INTRODUCTION
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with most reported cases occurring in Brazil.1 The fungus rarely leads to overt disease and asymptomatic infections have been recognized.2 For many years, different crude antigen preparations have been used in epidemiologic surveys to diagnose infection with Paracoccidioides brasiliensis.3 The majority of these surveys used the Fava Netto polysaccharide antigen, also known as paracoccidioidin,4 a crude preparation obtained from different samples of the fungus. Surveys carried out in the Amazon region using paracoccidioidin with simultaneous administration of histoplasmin have shown a wide variation in prevalence rates from 6.4% to 43.8%.5,6 Since standardized crude antigen preparations were not used in these surveys, it is difficult to determine the validity of these results. This is especially true since cross-reactivity with Histoplasma capsulatum, a common fungus thought to be more prevalent than P. brasiliensis in the Amazon region,6 has been shown to affect test specificity in the diagnosis of paracoccidioidomycosis.7 Ideally, more specific preparations need to be used in surveys to distinguish between these two fungal infections.
It has been recently shown that a purified component of P. brasiliensis antigen, a 43-kD glycoprotein (gp43) that is released by the fungus, can elicit delayed hypersensitivity in humans and infected animals.8–10 In experimentally infected guinea pigs, gp43 evokes a response similar to that of Fava Netto paracoccidioidin.8 When 25 patients with paracoccidioidomycosis were tested with both antigens, all but one patient reactive to paracoccidioidin were also reactive to gp43.8 However, there were five other patients who were reactive only to gp43, suggesting that this antigen may be more sensitive than Fava Netto paracoccidioidin.
Gp43 is the immunodominant antigen recognized by up to 100% of patients with paracoccidioidomycosis.11 This immunodominant antigen of P. brasiliensis also stimulates a humoral immune response, and is useful in diagnosis and post-therapeutic control.12 To identify the immunodominant epitopes in both humoral and cellular responses, the gene for gp43 has been cloned and sequenced. The gp43 open reading frame was found in a 1,329-basepair fragment containing two exons interrupted by an intron of 78 nucleotides.13 The component responsible for glycoprotein-mediated T cell activation and protection against paracoccidioidomycosis was mapped to a 15-amino-acid peptide (P10).14 The immune response in mice immunized with gp43 plus Freund’s complete adjuvant involves CD4+ Th-1 lymphocytes secreting interferon-gamma (IFN-γ) and interleukin 2 (IL-2) but not IL-4 and IL-10.14 In human paracoccidioidomycosis, lymphocyte hyporesponsiveness to gp43 has been observed in active disseminated disease with secretion of low levels of IL-2 and IFN-γ, but substantial amounts of IL-10.15 In addition, gp43 has an important effect in the fungus pathogenesis. The specific binding of gp43 surface protein to laminin by mechanism of adhesion to extracellular matrix may constitute the basis for the progression of the infection toward regional spreading and dissemination.16
There are no reports of epidemiologic surveys using gp43 in populations in rural Amazon regions, or any comparisons between crude paracoccidioidin and gp43 in field studies. In the present work, we designed a study to determine the prevalence of P. brasiliensis infection in children born and living in an endemic area in the Southern Amazon Basin. It was also our objective to evaluate the cross-reactivity of the polysaccharide-like antigen and gp43 skin tests with H. capsulatum infection.
METHODS
Study area.
Peixoto de Azevedo (Mato Grosso State) is a town in the Southern Amazon Basin of Brazil located 290 meters above sea level. Its climate is equatorial, with an average annual precipitation of approximately 2,500–3,000 mm. This study was performed in October 1996. The population estimate in 1996 was 29,333 inhabitants, and the most common occupations were gold mining and subsistence farming. Over the past 20 years, the town’s population had expanded rapidly due to the discovery of new gold deposits. It then decreased to approximately one-third of its peak as the deposits were depleted.
Subjects.
The study population consisted of 298 healthy school children between seven and 18 years of age who were enrolled into the study at two school sites. Subjects were randomized to four study groups in a ratio of 4:4:1:1, respectively. Group A (117 subjects) was tested with paracoccidioidin and histoplasmin intradermally, group B (115 subjects) was tested with gp43 and histoplasmin, group C (34 subjects) was tested with paracoccidioidin and placebo, and group D (32 subjects) was tested with gp43 and placebo. Groups A and B were disproportionately large so that the prevalence of P. brasiliensis infection stratified by the presence or absence of infection with H. capsulatum could be examined. Groups C and D received placebo instead of histoplasmin and were used as controls to determine the effects of histoplasmin on tests for P. brasiliensis antigens by comparing groups A and B versus C and D.
Participants were interviewed prior to skin testing and a physical examination was performed with special attention given to mucosal membranes, enlarged lymph nodes, and hepatosplenomegaly. Epidemiologic information collected from the subjects included date and place of birth, regions of residence, period of residence in the study area, and exposure to forests and rivers. Informed consent for participation in the study was obtained from patients or their parents or guardians. The study protocol was reviewed and approved by the Ethical and Research Committee of the Hospital das Clínicas of the Medical School of the University of Sao Paulo.
Antigens.
Paracoccidioidin (a polysaccharide-like antigen from P. brasiliensis) was prepared using seven strains of P. brasiliensis grown in Fava Netto agar medium for 20 days at 36°C as previously described.17 Yeast cells were collected, suspended in physiologic saline, centrifuged at 3,000 rpm for 10 minutes, and the supernatant fluids were discarded. Cells were then rinsed in acetone and ethyl ether, suspended in veronal buffer, pH 7.4, and autoclaved for 20 minutes at 120°C. Supernatant fluids were passed through sterilizing membranes, preserved in 1:10,000 thimerosal, distributed into flasks, and maintained at 4°C until use. Paracoccidioidin was diluted 1:3 in sterile saline for use in intradermal tests.
The exoantigen from P. brasiliensis B-339 was used as antigen source to obtain purified gp43. Supernatant fluids from seven day-old cultures were filtered, concentrated and dialyzed according to previous reports.18 Gp43 was purified by affinity chromatography on a Affigel 10 column (Bio-Rad Laboratories, Hercules, CA) coupled to an IgG anti-gp43 mouse monoclonal antibody. Protein contents were determined by the method of Bradford.19 For intradermal use, a solution of gp43 in saline containing 50 μg/mL was sterilized by filtration through disposable filters (Sigma, St. Louis, MO) and stored at −20°C until use.
Crude histoplasmin was prepared as previously described.20,21 Three different isolates of H. capsulatum were cultured in the Smith-asparagine semi-synthetic medium at room temperature for six months. The antigen was obtained from 20-fold concentrated culture filtrates. Thimerosal was added to a final concentration of 1:5,000 for preservation and the antigen was maintained at 4°C until use. The histoplasmin was diluted 1:1,000 in sterile saline for use in intradermal tests.
The placebo used for groups C and D was sterile 0.9% physiologic saline.
Skin tests were performed by injecting 0.1 mL of either antigen or placebo dilution by the intradermal route onto the anterior surface of patients’ forearms using standard techniques. Paracoccidioidin or gp43 was always injected onto the right forearm and histoplasmin or placebo onto the left forearm. Readings of reactions were taken 48 hours after inoculations. The reader was blinded with regard to specific study group. All test results were considered positive whenever a diameter of induration ≥ 5 mm was observed (diameter measured with a millimeter ruler).
Statistical analysis.
Results were analyzed using Epi-Info version 6.02 (Centers for Disease Control and Prevention, Atlanta, GA). Data were analyzed by analysis of variance (ANOVA), the chi-square test, and Fisher’s exact test. Results were considered significant when the P value was ≤ 0.05. Binomial calculations were used to determine confidence intervals (CIs) of proportions.
RESULTS
None of the subjects presented clinical signs of the disease. There were no significant differences among the four study groups administered different skin tests regarding sex (P = 0.14), age (P = 0.82, by ANOVA), period of residence in Peixoto de Azevedo (P = 0.349, by ANOVA), forest exposure (P = 0.56), river exposure (P = 0.66), and proportion of subjects enrolled from each school (P = 0.24) (Table 1).
Tests results are shown in Tables 2 and 3. In group A, there was a higher proportion of subjects who showed reactivity in the paracoccidioidin test among histoplasmin-reactive subjects (44%, 16 of 36; 95% CI = 28–62%). All other groups had similar, low proportions of subjects reactive to paracoccidioidin or gp43 (4–6%, overall prevalence = 4.6%; 95% CI = 2.5–7.7%). This proportion was significantly lower (P < 5 × 10−4) for each group than that of the histoplasmin-reactive subjects in group A. These data resulted in a higher risk ratio (RR) (histoplasmin reactivity as a risk factor for reactivity in the paracoccidioidin test) for group A (RR(A) = 9.0, 95% CI = 3.2–25), while the RR observed in group B was not statistically significant (RR(B) = 1.6, 95% CI = 0.3–9). No subjects in groups C and D showed reactivity in the skin test with placebo.
To investigate risk factors associated with P. brasiliensis-positive skin test results, we compared those with negative test results (n = 269) with those with positive test results, excluding those from group A who were reactive to both histoplasmin and paracoccidioidin (n = 13) (Table 4). We could not distinguish between false-positive and true-positive P. brasiliensis reactions in the latter group. There were significant differences between groups with regard to age (mean ages = 13.4 and 11.3 years for positive and negative subjects, respectively; P = 0.005, by ANOVA) and period of residence in the study area (mean periods = 9.6 and 7.6 years for positive and negative subjects, respectively; P = 0.036, by ANOVA). None of the other factors analyzed (sex, school, forest exposure, river exposure, enlarged lymph nodes, and hepatosplenomegaly) were significantly different between the two groups.
Having identified age and period of residence in the study area as risk factors for paracoccidioidomycosis, we next compared these factors between histoplasmin-reactive subjects in groups A and B to see if unequal distribution of either factor represents a marker for differences in exposure, which might explain differences in P. brasiliensis reactivity. The histoplasmin-positive subjects from groups A and B were comparable regarding the two risk factors for previous P. brasiliensis exposure (age, P = 0.43; period of residence in study area, P = 0.54).
With regard to the prevalence of histoplasmin reactions, overall histoplasmin reactivity was 31% (36 of 117) and 30% (34 of 115) in groups A and B, respectively (Table 2). However, in group A, there was a higher proportion of subjects who reacted to histoplasmin among those with a positive paracoccidioidin test result (80%, 16 of 20; 95% CI = 56–94%) compared with those with a negative paracoccidioidin test result (20%, 20 of 97; 95% CI = 13–29%). Histoplasmin-reactive subjects in groups A and B were comparable with regard to baseline and possible risk factors: age (P = 0.43, by ANOVA), period of residence in Peixoto de Azevedo (P = 0.54, by ANOVA), sex (P = 0.97), proportion of subjects enrolled from each school (P = 0.15), forest exposure (P = 0.64), and river exposure (P = 0.30).
DISCUSSION
Since the ecology of P. brasiliensis is poorly defined and screening tests for exposure to P. brasiliensis are not specific, it has been difficult to examine the epidemiology and geographic distribution of this organism. Paracoccidioides brasiliensis has been isolated from the soil on few occasions22,23 and infections in armadillos have been reported.24,25 To study the epidemiology of this infection, many surveys have been conducted using different preparations of paracoccidioidin to perform skin tests on populations and identify markers of previous infection. Because subjects previously infected with H. capsulatum may have positive paracoccidioidin skin test results,7 histoplasmin tests have been frequently included in such studies. Cross-reactivity between the two fungi using non-standardized antigen preparations has made it difficult to identify false-positive paracoccidioidin test results in those subjects previously sensitized by H. capsulatum. A more specific test for infection with P. brasiliensis is urgently needed, particularly in situations where infection with P. brasiliensis is much lower than that of infection with H. capsulatum. In such situations, cross-reactivity would lead to significant overestimations of true cases of the former while having relatively little effect on the latter.
Perhaps the great variation of P. brasiliensis prevalence in previous surveys carried out in the Amazon region (from 6.4% to 43.8%)5,6 was influenced by cross-reactivity in the different degrees of exposure to H. capsulatum among the different study groups. In these same studies, H. capsulatum prevalence was estimated by histoplasmin skin tests, and this prevalence varied from 5.8% to 80.5%. There was an indication that cross-reactivity may have occurred in other studies since Mok and Fava Netto observed a higher reactivity to paracoccidioidin in individuals with positive histoplasmin test results (24.6%) when compared with individuals with negative histoplasmin test results (3.2%).5 Note that this eight-fold increase is similar to the nine-fold increase observed by our group. However, they could not rule out that the association of reactivity to paracoccidioidin and histoplasmin might have been due to co-infection rather than cross-reactivity.
From the point of view of studying the prevalence of H. capsulatum infection, it is possible that simultaneous skin tests using histoplasmin and paracoccidioidin may also lead to false-positive results. In our study, as in others,6,7,26 since H. capsulatum infection has a much higher prevalence than P. brasiliensis infection, the distortion of the observed histoplasmosis prevalence is relatively small compared with the converse situation, in which one overestimates the P. brasiliensis infection prevalence. This is seen in our results. The H. capsulatum infection prevalence is relatively constant for both groups (approximately 30%, 36 of 117 in group A and 34 of 115 in group B; Table 2), despite a much larger number positive for histoplasmin in group A individuals positive for paracoccidioidin (80%, 16 of 20; Table 2). Some humoral cross-reactivity, mainly in enzyme-linked immunosorbent assays, was reported with gp43 and was abrogated by adsorption with H. capsulatum and Leishmania antigens.27 This cross-reactivity is mainly attributed to the polysaccharide component of the molecule.10,28
Our results show that the proportion of subjects who reacted to paracoccidioidin was significantly greater only in those reactive to histoplasmin antigen. All other groups (non-reactive to histoplasmin, not tested with histoplasmin, or tested with gp43) showed strikingly similar paracoccidioidin-or gp43-reactive proportions. These latter groups were used to determine the prevalence of previous P. brasiliensis exposure, as well as to identify age and period of residence in study area as risk factors. In other studies, the prevalence of P. brasiliensis infection increased with age.26,29 Similar P. brasiliensis infection prevalences in histoplasmin-positive and -negative individuals in group B suggest that co-infection is rare in these individuals, at least within the context of our study population/environment. This argument is also supported by the observation that groups C and D did not have higher proportions of paracoccidioidin-positive subjects than the histoplasmin-negative subjects in groups A and B. Since the risk factors for a paracoccidioidin-positive test result identified in this study (age and period of residence in study area) were similar for the histoplasmin-reactive subjects in groups A and B, it is conceivable that the difference in the paracoccidioidin reactivity results from the testing method itself, rather than confounded by infection risk. All of these findings are compatible with the interpretation that cross-reactivity (positive paracoccidioidin test result) occurs only when the immune memory of a previous H. capsulatum infection is “boosted” by histoplasmin skin testing. A false-positive test result does not occur when no histoplasmin challenge is given or if the histoplasmin test result is negative (presumably due to the absence of previous infection). Although the numbers are small, similar rates of reactivity between paracoccidioidin and gp43 for those not reactive to histoplasmin suggests similar sensitivities between preparations. If one takes into account the higher specificity of gp43, this antigen should be preferentially used when histoplasmin antigen testing is to be done, or if there is a chance that histoplasmosis infections could result in a high rate of cross-reactivity.
We hasten to point out that our findings may be specific only to our study population. Our study did not incorporate gold standard control groups. Previous studies were based on hospitalized patients undergoing treatment for paracoccidioidomycosis in whom reactions to gp43 were more frequently observed than reactions to paracoccidioidin.8 In populations such as ours, sensitivity may be low. Conversely, although our findings suggest very little cross-reactions in certain groups, one must remember that our subjects were children, whose histoplasmin reactions and associated cross-reactivity may be weak. Perhaps older individuals with longer and more frequent exposure to P. brasiliensis may show greater cross-reactivity in the groups in whom we did not see excessive numbers of positive reactions. In summary, our findings support the fact that there is a relative advantage (improved specificity) of gp43 compared with crude paracoccidioidin. Notwithstanding, the absolute measures of sensitivity and specificity (as for all diagnostic tests) need to be worked out with gold standard positive and negative groups for each target population.
Description of subjects according to age, period of residence in Peixoto, Brazil, sex, forest exposure, and river exposure*
| Groups | A (n = 117) | B (n = 115) | C (n = 34) | D (n = 32) | All groups (n = 298) | Test (P) |
|---|---|---|---|---|---|---|
| * ANOVA = analysis of variance. | ||||||
| Mean (SD) age, years | 11.47 (2.89) | 11.63 (2.95) | 11.18 (2.47) | 11.25 (2.41) | 11.48 (2.82) | ANOVA (0.822) |
| Mean (SD) period of residence in Peixoto, years | 8.10 (3.6) | 7.26 (3.83) | 7.91 (2.97) | 7.97 (3.4) | 7.74 (3.61) | ANOVA (0.349) |
| Sex, female no. (%) | 56 (47.5) | 69 (60) | 18 (52.9) | 21 (65.6) | 135 (45.2) | χ2 = 5.64 (0.13) |
| Forest exposure, no. (%) | 39 (33.1) | 48 (41.7) | 14 (41.2) | 12 (37.5) | 113 (37.8) | χ2 = 2.06 (0.56) |
| River exposure, no. (%) | 90 (76.3) | 94 (81.7) | 27 (79.4) | 27 (84.4) | 238 (79.6) | χ2 = 1.58 (0.66) |
Skin tests results in groups A and B*
| Group A | Paracoccidioidin (+) | Paracoccidioidin (−) | Total | Frequency (+)/total (95% CI) | Risk ratios |
|---|---|---|---|---|---|
| * CI = confidence interval; Hist = histoplasmin skin test. | |||||
| His + | 16 | 20 | 36 | 44% (28–62%) | 9.0 |
| Hist − | 4 | 77 | 81 | 5% (1.4–12%) | (95% CI = 3.2–25) |
| Total | 20 | 97 | 117 | 17% (11–25%) | |
| Group B | gp 43 (+) | gp 43 (−) | Total | Frequency (+)/total (95% CI) | Risk ratios |
| Hist + | 2 | 32 | 34 | 6% (0.7–20%) | 1.6 |
| Hist − | 3 | 78 | 81 | 4% (0.8–10%) | (95% CI = 0.3–9) |
| Total | 5 | 110 | 115 | 4% (1.4–10%) | |
Skin tests results in groups C and D*
| Group C | Paracoccidioidin (+) | Paracoccidioidin (−) | Total | Frequency (+)/total (95% CI) |
|---|---|---|---|---|
| * CI = confidence interval. | ||||
| Placebo (+) | 0 | 0 | 0 | 0 |
| Placebo (−) | 2 | 32 | 34 | 6% (0.7–20%) |
| Group D | gp 43 (+) | gp 43 (−) | Total | Frequency (+)/total (95% CI) |
| Placebo (+) | 0 | 0 | 0 | 0 |
| Placebo (−) | 2 | 30 | 32 | 6% (0.8–21%) |
Summary of all groups, excuding 16 subjects from group A with positive histoplasmin and positive paracoccidioidin test results*
| Groups A/B/C/D | Paracoccidioidin or gp43 (+) | Paracoccidioidin or gp43 (−) | Total | Frequency (+)/total (95% CI) |
|---|---|---|---|---|
| * CI = confidence interval. | ||||
| Total | 13 | 269 | 282 | 4.6% (2.5–7.7%) |
Authors’ addresses: E. M. N. Kalmar and F. E. C. Alencar, Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil. F. P. Alves, Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de Sao Paulo, Sao, Paulo, Brazil. L. W. Pang, United States Army Medical Research Unit-Brazil, Rio de Janeiro, Brazil. G. M. B. Del Negro Laboratório de Micologia Médica, Hospital das Clínicas, Faculdade de Medicina, Universidade de Sao Paulo, Av. Dr. Enéas Carvalho Aguiar, 500 Térreo, Sala 4, CEP 05403-000, Sao Paulo, SP, Brazil. Z. P. Camargo, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil. M. A. Shikanai-Yasuda, Laboratório de Imunologia Médica, Hospital das Clínicas, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil.
Acknowledgments: We thank the Health Department of the Municipality of Peixoto de Azevedo (Mato Grosso, Brazil) for their assistance, Dr. Rui Rafael Durlacher for field support, Dr. Crispim Cerutti, Jr. for helpful suggestions, and Dr. Gil Benard for critically reviewing the manuscript. This work was presented in part at the Eighth International Congress of Infectious Diseases, Boston, MA, May 15–18, 1998.
Financial support: This work was supported by the Centro de Estudos Walter Leser, Fundação Faculdade de Medicina.
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