• View in gallery

    ROC curves obtained for the ELISA assay. ( A ) ROC curves for the visceral L. donovani (thick solid line) and L. infantum (thin solid line) species. ( B ) ROC curves for the cutaneous L. amazonensis (thick dotted line) and L. major (thin dotted line) species. The dotted grey line represents the reference line.

  • View in gallery

    IgG antibody levels determined by ELISA against ( A ) L. donovani , ( B ) L. infantum , ( C ) L. amazonensis , and ( D ) L. major soluble extracts in sera of patients with cutaneous (CLO and CLN) or visceral leishmaniasis [VL(Ld) and VL(Li)], malaria (M), Leishmania -negative with dermal pathologies (DP), Leishmania -negative with other pathologies (OP), healthy endemic (EN), and healthy non-endemic individuals (NEN). Results are expressed as the optical densities at 492 nm. Dotted line represents cut-off values (as determined by the ROC curves) between negative and positive results. Each dot represents an individual serum; bars display median.

  • View in gallery

    ROC curves obtained for the flow cytometry assay. ( A ) ROC curves for the visceral L. donovani (thick solid line) and L. infantum (thin solid line) species. ( B ) ROC curves for the cutaneous L. amazonensis (thick dotted line) and L. major (thin dotted line) species. The dotted grey line represents the reference line.

  • View in gallery

    Flow cytometry analysis of IgG antibodies against ( A ) L. donovani , ( B ) L. infantum , ( C ) L. amazonensis , and ( D ) L. major in the sera of patients with cutaneous (CLO and CLN) or visceral leishmaniasis [VL(Ld) and VL(Li)], malaria (M), Leishmania -negative with dermal pathologies (DP), Leishmania -negative with other pathologies (OP), healthy endemic (EN), and healthy non-endemic individuals (NEN). Dotted line represents cut-off values (as determined by the ROC curves) between negative and positive results. Each dot represents an individual serum; bars display median.

  • 1

    Evans TG, 1993 . Leishmaniasis. Infect Dis Clin North Am 7 :527– 546.

  • 2

    Escobar MA, Martinez F, Scott Smith D, Palma GI, 1992 . American cutaneous and mucocutaneous leishmaniasis (tegumentary): a diagnostic challenge. Trop Doct 22 :69– 78.

    • Search Google Scholar
    • Export Citation
  • 3

    Spira AM, 2003 . Assessment of travellers who return home ill. Lancet 362 :83– 84.

  • 4

    Herwaldt BL, 1999 . Leishmaniasis. Lancet 354 :1191– 1199.

  • 5

    Reithinger R, Dujardin JC, 2007 . Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 45 :21– 25.

    • Search Google Scholar
    • Export Citation
  • 6

    Schallig HD, Schoone GJ, Kroon CC, Hailu A, Chappuis F, Veeken H, 2001 . Development and application of “simple” diagnostic tools for visceral leishmaniasis. Med Microbiol Immunol (Berl) 190 :69– 71.

    • Search Google Scholar
    • Export Citation
  • 7

    Schoone GJ, Hailu A, Kroon CC, Nieuwenhuys JL, Schallig HD, Oskam L, 2001 . A fast agglutination screening test (FAST) for the detection of anti- Leishmania antibodies. Trans R Soc Trop Med Hyg 95 :400– 401.

    • Search Google Scholar
    • Export Citation
  • 8

    Noya O, Patarroyo ME, Guzmán F, Alarcón de Noya B, 2003 . Immunodiagnosis of parasitic diseases with synthetic peptides. Curr Protein Pept Sci 4 :299– 308.

    • Search Google Scholar
    • Export Citation
  • 9

    Rocha RD, Gontijo CM, Eloi-Santos SM, Teixeira Carvalho A, Correa-Oliveira R, Marques MJ, Genaro O, Mayrink W, Martins-Filho OA, 2002 . Anti-live Leishmania (Viannia) braziliensis promastigote antibodies, detected by flow cytometry, to identify active infection in american cutaneous leishmaniasis. Rev Soc Bras Med Trop 35 :551– 562.

    • Search Google Scholar
    • Export Citation
  • 10

    Singh S, Dey A, Sivakumar R, 2005 . Applications of molecular methods for Leishmania control. Expert Rev Mol Diagn 5 :251– 265.

  • 11

    Santarém N, Tomás A, Ouaissi A, Tavares J, Ferreira N, Manso A, Campino L, Correia JM, Cordeiro-da-Silva A, 2005 . Antibodies against a Leishmania infantum peroxiredoxin as a possible marker for diagnosis of visceral leishmaniasis and for monitoring the efficacy of treatment. Immunol Lett 101 :18– 23.

    • Search Google Scholar
    • Export Citation
  • 12

    Silvestre R, Santarém N, Cunha J, Cardoso L, Nieto J, Carrillo E, Moreno J, Cordeiro-da-Silva A, 2008 . Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amas-tigote-flow cytometry. Vet Parasitol 158 :23– 30.

    • Search Google Scholar
    • Export Citation
  • 13

    van der Meide WF, Sabajo LO, Jensema AJ, Peekel I, Faber WR, Schallig HD, Fat RF, 2009 . Evaluation of treatment with pent-amidine for cutaneous leishmaniasis in Suriname. Int J Dermatol 48 :52– 58.

    • Search Google Scholar
    • Export Citation
  • 14

    Dantas-Torres F, 2006 . Leishmania infantum versus Leishmania chagasi : do not forget the law of priority. Mem Inst Oswaldo Cruz 101 :117– 118.

    • Search Google Scholar
    • Export Citation
  • 15

    Minodier P, Piarroux R, Gambarelli F, Joblet C, Dumon H, 1997 . Rapid identification of causative species in patients with Old World leishmaniasis. J Clin Microbiol 35 :2551– 2555.

    • Search Google Scholar
    • Export Citation
  • 16

    Shamsuzzaman SM, Furuya M, Choudhury AK, Korenaga M, HashiguchiY,2000 . Characterization of Bangladeshi Leishmania isolated from kala-azar patients by isoenzyme electrophoresis. Parasitol Int 49 :139– 145.

    • Search Google Scholar
    • Export Citation
  • 17

    Zemzoumi K, Sereno D, François C, Guilvard E, Lemesre JL, Ouaissi A, 1998 . Leishmania major : cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family. Biol Cell 90 :239– 245.

    • Search Google Scholar
    • Export Citation
  • 18

    Zweig MH, Campbell G, 1993 . Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem 39 :561– 577.

    • Search Google Scholar
    • Export Citation
  • 19

    Swets JA, 1988 . Measuring the accuracy of diagnostic systems. Science 240 :1285– 1293.

  • 20

    Garin YJ, Meneceur P, Pratlong F, Dedet JP, Derouin F, Lorenzo F, 2005 . A2 gene of Old World cutaneous Leishmania is a single highly conserved functional gene. BMC Infect Dis 5 :18 .

    • Search Google Scholar
    • Export Citation
  • 21

    Moody SF, 1993 . Molecular variation in Leishmania . Acta Trop 53 :185– 204.

  • 22

    Requena JM, Alonso C, Soto M, 2000 . Evolutionarily conserved proteins as prominent immunogens during Leishmania infections. Parasitol Today 16 :246– 250.

    • Search Google Scholar
    • Export Citation
  • 23

    Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, Sisk E, Rajandream MA, Adlem E, Aert R, Anupama A, Apostolou Z, Attipoe P, Bason N, Bauser C, Beck A, Beverley SM, Bianchettin G, Borzym K, Bothe G, Bruschi CV, Collins M, Cadag E, Ciarloni L, Clayton C, Coulson RM, Cronin A, Cruz AK, Davies RM, De Gaudenzi J, Dobson DE, Duesterhoeft A, Fazelina G, Fosker N, Frasch AC, Fraser A, Fuchs M, Gabel C, Goble A, Goffeau A, Harris D, Hertz-Fowler C, Hilbert H, Horn D, Huang Y, Klages S, Knights A, Kube M, Larke N, Litvin L, Lord A, Louie T, Marra M, Masuy D, Matthews K, Michaeli S, Mottram JC, Müller-Auer S, Munden H, Nelson S, Norbertczak H, Oliver K, O’neil S, Pentony M, Pohl TM, Price C, Purnelle B, Quail MA, Rabbinowitsch E, Reinhardt R, Rieger M, Rinta J, Robben J, Robertson L, Ruiz JC, Rutter S, Saunders D, Schäfer M, Schein J, Schwartz DC, Seeger K, Seyler A, Sharp S, Shin H, Sivam D, Squares R, Squares S, Tosato V, Vogt C, Volckaert G, Wambutt R, Warren T, Wedler H, Woodward J, Zhou S, Zimmermann W, Smith DF, Blackwell JM, Stuart KD, Barrell B, Myler PJ, 2005 . The genome of the kinetoplastid parasite, Leishmania major . Science 309 :436– 442.

    • Search Google Scholar
    • Export Citation
  • 24

    Stevens JR, Noyes HA, Schofield CJ, Gibson W, 2001 . The molecular evolution of Trypanosomatidae . Adv Parasitol 48 :1– 56.

  • 25

    Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, Quail MA, Peters N, Adlem E, Tivey A, Aslett M, Kerhornou A, Ivens A, Fraser A, Rajandream MA, Carver T, Norbertczak H, Chillingworth T, Hance Z, Jagels K, Moule S, Ormond D, Rutter S, Squares R, Whitehead S, Rabbinowitsch E, Arrowsmith C, White B, Thurston S, Bringaud F, Baldauf SL, Faulconbridge A, Jeffares D, Depledge DP, Oyola SO, Hilley JD, Brito LO, Tosi LR, Barrell B, Cruz AK, Mottram JC, Smith DF, Berriman M, 2007 . Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet 39 :839– 847.

    • Search Google Scholar
    • Export Citation
  • 26

    Lynn MA, McMaster WR, 2008 . Leishmania : conserved evolution-diverse diseases. Trends Parasitol 24 :103– 105.

  • 27

    Rocha RD, Gontijo CM, Elói-Santos SM, Teixeira-Carvalho A, Corrêa-Oliveira R, Ferrari TC, Marques MJ, Mayrink W, Martins-Filho AO, 2006 . Clinical value of anti-live Leishmania (Viannia) braziliensis immunoglobulin G subclasses, detected by flow cytometry, for diagnosing active localized cutaneous leishmaniasis. Trop Med Int Health 11 :156– 166.

    • Search Google Scholar
    • Export Citation
  • 28

    Pissinate JF, Gomes IT, Peruhype-Magalhães V, Dietze R, Martins-Filho OA, Lemos EM, 2008 . Upgrading the flow-cytometric analysis of anti- Leishmania immunoglobulins for the diagnosis of American tegumentary leishmaniasis. J Immunol Methods 336 :193– 202.

    • Search Google Scholar
    • Export Citation
  • 29

    Junqueira Pedras M, Orsini M, Castro M, Passos VM, Rabello A, 2003 . Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis 47 :477– 485.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Evaluation of Leishmania Species Reactivity in Human Serologic Diagnosis of Leishmaniasis

View More View Less
  • 1 Parasite Disease Group, IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal; Departamento de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Portugal; KIT (Koninklijk Instituut voor de Tropen/Royal Tropical Institute), KIT Biomedical Research, Amsterdam, The Netherlands

The sensitivities and specificities of IgG-ELISA and IgG flow cytometry based techniques using different Leishmania species were determined using sera collected from 40 cutaneous or visceral leishmaniasis patients. The flow cytometry technique, using promastigote parasite forms, performed better than total soluble extract IgG-ELISA. At the species level, the use of Leishmania amazonensis and Leishmania major as antigens in enzyme linked immunosorbent assay (ELISA) decreased the overall sensitivity. To assess the specificity of these tests, sera from malaria, toxoplasmosis, amoebiasis, schistosomiasis, and leprosy patients were used. We also included sera from Leishmania non-infected endemic individuals. The cutaneous species displayed a decreased specificity in both assays. Although more sensitive, flow cytometry using promastigote parasite forms generally presented lower levels of specificity when compared with total extract of IgG-ELISA. Overall, the results of the study show the potential of IgG flow cytometry for the diagnosis of leishmaniasis. Although highly sensitive, a refinement of the flow cytometry method should be performed to improve the overall specificity.

INTRODUCTION

Leishmaniasis is one of the major communicable diseases of the world, being the third most important vectorborne disease after malaria and sleeping sickness. Leishmaniasis is traditionally classified into three major clinical manifestations: cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL). 1 These distinct entities are characterized by a broad spectrum of non-pathognomonic clinical manifestations. Leishmaniasis symptoms can mimic diseases of other etiologies rendering the clinical diagnosis to be difficult. Furthermore, other pathologies with similar clinical manifestations (leprosy, tuberculosis, skin cancers, cutaneous mycoses for CL and malaria, African trypanosomiasis, brucellosis, toxoplasmosis, amoebiasis or schistosomiasis for VL) are common in Leishmania endemic areas. 2 Hence, a differential diagnosis is essential for confirming the clinical suspicion. 3

Ideally, all cases of leishmaniasis should be confirmed by direct detection and identification of infecting parasite species observation. Clinical specimens examined are usually infected skin biopsies (for CL and MCL) or aspirates from spleen, bone marrow or lymph nodes (for VL). The definitive diagnosis is achieved when parasites are detected by direct observation in stained microscopic preparations, in culture medium or in animal inoculation. 4 Although parasitologic diagnosis still remains the method of choice to confirm leishmaniasis, it is time-consuming and not feasible under field conditions. Moreover, the sensitivity of this technique can be low in sub-clinical infections depending on the clinical material, the sampling procedure and on the skills of the technical staff. Recently, the use of molecular biology techniques (polymerase chain reaction [PCR]-based) is becoming increasingly relevant to the diagnosis of leishmaniasis because it is capable of detecting DNA or RNA unique to the parasite, with a high degree of specificity and sensitivity just a few weeks after the appearance of the first clinical symptoms. 5 Although molecular biology techniques can be used in the confirmation of all leishmaniasis cases, these methodologies are frequently the most powerful diagnosis tests producing positive results in CL, MCL and human immunodeficiency virus (HIV)/VL co-infected patients in which the humoral response is generally weak. Nevertheless, the use of PCR as a routine diagnostic method requires high equipment and working costs, which limits the feasibility of PCR diagnosis in developing countries.

Serologic approaches are also sensitive and often applied as indirect diagnostic methods. They are more commonly used in the diagnosis of VL than in CL or MCL, because the latter are in most cases associated with low levels of Leishmania circulating antibodies. 3 Among the serologic methods, the agglutination tests (direct agglutination test [DAT] and fast agglutination screening test [FAST]) are specific and sensitive tests, suitable for both laboratory and field use. 6 , 7 These tests use whole trypsinized, coomassie stained promastigotes either as a suspension or in a freeze-dried form. Other serologic methods, such as indirect fluorescent antibody test (IFAT), immunoblotting, and enzyme-linked immunosorbent assay (ELISA) are classic methods used in the detection of Leishmania -specific antibodies. With the exception of ELISA, the latter are methods normally limited to laboratory conditions and require technical expertise. In contrast, ELISA-based techniques are valuable tools in the serodiagnosis of leishmaniasis and allow high throughput screening of a large number of samples. Most commonly used in ELISA are total crude soluble antigens derived from the promastigote stage of different Leishmania species. 8 These ELISA showed sensitivities and specificities ranging from 80% to 100% and 85% to 95%, respectively. More recently, alternative methodologies have been proposed to increase overall sensitivity and specificity of conventional serologic approaches. One such approach is IgG flow cytometry to detect anti-live Leishmania braziliensis antibodies in sera of active cutaneous leishmaniasis patients. 9 In theory, the flow cytometry technique can specifically detect the antibodies recognizing the Leishmania surface antigens, therefore helping to restrain the potential cross-reactivity against more conserved intracellular structures. In addition, flow cytometry advantageously allows the analysis of thousands of parasites per assay. Therefore, it is generally accepted that while the diagnostic sensitivity depends on the test and the serologic procedure used, the specificity will depend strictly on the antigen rather than the methodology itself. 10

The objective of this study was to evaluate Leishmania spp.-specific recognition using both Leishmania promastigote total soluble extract IgG-ELISA and Leishmania promastigote IgG flow cytometry techniques.

MATERIAL AND METHODS

Enzyme linked immunosorbent assay (ELISA) for immunoglobulin.

The soluble promastigote extracts and the ELISA technique were performed adapting the protocols described elsewhere with minor modifications. 11 , 12 Briefly, after blocking with milk buffer (3% of low-fat milk in phosphate buffered saline pH 7.4 [PBS]), 50 μL of an optimized 1/100 dilution in PBS/Tween 20 0.05% of each serum was incubated for 30 min at 37°C in wells previously coated with 10 μg/mL of each extract. A peroxidase labeled goat anti-human IgG (Sigma, St. Louis, MO) secondary antibody diluted at 1:5000 was added for a similar period of time, and the plates were developed with 0.5 mg/mL o-phenylenediamine dihydrochloride (OPD, Sigma) in citrate buffer.

Immunofluorescence by flow cytometry.

The flow cytometry analyses with intact live Leishmania spp. promastigotes were performed as follows. The parasites were recovered from stationary-phase (5 days) cultures. After three washing steps with PBS, 5 × 10 5 promastigotes were incubated in 96-well U-bottom polysterene plates (BD Falcon, San Jose, CA) with 25 μL of each serum diluted 1/50 in PBS/bovine serum albumin (BSA) 1% for 30 min at 4°C. Then, 10 μL of mouse fluorescein (FITC) labeled anti-human IgG (BD Pharmingen, San Diego, CA) were incubated in each well for 30 min at 4°C. After three washing steps, Leishmania spp.promastigotes were re-suspended in 300 μL of PBS/BSA 1% and analyzed by flow cytometry in a fluorescence-activated cell sorting (FACS) scan equipped with CellQuest Pro software (BD Bioscences, San Jose, CA). In all experiments we included positive and negative controls as previously described. 11 Parasites were identified on the basis of forward/side scatter values and a total of 10.000 events were acquired. For each experiment, on the basis of the histogram representing the binding of parasites incubated in the absence of human serum but in the presence of FITC conjugated mouse anti-human, an area was chosen to contain a maximum of 2.0% of positive fluorescent parasites (PFP). This area allowed for the measurement of the % PFP in all data analyzed.

Patients.

We recurred to both techniques to measure the levels of anti- Leishmania immunoglobulins in the sera of 10 Surinamese cutaneous leishmaniasis patients caused by Leishmania (Viannia) guyanensis (cutaneous leishmaniasis New World [CLN]) 13 , which presented one to three lesions on their extremities and 10 Afghan cutaneous leishmaniasis patients, with chronic single to few lesions on extremities caused by Leishmania major (cutaneous leishmaniasis Old World [CLO]). In addition, we included 10 Indian visceral leishmaniasis patients, caused by Leishmania donovani (VL [Ld]) and 10 Brazilian visceral leishmaniasis patients, caused by Leishmania infantum (= Leishmania chagasi ) 14 [VL (Li)], all patients with clinical symptoms. Written informed consent was obtained from all patients included in this study.

Parasites.

The soluble antigens and the live parasites were obtained from four distinct Leishmania species: L. major (MHOM/MA/81/LEM 265) 15 and Leishmania amazonensis (originated from the strain collection of WHO; MHOM/BR/76/LTB-012), the main ethiologic agents of cutaneous leishmaniasis in the Old World and in the New World, respectively, and L. donovani (reference strain MHOM/IN/83/ DD8) 16 and L.infantum (= L.chagasi ) (reference strain MHOM/ MA/67/ITMAP-263), both agents of visceral leishmaniasis in the Old World. Promastigotes of all Leishmania species were cultured as previously described. 17

RESULTS

Cut-off determination for the ELISA assays.

The ELISA cut-off values for the soluble extract of each Leishmania spp. were defined based on the receiver operating curves (ROC) 18 using sera obtained from non-endemic healthy individuals (Dutch volunteers) ( N = 10) as negative controls and the reactivity of all leishmaniasis patients, both CL and VL, as positive values ( Figure 1 ). Thus, we established as ELISA cut-off points the optical density (OD) reactivity at 492 nm values of 0.416 for L. donovani , 0.389 for L. infantum , 0.396 for L. amazonensis , and 0,486 for L. major . According to Swets, all of the Leishmania soluble extracts tested presented similar accuracies since proximal values were obtained for the area under (AUC) each ROC curve: L. donovani AUC = 0.96; 95% confidence interval (CI): 0.92–1.0; L. infantum AUC = 0.98; 95% CI: 0.95–1.0; L. amazonensis AUC = 0.96; 95% CI: 0.91–1.0; and L. major AUC = 0.95; 95% CI: 0.89–1.0. 19

VL species present better overall sensitivity in the ELISA technique.

Values of overall sensitivity varied from 87.5% to 95%, depending on the Leishmania spp. evaluated ( Table 1 ). Among all groups of leishmaniasis patients, higher Leishmania IgG recognition was observed for VL patients, regardless of the antigen used. Indeed, all 10 VL sera were found positive for Leishmania spp.-specific IgG, except when L. major antigen was used; in that case one serum sample was found below cut-off value ( Figure 2 ). However, the serologic diagnosis of CL remains problematic because of a lack of sensitivity and specificity when compared with their visceral counterparts 9 and an evident correlation between species-specific diagnosis and pathology could not be found in the present study.

L. major and L. amazonensis are both causative agents of CL, we anticipated a higher sensitivity for autologous antigens. Nonetheless, for the CL patients sera a better sensitivity was obtained when the visceral species, L. donovani and L. infantum (90%) were used as antigen, compared with their cutaneous counterparts, L. amazonensis and L. major (80%) in ELISA assay. Although surprising, there are several possible explanations for these results. First, we must consider that the reduced number of patients evaluated might contribute to an apparent lack of species-specific diagnosis. Second, the IgG reactivity of CL patients was found not to be significantly different in these two species, L. amazonensis and L. major , than when using visceral ones. The sera reactivity at 492 nm of CLO and CLN patients was 0.48 ± 0.22 and 0.78 ± 0.27, respectively, using L. amazonensis as antigen and 0.57 ± 0.23 and 0.99 ± 0.36, respectively, using L. major ( Figure 2 ). When visceral species were used as antigen in the ELISA technique, these values were 0.62 ± 0.30 and 1.16 ± 0.46, respectively, for L. donovani and 0.53 ± 0.24 and 1.10 ± 0.45, respectively, for L. infantum . This data may indicate that the sera of CL patients, which possess lower levels of Leishmania -specific antibodies, recognize preferably the most immunogenic Leishmania proteins, which should be present in higher quantities or differently transcribed/translated in visceral when compared with cutaneous species. For example, the amastigote stage specific A2 protein in CL isolates was lacking most of the nucleotide repeats that constitute the variable region at the 5′ end of the A2 sequences in VL that are responsible for its immunogenicity. 20 Further studies using a higher number of cutaneous disease patients and Leishmania species are needed to explore the lack of species-specific diagnosis in CL.

Determination of IgG ELISA specificity.

The cross-reactivity of the ELISA was assessed using sera from microscopically confirmed anonomized P. falciparum cases (malaria; M) ( N = 10), leprosy (dermal pathologies; DP) ( N = 5), and other pathologies ([OP; toxoplasmosis [ N = 5], amoebiasis [ N = 5], and schistosomiasis [ N = 2]) that are infections that frequently overlap with endemic areas of leishmaniasis. In addition, we included sera recovered from Leishmania non-infected individuals living in endemic areas. High frequency of cross-reactivity was observed from sera of malaria patients ( Table 1 ). However, all sera recovered from patients with leprosy were found to react below the cut-off value for any antigen used. A similar observation was performed for the sera recovered from patients with other pathologies (toxoplasmosis, amoebiasis, and schistosomiasis). Only one serum was found to be positive in all Leishmania soluble extracts, with the exception of L. infantum . Moreover, a significant cross-reactivity was found for healthy individuals living in endemic areas of leishmaniasis ( Table 1 ). The overall specificity of the ELISA technique was high, varying from 82.4% with both cutaneous species to 85.9% for L. donovani and 89.5% for L. infantum.

Flow cytometry cut-off determination.

The flow cytometry approach was initially developed to overcome the limitations of current ELISA diagnostic approaches. The flow cytometry technique has the advantage of using only outside membrane antigens as the epitope antigenic source for IgG binding. Hence, a decrease of cross-reactivity should be expected because the more conserved intra-cytoplasmatic epitope antigens would not be involved in the serologic detection. Similar to ELISA, the flow cytometry cut-off values for each Leishmania spp. were defined based on the ROC using sera obtained from non-endemic healthy individuals as negative controls and the reactivity of all leishmaniasis patients, both CL and VL, as positive values ( Figure 3 ). Thus, we established the % PF P values of 14.63 for L. donovani , 11.57 for L. infantum , 11.12 for L. amazonensis , and 10.9 for L. major as flow cytometry cut-off values. All of the Leishmania species presented excellent accuracies with similar AUC ([ L. donovani AUC = 0.97; 95% CI: 0.94–1.0]; [ L. infantum AUC = 0.97; 95% CI: 0.93–1.0]; [ L. amazonensis AUC = 0.98; 95% CI: 0.93–1.0]; [ L. major AUC = 0.99; 95% CI: 0.98–1.0]). 19

The flow cytometry methodology, although less specific, presents a higher sensitivity than ELISA technique efficiently diagnosing CL.

The flow cytometry analysis using promastigote parasite form improved the IgG recognition, with an overall sensitivity of 95% using L. major or the visceral species and 97.5% using L. amazonensis ( Table 2 ). All sera recovered from the 10 VL patients were found to be positive regardless of the antigen used ( Figure 4 and Table 2 ). Although no significant differences were found in overall sensitivity, a better species specificity was observed during the flow cytometry analysis. The measurement of flow cytometry % PFP using sera from CLO and CLN patients was mean 30.66 ± 13.70 and 34.37 ± 23.26, respectively, using L. amazonensis and 33.47 ± 23.69 and 35.86 ± 16.23, respectively, using L. major . In contrast to ELISA measurements, the % PF P values were higher than those obtained using as antigen the visceral species, 23.54 ± 8.96 and 33.81 ± 15.00, respectively, using L. donovani and 19.70 ± 8.98 and 31.89 ± 16.09, respectively, using L. infantum . Similarly, VL patient sera preferentially recognize visceral species (with the % PF P values of 66.82 ± 31.99 and 70.30 ± 23.37 using L. donovani and L. infantum , respectively) when compared with the cutaneous ones (with the % PF P values of 46.84 ± 24.78 and 59.20 ± 31.23 using L. amazonensis and L. major , respectively) (two-tailed student’s t test, P < 0.05 between L. donovani or L. infantum and L. amazonensis for the VL sera). Most likely, structural variation of major surface and released molecules between Leishmania species, which might lead to species-specific recognition, is responsible for this behavior. 21

DISCUSSION

Although a large improvement was observed in terms of sensitivity using the flow cytometry methodology, a loss of specificity was observed, especially with the cutaneous species used as antigen ( Table 2 ). This was mainly caused by cross-reactivity with sera from malaria, amoebiasis and schistosomiasis patients, with no positive reactivity observed with sera from toxoplasmosis patients. In addition, there was one leprosy serum that cross-reacted with L. major promastigotes. Interestingly, a high level of cross-reactivity was found against sera recovered from healthy endemic individuals. The observed cross-reactivity can be explained by the presence of several evolutionarily conserved antigens identified in the Leishmania genome database. 22 , 23

Therefore, the cross-reactivity observed with sera from malaria, amoebiasis and schistosomiasis patients might be the result of the presence of highly conserved epitopes of immunogenic Leishmania proteins present in both visceral and cutaneous forms. In opposition, the cross-reactivity detected with endemic healthy individuals was higher in flow cytometry than ELISA assays. This suggests previous contact(s) of healthy individuals living in Leishmania endemic areas with the parasite. Indeed, because not all infected sand-fly bites result in active infection, a transient and inconsequent contact with the parasite might allow the production of antibodies against easy accessible surface antigens. Overall, our results show that the cross-reactivity was higher when cutaneous species were used. Given the ancient evolutionary divergence in Leishmania species, 24 it is not surprising that different Leishmania species, in particular visceral versus cutaneous species, are recognized differently by the host immune system. Although the recent genome sequence completion of three Leishmania species show a high degree of conservation with less than 1% of species-specific genes, 25 it now seems clear that alternate translational control and protein stability will be responsible for the different phenotypical characteristics, such as Leishmania species tropism. 26 Similarly, one may consider that these mechanisms might lead to increased epitope conservation on cutaneous strains, which will explain their increased cross-reactivity observed in this study. Future comparative genetic and proteomic studies will certainly elucidate this point.

Recently, the determination of IgG subclasses by flow cytometry was proposed as an upgrading methodology to obtain overall improved performance. 27 , 28 In addition, another study showed considerably reduced cross-reactivity in the ELISA technique by measuring anti- Leishmania IgG1 antibodies. 29 Further studies are needed to investigate in detail the IgG subclass responses toward the different Leishmania species using both methodologies. Moreover, efforts should be made to develop a flow cytometry methodology using fixed promastigotes that present long-term stability. Because the manipulation of live parasites still constitute, although at a low extent, a risk of infection, this approach might be safer and also will assure a worldwide reproducible diagnostic test for all types of leishmaniasis.

In summary, on the basis of our results, we conclude that 1) the IgG flow cytometry methodology offers higher sensitivity than the ELISA technique; 2) promastigote visceral species offer better overall sensitivities in both techniques; 3) the diagnosis of CL, caused by Old and New World species, can be efficiently achieved recurring to flow cytometry; and 4) further studies focusing on IgG subclasses should be performed to improve the unexpected low specificity of flow cytometry.

T able 1

Sensitivity and specificity of anti- Leishmania donovani, Leishmania infantum, Leishmania amazonensis and Leishmania major enzyme-linked immunosorbent assay (ELISA) technique in the diagnostics of cutaneous and visceral leishmaniasis*

T
able
 1
T able 2

Sensitivity and specificity of anti- Leishmania donovani, Leishmania infantum, Leishmaniaamazonensis, and Leishmania major flow cytometry in the diagnostics of cutaneous and visceral leishmaniasis*

T
able
 2
F igure 1.
F
igure
 1.

ROC curves obtained for the ELISA assay. ( A ) ROC curves for the visceral L. donovani (thick solid line) and L. infantum (thin solid line) species. ( B ) ROC curves for the cutaneous L. amazonensis (thick dotted line) and L. major (thin dotted line) species. The dotted grey line represents the reference line.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 2; 10.4269/ajtmh.2009.81.202

F igure 2.
F
igure
 2.

IgG antibody levels determined by ELISA against ( A ) L. donovani , ( B ) L. infantum , ( C ) L. amazonensis , and ( D ) L. major soluble extracts in sera of patients with cutaneous (CLO and CLN) or visceral leishmaniasis [VL(Ld) and VL(Li)], malaria (M), Leishmania -negative with dermal pathologies (DP), Leishmania -negative with other pathologies (OP), healthy endemic (EN), and healthy non-endemic individuals (NEN). Results are expressed as the optical densities at 492 nm. Dotted line represents cut-off values (as determined by the ROC curves) between negative and positive results. Each dot represents an individual serum; bars display median.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 2; 10.4269/ajtmh.2009.81.202

F igure 3.
F
igure
 3.

ROC curves obtained for the flow cytometry assay. ( A ) ROC curves for the visceral L. donovani (thick solid line) and L. infantum (thin solid line) species. ( B ) ROC curves for the cutaneous L. amazonensis (thick dotted line) and L. major (thin dotted line) species. The dotted grey line represents the reference line.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 2; 10.4269/ajtmh.2009.81.202

F igure 4.
F
igure
 4.

Flow cytometry analysis of IgG antibodies against ( A ) L. donovani , ( B ) L. infantum , ( C ) L. amazonensis , and ( D ) L. major in the sera of patients with cutaneous (CLO and CLN) or visceral leishmaniasis [VL(Ld) and VL(Li)], malaria (M), Leishmania -negative with dermal pathologies (DP), Leishmania -negative with other pathologies (OP), healthy endemic (EN), and healthy non-endemic individuals (NEN). Dotted line represents cut-off values (as determined by the ROC curves) between negative and positive results. Each dot represents an individual serum; bars display median.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 2; 10.4269/ajtmh.2009.81.202

*

Address correspondence to Anabela Cordeiro-da-Silva, Parasite Disease Group, Biology of Infection and Immunology, Instituto de Biologia Molecular e Celular da Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. E-mail: cordeiro@ibmc.up.pt

These authors contributed equally to this work.

Authors’ addresses: Ricardo Silvestre, Nuno Santarém, Lúcia Teixeira, Joana Cunha, and Anabela Cordeiro-da-Silva, Parasite Disease Group, Biology of Infection and Immunology, IBMC—Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal, Tel: +351-226-074-900, Fax: +351-226-099-157, and Departamento de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Portugal, Rua Aníbal Cunha, 164, 4050-047 Porto, Tel: 00351-222-078-900, Fax: 00351-222-003-977, E-mails: rleal@ibmc.up.pt , nsantarem@portugalmail.pt , jcunha@ibmc.up.pt , luciat@ibmc.up.pt , and cordeiro@ibmc.up.pt . Henk Schallig, KIT (Koninklijk Instituut voor de Tropen/Royal Tropical Institute), KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands, Tel: 0031(0)20-5665447, Fax: 0031(0)20-6971841, E-mail: h.schallig@kit.nl .

Financial support: R. Silvestre and N. Santarém were supported by fellowships from FCT, POCI 2010, and co-funded by FEDER in the project SFRH/BPD/41476/2007 and POCI/SAU-FCT/59837/2004, respectively. The work was also supported by FCT, POCI 2010, and co-funded by FEDER in the project PTD/SAU-FCF/67351/2006 and PTDC/CVT/65047/2006

Disclosure: This study was performed as part of a reviewed and approved protocol by the Medical Ethical Committee of the Academic Medical Center in Amsterdam (MEC 03/228) in 2003.

REFERENCES

  • 1

    Evans TG, 1993 . Leishmaniasis. Infect Dis Clin North Am 7 :527– 546.

  • 2

    Escobar MA, Martinez F, Scott Smith D, Palma GI, 1992 . American cutaneous and mucocutaneous leishmaniasis (tegumentary): a diagnostic challenge. Trop Doct 22 :69– 78.

    • Search Google Scholar
    • Export Citation
  • 3

    Spira AM, 2003 . Assessment of travellers who return home ill. Lancet 362 :83– 84.

  • 4

    Herwaldt BL, 1999 . Leishmaniasis. Lancet 354 :1191– 1199.

  • 5

    Reithinger R, Dujardin JC, 2007 . Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 45 :21– 25.

    • Search Google Scholar
    • Export Citation
  • 6

    Schallig HD, Schoone GJ, Kroon CC, Hailu A, Chappuis F, Veeken H, 2001 . Development and application of “simple” diagnostic tools for visceral leishmaniasis. Med Microbiol Immunol (Berl) 190 :69– 71.

    • Search Google Scholar
    • Export Citation
  • 7

    Schoone GJ, Hailu A, Kroon CC, Nieuwenhuys JL, Schallig HD, Oskam L, 2001 . A fast agglutination screening test (FAST) for the detection of anti- Leishmania antibodies. Trans R Soc Trop Med Hyg 95 :400– 401.

    • Search Google Scholar
    • Export Citation
  • 8

    Noya O, Patarroyo ME, Guzmán F, Alarcón de Noya B, 2003 . Immunodiagnosis of parasitic diseases with synthetic peptides. Curr Protein Pept Sci 4 :299– 308.

    • Search Google Scholar
    • Export Citation
  • 9

    Rocha RD, Gontijo CM, Eloi-Santos SM, Teixeira Carvalho A, Correa-Oliveira R, Marques MJ, Genaro O, Mayrink W, Martins-Filho OA, 2002 . Anti-live Leishmania (Viannia) braziliensis promastigote antibodies, detected by flow cytometry, to identify active infection in american cutaneous leishmaniasis. Rev Soc Bras Med Trop 35 :551– 562.

    • Search Google Scholar
    • Export Citation
  • 10

    Singh S, Dey A, Sivakumar R, 2005 . Applications of molecular methods for Leishmania control. Expert Rev Mol Diagn 5 :251– 265.

  • 11

    Santarém N, Tomás A, Ouaissi A, Tavares J, Ferreira N, Manso A, Campino L, Correia JM, Cordeiro-da-Silva A, 2005 . Antibodies against a Leishmania infantum peroxiredoxin as a possible marker for diagnosis of visceral leishmaniasis and for monitoring the efficacy of treatment. Immunol Lett 101 :18– 23.

    • Search Google Scholar
    • Export Citation
  • 12

    Silvestre R, Santarém N, Cunha J, Cardoso L, Nieto J, Carrillo E, Moreno J, Cordeiro-da-Silva A, 2008 . Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amas-tigote-flow cytometry. Vet Parasitol 158 :23– 30.

    • Search Google Scholar
    • Export Citation
  • 13

    van der Meide WF, Sabajo LO, Jensema AJ, Peekel I, Faber WR, Schallig HD, Fat RF, 2009 . Evaluation of treatment with pent-amidine for cutaneous leishmaniasis in Suriname. Int J Dermatol 48 :52– 58.

    • Search Google Scholar
    • Export Citation
  • 14

    Dantas-Torres F, 2006 . Leishmania infantum versus Leishmania chagasi : do not forget the law of priority. Mem Inst Oswaldo Cruz 101 :117– 118.

    • Search Google Scholar
    • Export Citation
  • 15

    Minodier P, Piarroux R, Gambarelli F, Joblet C, Dumon H, 1997 . Rapid identification of causative species in patients with Old World leishmaniasis. J Clin Microbiol 35 :2551– 2555.

    • Search Google Scholar
    • Export Citation
  • 16

    Shamsuzzaman SM, Furuya M, Choudhury AK, Korenaga M, HashiguchiY,2000 . Characterization of Bangladeshi Leishmania isolated from kala-azar patients by isoenzyme electrophoresis. Parasitol Int 49 :139– 145.

    • Search Google Scholar
    • Export Citation
  • 17

    Zemzoumi K, Sereno D, François C, Guilvard E, Lemesre JL, Ouaissi A, 1998 . Leishmania major : cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family. Biol Cell 90 :239– 245.

    • Search Google Scholar
    • Export Citation
  • 18

    Zweig MH, Campbell G, 1993 . Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem 39 :561– 577.

    • Search Google Scholar
    • Export Citation
  • 19

    Swets JA, 1988 . Measuring the accuracy of diagnostic systems. Science 240 :1285– 1293.

  • 20

    Garin YJ, Meneceur P, Pratlong F, Dedet JP, Derouin F, Lorenzo F, 2005 . A2 gene of Old World cutaneous Leishmania is a single highly conserved functional gene. BMC Infect Dis 5 :18 .

    • Search Google Scholar
    • Export Citation
  • 21

    Moody SF, 1993 . Molecular variation in Leishmania . Acta Trop 53 :185– 204.

  • 22

    Requena JM, Alonso C, Soto M, 2000 . Evolutionarily conserved proteins as prominent immunogens during Leishmania infections. Parasitol Today 16 :246– 250.

    • Search Google Scholar
    • Export Citation
  • 23

    Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, Sisk E, Rajandream MA, Adlem E, Aert R, Anupama A, Apostolou Z, Attipoe P, Bason N, Bauser C, Beck A, Beverley SM, Bianchettin G, Borzym K, Bothe G, Bruschi CV, Collins M, Cadag E, Ciarloni L, Clayton C, Coulson RM, Cronin A, Cruz AK, Davies RM, De Gaudenzi J, Dobson DE, Duesterhoeft A, Fazelina G, Fosker N, Frasch AC, Fraser A, Fuchs M, Gabel C, Goble A, Goffeau A, Harris D, Hertz-Fowler C, Hilbert H, Horn D, Huang Y, Klages S, Knights A, Kube M, Larke N, Litvin L, Lord A, Louie T, Marra M, Masuy D, Matthews K, Michaeli S, Mottram JC, Müller-Auer S, Munden H, Nelson S, Norbertczak H, Oliver K, O’neil S, Pentony M, Pohl TM, Price C, Purnelle B, Quail MA, Rabbinowitsch E, Reinhardt R, Rieger M, Rinta J, Robben J, Robertson L, Ruiz JC, Rutter S, Saunders D, Schäfer M, Schein J, Schwartz DC, Seeger K, Seyler A, Sharp S, Shin H, Sivam D, Squares R, Squares S, Tosato V, Vogt C, Volckaert G, Wambutt R, Warren T, Wedler H, Woodward J, Zhou S, Zimmermann W, Smith DF, Blackwell JM, Stuart KD, Barrell B, Myler PJ, 2005 . The genome of the kinetoplastid parasite, Leishmania major . Science 309 :436– 442.

    • Search Google Scholar
    • Export Citation
  • 24

    Stevens JR, Noyes HA, Schofield CJ, Gibson W, 2001 . The molecular evolution of Trypanosomatidae . Adv Parasitol 48 :1– 56.

  • 25

    Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, Quail MA, Peters N, Adlem E, Tivey A, Aslett M, Kerhornou A, Ivens A, Fraser A, Rajandream MA, Carver T, Norbertczak H, Chillingworth T, Hance Z, Jagels K, Moule S, Ormond D, Rutter S, Squares R, Whitehead S, Rabbinowitsch E, Arrowsmith C, White B, Thurston S, Bringaud F, Baldauf SL, Faulconbridge A, Jeffares D, Depledge DP, Oyola SO, Hilley JD, Brito LO, Tosi LR, Barrell B, Cruz AK, Mottram JC, Smith DF, Berriman M, 2007 . Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet 39 :839– 847.

    • Search Google Scholar
    • Export Citation
  • 26

    Lynn MA, McMaster WR, 2008 . Leishmania : conserved evolution-diverse diseases. Trends Parasitol 24 :103– 105.

  • 27

    Rocha RD, Gontijo CM, Elói-Santos SM, Teixeira-Carvalho A, Corrêa-Oliveira R, Ferrari TC, Marques MJ, Mayrink W, Martins-Filho AO, 2006 . Clinical value of anti-live Leishmania (Viannia) braziliensis immunoglobulin G subclasses, detected by flow cytometry, for diagnosing active localized cutaneous leishmaniasis. Trop Med Int Health 11 :156– 166.

    • Search Google Scholar
    • Export Citation
  • 28

    Pissinate JF, Gomes IT, Peruhype-Magalhães V, Dietze R, Martins-Filho OA, Lemos EM, 2008 . Upgrading the flow-cytometric analysis of anti- Leishmania immunoglobulins for the diagnosis of American tegumentary leishmaniasis. J Immunol Methods 336 :193– 202.

    • Search Google Scholar
    • Export Citation
  • 29

    Junqueira Pedras M, Orsini M, Castro M, Passos VM, Rabello A, 2003 . Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis 47 :477– 485.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Anabela Cordeiro-da-Silva, Parasite Disease Group, Biology of Infection and Immunology, Instituto de Biologia Molecular e Celular da Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal, E-mail: cordeiro@ibmc.up.pt .
Save