A n to n io W a lte r F e rre ira , S a n d ra d o L a g o M o ra e s d e A v ila
L a b o r a t o r y
d i a g n o s i s o f C h a g a s ' h e a r t d i s e a s e
D iv is io n o f I n fe c tio u s D is e a s e s , I n s titu te o f T r o p ic a l M e d ic in e I m m u n o lo g y a n d S e r o e p id e m io lo g y L a b o r a to r y , F a c u ld a d e d e M e d ic in a d a
U n iv e r s id a d e d e S iio P a u lo . S iio P a u lo , B r a z il
The laboratory diagnosis of C hagas' disease is a com plex one. Factors relating to the host im m une response and the antigenic variability of T . c ru z im ust be considered in the final interpretation of test results. P arasitologic m ethods for detecting T . c ru z i, im m unologic m ethods for detecting T . c ru z iantigens in different biological fluids and serologic tests for detection and quantification
of different classes of im m unoglobulins are w ell standardized and used in the diagnosis of the acute or chronic phase of the disease. X enodiagnosis is the m ost com m on parasitologic test em ployed, although it detects only 50% of infections in the chronic
phase. Indirect im m unofluorescence for detecting IgG and IgM antibodies, hem agglutination and enzym e im m unoassay are the serologic tests m ost frequently em ployed for diagnosis, to screen blood donors and for seroepidem iologic studies. A n im portant caveat to be rem em bered is that serologic tests provide only a probable diagnosis, w hich depends on the prevalence of C hagas disease, as w ell as on the sensitivity and specificity of the test em ployed. The use of w ell defined specific antigens, obtained through recom binant m ethods or chrom atography, opens an im portant field for the developm ent of very specific tests, w ithout
significant loss of sensitivity.
U N ITE R M O S : C hagas. Laboratory. D iagnosis.
IN TR O D U C TIO N
T
r y p a n o s o m a c r u z i, t h e e t i o l o g i c a g e n t o f C h a g a s ' d i s e a s e , i s a p r o t o z o a n f o u n d i n d i f f e r e n t a n i m a ls p e c i e s . H u m a n d i s e a s e i s a c q u i r e d t h r o u g h i n s e c t
v e c t o r s b e l o n g i n g t o t h e s u b f a m i l y T r ia to m in a , f a m i l y R e d u v iid a e , o r d e r H e m ip te r a .
I n L a t i n A m e r i c a , t h e m o s t i m p o r t a n t v e c t o r s
i m p l i c a t e d i n t h e t r a n s m i s s i o n o f t h e r u r a l f o r m o f t h e
d i s e a s e a r e T r ia to m a in fe s ta n s , P a s tr o n g y lu s m e g is tu s , R h o d n iu s p r o lix u s , T r ia to m a d im id ia ta a n d T r ia to m a b r a s ilie n s is . T . c r u z i b e l o n g s t o t h e S te r c o r a r ia s e s s i o n o f
t h e f a m i l y T r y p a n o s o m a tid a e , w h i c h i n c l u d e s
A d d re s s fo r c o rre s p o n d e n c e : A n to n io W a lte r F e rre ira
In s titu to d e M e d ic in a T ro p ic a l d e S a o P a u lo A v . D r. E n e a s d e C a rv a lh o A g u ia r, 4 7 0 S a o P a u lo -S P - B ra s il - C E P 0 5 4 0 3 -0 0 0
t r y p a n o s o m e s w h i c h d e v e l o p i n t h e d i g e s t i v e t r a c t o f t h e v e c t o r s a n d a r e t r a n s m i t t e d t h r o u g h d i r e c t c o n t a c t w i t h
t h e f e c e s o f t h e t r i a t o m a b u g . T . c r u z i h a s g r e a t a n t i g e n i c
v a r i a t i o n , w h i c h m a k e s d i f f i c u l t t h e s t u d y o f t h e
e p i d e m i o l o g i c a l , c l i n i c a l , p a t h o l o g i c a l , l a b o r a t o r y a n d
t h e r a p e u t i c a s p e c t s o f C h a g a s ' d i s e a s e . I n u r b a n a r e a s ,
t r a n s m i s s i o n o f t h e d i s e a s e t h r o u g h b l o o d p r o d u c t s i s o f g r e a t c o n c e r n a n d r e s u l t s f r o m t h e m i g r a t i o n o f i n f e c t e d
i n d i v i d u a l s f r o m e n d e m i c a r e a s t o i n d u s t r i a l i z e d c e n t e r s .
C o n g e n i t a l a n d a c c i d e n t a l t r a n s m i s s i o n a r e a l s o b e e n d e s c r i b e d i n t h e l i t e r a t u r e .
LA B O R A TO R Y D IA G N O S IS
T h e l a b o r a t o r y d i a g n o s i s o f C h a g a s ' d i s e a s e i s
a c c o m p l i s h e d b y m e t h o d s w h i c h d e m o n s t r a t e t h e p r e s e n c e
o f t h e p a r a s i t e i n t h e b l o o d , d i r e c t l y o r i n d i r e c t l y ( p a r a s i t o l o g i c m e t h o d s ) a n d t h r o u g h t h e d e t e c t i o n o f s e r u m a n ti- T .c r u z i a n t i b o d i e s .
P A R A S IT O L O G IC
M E T H O D S
T hese m ethods can be em ployed to diagnose either
the acute or chronic phase of disease.
In the acute phase of C hagas' disease (first 6 w eeks),
m ethods w hich dem onstrate the presence of trypanosom es
in the bloodstream are em ployed. T his can be
accom plished through the exam ination of a thin peripheral
blood sm ear under a m icroscope. O ther variations of this
m ethod, such as preparing thick blood sm ears or
concentrating the parasite, increase the likelihood of
detection. A prom ising technique called Q uantitative B uffy
C oat (Q B C m ethod), w hich is w idely used to diagnose
infections w ith plasm odia, has been successfully applied
to detect trypanosom es, especially in patients w ith very
low levels of parasitem ia. In new borns w ith congenital
T.
cruzi
infection, m any authors recom m end the search fortrypanosom es in bone m arrow and spinal fluid. A m astigote
form s have also been dem onstrated in the chronic phase
of the disease, m ainly in m uscle tissue. T he sensitivity of
the m ethods w hich directly dem onstrate the presence T .
cruzi in the bloodstream ranges from 50 to 95% and is
influenced by several factors, ranging from the quality of
the m icroscopic equi pm ent to the expertise of the observer.
In the chronic phase of the disease, xenodiagnosis
and blood culture (indirect m ethods) are em ployed.
X enodiagnosis is done through the use of nym ph
vectors, bred in laboratory and fed w ith blood from fow l
resistant to
T.
cruzi
infection. In this technique, the nym phsare allow ed to have a blood m eal through the patient's
skin. A bout 40 nym phs are put in 4 boxes and kept at
28°C , w ith 85% air hum idity. A fter 4 to 6 w eeks, the gut
of the insect and its contents are exam ined for the presence
of
T.
cruzi,
under a m icroscopy. T he sensitivity of thism ethod is 85-100% in the acute phase and about 50% in
the chronic phase: A variant of the classical m ethod
(artificial xenodiagnosis) w as introduced to avoid
hypersensitivity reactions in susceptible patients. N ym ph
vectors are allow ed to feed on blood from the suspected
patients, w hich is kept in sm all dialysis bags, usually m ade
from the intestine of pigs. M any authors report that
sequential readings, at 30, 60, 90 days increase positive
results in the chronic phase of the disease.
B lood culture, though an insensitive technique, is
useful in isolating
T.
cruzi
strains for studies of biochem icaland im m unochem istry typing. W hen used for diagnostic
purposes, it detects approxim ately 50% of cases in the
chronic disease.
O ther diagnostic m ethods such as anim al
innoculation (m ice, guinea pigs) and in vitro cell culture
are seldom em ployed (7).
S E R O L O G IC D IA G N O S IS
S erologic tests are w idely used to: screen suitable
blood donors, as m arkers to m onitor therapy, to confirm
or exclude clinical suspicion of C hagas' disease, in
epidem iologic studies and to screen infected industry
w orkers. S erologic tests give only an estim ate of
probability of disease and their final interpretation is
influenced by num erous factors, like the sensitivity and
specificity of the test and the prevalence of C hagas' disease
in the population being tested.
M any standardized tests have been used to diagnose
C hagas' disease. It is im portant to bear in m ind the great
antigenic com plexity of
T. cruzi.
T his characteristicdictates the host's im m une response to the disease and
has led to researchers for different m arkers of disease.
T hrough peptide technology and m olecular biology
techniques, m any investigators are attem pting to identify
highly specific antigenic epitopes.
S tolf, in 1992, described the ideal antigen as the one
w hich w ould be present in all strains from different
endem ic areas, highly im m unogenic, not present in other
pathogenic m icroorganism s, stable and easy to be obtained
for use in serologic tests (19). N ext, w e w ill m ake a
discussion of the different tests em ployed in the serologic
diagnosis of C hagas disease. It is w orth rem em bering that,
for the different serologic tests, there is an overlap in the
reactivity curves of infected and non-infected individuals.
B y changing the cut-off point of the test, one can achieve
m axim um sensitivity or specificity. T he point w here the
curves intersect defines the values of sensitivity and
specificity (18).
1) Complement fixation
Introduced by G uerreiro and M achado in 1913, the
com plem ent fixation test has only historic value, though
it is still used by som e to screen blood donors and for
diagnostic purposes. T he technical com plexity of this test,
w hich requires the daily standardization of its com ponents
- antigen, hem olytic system and com plem ent - interferes
in its reproducibility. Its low level of sensiti vity and
specificity have also contributed to the low popularity of
this test.
2) Precipitation test
T he different variations of this test, though highly
It is m ainly used in the study of the different antigenic
com ponents of
T. cruzi.
C ounterim m unolectrophoresis has been the preferred m ethod for diagnosis andseroepidem iologic studies. B reniere, using serum w ith the
com ponent 5 of
T. cruzi
obtained from rabbits, obtained a sensitivity of 85% and specificity of 100% , even w hentesting sera from patients w ith leishm aniasis (1). R equejo
et al. recently standardized and tested the D IG -ELISA
assay, w hich is an association betw een the im m unoassay
and the agar diffusion test. The authors found the test to
have high sensitivity and specificity and recom m ended it
for screening and for seroepidem iologic surveys.
M ulticenter studies are necessary to validate this test (18).
3) Agglutination tests
This test, w ith its variants, has been w idely used in the diagnosis of C hagas disease.
3a) Direct Agglutination
Several authors have described the use of this test,
com paring it to im m unofluorescence. V attuone and
Y anow sky, em ploying a suspension of epim astigote form s
of
T. cruzi,
treated w ith enzym es and fixed w ith form alin,obtained good sensitivity in the detection of antibodies in
the acute phase of C hagas' disease (20). H arith et al.
standardized the m icroagglutination test, w hich em ployed
epim astigote form s of
T. cruzi,
treated w ith tripsin andstained w ith C oom asie-B lue. Treatm ent of the 2 sera w ith
m ercaptoethanol w as critical for the detection of specific antibodies. The sensitivity and specificity of the test w ere
very high. A draw back of the agglutination test is the
large am ount of parasites necessary to prepare the antigenic suspension (12).
3b) Hemagglutination
This test is w idely used for diagnosis, screening and
seroepidem iologic studies. In the test, erythrocytes from m am m als or fow l are treated w ith form alin and sensitized
w ith antigenic com ponents, partially or com pletely soluble.
The product, lyophilized or in suspension, has excellent
stability in adverse tem perature conditions (5). The test,
either quantitative or qualitative, is perform ed in m icrotiter plates (13), w ith the use of different antigenic extracts.
The best results
.
are obtained w ith alkaline, and sonicatedextracts. The treatm ent of the 2 sera w ith m ercaptoethanol increases the specificity of the test. The hem agglutination
test w as studied by N eal and M iles, w ho used the Y strain
ofT. crilzi,
grow n in LIT m edia. They tested diluted blood, collected in paper filter, from different populations fromLatin A m erican countries (16).
N o regional differences in antibody response w ere
observed. B ecause of its sim plicity and low cost, the
hem agglutination test is recom m ended for screening blood
donors. In B razil, w ell standardized kits from different
m anufacturers are com m ercially available.
3c) Latex agglutination
Though a very prom ising test, the latex agglutination
test w as released in B razil w ithout proper standardization.
False-positive and false-negative results, coupled w ith poor
reproducibility, led to the discontinuation of this test by
the m anufacturer. W ith the current possibility of creating
covalent bonds betw een
T. cruzi
antigens and free radicalspresent in the latex particles, new perspecti ves for
obtaining a new test w ith good stability, low cost, ease of
use and reliable sensitivity and specificity have appeared.
4) Immunofluorescence
The indirect im m unofluorescence test is usually
perform ed w ith epim astigote form s of the Y strain of
T.
cruzi,
obtained from cultures of the parasite in LIT m edium . The form alin treated trypanosom es are then fixedin glass slides and incubated in diluted serum for 30
m inutes at 37°C . A fter proper w ashings, the slides are
incubated w ith fluorescent conjugate (sheep or goat serum
anti-hum an IgG or IgM , labeled w ith fluorescein
isothiocyanate). A fter another incubation and w ashings,
the slide is read w ith the use of a fluorescence m icroscope
(6). The im m unofluorescence test for the detection' of IgG anti-To
cruzi
antibodies is considered to be the goldstandard in the serologic diagnosis of C hagas' disease (3).
A ntigenic variation has been observed in the different
parasitic stages of
T. cruzi.
C am argo found higherantibody titers w ith the tripom astigote than w ith epim astigote form s (2). Prim avera et al. com pared
epim astigote w ith tripom astigote form s of
T. cruzi
andconcluded that the am astigotes are m ore reactive for the
detection of IgA antibodies, especially in patients w ith the
digestive form of the disease (17). Levy standardized the in situ im m unofluorescence test w ith tripom astigote form s,
in order to detect m em brane epitopes and to follow persistent infections. The test has effectively substituted
the com plem ent lysis reaction (15).
D ifferent factors m ay affect the results of the
im m unofluorescence test: the quality of the optical equipm ent and of the antigens, the definition of w hat
constitutes a positive test. These factors should be taken
into account and the test should be rigorously standardized in order to obtain reliable results (10).
5) Enzyme immunoassay
In 1 9 7 5 , F erreira stan d ard ized th e im m u n o p ero x id ase
test, u sin g fo rm alin fix ed ep im astig o te fo rm s o f
T.
crud,
fo rm alin fix ed in g lass slid es as an tig en an d en zy m atic
co n ju g ate (sh eep o r g o at seru m co n ju g ated to p ero x id ase).
A fter in cu b atio n w ith d ilu ted seru m an d co n ju g ate,
th e co m p lex is rev ealed w ith su b strate an d h y d ro g en
d o n o rs. T h e m eth o d h ad th e sam e sen sitiv ity an d sp ecificity
o f th e im m u n o flu o rescen ce test. T h e ad v an tag e is th at th e
resu ltin g co lo r d ev elo p m en t m ay b e v isu alized w ith an
o p tical m icro sco p e, th u s red u ci n g th e co st o f th e test (1 1 ).
T h e u se o f th e en zy m e lin k ed im m u n o so rb en t assay
(E L IS A ) fo r th e d iag n o sis o f C h ag as d isease w as d escrib ed
b y V o ller et al. T h e test w as stan d ard ized in m icro titer
p lates, ad so rb ed w ith so lu b le
T.
cruzi
an tig en s. A fterin cu b atio n w ith seru m an d en zy m atic 'co n ju g ate, co lo r
d ev elo p s in th e su p ern ate after ad d itio n o f su b strate an d
h y d ro g en d o n o rs. T h e in ten sity o f co lo r d ev elo p m en t is
m easu red b y sp ectro p h o to m etry (2 1 ).
D u e to its g o o d sen sitiv ity an d sp ecificity an d b ecau se
it is au to m ated , th e stan d ard ized en zy m e im m u n o assay
h as o p en ed n ew p ersp ectiv es in th e sero lo g ic d iag n o sis
o f C h ag as' d isease. T h e p o ssib ility o f em p lo y in g v ery
sp ecific an tig en ic co m p o n en ts, o b tain ed th ro u g h p h y sico
-ch em ical o r reco m b in an t m eth o d s, is u n d er in v estig atio n
b y sev eral scien tists aro u n d th e w o rld (4 ). L ik e th e
h em ag g lu tin atio n test, w ell stan d ard ized reag en ts are
co m m ercially av ailab le an d are esp ecially u sefu l fo r
screen in g b lo o d d o n o rs (9 ).
M E T H O D S F O R D E T E C T I N G A N T I G E N I N
B O D Y F L U I D S
T h e d etectio n o f
T.
cruzi
an tig en s in b o d y flu id s iso f g reat im p o rtan ce in th e co n firm atio n o f in fectio n ,
esp ecially w h en th e sero lo g ic tests o r p arasito lo g ic
m eth o d s are n eg ati v e.
T ests b ased o n p recip itatio n , co u n terim m u n o electro
-p h o resis o r im m u n o d ifu sio n , th o u g h h ig h ly sp ecific, h av e
lo w sen sitiv ity , b eco m in g u sefu l in th e co n firm atio n an d
p ro g n o sis o f d isease. T h e en zy m e im m u n o assay , cap tu re
o r d o u b le san d w ich , h as p ro v ed u sefu l in th e d etectio n o f
T.
cruzi
an tig en s in b lo o d o r u rin e, co n firm in g a su sp ectedd iag n o sis an d m o n ito rin g th e efficacy o f an ti-C h ag as
th erap y . S o m e au th o rs b eliev e th at th e d etectio n 0
T.
cruzi
an tig en s in u rin e is u sefu l in th e d iag n o sis o f co n g en ital
C h ag as' d isease. K atzin et al. h av e d escrib ed a q u ick
ag g lu tin atio n test to d etect
T.
crud
an tig en s in p atien ts inth e ch ro n ic p h ase o f th e d isease. In th is test, h o rse seru m
w ith an ti-
T.
cruzi
an tib o d ies lin k ed to n itro cellu lo sem icelles reacts w ith sm all am o u n ts o f an tig en s p resen t in
b io lo g ic flu id s. T h o u g h th e test ap p eared to b e v ery
sen sitiv e, fu rth er stu d ies are n ecessary to co n firm th e
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