• View in gallery

    Optical density values of the serologic tests according to test and group (—: test cut-off; __: group mean). (A) Indirect immunofluorescence test (cut-off: 1:80); (B) enzyme-linked immunosorbent assay (ELISA) using promastigote antigen (cut-off: 0.170); (C) ELISA using rK39 recombinant antigen (cut-off: 0.623); (D) ELISA using rK26 recombinant antigen (cut-off: 0.552). G1: kala-azar; G2: Argentina; G3: endemic area/no contact with visceral leishmaniasis; G4: endemic area/contact with visceral leishmaniasis; G5: other endemic disease; G5.1: cutaneous leishmaniasis/ Brejo do Mutambal; G5.2: cutaneous leishmaniasis/Manaus; G5.3: P. vivax malaria/Manaus; G5.4: P. falciparum malaria/Manaus.

  • View in gallery

    Spearman’s correlation coefficient between age and positive ELISAp and IIFT in subjects from an endemic area. (A) Correlation between ELISAp and age of subjects from G3. (B ) Correlation between ELISAp and age of subjects from G4. (C) Correlation between IIFT and age of subjects from G3. (D) Correlation between IIFT and age of subjects from G4. G3: Serum samples of subjects from an endemic area without contact with visceral leishmaniasis; G4: serum samples of subjects from an endemic area with domiciliary contact with visceral leishmaniasis.

  • View in gallery

    Positive serologic test for visceral leishmaniasis and positive serology for Chagas disease in subjects from an endemic area (G3 + G4). G3: Serum samples of subjects from an endemic area without contact with visceral leishmaniasis; G4: serum samples of subjects from an endemic area with domiciliary contact with visceral leishmaniasis.

  • 1

    Abramo C, Fontes CJ, Krettli AU, 1995. Cross-reactivity between antibodies in the sera of individuals with leishmaniasis, toxo-plasmosis, and Chagas’ disease and antigens of the blood-stage forms of Plasmodium falciparum determined by indirect immunofluorescence. Am J Trop Med Hyg 53 :202–205.

    • Search Google Scholar
    • Export Citation
  • 2

    Araujo FG, Mayrink W, 1968. Fluorescent antibody test in visceral leishmaniasis. II. Studies on the specificity of the test. Rev Inst Med Trop Sao Paulo 10 :41–45.

    • Search Google Scholar
    • Export Citation
  • 3

    Bray RS, Lainson R, 1965. The immunology and serology of leishmaniasis: I. The fluorescent antibody staining technique. Trans R Soc Trop Med Hyg 59 :535–544.

    • Search Google Scholar
    • Export Citation
  • 4

    Camargo MA, Rebonato C, 1969. Cross-reactivity in fluorescent test for Trypanosoma and leishmania antibody. Am J Trop Med Hyg 18 :500–505.

    • Search Google Scholar
    • Export Citation
  • 5

    Edrissian H, Darabian P, 1979. A comparison of enzyme-linked immunosorbent assay and indirect fluorescent antibody test in the serodiagnosis of cutaneous and visceral leishmaniasis in Iran. Trans R Soc Trop Med Hyg 73 :289–292.

    • Search Google Scholar
    • Export Citation
  • 6

    Evans TG, Krug EC, Wilson ME, Vasconcelos AW, De Alencar JE, Pearson RD, 1989. Evaluation of antibody responses in American visceral leishmaniasis by ELISA and immunoblot. Mem Inst Oswaldo Cruz 84 :157–166.

    • Search Google Scholar
    • Export Citation
  • 7

    Guimarães MCS, Celeste BJ, Castilho EA, Mineo JR, Diniz JMP, 1981. Immunoenzymatic assay (ELISA) in mucocutaneous leishmaniasis, kala-azar, and Chagas’ disease: an epimastigote Trypanosoma cruzi antigen able to distinguish between anti-Trypanosoma and anti-leishmania antibodies. Am J Trop Med Hyg 30 :942–947.

    • Search Google Scholar
    • Export Citation
  • 8

    Harit EA, Kolk AHJ, Kager PA, Leeuwenburg J, Faber FJ, Muigai R, Kiugu S, Laarman JJ, 1987. Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and seroepidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Trans R Soc Trop Med Hyg 81 :603–606.

    • Search Google Scholar
    • Export Citation
  • 9

    Hommel M, Peters W, Ranque J, Quilici M, Lanotte G, 1978. The micro-ELISA technique in the serodiagnosis of visceral leishmaniasis. Ann Trop Med Parasitol 72 :213–218.

    • Search Google Scholar
    • Export Citation
  • 10

    Mohammed AR, Wright EP, Kager PA, Laarman JJ, Pondman KW, 1985. ELISA using intact promastigotes for immunodiagnosis of kala-azar. Trans R Soc Trop Med Hyg 79 :344–350.

    • Search Google Scholar
    • Export Citation
  • 11

    Walton BC, Brooks WH, Arjona I, 1972. Serodiagnosis of American leishmaniasis by indirect fluorescent antibody test. Trans R Soc Trop Med Hyg 21 :296–299.

    • Search Google Scholar
    • Export Citation
  • 12

    Ho M, Leeuwenburg J, Mbugua G, Wamachi A, Voller A, 1983. An enzyme-linked immunosorbent assay (ELISA) for field diagnosis of visceral leishmaniasis. Am J Trop Med Hyg 32 :943–946.

    • Search Google Scholar
    • Export Citation
  • 13

    Jahn A, Lelmett M, Diesfeld HJ, 1986. Seroepidemiological study on kala-azar in Baringo District, Kenya. J Trop Med Hyg 89 :91–104.

  • 14

    Burns JM Jr, Shreffler WG, Benson DR, Ghalib HW, Badaró R, Reed SG, 1993. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci USA 90 :775–779.

    • Search Google Scholar
    • Export Citation
  • 15

    Badaró R, Benson D, Eulalio MC, Freire M, Cunha S, Netto EM, Pedral-Sampaio D, Madureira C, Burns JM, Houghton RL, David JR, Reed SG, 1996. rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis 173 :758–761.

    • Search Google Scholar
    • Export Citation
  • 16

    Bern C, Jha SN, Joshi AB, Thakur GD, Bista MB, 2000. Use of the recombinant K39 dipstick test and the direct agglutination test in a setting endemic for visceral leishmaniasis in Nepal. Am J Trop Med Hyg 63 :153–157.

    • Search Google Scholar
    • Export Citation
  • 17

    Braz RFS, Nascimento ET, Martins DRA, Wilson ME, Pearson RD, Reed SG, Jerônimo SMB, 2002. The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection. Am J Trop Med Hyg 67 :344–348.

    • Search Google Scholar
    • Export Citation
  • 18

    Guimarães SF, Lemos EM, Corey R, Dietze R, 2003. Performance of recombinant K39 antigen in the diagnosis of Brazilian visceral leishmaniasis. Am J Trop Med Hyg 68 :321–324.

    • Search Google Scholar
    • Export Citation
  • 19

    Kumar R, Pai K, Pathak K, Sundar S, 2001. Enzyme-linked immunosorbent assay for recombinant K39 antigen in diagnosis and prognosis of Indian visceral leishmaniasis. Clin Diagn Lab Immunol 8 :1220–1224.

    • Search Google Scholar
    • Export Citation
  • 20

    Qu JQ, Zhong L, Masoom-Yasinzai M, Abdur-Rab M, Aksu HS, Reed SG, Chang KP, Gilman-Sachs A, 1994. Serodiagnosis of Asian leishmaniasis with a recombinant antigen from the repetitive domain of a Leishmania kinesin.Trans R Soc Trop Med Hyg 88 :543–545.

    • Search Google Scholar
    • Export Citation
  • 21

    Ritmeijer K, Melaku Y, Mueller M, Kipngetich S, O’Keeffe C, Davidson RN, 2006. Evaluation of a new recombinant K39 rapid diagnostic test for Sudanese visceral leishmaniasis. Am J Trop Med Hyg 74 :76–80.

    • Search Google Scholar
    • Export Citation
  • 22

    Singh S, Gilman-Sachs A, Chang KP, Reed SG, 1995. Diagnostic and prognostic value of K39 recombinant antigen in Indian leishmaniasis. J Parasitol 81 :1000–1003.

    • Search Google Scholar
    • Export Citation
  • 23

    Sundar S, Reed SG, Singh VP, Kumar PCK, Murray HW, 1998. Rapid accurate field diagnosis of Indian visceral leishmaniasis. Lancet 351 :563–565.

    • Search Google Scholar
    • Export Citation
  • 24

    Singh S, Kumari V, Singh N, 2002. Predicting kala-azar disease manifestation in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen. Clin Diagn Lab Immunol 9 :568–572.

    • Search Google Scholar
    • Export Citation
  • 25

    Chappuis F, Rijal S, Soto A, Menten J, Boelaert M, 2006. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ 333 :723–728.

    • Search Google Scholar
    • Export Citation
  • 26

    Zijlstra EE, Nur Y, Desjeux P, Kahill EAG, El-Hassan AM, Groen J, 2001. Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan. Trop Med Int Health 6 :108–113.

    • Search Google Scholar
    • Export Citation
  • 27

    Bhatia A, Daifalla NS, Jen S, Badaró R, Reed SG, Skeiky YAW, 1999. Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi. Mol Biochem Parasitol 102 :249–261.

    • Search Google Scholar
    • Export Citation
  • 28

    Corkill NL, 1949. The activation of latent kala-azar in relation to protein metabolism. Ann Trop Med Parasitol 43 :261.

  • 29

    Alvar J, Canavate C, Gutiérrez-Solar B, Jiménez M, Laguna F, López-Vélez R, Molina R, Moreno J, 1997. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin Microbiol Rev 10 :298–391.

    • Search Google Scholar
    • Export Citation
  • 30

    Badaró R, Jones TC, Carvalho EM, Sampaio DP, Reed SG, Barral A, Teixeira R, Johnson WD Jr, 1986. New perspectives on a sub clinical form of visceral leishmaniasis. J Infect Dis 154 :1003–1011.

    • Search Google Scholar
    • Export Citation
  • 31

    Evans TG, Teixeira MJ, Mcauliffe IT,Vasconcelos IAB, Vasconcelos AW, Sousa AQ, Lima JWO, Pearson RD, 1992. Epidemiology of visceral leishmaniasis in northeast Brazil. J Infect Dis 166 :1124–1132.

    • Search Google Scholar
    • Export Citation
  • 32

    Cabello PH, Lima AMVM, Azevedo ES, Krieger H, 1995. Familial aggregation of Leishmania chagasi infection in northeastern Brazil. Am J Trop Med Hyg 53 :364–365.

    • Search Google Scholar
    • Export Citation
  • 33

    Shido SA, Akuffo HO, Mohamed AA, Huldt G, Nilsson LA, Ouchterlony O, Thorstensson R, 1995. Visceral leishmaniasis in Somalia: prevalence of leishmanin-positive and seropositive inhabitants in an endemic area. Trans R Soc Trop Med Hyg 89 :21–24.

    • Search Google Scholar
    • Export Citation
  • 34

    D’Oliveira A Jr, Costa SRM, Barbosa AB, Orge MGO, Carvalho EM, 1997. Asymptomatic Leishmania chagasi infection in relatives and neighbors of patients with visceral leishmaniasis. Mem Inst Oswaldo Cruz 92 :15–20.

    • Search Google Scholar
    • Export Citation
  • 35

    Jerônimo SMB, Oliveira RM, Mackay S, Costa RM, Sweet J, Nascimento ET, Luz KG, Fernandes MZ, Jernigan J, Pearson RD, 1994. An urban outbreak of visceral leishmaniasis in Natal, Brazil. Trans R Soc Trop Med Hyg 88 :386–388.

    • Search Google Scholar
    • Export Citation
  • 36

    Hailu A, Menon JN, Berhe N, Gedamu L, Hassard TH, Kager PA, Olobo J, Bretscher PA, 2001. Distinct immunity in patients with visceral leishmaniasis from that in subclinically infected and drug-cured people: implications for the mechanism underlying drug cure. J Infect Dis 184 :112–115.

    • Search Google Scholar
    • Export Citation
  • 37

    Le Fichoux Y, Quaranta JF, Aufeuvre JP, Lelievre A, Marty P, Suffia I, Rousseau D, Kubar J, 1999. Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. J Clin Microbiol 37 :1953–1957.

    • Search Google Scholar
    • Export Citation
  • 38

    Riera C, Fisa R, Udina M, Gállego M, Portus M, 2004. Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans R Soc Trop Med Hyg 98 :102–110.

    • Search Google Scholar
    • Export Citation
  • 39

    Sakru N, Korkmaz M, Ozbel Y, Ertabaklar H, Sengul M, Toz SO, 2007. Investigation of asymptomatic visceral leishmaniasis cases using western blot in an endemic area in Turkey. New Microbiol 30 :13–18.

    • Search Google Scholar
    • Export Citation
  • 40

    Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, Smith PG, Sriram N, Wongsrichanalai C, Linke R, O’Brian R, Perkins M, Cunningham J, Matsoso P, Nathanson CM, Olliaro P, Peeling RW, Ramsay A, 2006. Evaluation of diagnostic tests for infectious diseases: general principles. Nat Rev Microbiol 4 (12 Suppl):S21–S31.

    • Search Google Scholar
    • Export Citation
  • 41

    Brazil. Ministry of Health. National Research Council. Resolution 196/96. October 10, 1996.

  • 42

    Badaró R, Reed SG, Barral A, Orge MGO, Jones TC, 1986. Evaluation of micro enzyme-linked immunosorbent assay (ELISA) for antibodies in American visceral leishmaniasis: antigen selection for detection of infection-specific response. Am J Trop Med Hyg 35 :72–78.

    • Search Google Scholar
    • Export Citation
  • 43

    Voller A, Bidwell DE, Bartlett A, 1976. Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull World Health Organ 53 :55–65.

    • Search Google Scholar
    • Export Citation
  • 44

    Camargo ME, 1966. Fluorescent antibody test for the serodiagnosis of American trypanosomiasis. Technical modification employing preserved culture forms of Trypanosoma cruzi in a slide test. Rev Inst Med Trop Sao Paulo 8 :227–234.

    • Search Google Scholar
    • Export Citation
  • 45

    Cerisola JA, 1970. Immunodiagnosis of Chagas’ disease: hemagglutination and immunofluorescent test. J Parasitol 50 :409–410.

  • 46

    Cerisola JA, Alvarez M, De Rissio AM, 1970. Imunodiagnóstico da doença de Chagas. Evolução sorológica de pacientes com doença de Chagas. Rev Inst Med Trop Sao Paulo 12 :403–411.

    • Search Google Scholar
    • Export Citation
  • 47

    Jelinek T, Eichenlaub S, Löscher T, 1999. Sensitivity and specificity of a rapid immunochromatographic test for diagnosis of visceral leishmaniasis. Eur J Clin Microbiol Infect Dis 18 :669–670.

    • Search Google Scholar
    • Export Citation
  • 48

    Veeken H, Ritmeijer K, Seaman J, Davidson R, 2003. Comparison of an rK39 dipstick rapid test with direct agglutination test and splenic aspiration for the diagnosis of kala-azar in Sudan. Trop Med Int Health 8 :164–167.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Comparative Study of Serologic Tests for the Diagnosis of Asymptomatic Visceral Leishmaniasis in an Endemic Area

View More View Less
  • 1 Department of Tropical Medicine and Infectology, Federal University of Triângulo Mineiro, Brazil; Laboratory of Leishmaniasis and Vaccines, Department of Parasitology, Institute of Biological Sciences, Federal University of Minas Gerais, Brazil; Clemente Faria Hospital, State University of Montes Claros, Brazil; Amazonas Institute of Tropical Medicine, Manaus, Brazil

Serologic tests have been widely used for the diagnosis of asymptomatic visceral leishmaniasis. This study evaluated five serologic tests used for the diagnosis of asymptomatic infection: enzyme-linked immunosorbent assay (ELISA) using promastigote antigen (ELISAp), ELISA using recombinant K39 (ELISA rK39), and K26 (ELISA rK26) antigens, an indirect immunofluorescence test using Leishmania (Leishmania) amazonensis promastigote antigen (IIFT), and an immunochromatographic test using rK39 antigen (TRALd). As a reference regarding the performance of the tests, patients with classic visceral leishmaniasis originating from Minas Gerais, Brazil (N = 36), were defined as the positive group and samples of healthy individuals from nonendemic areas (Argentina) (N = 127) were used as negative controls. Patients with other diseases such as cutaneous leishmaniasis (N = 53) and malaria (N = 56) were also studied to evaluate the chance of cross-reactivity in these tests. Finally, subjects from an area endemic for visceral leishmaniasis in Brazil (Porteirinha, northern Minas Gerais) (N = 1241) were screened for asymptomatic infection with Leishmania and Chagas disease. The sensitivity of the serologic tests was 50% (18/36), 66.7% (24/36), 69.4% (25/36), 83.3% (30/36), and 88.9% (32/36) for ELISAp, ELISA rK26, ELISA rK39, IIFT, and TRALd, respectively. Specificity, calculated using the truly negative group, was 96% (122/127) for TRALd, 97.6% (124/127) for ELISAp and IIFT, and 100% (127/127) for ELISA rK39 and rK26. Positivity in at least one test employing recombinant antigen was observed in 24 (45%) patients with cutaneous leishmaniasis and 47 (82.4%) with malaria. In the visceral leishmaniasis-endemic area, the positivity of the serologic tests ranged from 3.9% to 37.5%. The enzyme-linked immunosorbent assay (ELISA) tests using recombinant antigens were more frequently positive in subjects with a history of exposure to human or canine visceral leishmaniasis (ELISArK39: 14.6% [149/1017] versus 37.5% [84/224]; ELISA rK26: 12.7% [129/1017] versus 21.4% [48/224], P < 0.001 for both). Kappa agreement was low, with a maximum value of 0.449 between ELISAp and IIFT. In addition, among the 112 IIFT-positive subjects, 75 (67%) also presented positive serology for Chagas disease. In conclusion, IIFT and TRALd presented the best performance to diagnose classic cases of visceral leishmaniasis in an endemic area. Cross-reactivity of the tests with Chagas disease, cutaneous leishmaniasis, and malaria should be taken into account. However, the differences in the positivity of the tests used, together with the low agreement between results, do not permit to select the best test for the diagnosis of asymptomatic Leishmania infection.

INTRODUCTION

Visceral leishmaniasis (VL) is a neglected disease and its definitive diagnosis requires qualified professionals who rarely live in endemic areas. Consequently, serologic tests have been widely used for the diagnosis of VL since antibody titers are high and therefore easily detectable.

In Brazil, the serologic diagnosis of human VL is made by indirect immunofluorescence testing (IIFT), a technique recommended by the Brazilian Ministry of Health or by ELISA using promastigote antigens. However, the main disadvantage of these techniques is the occurrence of cross-reactivity with other diseases, such as Chagas disease, cutaneous leishmaniasis, malaria, and tuberculosis.1–13

The K39 antigen, which corresponds to a 39-amino acid repeat of a Leishmania donovani protein (specific kinesin), was developed in an attempt to improve the sensitivity and specificity of serum reactions. 14 The recombinant product (rK39) has been used as antigen in ELISA assays and also in strip tests. Both methods have shown sensitivity and specificity above 90% in Brazil, China, Sudan, Pakistan, Turkey, and India. 1524 However, Chappuis and others 25 and Zijlstra and other, 26 in Sudan, reported sensitivities of only 49% and 57%, respectively, for immunochromatographic tests using rK39. Furthermore, rK26, another recombinant antigen, showed good performance in the diagnosis of symptomatic VL, but its use has been recommended in association with rK39. 27

Most studies regarding the serologic diagnosis of VL use one or two tests as reference. Thus, a positive serologic test in the presence of compatible signs and symptoms is interpreted as VL. However, other diseases may present clinical outcomes similar to those of VL and, therefore, a single positive serologic test associated with compatible clinical signs may result in an incorrect diagnosis.

In addition to these difficulties, several authors have called attention to patients who have a benign or asymptomatic form of the disease, also called latent, asymptomatic, inapparent, or subclinical disease. These forms are much more frequent than the disease itself, with severely ill patients accounting for only 10–20% of infected individuals according to the World Health Organization (WHO). 28,29 The diagnostic elucidation of asymptomatic cases would contribute to a more adequate characterization of the epidemiology of VL and the evaluation of control actions and would serve as a marker of possible progression to the disease. 15,22

In endemic areas, as done for symptomatic disease, the diagnosis of asymptomatic VL is frequently made by serologic tests. In this respect, a positive serologic test of an asymptomatic individual living in an endemic area is interpreted as asymptomatic Leishmania infection.15,3039 This interpretation is prone to errors caused by cross-reactivity with other subclinical diseases and possible limitations of serology in terms of diagnosing asymptomatic infection. The difficulty in selecting the best serologic method for this purpose resides in the limitation of establishing a gold standard for the diagnosis of asymptomatic infected individuals, because invasive parasitologic examination (such as bone marrow puncture) is not justified in these cases.

Nowadays, several serologic tests are available for the diagnosis of VL. Selection of the best test is important to permit diagnostic differentiation between symptomatic and asymptomatic patients and to reduce cross-reactivity. Thus, the aim of this study was to evaluate the effectiveness of different serologic tests in the diagnosis of asymptomatic VL concomitantly performed in an endemic area.

SUBJECTS AND METHODS

A total of 1,513 serum samples obtained from different subjects were analyzed. Patients with classic VL (truly positive) and healthy subjects from a non-endemic area (truly negative) were studied. 40 In addition, serum samples from patients with a diagnosis of cutaneous leishmaniasis and malaria were tested to quantify the cross-reactivity with these diseases. Finally, serum samples from subjects living in VL-endemic areas were screened for the identification of asymptomatic infection. The following five groups were thus defined:

G1: patients with a recent parasitologic diagnosis of VL made at the Clemente Faria Hospital, Montes Claros, Minas Gerais, Brazil (N = 36);

G2: subjects with negative serologic tests for Chagas disease from Cordoba and Buenos Aires, Argentina (N = 127);

G3: subjects from a VL-endemic area (Porteirinha, Minas Gerais, Brazil) without a history of leishmaniasis or domiciliary contact with cases of human or canine VL (N = 1017);

G4: healthy subjects from a VL-endemic area (Porteirinha, Minas Gerais, Brazil) with a history of leishmaniasis or domiciliary contact with cases of human or canine VL (N = 224);

G5: subjects with other endemic diseases;

G5.1: subjects with cutaneous leishmaniasis from Brejo do Mutambal in Varzelândia, Minas Gerais, Brazil, where Leishmania (Viannia) braziliensis is endemic (N = 26);

G5.2: patients with confirmed cutaneous leishmaniasis from Manaus, Amazonas, Brazil, where Leishmania (Viannia) guyanensis is endemic (N = 27);

G5.3: patients with Plasmodium vivax malaria from Manaus, Amazonas, Brazil (N = 30); and

G5.4: patients with Plasmodium falciparum malaria from Manaus, Amazonas, Brazil (N = 26).

All subjects from a VL-endemic area (Porteirinha, Minas Gerais, Brazil), G3 and G4, were submitted to anamnesis and clinical examination by an infectologist. Subjects presenting no clinical history or physical signs compatible with VL (fever, paleness, visceromegaly, adynamia, weight loss) were classified as asymptomatic.

The serum samples were collected between January 1998 and May 1999 and between January and October 2001, and were processed in 1999 and 2001, respectively. The TRALd, ELISA rK39, and ELISA rK26 tests were performed at the Laboratory of Immunology, Federal University of Triângulo Mineiro (UFTM). Another aliquot stored at −20°C was kept on dry ice and sent to the Laboratory of Leishmaniasis and Vaccines, Institute of Biologic Sciences, Federal University of Minas Gerais (ICB/UFMG), Belo Horizonte, for ELISA and IIFT using promastigote antigen. All laboratory researchers were unaware of the origin of the samples.

All serum samples were analyzed by ELISAp, ELISA rK26, ELISA rK39, IIFT, and TRALd as described below. In addition to these tests, samples originating from the VL-endemic area, G3 (N = 1017) and G4 (N = 224), were also submitted to serology for Chagas disease.

Informed consent was obtained from all participants and the project was approved by the Board of UFTM, according to Resolution 196/96 of the National Research Council, Brazilian Ministry of Health. 41

Statistical analysis was performed using the Statistica 6.0 program and the figures were prepared with the SigmaPlot 2004 for Windows program, version 9.0 (Systat Software, Inc., San Jose, CA). Agreement between results was assessed using the kappa coefficient calculated with the SPSS 16.0 program (SPSS Inc., Chicago, IL, 2007). Frequencies were compared using the χ2 test, with the level of significance set at 95% (P < 0.05). Spearman’s correlation test was used to evaluate the correlation between age and positivity of the serologic tests, with the level of significance set at 95% (P < 0.05).

Serologic tests for visceral leishmaniasis.

TRAL-d (rK39).

For the immunochromatographic test, a kit (InBios International, Seattle, WA) consisting of paper strips coated with rK39 antigen was used according to manufacturer instructions. The strips were stored at room temperature (28–30°C) or refrigerated (2–8°C) and protected from humidity. The buffer solution was stored at 2–8°C. Readings were obtained 10 minutes later according to manufacturer recommendations and the results are reported as positive or negative.

ELISA using recombinant antigens (ELISA rK39 and ELISA rK26).

The assays were carried out according to Badaró and others 42 using recombinant K39 and K26 antigens of Leishmania (Leishmania) chagasi produced by InBios International. The serum samples were diluted 1:50 and 1:100 and developed with peroxidase-conjugated protein A (Sigma Co., St. Louis, MO). The cut-off value was established as the mean absorbance plus 3 standard deviations of the negative controls (G2).

ELISA using promastigote antigen (ELISAp).

The assay was carried out as described by Hommel and others9 and Voller and others 43 using as antigen L. (L.) amazonensis (MHOM/BR/60/BH6) promastigotes in the stationary growth phase cultured in liver infusion tryptose (LIT) medium and lysed by sonication. Serum samples were tested at an initial dilution of 1:80. The cut-off value for anti-Leishmania IgG reactivity was established using 40 sera obtained from subjects truly negative for American cutaneous leishmaniasis stored at the Laboratory of Leishmaniasis and Vaccines, UFMG, Belo Horizonte. The cut-off was defined as the mean absorbance of these negative control sera plus 3 standard deviations.

Indirect immunofluorescence test (IIFT).

The assay was performed as described by Camargo 44 using L. (L.) amazonensis (MHOM/BR/60/BH6) promastigotes in the exponential growth phase cultured in LIT medium as antigen. Fluorescein isothiocyanate-labeled human IgG anti-globulin obtained from rabbit immune serum was used as conjugate (Biomanguinhos, Rio de Janeiro, Brazil). All samples showing positivity at a dilution of 1:80 were considered to be reactive.

Serologic tests for Chagas disease.

Subjects of G2 were characterized using an indirect hemagglutination assay (IHA) and IIFT for T. cruzi antibodies performed at the Laboratory of Analysis, Ministry of Health of Cordoba, Argentina. The subjects from the VL-endemic area were tested by IHA and IIFT according to the techniques described by Camargo 44 and Cerisola. 45,46 The samples were considered to be positive for Chagas disease when both tests were positive.

RESULTS

The absorbance results of the ELISA tests and IIFT titers are shown in Figure 1. The mean IIFT titer was approximately 1/320 (two dilutions above the cut-off of 1/80) in the group with VL (G1), whereas the mean titer was close to zero in the control group from Argentina (G2), with this test thus permitting good discrimination between positive and negative controls. The same was observed for the ELISA rK39 test, with a mean absorbance of 2.129 for the positive control group. This value was higher than the mean obtained for the negative control (0.135) plus 12 standard deviations (3 standard deviations were used for calculation of the cut-off). The discrimination between positive and negative controls was not as clear for the ELISAp and ELISA rK26 tests, with the mean optical densities of the positive controls being similar to the cut-off values (ELISAp, mean in G1: 0.209, cut-off: 0.170; ELISA rK26, mean in G1: 0.822, cut-off: 0.552).

In the endemic area, mean absorbance values obtained for samples from endemic areas were lower than the cutoff for all tests (Figure 1). However, the discrimination between the mean and cut-off of the tests was not as clear, especially when analyzing ELISAp in G3 (mean: 0.142 and cut-off: 0.170) and ELISA rK39 in G4 (mean: 0.599 and cutoff: 0.623).

With respect to the other diseases (G5), an interesting finding was the high mean absorbance obtained for patients with malaria in the tests using recombinant antigens, with absorbance ranging from 1.200 to 1.666 and a cut-off of 0.623 for ELISA rK39 and 0.552 for ELISA rK26.

The results of positivity in the different tests obtained for each group are shown in Table 1. Sensitivity and specificity of the tests were calculated using the results of the positive and negative controls as reference. Sensitivity was 50% (18/36), 66.7% (24/36), 69.4% (25/36), 83.3% (30/36), and 88.9% (32/36) for ELISAp, ELISA rK26, ELISA rK39, IIFT, and TRALd, respectively. Specificity was 96% (122/127) for TRALd, 97.6% (124/127) for ELISAp and IIFT, and 100% (127/127) for ELISA rK39 and rK26.

Among patients with cutaneous leishmaniasis, serum samples from Varzelândia (G5.1) showed higher reactivity in the tests using conventional antigens than those from Manaus (G5.2) (ELISAp: G5.1 = 46.1% [12/26] versus G5.2 = 29.6% [8/27] and IIFT: G5.1 = 34.6% [9/26] versus G5.2 = 22.2% [6/27]), whereas opposite results were obtained with the assays using recombinant antigens (ELISArK39: G5.1 = 19.2% [5/26] versus G5.2 = 51.8% [14/27] and ELISA rK26: G5.1 = 19.2% [5/26] versus G5.2 = 29.6% [8/27]). However, these differences were not statistically significant. In addition, serum samples from patients with malaria presented more than 80% reactivity in ELISA using recombinant antigens (Table 1).

In the endemic area, medical evaluation, clinical history, and physical examination revealed no signs compatible with VL in any of the subjects. Age ranged from 1 to 93 years, with a mean age of 26.2 years (SD = ±19.2) in G3 and of 25.9 years (SD = ±19.4) in G4. A correlation was observed between increased age and an increase in the number of subjects who tested seropositive by ELISAp and IIFT (P ≤ 0.05) (Figure 2). Positivity of the serologic tests ranged from 3.9% to 37.5% depending on the test and group (Table 1).

Among subjects from the endemic area, the frequency of positive reactions to the recombinant antigens was significantly higher among subjects previously in contact with VL than among those with no contact (ELISArK39: 14.6% [149/1017] versus 37.5% [84/224] and ELISA rK26: 12.7% [129/1017] versus 21.4% [48/224], P < 0.001 for both). Chagas disease was diagnosed by IHA and IIFT in 96 (9.4%) subjects from G3 and 25 (11.2%) from G4. No significant difference in the percentage of subjects who were seropositive for Chagas disease was observed between G3 and G4. Of these, 117 (96.7%) were older than 20 years of age, with a mean age of 40.7 years (SD = ±15.2). Thus, considering all subjects from the endemic area, a positive serologic test for Chagas disease was observed among 75 (67.0%) of subjects who also tested positive for Leishmania by IIFT, 90 (28.7%) with positive ELISAp, 43 (18.4%) with positive ELISA rK39, 28 (15.8%) with positive ELISA rK26, and 6 (11.3%) with positive TRALd (Figure 3).

The lowest kappa coefficient of agreement was observed between ELISA rK26 and ELISAp (0.035) and the highest value between ELISAp and IIFT (0.449) (Table 2). The best agreement was observed between tests that used similar antigens: ELISAp and IIFT (0.449) and ELISA rK39 and ELISA rK26 (0.403).

DISCUSSION

The sensitivity of the serologic tests used for the diagnosis of VL in this study was lower than expected. This finding might be attributed to the relatively small group of positive VL assays. Low sensitivity of the strip test using rK39 antigen has been reported in some studies 25,26,34,47; however, we observed a higher sensitivity in this study. Evaluation of ELISA using recombinant antigens showed that rK39 better discriminated cases of VL than rK26. The lowest sensitivity was observed for ELISA using promastigote antigen, a finding that might be explained by the preparation of the antigen; lack of a standardized preparation protocol and use of L. (L.) amazonensis. The IIFT showed good performance in detecting subjects with VL and, therefore, has also been adopted by the Brazilian Ministry of Health.

The specificity of the tests calculated only for asymptomatic subjects from Argentina ranged from 96.1% to 98.4%. However, this selected group does not represent the reality in VL-endemic areas, especially Brazil where subjects are in contact with Leishmania spp. and other diseases. As evidence, mean absorbance values obtained for serum samples from the endemic area were higher than those observed for the Argentina samples for all serologic tests. In this respect, it is difficult to select the best control group for population studies. If, instead, we would choose a group of asymptomatic subjects from an endemic area using one test as a reference (IIFT or ELISAp), this “negative control” group would include subjects who are seroreactive in other tests (ELISA rK39, ELISArK26, etc.), because there is no agreement between the results of the tests. There is currently no gold standard for the serologic diagnosis of asymptomatic Leishmania infection.

In the endemic area, one interesting finding was the correlation between the increase in age and positivity by ELISA and IIFT. This result seems to be coherent with the diagnosis of asymptomatic infection made by these tests, because a longer period of exposure in the endemic area would increase the chance of acquisition of this infection. However, this prolonged exposure would also increase the chance of contracting other infections that potentially result in cross-reactivity with Leishmania, such as Chagas disease. In fact, we found a large number of subjects with a positive serologic test for Chagas disease among those who were diagnosed with asymptomatic VL by conventional tests, especially IIFT. We were unable to determine whether this finding was caused by cross-reactivity with Chagas disease or to asymptomatic infection. Thus, these results suggest that serologic tests using recombinant antigens might be more specific for the diagnosis of Leishmania infection in endemic areas. Furthermore, positivity was higher among subjects from endemic areas who are in contact with VL for the assays using recombinant antigen than for conventional tests.

However, we also observed a significant percentage of positive reactions to recombinant antigens among patients with cutaneous leishmaniasis and malaria. Curiously, patients from Manaus (with cutaneous leishmaniasis and malaria) showed important reactivity to rK39 antigen. In this respect, other investigators also reported cross-reactivity with rK39 in patients with cutaneous leishmaniasis. 17,25 This fact might be a result of the possible homology between K39 and other molecules present in dermatotropic Leishmania species. We are unable to explain the cross-reactivity with malaria; however, these results agree with Veeken and others 48 who found 70% specificity when testing samples from patients with brucellosis or malaria. It is important to note that Burns and others, 14 in their original work describing rK39, did not include malaria samples in the control group. On the other hand, Guimarães and others 18 analyzed samples from 10 patients with malaria and none of them was reactive to rK39.

The rK39 antigen used in the ELISA or strip test showed no advantages for the diagnosis of VL in endemic areas compared with IIFT, with a similar performance in the diagnosis of cases of VL. The IIFT presented high cross-reactivity with Chagas disease and rK39 cross-reacted with malaria samples. Therefore, because tests using rK39 are more expensive than IIFT, our results are in accordance with the Brazilian Ministry of Health which recommends the use of IIFT as a conventional test financed by the government.

With respect to the diagnosis of asymptomatic infection with Leishmania, the low agreement between the results of the different tests did not permit to select a method that would identify infected individuals. The reactivity of the tests may also indicate cross-positivity with other subclinical diseases such as the indeterminate form of Chagas disease. Thus, the serologic diagnosis of asymptomatic infection in endemic areas using only one serologic test may yield a false result and may over- or underestimate the number of infected individuals. Further studies are necessary to identify a serologic test that shows good sensitivity and specificity in endemic areas and can be used for the diagnosis of asymptomatic Leishmania infection.

Table 1

Positivity of the serologic tests for the diagnosis of visceral leishmaniasis in the different groups studied*

Table 1
Table 2

Results of the analysis of kappa agreement between serologic tests for the diagnosis of visceral leishmaniasis

Table 2
Figure 1.
Figure 1.

Optical density values of the serologic tests according to test and group (—: test cut-off; __: group mean). (A) Indirect immunofluorescence test (cut-off: 1:80); (B) enzyme-linked immunosorbent assay (ELISA) using promastigote antigen (cut-off: 0.170); (C) ELISA using rK39 recombinant antigen (cut-off: 0.623); (D) ELISA using rK26 recombinant antigen (cut-off: 0.552). G1: kala-azar; G2: Argentina; G3: endemic area/no contact with visceral leishmaniasis; G4: endemic area/contact with visceral leishmaniasis; G5: other endemic disease; G5.1: cutaneous leishmaniasis/ Brejo do Mutambal; G5.2: cutaneous leishmaniasis/Manaus; G5.3: P. vivax malaria/Manaus; G5.4: P. falciparum malaria/Manaus.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 1;

Figure 2.
Figure 2.

Spearman’s correlation coefficient between age and positive ELISAp and IIFT in subjects from an endemic area. (A) Correlation between ELISAp and age of subjects from G3. (B ) Correlation between ELISAp and age of subjects from G4. (C) Correlation between IIFT and age of subjects from G3. (D) Correlation between IIFT and age of subjects from G4. G3: Serum samples of subjects from an endemic area without contact with visceral leishmaniasis; G4: serum samples of subjects from an endemic area with domiciliary contact with visceral leishmaniasis.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 1;

Figure 3.
Figure 3.

Positive serologic test for visceral leishmaniasis and positive serology for Chagas disease in subjects from an endemic area (G3 + G4). G3: Serum samples of subjects from an endemic area without contact with visceral leishmaniasis; G4: serum samples of subjects from an endemic area with domiciliary contact with visceral leishmaniasis.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 1;

*

Address correspondence to Luciana de Almeida Silva, Department of Tropical Medicine and Infectology, Federal University of Triângulo Mineiro, Caixa Postal: 118, CEP 38001-970, Uberaba, Brazil. E-mail: lalmeidas@dcm.uftm.edu.br

In memoriam.

Authors’ addresses: Héctor Dardo Romero, Luciana de Almeida Silva, Mario Leon Silva-Vergara, Virmondes Rodrigues, and Aluízio Prata, Department of Tropical Medicine and Infectology, Federal University of Triângulo Mineiro, Caixa Postal: 118, CEP 38001-970, Uberaba, MG, Brazil, Tel: 34-3318-5254, Fax: 34-3318-5229. Helio Moraes-Souza, Department of Hematology, Federal University of Triângulo Mineiro, Caixa Postal: 118, CEP 38001-970, Uberaba, MG, Brazil, Tel: 34-3318-5254, Fax: 34-3318-5229. Sílvio Fernandes Guimarães, Clemente Faria Hospital, State University of Montes Claros, Av. Cula Mangabeira 562, CEP 39401-002 Montes Claros, MG, Brazil. Wilson Alecrim, Amazonas Institute of Tropical Medicine, Av. Pedro Teixeira 25, CEP 69040-000, Manaus, AM, Brazil.

Acknowledgments: We thank Antônio Campos-Neto for providing the rK39 and rK26 antigens, Roberto Badaró for providing the TRALd strips, and Gabriel Nascentes for help with the statistical analysis and revision of the manuscript.

Financial support: The study was supported by the National Council for Scientific and Technological Development and the National Health Foundation.

REFERENCES

  • 1

    Abramo C, Fontes CJ, Krettli AU, 1995. Cross-reactivity between antibodies in the sera of individuals with leishmaniasis, toxo-plasmosis, and Chagas’ disease and antigens of the blood-stage forms of Plasmodium falciparum determined by indirect immunofluorescence. Am J Trop Med Hyg 53 :202–205.

    • Search Google Scholar
    • Export Citation
  • 2

    Araujo FG, Mayrink W, 1968. Fluorescent antibody test in visceral leishmaniasis. II. Studies on the specificity of the test. Rev Inst Med Trop Sao Paulo 10 :41–45.

    • Search Google Scholar
    • Export Citation
  • 3

    Bray RS, Lainson R, 1965. The immunology and serology of leishmaniasis: I. The fluorescent antibody staining technique. Trans R Soc Trop Med Hyg 59 :535–544.

    • Search Google Scholar
    • Export Citation
  • 4

    Camargo MA, Rebonato C, 1969. Cross-reactivity in fluorescent test for Trypanosoma and leishmania antibody. Am J Trop Med Hyg 18 :500–505.

    • Search Google Scholar
    • Export Citation
  • 5

    Edrissian H, Darabian P, 1979. A comparison of enzyme-linked immunosorbent assay and indirect fluorescent antibody test in the serodiagnosis of cutaneous and visceral leishmaniasis in Iran. Trans R Soc Trop Med Hyg 73 :289–292.

    • Search Google Scholar
    • Export Citation
  • 6

    Evans TG, Krug EC, Wilson ME, Vasconcelos AW, De Alencar JE, Pearson RD, 1989. Evaluation of antibody responses in American visceral leishmaniasis by ELISA and immunoblot. Mem Inst Oswaldo Cruz 84 :157–166.

    • Search Google Scholar
    • Export Citation
  • 7

    Guimarães MCS, Celeste BJ, Castilho EA, Mineo JR, Diniz JMP, 1981. Immunoenzymatic assay (ELISA) in mucocutaneous leishmaniasis, kala-azar, and Chagas’ disease: an epimastigote Trypanosoma cruzi antigen able to distinguish between anti-Trypanosoma and anti-leishmania antibodies. Am J Trop Med Hyg 30 :942–947.

    • Search Google Scholar
    • Export Citation
  • 8

    Harit EA, Kolk AHJ, Kager PA, Leeuwenburg J, Faber FJ, Muigai R, Kiugu S, Laarman JJ, 1987. Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and seroepidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Trans R Soc Trop Med Hyg 81 :603–606.

    • Search Google Scholar
    • Export Citation
  • 9

    Hommel M, Peters W, Ranque J, Quilici M, Lanotte G, 1978. The micro-ELISA technique in the serodiagnosis of visceral leishmaniasis. Ann Trop Med Parasitol 72 :213–218.

    • Search Google Scholar
    • Export Citation
  • 10

    Mohammed AR, Wright EP, Kager PA, Laarman JJ, Pondman KW, 1985. ELISA using intact promastigotes for immunodiagnosis of kala-azar. Trans R Soc Trop Med Hyg 79 :344–350.

    • Search Google Scholar
    • Export Citation
  • 11

    Walton BC, Brooks WH, Arjona I, 1972. Serodiagnosis of American leishmaniasis by indirect fluorescent antibody test. Trans R Soc Trop Med Hyg 21 :296–299.

    • Search Google Scholar
    • Export Citation
  • 12

    Ho M, Leeuwenburg J, Mbugua G, Wamachi A, Voller A, 1983. An enzyme-linked immunosorbent assay (ELISA) for field diagnosis of visceral leishmaniasis. Am J Trop Med Hyg 32 :943–946.

    • Search Google Scholar
    • Export Citation
  • 13

    Jahn A, Lelmett M, Diesfeld HJ, 1986. Seroepidemiological study on kala-azar in Baringo District, Kenya. J Trop Med Hyg 89 :91–104.

  • 14

    Burns JM Jr, Shreffler WG, Benson DR, Ghalib HW, Badaró R, Reed SG, 1993. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci USA 90 :775–779.

    • Search Google Scholar
    • Export Citation
  • 15

    Badaró R, Benson D, Eulalio MC, Freire M, Cunha S, Netto EM, Pedral-Sampaio D, Madureira C, Burns JM, Houghton RL, David JR, Reed SG, 1996. rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis 173 :758–761.

    • Search Google Scholar
    • Export Citation
  • 16

    Bern C, Jha SN, Joshi AB, Thakur GD, Bista MB, 2000. Use of the recombinant K39 dipstick test and the direct agglutination test in a setting endemic for visceral leishmaniasis in Nepal. Am J Trop Med Hyg 63 :153–157.

    • Search Google Scholar
    • Export Citation
  • 17

    Braz RFS, Nascimento ET, Martins DRA, Wilson ME, Pearson RD, Reed SG, Jerônimo SMB, 2002. The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection. Am J Trop Med Hyg 67 :344–348.

    • Search Google Scholar
    • Export Citation
  • 18

    Guimarães SF, Lemos EM, Corey R, Dietze R, 2003. Performance of recombinant K39 antigen in the diagnosis of Brazilian visceral leishmaniasis. Am J Trop Med Hyg 68 :321–324.

    • Search Google Scholar
    • Export Citation
  • 19

    Kumar R, Pai K, Pathak K, Sundar S, 2001. Enzyme-linked immunosorbent assay for recombinant K39 antigen in diagnosis and prognosis of Indian visceral leishmaniasis. Clin Diagn Lab Immunol 8 :1220–1224.

    • Search Google Scholar
    • Export Citation
  • 20

    Qu JQ, Zhong L, Masoom-Yasinzai M, Abdur-Rab M, Aksu HS, Reed SG, Chang KP, Gilman-Sachs A, 1994. Serodiagnosis of Asian leishmaniasis with a recombinant antigen from the repetitive domain of a Leishmania kinesin.Trans R Soc Trop Med Hyg 88 :543–545.

    • Search Google Scholar
    • Export Citation
  • 21

    Ritmeijer K, Melaku Y, Mueller M, Kipngetich S, O’Keeffe C, Davidson RN, 2006. Evaluation of a new recombinant K39 rapid diagnostic test for Sudanese visceral leishmaniasis. Am J Trop Med Hyg 74 :76–80.

    • Search Google Scholar
    • Export Citation
  • 22

    Singh S, Gilman-Sachs A, Chang KP, Reed SG, 1995. Diagnostic and prognostic value of K39 recombinant antigen in Indian leishmaniasis. J Parasitol 81 :1000–1003.

    • Search Google Scholar
    • Export Citation
  • 23

    Sundar S, Reed SG, Singh VP, Kumar PCK, Murray HW, 1998. Rapid accurate field diagnosis of Indian visceral leishmaniasis. Lancet 351 :563–565.

    • Search Google Scholar
    • Export Citation
  • 24

    Singh S, Kumari V, Singh N, 2002. Predicting kala-azar disease manifestation in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen. Clin Diagn Lab Immunol 9 :568–572.

    • Search Google Scholar
    • Export Citation
  • 25

    Chappuis F, Rijal S, Soto A, Menten J, Boelaert M, 2006. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ 333 :723–728.

    • Search Google Scholar
    • Export Citation
  • 26

    Zijlstra EE, Nur Y, Desjeux P, Kahill EAG, El-Hassan AM, Groen J, 2001. Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan. Trop Med Int Health 6 :108–113.

    • Search Google Scholar
    • Export Citation
  • 27

    Bhatia A, Daifalla NS, Jen S, Badaró R, Reed SG, Skeiky YAW, 1999. Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi. Mol Biochem Parasitol 102 :249–261.

    • Search Google Scholar
    • Export Citation
  • 28

    Corkill NL, 1949. The activation of latent kala-azar in relation to protein metabolism. Ann Trop Med Parasitol 43 :261.

  • 29

    Alvar J, Canavate C, Gutiérrez-Solar B, Jiménez M, Laguna F, López-Vélez R, Molina R, Moreno J, 1997. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin Microbiol Rev 10 :298–391.

    • Search Google Scholar
    • Export Citation
  • 30

    Badaró R, Jones TC, Carvalho EM, Sampaio DP, Reed SG, Barral A, Teixeira R, Johnson WD Jr, 1986. New perspectives on a sub clinical form of visceral leishmaniasis. J Infect Dis 154 :1003–1011.

    • Search Google Scholar
    • Export Citation
  • 31

    Evans TG, Teixeira MJ, Mcauliffe IT,Vasconcelos IAB, Vasconcelos AW, Sousa AQ, Lima JWO, Pearson RD, 1992. Epidemiology of visceral leishmaniasis in northeast Brazil. J Infect Dis 166 :1124–1132.

    • Search Google Scholar
    • Export Citation
  • 32

    Cabello PH, Lima AMVM, Azevedo ES, Krieger H, 1995. Familial aggregation of Leishmania chagasi infection in northeastern Brazil. Am J Trop Med Hyg 53 :364–365.

    • Search Google Scholar
    • Export Citation
  • 33

    Shido SA, Akuffo HO, Mohamed AA, Huldt G, Nilsson LA, Ouchterlony O, Thorstensson R, 1995. Visceral leishmaniasis in Somalia: prevalence of leishmanin-positive and seropositive inhabitants in an endemic area. Trans R Soc Trop Med Hyg 89 :21–24.

    • Search Google Scholar
    • Export Citation
  • 34

    D’Oliveira A Jr, Costa SRM, Barbosa AB, Orge MGO, Carvalho EM, 1997. Asymptomatic Leishmania chagasi infection in relatives and neighbors of patients with visceral leishmaniasis. Mem Inst Oswaldo Cruz 92 :15–20.

    • Search Google Scholar
    • Export Citation
  • 35

    Jerônimo SMB, Oliveira RM, Mackay S, Costa RM, Sweet J, Nascimento ET, Luz KG, Fernandes MZ, Jernigan J, Pearson RD, 1994. An urban outbreak of visceral leishmaniasis in Natal, Brazil. Trans R Soc Trop Med Hyg 88 :386–388.

    • Search Google Scholar
    • Export Citation
  • 36

    Hailu A, Menon JN, Berhe N, Gedamu L, Hassard TH, Kager PA, Olobo J, Bretscher PA, 2001. Distinct immunity in patients with visceral leishmaniasis from that in subclinically infected and drug-cured people: implications for the mechanism underlying drug cure. J Infect Dis 184 :112–115.

    • Search Google Scholar
    • Export Citation
  • 37

    Le Fichoux Y, Quaranta JF, Aufeuvre JP, Lelievre A, Marty P, Suffia I, Rousseau D, Kubar J, 1999. Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. J Clin Microbiol 37 :1953–1957.

    • Search Google Scholar
    • Export Citation
  • 38

    Riera C, Fisa R, Udina M, Gállego M, Portus M, 2004. Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans R Soc Trop Med Hyg 98 :102–110.

    • Search Google Scholar
    • Export Citation
  • 39

    Sakru N, Korkmaz M, Ozbel Y, Ertabaklar H, Sengul M, Toz SO, 2007. Investigation of asymptomatic visceral leishmaniasis cases using western blot in an endemic area in Turkey. New Microbiol 30 :13–18.

    • Search Google Scholar
    • Export Citation
  • 40

    Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, Smith PG, Sriram N, Wongsrichanalai C, Linke R, O’Brian R, Perkins M, Cunningham J, Matsoso P, Nathanson CM, Olliaro P, Peeling RW, Ramsay A, 2006. Evaluation of diagnostic tests for infectious diseases: general principles. Nat Rev Microbiol 4 (12 Suppl):S21–S31.

    • Search Google Scholar
    • Export Citation
  • 41

    Brazil. Ministry of Health. National Research Council. Resolution 196/96. October 10, 1996.

  • 42

    Badaró R, Reed SG, Barral A, Orge MGO, Jones TC, 1986. Evaluation of micro enzyme-linked immunosorbent assay (ELISA) for antibodies in American visceral leishmaniasis: antigen selection for detection of infection-specific response. Am J Trop Med Hyg 35 :72–78.

    • Search Google Scholar
    • Export Citation
  • 43

    Voller A, Bidwell DE, Bartlett A, 1976. Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull World Health Organ 53 :55–65.

    • Search Google Scholar
    • Export Citation
  • 44

    Camargo ME, 1966. Fluorescent antibody test for the serodiagnosis of American trypanosomiasis. Technical modification employing preserved culture forms of Trypanosoma cruzi in a slide test. Rev Inst Med Trop Sao Paulo 8 :227–234.

    • Search Google Scholar
    • Export Citation
  • 45

    Cerisola JA, 1970. Immunodiagnosis of Chagas’ disease: hemagglutination and immunofluorescent test. J Parasitol 50 :409–410.

  • 46

    Cerisola JA, Alvarez M, De Rissio AM, 1970. Imunodiagnóstico da doença de Chagas. Evolução sorológica de pacientes com doença de Chagas. Rev Inst Med Trop Sao Paulo 12 :403–411.

    • Search Google Scholar
    • Export Citation
  • 47

    Jelinek T, Eichenlaub S, Löscher T, 1999. Sensitivity and specificity of a rapid immunochromatographic test for diagnosis of visceral leishmaniasis. Eur J Clin Microbiol Infect Dis 18 :669–670.

    • Search Google Scholar
    • Export Citation
  • 48

    Veeken H, Ritmeijer K, Seaman J, Davidson R, 2003. Comparison of an rK39 dipstick rapid test with direct agglutination test and splenic aspiration for the diagnosis of kala-azar in Sudan. Trop Med Int Health 8 :164–167.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Luciana de Almeida Silva, Department of Tropical Medicine and Infectology, Federal University of Triângulo Mineiro, Brazil, Caixa Postal: 118, CEP 38001-970, Uberaba, MG, Brazil, E-mail: lalmeidas@dcm.uftm.edu.br.
Save