use of a simplified polymerase chain reaction

Am. J. Trop. Med. Hyg., 51(6). 1994. pp. 771—777 Copyright ©1994 by The American Society of Tropical

Medicine

and Hygiene

USE OF A SIMPLIFIED POLYMERASE CHAIN REACTION PROCEDURE TO DETECT TRYPANOSOMA CRUZI IN BLOOD SAMPLES FROM CHRONIC CHAGASIC PATIENTS IN A RURAL ENDEMIC AREA PATRICK

WINCKER,

CONSTANCA

BRITFO,

JOSE BORGES

ANGELICA CARDOSO, WALTER OELEMANN,

PEREIRA,

MARIA

@r@iD CARLOS M. MOREL

Laboratorio de Biologia Molecular e Doencas Endemicas, Departamento de Bioquimica Molecular,e Departamento de Medicina Tropical,Instituto Oswaldo Cruz,FIOCRUI Janeiro, Brazil

e Biologia Rio de

Abstract. The feasibility of using DNA amplification by the polymerase chain reaction (PCR) for specific detection of Trypanosoma cruzi in human blood specimens was inves tigated. One hundred blood samples were collected in an endemic area of Minas Gerais, Brazil. They were submitted to DNA extraction and PCR amplification with kinetoplast DNA—specific primers using a simplified boiling procedure that linearized most minicircle molecules without the aid of chemical reagents. Samples that gave negative results were checked

for

possible

inhibition

of amplification

using

primers

derived

from

a human

specific sequence, and those showing some level of inhibition were retested after a new DNA extraction. Of 86 patients previously diagnosed as chagasic by serologic techniques, 83 were

positive

in our PCR

test

(sensitivity

= 96.5%),

including

all the xenodiagnosis

positive patients and 21(87.5%) of 24 xenodiagnosis-negative individuals. In addition, four of six patients with doubtful serologic results were confirmed as positive by PCR. Our results suggest that the PCR may be a useful complement to serology in the diagnosis of Chagas' disease, and that it is the most powerful technique available for parasite de tection in patients with chronic disease. Chagas' disease is a major health problem throughout Latin America, with approximately 16—18 million people infected.' Its causative agent, the protozoan parasite Trypanosoma cru

zi, is usually transmitted to humans by reduviid bugs, although blood transfusion is increasingly implicated in the appearance of new cases. After an initial acute phase, in which diagnosis is gen erally straightforward due to a high level of cir culating parasites, infected patients enter a chronic phase without clinical symptoms. Some of these patients will then go on to develop clin ical forms of the disease that can lead to pro gressive morbidity and death.2 Direct diagnosis of chronic Chagas' disease has always been problematic due to the very low level of parasites encountered in the blood of patients with chronic disease. As a result, the technique most widely used to confirm T. cruzi infection has been serologic testing, despite the generally low specificity of this method due to cross-reactivity

with

sites.3 Xenodiagnosis, fect specificity, but

antigens

from

other

disease

is endemic.

para MATERIALS

meanwhile, ensures per its sensitivity, although

higher than in any other commonly

sitologic technique, is only 17—70% of that at tainable with serologic methods, depending on the endemic area under study.4 Recently, studies have been carried out using sensitive T. cruzi—specific polymerase chain re action (PCR) tests for detection of DNA in blood samples.5 Either kinetoplast DNA (kDNA) or nuclear satellite DNA has been used for this purpose. The sensitivity of the amplification pro cess appears to be sufficient to detect a single parasite in 20 ml of solutionP9 However, since the concentration of trypanosomesin the blood of chagasic individuals is highly variable, the efficiency of such tests needs to be further in vestigated using large numbers of blood sam ples. In the present study, we tested the efficacy of PCR amplification of kDNA sequences using 100 blood samples from individuals with posi tive, negative, and indeterminate serologic re sults inhabiting a single area in which Chagas'

AND

METHODS

Sample collection. The samples used in this study were collected in July 1992 in Virgem da

used para 771

772

WINCKER AND OThERS

Lapa, Minas Gerais, Brazil, a region in which Chagas' disease is endemic. Serologic analysis was performed using three different techniques: indirect immunofluorescence, hemagglutination, and enzyme-linked immunosorbent assay (the positivity threshold was > a 1:40 dilution for each technique). Most of these patients had al ready been examined in the previous 18 years using serologic methods, xenodiagnosis, and electrocardiography.'° Blood (10 ml) was im mediately mixed after collection with one vol ume of a 6 M guanidine hydrochloride/200 mM EDTA solution.7 The samples were stored at am bient temperature for 7—10 days, and subse quently at 4°C. Preparation of DNA. The method used to treat the samples prior to PCR amplification has been previously bed― Briefly, the poly propylene tubes containing the guanidine EDTA-blood lysates were immersed for 15 mm in boiling water to break up the majority of the minicircle DNA molecules forming the kDNA network. After cooling to room temperature, two aliquots of 100 p.1 were taken from each sample, and extracted once with phenol-chloroform, then once with chloroform, prior to precipitation with two volumes of ethanol in 100 mM sodium ac etate. The pellets obtained after centrifugation were each resuspended in 50 p.1 of distilled wa ter. PCR conditions. All amplifications were car ried out in a final volume of 75 p.1 using the hot start

procedure

(where

only

the DNA

the DNA sample, 2.5 p.1 of lOX Taq polymerase buffer, and 2.5 units of Tag DNA polymerase [Perkin-Elmer

Cetus])

was added

and water

was

added to give a final volume of 25 p.1. The PCR was then immediately started using a DNA Thermal Cycler 480 (Perkin-Elmer Cetus) and the following conditions: two cycles at 98°Cfor I mm and 64°Cfor I mm, 33 cycles at 94°Cfor I mm and 64°Cfor I mm, and a final extension at 72°Cfor 10 mm. The same protocol was used for amplification of the human @3-g1obingene sequences except that the primers used were #PCO3 5'-ACA CAAACTGTGTFCACTAGC-3' and #PCO4 5'TFTF'3 Oligonucleotide hybridization. Oligonucleo tide #1 19 (5'-TGGITiIGGGAGGGG(C/G)(G/ C)(T/G)TCAA(A/C)TIT-3'), derived from the sequence of the internal region of the amplified minicircle product,'4 was used as a probe for hy bridization after 5' end labeling with -y32P-ATP and polynucleotide kinase. Hybridization was carried out in 6X SSC (I X SSC = 150 mM NaCl, 15 mM trisodium citrate), 1X Denhardt's solution (0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 0.5% so dium dodecylsulfate (SDS), and 50 p.g/ml of de natured salmon sperm DNA. The filters were hy bridized at 37°Covernight and then washed at 55°C in 3X SSC and 0.1% SDS. RESULTS

polymer

ase and the oligonucleotides are in contact at high temperature, thus avoiding the synthesis of unspecific products), with physical separation of the primers and the DNA polymerase by a solid paraffin layer.'2 In this method, the lower solu tion consists of 5 p.1 of lOX Taq polymerase

Simplification of the DNA preparation method for the PCR amplification. Due to the low concentration of parasites in chagasic blood, detection of T. cruzi is possible only in samples with a volume of several milliliters. One way to overcome the problems of large-scale DNA

reaction

preparation

buffer

(100

mM

Tris-HC1,

pH 8.3, 500

mM KC1), 7.2 p1 of a dNTPs mixture (10 mM each), 13.5 p.1 of 25 mM MgC12, 200 ng of the T cruzi—specific primers (#121: 5'-AAATA ATGTACGGG(T/G)GAGATGCATGA-3'; #122: 5'-GGTTCGATFGGGGTrGGTGTAA TATA-3'), and water to give a final volume of 50 p.1, in a thin-walled reaction tube. After ad dition of one Ampliwax PCR Gem bead (Perkin Elmer Cetus, Norwalk, CT), the tube was placed in a 80°C heating block to melt the paraffin bead, and then chilled to room temperature. Af tsr complete solidification of the wax barrier, 25 p.1 of the upper mixture (consisting of 7.5 p.1 of

is to split the kDNA

networks

(each

of which contains about 10@interlocked minicir des) into single minicircles by moderate chem ical hydrolysis of the DNA.5 However, we have found

that

heat

treatment

is equally

effective,

with the added advantage of avoiding the risk of contamination that is inevitably associated with the addition of chemical reagents.― When ap plied to samples from chronic chagasic patients, the heat method enables the detection of the ex pected 330-basepair amplification product from original material of as little as 15 p.1 of blood lysate (Figure 1). We have found that when stored at 4°C,the lysates remain susceptible to

PCR DIAGNOSIS

OF CHAGAS'

two primers (Figure 2B). However, the optimi zation of the reaction conditions resulting from our use of the hot-start protocol enabled us to analyze the results by agarose gel electrophore sis alone, since a single band of the expected size was clearly visible in positive samples. When the duplicate experiments yielded con

I 330

773

DISEASE

flicting results, two new performed using material

bp a...

DNA extractions were from the same sample,

and the new preparations tails of the amplification Table 1.

were then tested. De results are shown in

Amplification of a human DNA sequence in i: cruzi-negative cation

FIGURE

1.

Ethidium

bromide—stained

2%

agarose

gel containing Trypanosoma cruzi—specificpolymerase chain reaction products of DNA isolated from a cha gasic inhabitant

of Virgem

da Lapa at different

times

after boiling. Lane I, Hae Ill-digested 4X174 DNA; lanes 2 and 4, negative controls from a nonchagasic individual

processed

together with the positive samples

shown, respectively, in lanes 3 and 5; lane 3, sample from chagasic

patient #3 processed

immediately

after

boiling and amplified for 30 cycles; lane 5, same sam ple from patient #3 reprocessed 10 months after boil ing and amplified for 35 cycles. bp = basepairs.

DNA preparation and PCR amplification for at least 10 months after being boiled (Figure 1). Detection of 7'. cruzi DNA in chronic cha gasic patients. One hundred blood specimens from chagasic and nonchagasic inhabitants of a single endemic area in southeastern Brazil were collected and submitted to PCR diagnosis using the technique described above. We routinely pre pared one negative control (consisting of a blood lysate from an apparently healthy individual who never lived in an endemic area) and one positive control (consisting of a blood lysate from a xenodiagnosis-positive chagasic patient) for every five samples tested. Each DNA ex traction was performed in duplicate for each pa tient, and the PCR was performed independently on these two preparations. Figure 2 shows a typ ical amplification experiment. The nature of the amplified product could be confirmed by prob ing with a labeled oligonucleotide that was com plementary to T. cruzi minicircle DNA and whose sequence was different from those of the

of our

samples.

100 blood

The PCR amplifi

samples

using

T. cruzi

kDNA specific primers yielded 14 negative re sults (Table 1). To check for the possible pres ence of a PCR inhibitor in these samples, we performed an amplification reaction using prim ers specific for the human f3-globin gene, but otherwise maintaining the same conditions as before. This second type of PCR should yield a I lO-basepair product of strong intensity,'3 and we obtained such a result with 11 of the 14 sam pies. The other three samples, however, either gave no amplification (#18) or a low level of the expected product (#38 and #65; compare with the positive control, lane 8 of Figure 3). Aliquots from these three blood lysates were extracted in duplicate again, and the resulting DNA prepa rations subjected to amplification with 7'. cruzi and @3-g1obin—specificprimers, in turn. All the samples were strongly positive with the f3-globin primers. Meanwhile, with the minicircle prim ers, samples #38 and #65 remained negative, but sample #18 appeared to be positive. Some sam ples that remained negative were further tested for PCR amplification with the minicircie prim ers after introduction of purified kDNA to check for a possible primer-specific inhibition. All of them could be successfully amplified. DISCUSSION

This study addresses the feasibility of detect ing T cruzi DNA by PCR amplification in blood samples from chronic chagasic patients. The only previous large-scale study on this subject made use of a complex chemical treatment for splitting the kDNA.'5 This treatment proved ef ficient, but required expensive reagents and in troduced a risk of blood sample contamination resulting from the addition of external solutions.

774

WINCKER

A

@

AND

123456789101112101415

OTHERS

B

1234

5678

91011

_______

330 bp —

FIGURE

12131415

2.

Typical

diagnosis

experiment

using

samples

from

five

inhabitants

of

Virgem

da

Lapa.

Samples

were processed and amplified in duplicate, and products were revealed by staining with ethidium bromide after migration on a 2% agarose gel (A) or by subsequent hybridization of the same gel with the specific oligonucle otide #119 (B). Lane 1, Hae Ill-digested @Xl74DNA; lane 2, negative control (no DNA added); lanes 3 and 4, negative control sample (from non-chagasic individual processed in duplicate together with the samples from the five patients); lanes 5 and 6, patient #99; lanes 7 and 8, patient #100; lanes 9 and 10, patient #77; lanes 11 and 12, patient #78; lanes 13 and 14. patient #79; lane 15, positive control (confirmed chagasic patient with a positive xenodiagnosis). Fifteen microliters of the 75 p.1total reaction volume was loaded into each slot. bp = basepairs.

The boiling method used in the present study, in addition to being less expensive, quicker, and simpler, does not involve addition of any new component to the blood lysate, limiting the pos 123456789

I. 0

110 bp@

FIGURE

3.

Ethidium

bromide—stained

2%

agarose

gel containing human f3-globin polymerase chain re action products of DNA isolated from three of the pa tients that failed to amplify the expected Trypanosoma cruzi product. Lane 1, Hae Ill-digested 4iX 174 DNA; lanes 2 and 3, duplicate experiment with patient #18; lanes 4 and 5, duplicate experiment with patient #38; lanes 6 and 7, duplicate experiment with patient #65; lane 8, positive control (chagasic patient exhibiting a positive signal with kinetoplast DNA—specificprim ers); lane 9, negative control (no DNA added). bp = basepairs.

sibilities

of contamination

to the DNA

extraction

stage, at which the risk can be monitored with appropriate controls.― The patients diagnosed by PCR in this study inhabit a rural endemic area in which Chagas' disease has been systematically researched for 18 years. During that time, a large section of the population has been repeatedly probed for an immune response against T. cruzi using serolog ic techniques.'6 In addition, part of the serology positive population has been submitted to one or more xenodiagnostic tests. When we compared the results of the present study with the serologic diagnoses of the last 18 years, we found that of 86 patients previously diagnosed as chagasic, 83 were positive in our PCR test. Sixty of these chagasic individuals had undergone one or more xenodiagnoses; those that gave positive results with xenodiagnosis were all detected by PCR, while 21 of the 24 xenodiagnosis-negative cha gasic patients were positive with the PCR (Table 2). Six patients had serologic results that were difficult to interpret due either to a very low titer of anti-T. cruzi antibodies or to discrepancies of the results between the three techniques that were used. The PCR technique detected four of them as positive, including one that had already developed cardiac problems. Finally, all the eight individuals that had been serologically di

PCR

DIAGNOSIS

OF CHAGAS'

TABLE

DISEASE

775

1

Detailedresultsof thepolymerase chain reaction(PCR) performed on samples of the 100 inhabitants of the area endemic for Trypanosoma cruzi* Patient

Serology

T. cruzi PCR #1

T. cruzi PCR #2

T. cruzi PCR #3

@-globin PCR

Patient

1 2 3 4

+ + + +

+,+ +,+ +,+ +,+

51 52 53 54

5

Serology

T. cruzi PCR#l

+ + D +

+,+ +,+ -,+,—

@-globinPCR T. cruzi T. cruzi #2 PCR #3

+,+ +,+

+

+,+

55

+

—,—

6 7 8 9 10 11

+ + — + + +

+,+ +,+ —,— +,— +,+ +,— +,+ +,+

56 57 58 59 60 61

D + + + + +

+,+ +,+ +,+ +,+ +,+ +,+

12

+

+,+

62

+

+,+

13 14

+ +

+,+ +,+

63 64

— +

+,+

15

+

+,+

65

+

—,—

16 17 18 19 20

+ + + + +

+,— +,+ +,+ —,— +,+ +,+

66 67 68 69 70

+ + + + +

+,+ +,+ +,+ +,+ +,+

21

+

+,—

71

+

+,+

22

+

+,+

72

+

+,+

23 24

+ +

+,— +,+

73 74

+ +

+,— +,+

+,+

25 26 27

— + +

—,— +,+ +,+

75 76 77

+ + +

+,— +,+ +,+

+,+

28

+

+,+

78

D

+,+

29 30 31

+ + D

+,— +,— +,+ —,—

79 80 81

+ + +

+,+ +,+ +,+

32

+

+,+

82

+

+,—

+,+

33 34

+ +

+,+ +,—

83 84

+ +

+,— +,—

+,+ +,+

35

+

+,+

85

D

+,—

+,—

36

+

+,—

86

D

+,+

37

+

+,+

38 39 40 41

— + — +

—,— +,+ +,— +,+

+,+

—,—

+,+ +,+

+,+

+,+ +,+

+,+ +,+ +1—,+1— —,—

+,+

87

+

—,—

88 89 90 91

+ + + +

+, — +,+ +,+ +,+

92

+

+,+

93

+

+,+

42

+

+,+

43

—

—,—

44

+

+,+

94

+

+,+

45

+

+,+

95

+

+,+

46

—

—,—

96

+

+,+

47

+

+,—

+,+

97

+

+,+

48 49

+ +

+,+ +,—

+,+

98 99

+ +

+,— +,+

50

+

+,—

+,—

+,+

—

—,—

using

different

* Serology

was

performed

+,+

+,+

three

100 techniques

(hemagglutination.

enzyme-linked

PCR

+,+

+,+ +,+

+, +

+,+

immunosorbent

+,+ assay,

and

indirect

immunofluores

cence). Sera were considered positive (+) when at least two of the three techniques gave a result above the respective cutoff value, doubtful (D) when only one out of the three techniques gave a positive result, and negative (—) when all three test results were negative. +1— indicates cases in which control amplification with @-globin—specificprimers yielded a signal of low intensity.

776

WINCKER

TABLE

2

AND OThERS

26

26

0

agnosis

24

21

3

number of studies that show that T cruzi can be detected in a large number of heart biopsies from cases with chronic disease when the im munoperoxidase method of parasite antigen de tection is used. 9.20 More recently, the PCR has been successfully used to detect T. cruzi se quences in heart biopsies from patients that died of chronic chagasic cardiomyopathy.2' Taken to gether, all these results point to a long-standing presence of T. cruzi in chronic chagasic patients. The possible direct role of the parasite in the pathology of chronic disease may therefore be

Total36

8636

830

3

expected

Comparison of thepolymerase chain reaction(PCR) with xenodiagnosisfor the serology-positive pa tients* PCRPatient of casesPosi

statusNo.

tivePatients

with positive xenodi agnosis Patients who were not subject ed to xenodiagnosis Patients with negative xenodi

* Patients

who

underwent

senodiagnosis

wree

tiveNega

considered

positive

when at least one of the tests carried out during the last 18 years gave a positive result.

agnosed as negative for Chagas' disease showed no specific band following PCR amplification. The sensitivity of the PCR technique used in this study was 96.5% when compared with se rology. In a previous study carried out in the same region using the chemical method for split ting kDNA, PCR tests detected 100% of the cha gasic individuals in the sample group.'5 How ever, the patients used in the two studies were different, and therefore no comparision is pos sible. Clearly, to draw more definitive conclusions, we need to continue these studies using a larger number of samples, but the present results in dicate that the PCR may be a powerful comple ment to serology for Chagas' disease diagnos tics. This potential use of the PCR would be of particular

importance

in cases

in which

serology

cannot serve as a good indicator of the presence or absence of T. cruzi, for example, in patients undergoing specific treatment.'7 One important conclusion of this study is that the performance of our PCR test far exceeds that of xenodiagnos is, and may become the main technique for par asite detection in chronic chagasic patients. The long and indeterminate chronic phase of the disease, coupled with the frequency of au toimmune responses in chagasic patients, has led some researchers to question the role of the par asite in the development of disease pathology.'8 Such hypotheses have been partly sustained by the difficulties in detecting T. cruzi in blood us ing current techniques of parasite isolation. Our study shows that parasite DNA is detectable in most chagasic patients when a sensitive tech nique such as the PCR is used. It corroborates a

to attract

increased

attention

in future

studies. Acknowledgments: We are grateful to Dr. Larry Simp son and Herbert Avila for helpful discussions, Mozar Jose Coelho and Joaquim Mendes for assistance during the field work, and Alexander W. Bullock for correc tion of the manuscript. Financial support: This investigation received financial support from the UNDPIWor1d Bank/WHO Special Programme for Research and Training in Tropical Dis eases, the International Atomic Energy Agency, Con selho Nacional de Pesquisa e Desenvolvimento Cien

tifico e Technologico, Institut National de la Sante et de la Recherche Medicale (Reseau Nord-Sud 492NS5), Programa de Apoio a Pesdquisa Estrategica em Saude/ Fundacao Oswaldo Cruz, and Financiadora de Estudos e Projetos. Authors'

addresses:

Patrick WIncker, Constanca

Britto,

Maria Angelica Cardoso, Walter Oelemann, and Carlos M. Morel, Laboratorio de Biologia Molecular e Doen cas Endemicas, DBBM, Instituto Oswaldo Cruz, HO CRUZ, Avenida Brasil 4365, 21045-900 Rio de Ja neiro, RI, Brazil.

Jose Borges

Pereira,

Departamento

de Medicina Tropical, Instituto Oswaldo Cruz, FlO CRUZ, Avenida Brasil 4365, 21045-900 Rio de Ja neiro, RI, Brazil. REFERENCES

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World Health

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3. Schmunis G, 1991. Trypanosoma cruzi, the etio logic agent of Chagas' disease: status in the blood supply in endemic and nonendemic coun tries. Transfusion

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L,

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ates: diagnosis of chronic Chagas' disease. Mol Biochem Parasitol 48: 211—222. 8. Russomando G, Figueredo A, Almiron M, Saka moto M, Morita K, 1992. Polymerase chain re

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means of assessing cure after treatment of Cha gas' disease: a 10 year follow-up study. Trans R Soc Trop Med Hyg 87: 220—223. Brener Z, Krettli AU, 1990. Immunology of Cha gas' disease. Wyler DJ, ed. Modern Parasite Biology: Cellular, Immunologic and Molecular Aspects. New York: WH. Freeman and Com pany. 247—261. Barbosa AJA, Gobbi H, Lino BT, Lages-Silva E, Ramirez LE, Teixeira VPA, Almeida HO. 1986. Estudo comparativo entre o metodo convencion al e o metodo da peroxidase anti-peroxidase na pesquisa do parasitismo tissular na cardiopatia chagasica cronica. Rev Inst Med Trop Sao Pau lo 28: 9 1—96. Higuchi ML, Dc Brito T, Reis MM, Barbosa A, Belloti G, Pereira-Barreto AC, Pileggi F, 1993. Correlation between Trypanosoma cruzi parasit ism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: light mi croscopy and immunohistochemical findings. Cardiovasc Pathol 2: 101—106. Jones EM, Colley DG, Tostes 5, Reis Lopes E, Vnencak-Jones CL, McCurley TL, 1993. Am plification

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quence from inflammatory lesions in human chagasic cardiomyopathy. Am J Trop Med Hyg 48: 348—357.