with all mammals being susceptible to infection,
and humans becoming a later host following the historic peopling of the Americas.
The association between T. cruzi lineages, vectors and domestic, peridomestic and
sylvatic animals and ecological cycles is complex [6,7].
Investigating the association between infecting lineage(s) and clinical outcome or
ecological cycles has faced significant confounding challenges: the sequestration
of the parasite in host tissues during the chronic phase, possibly in a lineage-dependent
manner, hampers lineage identification by direct genotyping; in vitro culture of isolates
may favor the selection of certain lineages. Current serological techniques identify
T. cruzi specific antibodies (usually IgG), but are not designed to identify infecting
lineage. The possibility therefore arose that serology based on lineage-specific T.
cruzi antigens could overcome these difficulties, as it would allow the identification
of an individual's history of lineage infection without the need to genotype or isolate
In 2002, Di Noia and colleagues  published a pioneering report on TSSA, a mucin
expressed on the mammalian bloodstream form of T. cruzi. This allowed researchers
to enter a new era of lineage-specific serology for this parasite. Following the initial
characterization, greater diversity of the protein core of TSSA was revealed .
There is a short region of the protein core of T. cruzi TSSA where amino acid residues
vary according to lineage. Thus, TcI, TcIII and TcIV each have their own potential
lineage-specific TSSA epitope; TcII, TcV and TcVI share a common epitope, and the
hybrid lineages TcV and TcVI share an additional epitope.
Initially, recombinant TSSA proteins encompassing the TcI or TcII/V/VI common epitopes
were produced in E. coli, for use in ELISA and western blot. Those reports used sera
from mainly southern cone countries, principally Argentina, including those from chronic
symptomatic infections , pregnant chagasic women  and pediatric diagnosis
. An alternative approach is to use synthetic peptides (TSSApep-I, -II/V/VI, -III,
-IV and -V/VI) representing the lineage-specific epitopes in serological assays. When
these were first used in ELISA with sera from a range of South American countries,
several novel and unexpected results were found : an association between serological
recognition of TSSApep-II/V/VI and degree of clinical symptoms; reaction to TSSApep-II/V/VI
was observed in samples from Ecuador and these lineages have rarely been reported
in northern South America; specific TSSApep-IV reactions were found in Venezuela and
Antibody recognition of the current TSSA-I-specific epitope is rare. This may be due
to lack of antigenicity or partially because most assayed sera have originated from
regions where TcII, TcV and TcVI are predominant (based on genotyping). This observation
stimulated research into the function of the native TSSA isoforms, and assessment
of their antigenic properties. Canepa and collegues  demonstrated that although
the TSSA-II/V/VI isoform had a cell binding and entry capacity, causing the authors
to ascribe an ‘adhesin’ function, these properties were lacking in the TSSA-I isoform;
a subsequent report also proposed that TSSA-II/V/VI had an additional role in T. cruzi
differentiation . In terms of antigenicity, both bioinformatic  and peptide
mapping  studies have reinforced the strong antigenicity of the TSSA-II/V/VI common
The proven efficacy of TSSApep-II/V/VI in ELISA stimulated the development of a lateral
flow, immunochromatographic rapid diagnostic test (RDT) called Chagas Sero K-SeT,
in which this peptide was immobilized on a nitrocellulose membrane, and specific IgG
could be detected by protein G conjugate, within 15 min . This novel T. cruzi
lineage-specific RDT revealed that in Bolivian patient groups stratified by severity
of chagasic cardiomyopathy, RDT seropositivity was five-times higher among patients
with severe cardiomyopathy compared with those with no evidence of cardiomyopathy.
This was proposed to be due to repeated parasite exposure over time increasing inflammatory
cardiac damage in conjunction with an increase in anti-TSSApep-II/V/VI IgG. The same
study using Chagas Sero K-SeT also identified sporadic TcII/V/VI infections from Peru.
Trypanosoma cruzi has an extremely broad and diverse pattern of circulation among
mammals throughout the Americas. Investigation of these natural cycles of infection
has led to greatly increased understanding of their natural ecologies, and the transmission
risk to human populations. However, the limitations of T. cruzi lineage identification
described above apply equally to animals and humans. Thus, lineage-specific serology
has also been applied to mammals, initially identifying reactions to the TSSA-II/V/VI
isoform in Argentine dogs . Serology using the TSSA synthetic peptides has been
extended to sylvatic Brazilian primates, identifying Leontopithecus chrysomelas (golden-headed
lion tamarin) and L. rosalia (golden lion tamarin) as natural hosts of TcII and/or
TcV/VI in the Atlantic forest of Brazil . The use of Chagas Sero K-SeT with sympatric
humans and dogs from northern Argentina has shown the efficacy of protein G for detection
across several mammalian orders , including primates and rodents (McClean
et al. Unpublished Observations), thus reducing the need for species-specific secondary
antibodies. The use of Protein G and/or Protein A for IgG detection may further extend
this range. However, as with human sera, a test for TSSA-I remains elusive. The lack
of reactivity to TSSA-I has led to further efforts to identify an alternative robust
TcI antigen. Further studies on the TcIII and TcIV epitopes are clearly warranted.
This program of research on T. cruzi demonstrates the value and impact of lineage-specific
serology. The point-of-care/capture format of the test provides a result in 15 min
and is applicable to animals or patients in rural field locations, without access
to a laboratory. This enables low cost rapid surveillance for T. cruzi lineages among
potential animal reservoirs of infection, to assess the risk of emergent endemic regions
and to guide control strategies, without the need to isolate T. cruzi from the animals.
Furthermore, we can far more efficiently investigate whether the genetically distinct
T. cruzi lineages may be responsible for the different clinical presentations and
prognoses of Chagas disease. This approach also clearly has potential wider application
to other infectious diseases.