Inaccuracy of enzyme-linked immunosorbent assay using soluble and recombinant antigens to detect asymptomatic infection by Leishmania infantum.
BACKGROUND
One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period.


METHODS
Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve.


RESULTS
The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10).


CONCLUSIONS
Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.
Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Programa de Pós-Graduação em Parasitologia, Minas Gerais, Brasil.
Elizabeth Castro
EC
Moreno
Andréa Vieira
AV
Gonçalves
Anderson Vieira
AV
Chaves
Maria Norma
MN
Melo
José Roberto
JR
Lambertucci
Antero Silva Ribeiro
AS
Andrade
Deborah
D
Negrão-Corrêa
Carlos Mauricio
CM
de Figueiredo Antunes
Mariângela
M
Carneiro
D000328:Adult; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology; D015331:Cohort Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D018314:Leishmania infantum / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis* / Q000276:immunology / Q000469:parasitology; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D055815:Young Adult
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D011994:Recombinant Proteins