Original Article
Diagnosis of paracoccidioidomycosis by a dot blot assay using a recombinant Paracoccidioides brasiliensis p27 protein M. M. Correa,1 A. M. Bedoya,1 M. P. Guerrero,1 J. Me´ndez,3 A. Restrepo2 and J. G. McEwen3 1 Grupo de Microbiologı´a Molecular, Escuela de Bacteriologı´a y Laboratorio Clı´nico, Universidad de Antioquia, Medellı´n, Colombia 2Micologı´a Me´dica y Experimental, Corporacio´n para Investigaciones Biolo´gicas, Medellı´n, Colombia and 3Facultad de Medicina, Universidad de Antioquia and Corporacio´n para Investigaciones Biolo´gicas, Medellı´n, Colombia
Summary
A variety of immunological methods have proven useful for Paracoccidioidomycosis (PCM) diagnosis; however, they are often time consuming and many lack sensitivity and specificity, partially attributed to the use of crude antigens, which give cross reactivity. Until now, attempts to clone and express Paracoccidioides brasiliensis immunodominant antigens have presented difficulties of process and problems of cost. In an attempt to obtain a more rapid, sensitive, and specific test for PCM diagnosis, we subcloned the P. brasiliensis p27 gene and used the recombinant protein as the antigen in dot blot assays to evaluate its usefulness in paracoccidioidomicosis diagnosis. The development of an optimised procedure for p27 recombinant protein purification and production led to an easier and less expensive process than the one previously used in our laboratory and allowed the availability of enough purified protein for its evaluation as the antigen in the dot blot assays. In these assays, antibodies present in ten serum samples from seven patients with PCM recognised the recombinant protein showing a sensitivity of 100% with a specificity of 98%. These results confirm the value of the 27-kDa recombinant antigen in the serodiagnosis of paracoccidioidomycosis and that the dot blot format is an alternative to the immunoenzymatic assay procedure.
Key words: Paracoccidioidomycosis, Paracoccidioides brasiliensis, dot blot assay, recombinant protein, immunogenic p27 protein, diagnostic test, mycosis.
Introduction Paracoccidioidomycosis (PCM), a disease caused by the dimorphic fungus Paracoccidioides brasiliensis, is one of the most important systemic mycoses in Latin America.1 This mycosis affects preferentially farmers in their productive years residing in endemic countries; the latter spans from Mexico to Argentina. The disease affects mainly the lungs from where it disseminates to other organs producing secondary injuries in the mucosae, skin, lymphoid nodules and adrenal glands; frequently, pulmonary fibrosis can appear as incapaciCorrespondence: Margarita M. Correa, Calle 67 No. 53-108, Of. 5-437, Escuela de Bacteriologı´a y Laboratorio Clı´nico, Universidad de Antioquia, Medellı´n, Colombia. Tel.: 574 210 5495. Fax: 574 210 5481. E-mail:
[email protected] Accepted for publication 26 June 2006
2006 The Authors Journal Compilation 2006 Blackwell Publishing Ltd • Mycoses, 50, 41–47
tating sequelae of the disease, and in absence of an effective therapy, PCM progresses and may be lethal.2 The importance of a precise and rapid diagnosis of this mycosis resides on the prompter initiation of the specific therapy in order to avoid both increasing lung damage and dissemination of the fungus to other organs and the development of fibrosis. Various methods have been used for PCM diagnosis; they can be divided into microbiological and immunological; the first, include direct identification of the fungus in clinical samples, biopsies and cultures.1,3 Immunological tests are based on the detection of specific antibodies and include methods such as complement fixation, immunodiffusion and immunoenzymatic tests.4–6 Serological tests have been widely used in PCM diagnosis; however, one of the main problems of such tests is the high cross reactivity with agents causing other mycoses, e.g. Histoplasma capsulatum, because of the use of crude antigens prepared from the complete
doi:10.1111/j.0933-7407.2006.01306.x
M. M. Correa et al.
microorganism and its metabolic products. Another disadvantage is that the preparation of these antigens is a very complex and time consuming process.7 Additionally, these antigens show a great variability, making it very difficult to standardise diagnostic techniques on different laboratories.8 An alternative to obtaining suitable antigens is the cloning, expression and characterisation of inmunodominant antigens derived from the fungus.9–11 Cisalpino et al. [9] cloned a P. brasiliensis immunodominant antigen, the glycoprotein of 43 kDa (gp43); this recombinant antigen showed high reactivity when evaluated with sera from patients with PCM.12 McEwen et al. [13] cloned the protein of 27 kDa (p27) on the plasmid pBluescript; immunological characterisation of this recombinant protein showed that it was recognised by a high proportion of sera of patients with PCM.10 When this p27 recombinant protein was evaluated on an ELISA format, the values of sensitivity and specificity obtained were of 73.4% and 87.5% respectively.11 One of the main advantages of using recombinant antigens is the reduction of the cross-reactions that occur with other mycoses when crude antigens are used, as previously indicated. Additionally, recombinant proteins facilitate production of antigenic preparations that display a little variability and can be used in different tests and different laboratories. The problems found when P. brasiliensis recombinant proteins were initially produced were their lack of solubility in the case of gp43, and high production costs, as well as the complexity of the process in producing the p27 protein. Considering the difficulties found with PCM diagnosis, in this work, we subcloned and expressed the gene that codifies for the p27 protein and searched for an easy-to-perform test showing high sensitivity and specificity, as well as giving reproducible results in different laboratories. To this effect, we used this recombinant p27 protein as the antigen in a dot blot test, in order to achieve an assay that was sensitive, specific and relatively fast and inexpensive, convenient to be standardised and used in different laboratories as a PCM diagnostic assay and for follow-up studies in endemic areas.
Materials and methods Serum samples
Samples were collected between 1999 and 2001 at the Mycology Laboratory, Corporacio´n para Investigaciones Biolo´gicas (CIB), Medellı´n, Colombia; except some of the sera from aspergillosis patients which were collected in
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the previous years. All serum samples came from a bank of sera obtained from patients who were previously diagnosed with PCM, other mycoses or tuberculosis, according to the standardised diagnostic methodology (direct examination, isolation by culture and positive serological test) at CIB. These sera were properly aliquoted and stored, avoiding repeated freezing and thawing, which is important for obtaining reproducible results. A total of 50 individual serum specimens were examined, 10 from patients with PCM (Table 1 shows information on individual PCM patients), 10 each from histoplasmosis, aspergillosis, tuberculosis patients and from healthy individuals negative for PCM antibodies, which had been previously tested by agar gel immunodiffusion in our laboratory. Pools of sera from PCM, histoplasmosis, aspergillosis, chromoblastomycosis, cryptococcosis, sporotrichosis, tuberculosis patients and from healthy individuals. Each consisted of equal amounts of individual serum samples. Expression and purification of P. brasiliensis p27 recombinant protein
The gene coding the p27 protein had been initially cloned in the pBluescript II plasmid but we subcloned it in the vector pThioC (Invitrogen, Carlsbad, CA, USA) and expressed in Escherichia coli Top10 cells. The recombinant bacteria were scale-grown in an erlenmeyer containing 500 ml of Luria-Bertani medium with ampicillin added to a final concentration of 50 lg ml)1 and incubated at 37 C under continuous shaking (180 rpm). After 3 h of incubation, gene expression was induced by adding IPTG (isopropyl-b-D-thiogalactopyranoside; SIGMA, St Louis, MO, USA) to a 0.5 mmol l)1 Table 1 Characterisation of the patients with paracoccidioidomycosis (PCM) and source of the serum samples tested by dot blot
Patient
Serum numbers*
Sex
Age (years)
PCM clinical form
Outcome
1 2 3 4 5 6 7
1, 4 2 3, 9 5 6, 10 7 8
M M M M M M M
41 37 30 64 47 71 45
C C C C C CM C
S S S S S F S
M, male; C, chronic; CM, chronic with meningeal compromise; S, successful therapy; F, therapeutic failure. *Serum sample no. 4 was collected 4 weeks before serum sample no. 1; serum sample no. 9 was collected 17 weeks before no. 3; serum sample no. 6 was collected 13 weeks before no. 10.
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final concentration and incubated overnight. Cells were collected by centrifugation at 5488 g on a centrifuge (IEC Centra MP4R; Fisher Scientific, Pittsburgh, PA, USA), at 4 C, for 10 min. The pellet was resuspended in 10 ml of buffer (20 mmol l)1 Na2HPO4, 500 mmol l)1 of NaCl, pH 7.8) and lysed with a cell disruptor (BIONEB Cell & DNA disruptor; GlasCol, Terre Haute, IN, USA) by adding PMSF (phenylmethylsulphonylfluoride; ICN Biomedicals, Irvine, CA, USA) to 0.1 mmol l)1 final concentration; the cell extract was centrifuged at 7000 rpm, at 4 C, for 20 min. For recombinant protein purification, the cell extract supernatant was fractionated by a Sephadex G100 column [Pharmacia Fine Chemicals AB, Uppsala, Sweden; now GE Healthcare (formerly Amersham Biosciences/Pharmacial Biotech), Uppsala, Sweden], at 4 C, the resulting fractions were visualised in a 12% sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS-PAGE). The best two fractions were selected for further purification by affinity column (Ni NTA-Agarosa, Qiagen, Hilden, Germany). Recombinant protein p27 was eluted using Imidazole (SIGMA, St Louis, MO, USA) at different concentrations (20, 40, 60 and 100 mmol l)1) to determine the concentration that yield the greater amount of pure protein. Eluted products were visualised in a 12% SDS-PAGE. Protein concentration was calculated by the Bradford technique (Bio-Rad, Hercules, CA, USA). The antigenic activity of the proteins was assessed by Western blot analysis using a pool of sera from patients with active PCM as well as a pool of sera from patients with histoplasmosis and tuberculosis as negative controls.
had proved negative for PCM antibodies. Each serum diluted 1 : 2000 in TTBS plus 1% non-fat dry milk (TTBSM) was added to the corresponding membrane for 1 h. The membrane was washed again and immersed in a 1 : 2000 dilution of secondary antibody: goat anti-human IgG H + L immunoglobulin conjugated to peroxidase (Jackson Immunoresearch, Baltimore, MD, USA) in TTBSM was added for 1 h, and the membrane was washed again. Then, the membrane was immersed in a fresh mixture prepared using 10 mg of diaminobenzidine tetrahydrochloride (DAB) (Sigma) dissolved in a 1 ml of ethanol, 30 mg of 4-Chloro-1-Naphthol (4C1N) (Sigma) dissolved in 1 ml of ethanol; both suspensions were mixed with TBS plus 30 ll of H2O2 for a final volume of 100 ml. After 3 min of soft shaking, the reaction was stopped with distilled water. The specific positive reactions were visualised as yellow coloured spots. Additionally, some assays were performed using the secondary antibody conjugated to alkaline phosphatase (Southern Biotechnology Associates, Inc., Birmingham, AL, USA), to test for the format that could allow easier readings and interpretation of the results.
Dot blot assays
Results
One hundred microlitres of the p27 recombinant protein at a final concentration of 0.25 lg, diluted in 20 mmol l)1 Tris-HCL, pH 7.5, 500 mmol l)1 NaCl (TBS) buffer was applied to a membrane (PDVF; Millipore, Bedford, MA, USA) in each well of a 96-well microfiltration apparatus (Bio-Dot, Bio-Rad). The membrane was washed with 200 ll per well of 0.05% Tween-20 in TBS (TTBS). After the washes, the membrane was removed from the apparatus and blocked with 100 ml of TTBS plus 5% non-fat dry milk (TBSM) for 1 h, to avoid non-specific binding. Next, the membrane was washed with TTBS, three times. Depending on the assay, the primary antibody consisted of pools or individual sera from patients with PCM, tuberculosis or with other mycoses such as: histoplasmosis, aspergillosis, cryptococcosis, chromoblastomycosis and sporotrichosis or sera from healthy individuals whose sera
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Statistical methods
Statistical analysis of sensitivity, specificity and predictive values of dot blot assays were calculated by the method of Galen and Gambino [14]. Data used to compare the results of the dot blot assays and to calculate these values were obtained from a CIB database where confirmatory diagnostic test and information on patients are stored.
Expression and purification of p27 recombinant protein from P. brasiliensis
The subcloning of the gene encoding the p27 protein in the pThioC vector and its expression in E. coli Top10 cells was successful. The purification of protein on a Sephadex column produced 19 fractions that were visualised on the 12% SDS-PAGE electrophoresis (data not shown). Fractions 7 to 13 contained the greatest amount of recombinant protein with a very little background of bacterial proteins. Fractions 9 and 10 were selected for further purification on an affinity column. Samples eluted with different concentrations of imidazole were visualised on a 12% SDS-PAGE; fractions eluted with 40 mmol l)1 and 60 mmol l)1 imidazole contained the greatest amount of recombinant protein and minimum background. In the SDS-PAGE, a band of
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approximately 34 kDa was observed, corresponding to the p27 recombinant protein bound to thioredoxin. Protein concentration eluted with 40 mmol l)1 imidazole measured by the Bradford method (Bio-Rad) was of 0.136 lg ll)1. Immunogenicity of this p27 recombinant protein was demonstrated by Western blot, this protein was recognised by the sera from patients with PCM, while no signal was observed in the membranes treated with control sera from histoplamosis and tuberculosis patients (data not shown). Dot blot assays
Initial experiments allowed the determination of the optimal conditions to perform the dot blot assays. The conditions established were: i) recombinant p27 protein concentration of 0.25 ug well)1, ii) a 1 : 2000 dilution of sera from patients with PCM or patients with other mycoses, and iii) a 1 : 2000 dilution of the secondary antibody labelled with peroxidase. Using this label, positive reactions are visualised as yellow spots; depending on the intensity of the reaction, the signal varies from dark yellow or brown for strong positive reactions to pale yellow for weak positive reactions. In the dot blot assays, the recombinant p27 protein was recognised by the antibodies present in sera from patients with PCM, when used pooled (Fig. 1b) or
individually (Fig. 1a), while no reaction was observed with sera from healthy individuals negative for PCM antibodies which had been previously tested by agar gel immunodiffusion in our laboratory (Fig. 1e). In addition, the recombinant p27 protein was evaluated using sera from patients diagnosed with other pathologies, shown to cross react with PCM in immunological tests: histoplasmosis, aspergillosis, cryptococcosis, chromoblastomycosis, sporotrichosis and tuberculosis. Depending on the assay, sera were tested pooled and individually. No cross reaction was observed with pooled sera from individuals with these mycosis (Fig. 2) or tuberculosis (not shown), or when using individual serum from patients with histoplasmosis (Fig. 1c), aspergillosis (not shown), tuberculosis (Fig. 1d) and healthy individuals negative for PCM antibodies (Fig. 1e), except on one occasion when a serum from a patient with aspergillosis showed a positive signal. In addition, some dot blot assays were carried out using the conditions described previously and a secondary antibody conjugated with alkaline phosphatase. The intensity of the signal produced by this system was compared with the one obtained with peroxidase. Results obtained when evaluating the p27 recombinant protein with pools (Fig. 2) and individual sera (results not shown) from patients with PCM and other mycoses were similar
Figure 1 Dot blot assays using individual sera from patients with different pathologies. (a) with paracoccidioidomycosis (PCM); the characteristics of patients are described in Table 1. Serum numbers: 1– 10 (tested in duplicate; patient 1: serum sample no. 4 was collected 4 weeks before sample no. 1; patient 3: serum sample no. 9 was collected 17 weeks before no. 3; patient 5: serum sample no. 6 was collected 13 weeks before no. 10), (b) Positive control of PCM: pooled sera tested in duplicate (c) histoplasmosis, (d) tuberculosis and (e) sera from healthy individuals negative for PCM antibodies. Sera were added at a dilution of 1 : 2000; the p27 recombinant protein was added at a concentration of 0.25 lg well)1; the secondary antibody labelled with peroxidase was added on a dilution of 1 : 2000.
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Paracoccidioidomycosis diagnosis by dot blot assay
Figure 2 Dot blot assays using pooled sera from patients with: 1: paracoccidioidomycosis (PCM), 2: aspergillosis, 3: histoplasmosis, 4: sporotrichosis, 5: cryptococcosis, 6: chromoblastomycosis, 7: sera from healthy individuals negative for PCM antibodies. Sera were added on a dilution of 1 : 2000; the p27 recombinant protein was added at a concentration of 0.25 lg well)1; the secondary antibody was added at a dilution of 1 : 2000. Pooled sera were tested in duplicate. (a) Secondary antibody labelled with alkaline phosphatase, (b) with peroxidase.
to those obtained with the secondary antibody conjugated to peroxidase, the only difference was that the signal produced with alkaline phosphatase was purple. Values of sensitivity, specificity, and positive and negative predictive value were calculated. Sera that were previously confirmed as positive for PCM according to the CIB sera database were also positive in the dot blot assay, corresponding to 100% sensitivity. The specificity value was of 98%, and this was because of a serum from a patient with aspergillosis that showed a positive result in the dot blot test. The positive and negative predictive values for these assays were 91% and 100% respectively.
Discussion One of the main problems in PCM diagnosis is the lack of a standardised antigen. The Gp43 glycoprotein, one of the main immunodominant antigens of P. brasiliensis, have been extensively used as antigens in serological diagnosis, in the form of crude extracts obtained directly from the cultured fungus;2,15 however, as is well known, these crude antigens cross react with the agents of other mycoses. Expression and purification of recombinant antigens have appeared as a valuable alternative to the
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use of crude antigenic extracts.9,10,11 In our search for more reproducible results, we subcloned the gene coding for the p27 immunodominant antigen of P. brasiliensis and evaluated this recombinant protein in a dot blot assay, to explore its usefulness in the diagnosis of this mycosis. The use of a well-characterised antigen in the form of a recombinant protein and the appropriate test format to be used in PCM diagnosis would allow more reproducible results, making possible the standardisation of those diagnostic techniques in different laboratories. Several groups have cloned, expressed and characterised various PCM immunodominant antigens in an attempt to improve the sensibility and specificity of diagnostic tests;9,10,11 however, until recently, these have suffered difficulties in processing and cost problems. The p27 recombinant protein was previously cloned in pBluescript IISK and expressed in E. coli DH5a cells, produced by fermentation techniques and purified by preparative electrophoresis.10,11,13 Although, protein obtained in this system showed to be reactive when evaluated with sera from PCM patients, an important cross-reactivity was detected with sera from patients with aspergillosis and histoplasmosis; in addition, this process was expensive and time consuming. This initial cloning and expression of the p27 P. brasiliensis immunodominant protein was performed at a time when the knowledge of molecular biology of the fungus was limited and the cloning systems were less-developed and less-efficient than the ones available today. To optimise in the process of protein purification and production, we subcloned the gene coding for the p27 protein in a new vector, pThio C and expressed the protein in E. coli Top 10 cells. This new system led to an easier and less expensive purification process and allowed the availability of enough purified protein for its evaluation as the antigen in diagnostic assays. The newly produced p27 protein could then be used for standardisation of different assays, allowing testing in different laboratories and enabling comparisons. The immunogenicity of the newly produced protein was tested by Western blot analysis; the protein was recognised by the sera from patients with PCM, while no signal was observed in the membranes treated with control sera from histoplasmosis and tuberculosis patients. Our next approach was to select the test format which would lead to more reproducible results. It is known that PCM diagnosis has been based mostly on microbiological and immunological methods,1,5,6,16,17 but both these alternatives present problems and, although, immunological tests have proved useful, one of the difficulties encountered has been the high level of cross reactivity found among the patients with other
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mycosis, especially histoplasmosis, mostly because of the use of crude antigens and their high associated variability.2,8,10,11 Being aware of the need for more sensitive and specific methods and also the need of more cost-efficient and less time-consuming tests, we used the newly produced p27 protein on a dot blot format. The availability of an appropriate antigen used in combination with a standardised test, would allow its evaluation with a greater number of sera from patients with PCM in different stages of the disease to validate its use in diagnosis. Much emphasis has been made on the importance of the test format of the immunological assays for the proper reactivity of the antigen; activity can vary depending on several aspects: if the protein is assayed in solution, bound to a carrier or absorbed to a solid surface,18,19 variations could also occur because of the nature of the surface of the different membrane types used. The Dot blot assay was chosen for its evaluation as the format assay of the diagnostic test using the antigenic p27 recombinant protein based on multiple considerations; it is an easy technique that could be carried out by personnel with basic training; it requires as much time as other immunological test such as ELISA; but in contrast to some of these tests, it can be performed on laboratories with unsophisticated equipment, and the results are read by the naked eye using standards as positive and negative controls. A further advantage is that the antigen can be fixed on a membrane that can be stored at minus 20 C for long periods of time until the assay is performed; also the membrane with a fixed antigen can be transported to a reference laboratory, enabling comparison of results. All these features make the Dot blot assay a valuable technique not only for diagnostics but also for follow-up and epidemiological studies.4 Results of the dot blot assays showed that antibodies present in the sera from patients with PCM, when used pooled or individually, recognise the recombinant protein with no cross-reaction being observed with sera from patients with other mycoses or tuberculosis; the results were similar using sera from patients with aspergillosis, except on one occasion when a serum from one of these patients showed a positive signal. The sensitivity obtained using the p27 and dot blot was 100%, the specificity 98%. We consider this cross reactivity to be acceptable, mostly when compared with results of other studies where complex or recombinant antigens are used and different methodology for the assay is employed.4,9,10,11,17,20 The dot blot test has also been used by Taborda y Camargo [4] to detect antibodies against purified gp43
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glycoprotein, the other main antigenic determinant of P. brasiliensis; they also found reasonably good sensitivity and specificity; however, we consider that our test was an advanced one, probably because of the use of the recombinant antigen as opposite to the purified protein used by these authors. Although, we found that the dot blot used in combination with a recombinant antigen is an ideal test to standardise in different laboratories with an unsophisticated infrastructure, some considerations should be made in order to obtain reproducible results; these include, use of sera that have been properly aliquoted and stored, avoiding repeated freezing and thawing; each new antigen batch should be tested against known controls and the personnel should have a basic training on the procedure. As reported in the literature,21 we also found that even minimum differences in procedure, equipment and reagents may affect the results of the assays, even if the protocol continues to be the same. In this work we used a secondary antibody labelled with peroxidase; with this system, positive reactions are visualised as dark yellow or brown coloured spots. Some of our sera produced a pale yellow signal on the blots (Fig. 1a, 1 and 8), which we interpreted as a weak reaction. Sera sample 1 came from the same patient than sample 4, which produced a stronger signal; the difference between them was that the samples were taken at different times during treatment; sample 1 was taken 6 week after sample 4. For this reason, we recommend that in cases where the reactions are weak, a quantitative immunoenzymatic assay (EIA) be performed to elucidate or confirm the results of the blot; this criterion is also recommended when a weak signal should be differentiated from a background signal that is some times observed in the blots where the reagents are placed. We also performed some Western blot assays (results not shown) to test the recombinant p27 protein; in these assays, we also found good sensitivity and specificity; however, we recommend the use of the dot blot because the Western blot will be more difficult to implement in different laboratories where no special equipment is available. In addition, it is more labour intensive and time-consuming. This study demonstrated that the use of a wellcharacterised recombinant protein as the antigen on a dot blot format allowed attaining reproducible results using this test in our laboratory. Our optimisation of the process for p27 recombinant protein purification and production led to an easier and less expensive purification process than the one previously used in our
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Paracoccidioidomycosis diagnosis by dot blot assay
laboratory;10,11,13 in addition, it allowed the availability of enough purified protein for its evaluation as the antigen in a dot blot format. The results of these assays, in terms of sensitivity and specificity are encouraging, corresponding to 100% and 98%, respectively; the utilisation of the p27 recombinant antigen in a dot blot format should be evaluated further using a greater number of sera from patients with PCM in different stages of the disease, along with sera from patients with other mycoses. It is expected that in the future this assay can be standardised and used in different laboratories even with unsophisticated infrastructure and as a qualitative, presumptive PCM diagnostic assay and for follow-up studies in endemic areas. The results of this study confirm the value of the 27 kDa recombinant antigen in the serodiagnosis of paracoccidioidomycosis and that the dot blot format is an alternative to the EIA procedure.
Acknowledgements This work was supported by a grant from the Comite´ para el Desarrollo de la Investigacio´n (CODI), Universidad de Antioquia, modalidad Investigacio´n Aplicada. We are indebted to Beatriz Go´mez and Andre´s Higuita for the technical support.
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8 Franco M, Bagagli E, Cunha M, Chamma L, Fecchio D. Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences. Mycopathologia 1996; 135: 13–19. 9 Cisalpino P, Puccia R, Yamauchi L, Cano M., da Silveira JF, Travassos LR. Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis. J Biol Chem 1996; 271: 4553–60. 10 Ortiz BL, Garcia AM, Restrepo A, McEwen JG. Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis. Clin Diagn Lab Immunol 1996; 3: 239–41. 11 Ortiz BL, Dı´ez S, Ura´n ME et al. Use of the 27-kilodalton recombinant protein from Paracoccidioides brasiliensis in serodiagnosis of paracoccidioidomycosis. Clin Diagn Lab Immunol 1998; 5: 826–30. 12 Stambuk BU, Puccia R, de Almeida ML, Travassos LR, Schenkman S. Secretion of the 43 kDa glycoprotein antigen by Paracoccidioides brasiliensis. J Med Vet Mycol 1988; 26: 367–73. 13 McEwen JG, Ortiz BL, Garcia AM, Florez AM, Botero S, Restrepo A. Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. Fungal Genet Biol 1996; 20: 125–31. 14 Gallen RS, Gambino SR.. Beyond Normality. The Predictive Value and Efficiency of Medical Diagnosis. New York: John Wiley & Sons Inc., 1975. 15 Camargo Z, Unterkircher C, Travassos L. Identification of antigenic polypeptides of Paracoccidioides brasiliensis by immunoblotting. J Med Vet Mycol 1989; 27: 407–12. 16 Camargo SP, Cano LE. Humoral immunity in paracoccidiodomycosis. In: Franco M, Da Silva Lacaz C, Restrepo A, Del Negro G (eds), Paracoccidioidomycosis. Boca Rato´n: CRC Press, 1994: 187–201. 17 Dı´ez S, Gomez B, McEwen J, Restrepo A, Hay R, Hamilton A. Combined use of Paracoccidioides brasiliensis recombinant 27-kilodalton and purified 87-kilodalton antigens in an enzyme-linked immunosorbent assay for serodiagnosis of paracoccidioidomycosis. J Clin Microbiol 2003; 41: 1536–42. 18 Van Regenmortel MH. Antigenic cross-reactivity between proteins and peptides: new insights and applications. TIBS 1987; 12: 237–40. 19 Van Regenmortel MH. Synthetic peptides help in diagnosing viral infections. ASM News 1998; 64: 332–8. 20 Do Valle AC, Costa RL, Fialho Monteiro PC, Von Helder J, Muniz MM, ZancopeÕ-Oliveira RM. Interpretation and clinical correlation of serological tests in paracoccidioidomycosis. Med Mycol 2001; 39: 373–7. 21 Garaizar J, Lo´pez N, Laconcha I et al. Suitability of PCR fingerprinting, infrequent-restriction-site PCR and pulsed field gel electrophoresis, combined with Computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis. Appl Environ Microbiol 2000; 66: 5273–81.
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