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ABSTRACT |
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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In Peru, there are two forms of leishmaniasis: sylvatic leishmaniasis (SL) and Andean cutaneous leishmaniasis (ACL). SL, caused by Leishmania (Viannia) brazilensis, predominates in the Amazonian forest and is characterized by aggressive cutaneous lesions that can metastasize, resulting in mucocutaneous leishmaniasis (known locally as "Espundia"), which causes severe tissue destruction of the face.3 ACL caused by Leishmania (Viannia) peruviana (known locally as "Uta") is found in the western Andean and inter-Andean valleys and results in cutaneous lesions that are generally benign.4 Limited areas of overlap exist between both diseases, but these are becoming increasingly complicated by migration of people back and forth between endemic areas from each disease.
Because leishmaniasis has a broad clinical spectrum, diagnosis of both present and past cases may be difficult. Differential diagnosis is important, because diseases of other etiologies (e.g., Chagas’ disease, leprosy, sporotrichosis, skin cancers, cutaneous tuberculosis) with clinical symptoms similar to those of leishmaniasis are often present in leishmaniasis-endemic areas.5 Parasitologic diagnosis, the "gold standard" for leishmaniasis patients, is generally based on microscopic examination of a Giemsa-stained lesion scraping, aspirate or biopsy impression smears, histopathological examination of fixed lesion biopsies, or in vitro culture of biopsy triturates or aspirates. Therefore, the identification can be accomplished by isoenzyme analysis of cultured promastigotes or with various molecular methods.6 However, the sensitivity of this technique may be highly variable, depending on the number and dispersion of parasites in the biopsy samples.
Recently, several protocols based on polymerase chain reaction (PCR) have been developed for diagnosis, and most studies have found them to be consistently more sensitive (88% on average for ACL) than other parasitologic diagnostic methods.4 Nevertheless, successful PCR diagnosis is comparatively expensive, and its sensitivity depends on the origin of sample biopsy, DNA extraction methods, and the PCR primers used.7 Thus, further assessment of its performance (sensitivity and specificity) and field applicability are needed. Other techniques are the immunodiagnostic methods, such as an indirect inmunofluorescence assay (IFA), an enzyme-linked immunosorbent assay (ELISA), a complement fixation test, or indirect hemagglutination (IHA). However, the use of whole parasite extracts in these serologic tests is limited because of assay reproducibility and specificity mainly because of the cross-reactivity with other diseases such as Chagas’ disease.8 At the moment, serology is helpful for visceral forms because antibodies in cutaneous disease tend to be undetectable or present in low titer. For improvement of the specificity of the serodiagnostic tests, a search for defined Leishmania-specific antigens has been undertaken, and various candidates have been proposed; the k39 antigen from Leishmania chagasi and its use in the serodiagnosis of visceral leishmaniasis 9,10: a membrane antigen of 32 kd (P32) present in L. donovani infantum with a high sensitivity and specificity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis, 11 two hydrophilic antigens (k9 and k26) of L. chagasi,12 a secreted acid phosphatase, 13 an A2 antigen, 14 an excreted antigen from the culturing of the parasite in medium free of proteins and serum, 15 two thermal-shock proteins (Hsp 70 and 83), 16 a recombinant proteinase cistein, 17 three recombinant antigens (rH2A, KMP11, and Protein Q 18) rk26 and rK39 antigens from Leishmania infantum used in ELISAs provided very high sensitivities for the detection of symptomatic dogs, meanwhile, the rA2 protein from Leishmania donovani is more sensitive for asymptomatic dogs , 19 more recently, an antigen excreted by amastigote forms of L. amazonensis20 and, an excreted superoxide dismutase by L. infantum useful in diagnosing canine visceral leishmaniasis. 21
The aim of this study was to identify a new antigen excreted (FeSOD) by L. (V.) peruviana, L. (V.) brazilensis, and L. (L.) amazonensis , with the aim of developing a useful serologic assay to differentially diagnose the Andean cutaneous form (Uta) and sylvatic leishmaniasis (Espundia) in Latin America, where there are several circulating Leishmania species. Leishmania SODe was shown by ELISA to be a better antigen than that homogenate parasite fraction, showing an overall sensitivity and high specificity for routine use in serodiagnosis.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Antigen preparation. 1) Promastigote soluble lysate (homogenate fraction). The pellet of cells was washed twice, resuspended in 3 mL ice-cold STE buffer (0.25 mol/L sucrose, 25 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.8; Buffer 1), and disrupted by three cycles of sonic disintegration, 30 s at 60 V. The sonicated pellet was centrifuged at 2,500 x g for 10 minutes at 4°C, and the supernatant was recovered and desalted in a Sephadex G-25 column (Disposable PD 10 desalting columns; GE Healthcare, Barcelona, Spain), previously balanced with 20 mmol/L potassium phosphate buffer, pH 7.8, containing 1 mM EDTA (Buffer 2), bringing it to a final volume of 2.5 mL (homogenate fraction).
2) Isolation of SOD-excreted protein. For obtaining the SOD excreted (SODe), the pellet of promastigote cells was washed twice in MTL medium without serum, and the number of cells was counted using a hemocytometric chamber and distributed into aliquots of 5 x 109 parasites/mL. Afterward, they were again grown in MTL medium without serum for 24 hours. Afterward, the supernatant was collected by centrifugation at 1,500 x g for 10 minutes and passed through a filter of 0.45-µm pore size. The filtrate was subjected to two ice-cold consecutive ammonium sulphate additions between 35% and 85% (wt/vol) salt concentration and centrifuged (9,000 x g for 20 minutes at 4°C) to obtain a precipitate that was finally redissolved in 2.5 mL of Buffer 2 and desalted in a Sephadex G-25 column (PD 10; Pharmacia, Barcelona, Spain), previously balanced with Buffer 2, raising it to a final volume of 2.5 mL (fraction SODe).
In both fractions, the protein content was determined by using the Bio-Rad test, based on the Bradford method (Sigma, Madrid, Spain), with bovine serum albumin (BSA) as a standard.
As a means of confirming that there had been no cell lysis in the medium where the SODe was obtained, marker enzymes, pyruvate kinase, and hexokinase were assayed following the methodology of Bergmeyer. 22
Determination of isoelectric point. For the determination of SOD activity, the fractions of the homogenate and SODe obtained as described above were electrophoresed by isoelectric focusing in polyacrylamide Phast gel pI 3–9 using previously reported methodology. 23 The protein markers for pI were provided by Pharmacia (Uppsala, Sweden). The SOD activity was visualized by staining according to Beyer and Fridovich, 24 and for the protein markers, the lanes were stained with silver nitrate.
Polyclonal serum. To obtain the specific antibodies against the SODe of L. (V.) peruviana (SODe-Lp), we immunized two female 4-week-old Balb-C mice.
The antigen separation (concentration of proteins of 2 mg/ mL) was performed by electrophoresis of IEF 3–9 in polyacrylamide gels, as described previously. Afterward, the lane corresponding to the first well was cut, and the SOD activity was shown. 24 From the remaining gel, the zones corresponding to the activity bands of SODe (pI: 4.0) were cut, ground, and homogenized with sterile buffer phosphate (PBS).
The mice were each injected intraperitoneally in four immunizations, the first with complete Freund adjuvant (CFA) and the following three (booster immunizations) with incomplete Freund adjuvant (IFA) at 10-day intervals. The sera were collected 15 days after the last booster immunization by cardiac puncture. In the same way, we colleted control serum from the mouse that was immunized with polyacrylamine homogenized with CFA and IFA.
Western blot analysis. The antigen fraction (SODe-Lp) was run on IEF 3–9 gels (concentration of proteins of 2 mg/mL) and afterward transferred to nitrocellulose, for 30 minutes, as described in the Phast-System manual. The membrane was blocked for 2 hours at room temperature using 0.4% gelatine and 0.2% Tween 20 in PBS, followed by three washes in 0.1% Tween 20 in PBS (PBS-T); then it was incubated for 2 hours at room temperature, on the one hand with negative mouse serum at a dilution of 1/100 and on the other with anti-SODe mouse serum at dilutions of 1/100 and 1/500. After being washed as above, the membrane was further incubated for 2 hours at room temperature with the second antibody, anti-mouse IgG (Fc specific) peroxidase conjugate (Sigma; dilution 1/1,000). After washing, the substrate diaminobenzidine [DAB, 0.5 mg/mL in buffer Tris/HCl 0.1 mol/L, pH 7.4, containing 1/5,000 H2O2 (10 vol/vol)] was added, and the reaction was stopped with several washes in distilled water.
Sera. Blood samples were obtained by the Instituto Nacional de Salud of Lima (Peru) from two groups of leishmaniasis patients from different geographical areas of Peru: 1) 45 serum samples were from patients with SL and 2) 68 serum samples were from patients with ACL. All patients had clinical signs of leishmaniasis, and furthermore, the parasites were shown in biopsy samples of skin lesions.
Also included in the study were 20 serum samples from individuals diagnosed for Chagas’ disease: 10 serum samples provided by Instituto Nacional de Salud de Lima, and another 10 serum samples from individuals living in a Leishmaniasis non-endemic region (Santiago de Chile, Chile) with treatment of 10 years, provided by the Dr. Werner Apt. In the same way, sera from 12 healthy subjects living in a non-endemic area (Spain) were used as control sera to determine the cut-off for individual runs of the ELISA assays.
Indirect immunofluorescence. All sera were tested at a final dilution of 1/40 in-house test according to the method of Camargo 25 by examination with an Olympus BH-2 fluorescence microscope. For this assay in all cases, the antigen fraction consisted of promastigotes of L. (V.) peruviana, L. (V.) brazilensis, and L. (L.) amazonensis, which were cultured and processed as described above. All the samples were analyzed in triplicate in immunofluorescence slides, and the titers greater than 1/40 were considered positive.
Enzyme-linked immunosorbent assay.
For the ELISA tests, we used as the antigen the fractions: obtained as described previously, a homogenate and SODe, from L. (V.) peruviana (SODe-Lp), L. (V.) brazilensis (SODe-Lb), L. (L.) amazonensis (SODe-La), and L. (L.) infantum (SODe-Li). The optimal antigen concentrations were determined by checkerboard titration. Thus, the homogenate and SODe antigen fractions were used at a concentration of 10 µg and 2 µg, respectively, to coated onto polystyrene microtitre plates (Nunc, Dermark) considering samples positive at dilutions 
1/100. In all cases, the positive and negative controls were run simultaneously, and all the samples were analyzed in triplicate in polystyrene microtiter plates. Mean and SD of the optical densities (ODs) of the control sera were used to calculate the cut-off value (mean + 3 x SD).
Statistical analysis. To corroborate the prevalence of each test, a comparative study of proportions was performed with the program Statgraphic, making the appropriate hypothesis tests. The accuracy parameters (sensitivity, specificity, positive (PPV) and negative (NPV) predictive values, and positive (LR+) and negative (LR-) likelihood ratio) were calculated to the tests SODe-Lb and SODe-Lp, while a hypothesis test was used to determine whether the differences between them were significant (P < 0.05 considered significant). The data are presented as a descriptive table with calculation of the respective 95% confidence interval (CI).
Ethical consideration. Informed consent was obtained individually from all participants before the collection of blood samples. This study was approved by the Ethics Committee (CEIH) of the University of Granada (Spain).
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RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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, line 1); 5 SOD bands of the activity were detected by L. (V.) brazilensis, and L. (L.) amazonensis, although each with a different isoelectric point (Figure 1, Lanes 3–5).
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, Lanes 2, 4, and 6). The iso-electric point of this SODe was 4.0 for all three Leishmania species studied, coinciding with one of the cellular enzymatic forms and similar to the isoenzyme excreted by L. infantum (pI = 3.75). The marker-enzyme determination (pyruvate kinase and hexokinase) was negative (data not shown), indicating that the SODe was not a product of cell lysis during this culture period and that their presence was only because of excretion by the parasite.
Specific antibodies against the band of pI = 4.0 of L. (V.) peruviana (SODe-Lp), obtained from immunized Balb-C mice by Western blot using SODe as the antigen fraction, were used to show its immunogenic capacity (Figure 1B, Lanes 7 and 8). With the control serum (Figure 1B, Lane 9), the reaction was negative at a dilution of 1/100. Meanwhile, in the case of the anti-SODe-Lp serum ( Figure 1B , Lanes 7 and 8), the reactions proved positive at dilutions of 1/100 and 1/500.
The IFA assay result for antibodies to L. (V.) peruviana was positive for 75 of the 113 sera banked at a dilution of 1:40 (seroprevalence total of 66%; Table 1). The comparison of the positivity percentage at a dilution of 1:200 of the total result changes to 7% by IFA as opposed to 31%, 83%, 78%, 54%, and 10% by ELISA assays with homogenates from L. (V.) peruviana, with SODe-Lp, SODe-Lb, SODe-La, and SODe-Li, respectively (Table 1). That is, 35 sera tested positive against the homogenate fraction antigen (Figure 2); 94 sera were positive when the antigen fraction used was SODe-Lp (Figure 2A). The overall analysis of the 113 sera gave practically the same seroprevalence index as when the SODe-Lp and SODe-Lb antigen fractions were used. On the contrary, the difference was significant when the antigen fraction used was SODe-La, because the seroprevalence dipped to 54% at the same dilution. Meanwhile, when SODe from L. (L.) infantum (SODe-Li) was used as the antigen, the seroprevalence reached only 10% (11 positive sera) at the 1/200 dilution.
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After the sensitivity and specificity analysis of the ELISA using as antigen fractions SODe-Lp and SODe-Lb, both tests apparently showed different sensitivity (88.2% and 93.3%, respectively) and specificity (33.3% and 32.4%, respectively), but not being significant according to the sample data (P > 0.1; Table 2). In contrast, the assays made with the chagasic sera against SODe-Lp and SODe-Lb were negative. Therefore, there was no crossed reaction, and it was shown that there were no antibodies against this SODe in Chagas patients at a serum dilution of 1/200 (Figure 2B).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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In prior studies, we showed that epimastigote forms of T. cruzi and promastigotes of L. infantum and Phytomonas cultured in a medium without serum for 24 hours excreted an iron SOD with a pI close to 4.0. This FeSODe was immunogenic and capable of detecting specific antibodies against it in serum at a dilution of 1/500 by Western blot. 18,27 On this occasion, we identified SOD activity excreted into the culture medium by promastigotes of L. (V.) peruviana, L. (V.) brazilensis, and L. (L.) amazonensis. This activity is a single band of pI = 4.0, which has been partially purified and specific antibodies against this SODe proved reactive at dilutions of 1/500 by Western blot.
The principal objective of this study was to develop an ELISA that can detect antibodies in patients with SL and ACL. These assays are based on soluble antigens (SODe) from promastigotes of L. (V.) peruviana, L. (V.) brazilensis, and L. (L.) amazonensis cultivated in a protein-free medium. Many works have focused on the search for soluble antigens capable of being used to diagnose these diseases. 14,15,18,20,28 The excretion of antigens may constitute part of a strategy used by parasites to subvert or evade the host immune response, thereby achieving intracellular survival. 20 The results presented here showed that ELISA tests based on SODe antigen preparation from L. peruviana and L. brazilensis performed better than conventional serologic methods. A sensitivity of 83% was attained using SODe-Lp antigen, and there was 78% sensitivity using SODe-Lb antigen. These values are similar to those found by Carvalho and others 14 in sera of VL humans and dogs, using the recombinant L. donovani A2 antigen and comparable to those of Romero and others, 15 when analyzing the serum of 100 cutaneous leishmaniasis case-patients on the basis of the use of a novel excreted antigen from L. mexicana. The sensitivity found in our study is superior compared with the conventional diagnostic tests (IFA) performed in the same individuals (66% sensitivity at a significantly lower serum dilution 1/40); this sensitivity is far higher than that found when using parasite homogenates as the antigen.
Until now, different authors that use new molecules such as antigens have pointed out that the ACL was difficult to diagnose, for several reasons, including low antibody titers. Our data agree with previously published findings that show that the sensitivity and specificity of diagnostic methods depend on the type, source, and purity of antigen used, because some of the leishmanial antigens have common cross-reactive epitopes shared with other microorganisms, particularly Trypanosoma, Mycobacteria, Plasmodia, and Schistosoma.29 When we assayed sera from patients with ACL, the sensitivity of the SODe antigen from L. (V.) peruviana was far more specific and significantly greater (94%) than when SODe-Lb was used as the antigen fraction (68%). This may be because L. (V.) peruviana is more involved in the ACL, although not necessarily the only species of Leishmania involved. This hypothesis is strengthened by the results with patients affected by SL where SODe-Lb is much more specific (sensitivity of 93%) than when the antigen used is SOD excreted by L. (V.) peruviana (sensitivity 67%); it is generally understood that L. (V.) brazilensis is more aggressive, often leading to mucosal involvement. 13 The fact that antigens from both Leishmania species were prepared under identical technical conditions suggests that differences in antigenic quality may depend on the type and source of the antigen used for the particular species of the parasite. 29 To define the protein determinants of the SODe that could discriminate between leishmaniasis and Chagas’ disease, our analysis enabled us to confirm that the SOD excreted by Leishmania species is recognized only by leishmaniasis sera and not by the sera from Chagas’ disease patients. The absence of cross-reactivity with Chagas’ disease sera is important, because the endemic areas of the two diseases often overlap. 16
In summary, we developed an ELISA that can detect antibodies in human patients with SL and ACL. Leishmania SODe was shown to be a good antigen, showing an overall sensitivity and high specificity for routine use for serodiagnosis of diseases.
Received December 21, 2007. Accepted for publication August 13, 2008.
Financial support: This work was supported by the CGL2006-27889-E/BOS: Biobank and Unity of Characterization of trypanosoma-tid species and strains responsible of human, animal and vegetable pathologies.
* Address correspondence to Manuel Sánchez-Moreno, Departamento de Parasitología, Facultad de Ciencias, Instituto de Biotecnología, Universidad de Granada, Granada 18071, Spain. E-mail: msanchem{at}ugr.es 
Authors’ addresses: Clotilde Marín, Silvia S. Longoni, Jesús Urbano, Hector Mateo, María José Rosales, Gregorio Pérez-Cordón, and Manuel Sánchez-Moreno, Instituto de Biotecnología, Departamento de Parasitología, Facultad de Ciencias, Universidad de Granada, Granada, Spain. Gloria Minaya, Laboratorio de Leishmaniosis y Chagas, Instituto Nacional de Salud, Lima, Perú. José A. De Diego, Unidad de Parasitología y Medicina Tropical, Departamento de Medicina Preventiva y Salud Pública Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain. Desiré Romero, Departamento de Estadística e Investigación Operativa, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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