Serology of Paracoccidioidomycosis
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositório Institucional UNIFESP Mycopathologia (2008) 165:289–302 DOI 10.1007/s11046-007-9060-5 Serology of paracoccidioidomycosis Zoilo Pires de Camargo Received: 10 July 2007 / Accepted: 30 August 2007 Ó Springer Science+Business Media B.V. 2007 Abstract This review provides the background for Keywords Antigens Á Mycosis Á understanding the role of a battery of diagnostic Paracoccidioidomycosis Á Paracoccidioides methods in paracoccidioidomycosis (PCM). This brasiliensis Á Serology systemic mycosis is a disease endemic in many regions of Latin America, with sporadic cases also occurring throughout the world (mycosis of importa- Introduction tion). Although excellent laboratory methods for diagnosis are available, there are deficiencies that The fungus Paracoccidioides brasiliensis is must be met by continued research. Understanding ensconced in the nature in Latin America, but its the uses and limitations of a battery of laboratory exact niche is not known. The expanding human and methods is essential to diagnose PCM. Clinicians and non-human populations of endemic areas provide a laboratory directors must be familiar with the uses continuing supply of individuals who are susceptible and limitations of a battery of serologic and myco- to infection with P. brasiliensis. Paracoccidioidomy- logical tests to accurately diagnose of PCM. cosis (PCM) is caused by the inhalation of conidia Antibody and antigen detections are valuable found in nature and under adverse nutritional condi- adjuncts to histopathology and culture. More tions, the fungus sporulates forming conidia which recently, the gp43 and gp70 antigen detection assay are less than 5 lm in diameter and can easily reach have improved the methodology of diagnosis of this the alveoli when inhaled, and a lung-lymph node mycosis, which improves reproducibility and facili- primary complex develops. These early lesions may tates monitoring antigen clearance during antifungal remain silent for many years and in a manner similar treatment. Furthermore, detection of antigen in cere- to tuberculosis and other systemic mycoses, they brospinal fluid and in bronchoalveolar lavage fluid may, at any time, progress in the lungs or disseminate increases the sensitivity for diagnosis of PCM in by lymphohematogenic route. central nervous system and in pulmonary infections, Serologic tests have served for several decades as respectively. aid in the diagnosis and management of paracoccid- ioidomycosis. Among the serologic tests available for Z. P. de Camargo (&) mycosis, those for paracoccidioidomycosis have been Department of Microbiology, Immunology and the most reliable and have been useful in human Parasitology, Cellular Biology Division, Federal medicine. University of Sa˜o Paulo (UNIFESP), Rua Botucatu 862/8° andar, Sao Paulo, SP 04023-062, Brazil The following serologic tests have been used for e-mail: [email protected] diagnosing PCM: tube precipitin, complement 123 290 Mycopathologia (2008) 165:289–302 fixation (CF), double immunodiffusion, counter- In 1988, we reported that a diagnostically immunoelectrophoresis (CIE), immunofluorescence, useful exocellular antigen preparation, Ag7, could radioimmunoassay, passive hemagglutination inhibi- reproducibly be produced from yeast forms of tion, immunoenzyme assays, such as ELISA, P. brasiliensis [12]. We suggested that this could be MELISA, Dot Blot, Western Blot, and more recently used as a standard method in ID tests. The method inhibition-ELISA. required, first, the determination of the number of viable cells in a 3-day old pre-inoculum, the transfer of this growth to Fernbach flask culture for 7 days, Antigen preparation the pooling of six lots consisting of three flasks each in a total of 9.9 l medium, dialysis of this filtrate, In 1916, Moses [1] was the first to use P. brasiliensis concentration by vacuum evaporation, new dialysis, antigens for serologic testing, using a saline extract and finally lyophilization. The successful use of this from the fungus, for successful testing in CF. Since this Ag7 is based on the presence of abundant amounts of study, a number of different techniques for the use and gp43, a 43,000-Da protein that is the immunodom- preparation of antigens have been described. However, inant antigen of the system and that is considered in 1955, Fava Netto [2] standardized a polysaccharide- specific for P. brasiliensis in ID tests. Since many rich P. brasiliensis antigen to use in CF tests. With this steps are necessary to obtain the final antigen test and this kind of antigen, the serology for PCM preparation, efforts were made to simplify the achieved a 90% sensitivity, thus demonstrating the methodology in order to circumvent the material usefulness of the technique not only in diagnosis but and economic difficulties found in most Latin Amer- also in monitoring the course of the disease. ican laboratories. Double immunodiffusion (ID), that involves agar Since 1993, a simplified Ag7 antigen preparation or agarose gel support, is the most used test in technique as described here has been reproduced immunodiagnosis of PCM. The preparation of culture monthly in our laboratory with very good results. The filtrate antigens and their use for immunodiagnosis of fungus was initially grown on Sabouraud medium PCM by ID were pioneered by Restrepo [3] and (SAB) slant tubes for 3 days at 35°C. At that time, Negroni [4], who observed a level of sensitivity close growth (consisting entirely of yeast cells) was to 90%. During many years their antigens were collected from at least 10 tubes, yielding an inoculum kindly supplied by them and used by almost all of approximately 2 · 106 cells. These cells were laboratories working in this area. inoculated into 500-ml Erlenmeyer flasks containing For several years, many different antigenic prep- 100 ml yeast extract-peptone-dextrose (YPD) broth arations have been used for the serodiagnosis of PCM (Difco). This culture was incubated for 3 days at by ID. These various antigens lacked a standardized 35°C on a rotatory incubator at 50 r.p.m. The growth preparation from one laboratory to another, and obtained was transferred to 1,800-ml Fernbach flasks included sonic extracts from the yeast forms, con- containing 500 ml YPD broth. The flasks were centrated filtrates, and lyophilized to cite just a few further incubated as above for 7 days. Supernatant approaches. Moreover, each antigen was prepared fluids were collected following paper filtration, from cells grown in different culture media and under concentrated under vacuum at 45°C, and dialyzed different growth conditions (such as incubation time, against distilled water. The presence of gp43 was growth temperature, size of initial inoculum, and monitored by sodium dodecyl sulfate/polyacrylamide shaken or stationary cultures). It is possible for gel electrophoresis (SDS-PAGE) followed by silver different antigenic preparations to vary considerably staining. After this step, the antigen was tested by ID. in activity and quality as a result of lack of proper lot- If positive reactions were obtained with the control to-lot production standards even within the same sera, the antigen was aliquoted and maintained at laboratory. With this variety of problems in produc- 5°C. If the reactions were negative, the antigen was ing a proper P. brasiliensis antigen, it is not concentrated again and retested. In general, concen- surprising that there is considerable disagreement tration of the filtrate 10- to 15-fold is necessary. regarding the sensitivity of the ID test for the This simplified protocol for Ag7 production has serodiagnosis of PCM [5–11]. shown a high degree of sensitivity, specificity, and 123 Mycopathologia (2008) 165:289–302 291 reproducibility in the serodiagnosis of PCM. We tissue invasion. However, these ideal situations are believe this procedure can be performed by most not always possible, and so one must often employ laboratories, even those with relatively few diagnostic approaches that are based on serologic resources. Figure 1 shows the antigenic profile of testing. In the case of PCM, serologic testing, such as Ag7. immunodiffusion, is so reliable that a positive test, even if the titer is low, is indicative of infection. The last decades show a rapidly expanding field of immunology permitting the development of several Antibody detection new techniques, which were gradually being adopted by researchers interested in the diagnosis of fungal During the process of infection, the antigens from diseases, and several methods were currently being P. brasiliensis can activate B lymphocytes which used as possible aids in diagnosing some mycotic main function is to produce immunoglobulins (Ig). infections, mainly PCM. Much of those serologic These proteins play a crucial role in defense against a assays utilize polyclonal/monoclonal antibodies or variety of infectious microorganisms; they are present antigens which can be easily quantified with high in high concentrations in body fluids and in stable sensitivity and have resulted in methods with more form. These properties led to their relative ease of sensitivity and specificity. detection and quantitation, providing major criteria The successful application of serological tests for for the presence and activity of the disease (serodi- diagnosing mycotic infections is very important agnosis), and for monitoring both evolution of the because the incidence and mortality of the mycoses, disease and response to treatment.