Abstract
Penicilliosis, caused by the dimorphic fungus Penicillium marneffei, is an important opportunistic systemic fungal infection affecting immunocompromised individuals living in areas where penicilliosis is endemic. We have demonstrated previously that a urinary enzyme-linked immunosorbent assay (ELISA) with purified rabbit polyclonal antibody against killed whole-fission-form arthroconidia of P. marneffei was specific and highly sensitive for the diagnosis of penicilliosis. In this study, a dot blot ELISA and a latex agglutination (LA) test were developed with the same polyclonal antibody and compared with the ELISA for the detection of P. marneffei urinary antigen. Urine specimens from 37 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects and 248 hospitalized patients without penicilliosis) were tested. Antigen was detected in urine from all 37 (100%) penicilliosis patients by the LA test, 35 (94.6%) penicilliosis patients by the dot blot ELISA, and 36 (97.3%) penicilliosis patients by the ELISA. False-positive results were found by the three assays for 2 (0.7%), 8 (2.7%), and 6 (2%) of 300 controls, respectively. The overall sensitivities of the diagnostic tests were as follows: dot blot ELISA, 94.6%; ELISA, 97.3%; and LA test, 100% (specificities, 97.3, 98, and 99.3%, respectively). The LA test is simple, robust, rapid, and convenient and should prove to be an important addition to the existing diagnostic tests for penicilliosis.
Penicilliosis, caused by the dimorphic fungus Penicillium marneffei, is increasingly common in southeast Asia, particularly Thailand (4, 7). Human infection with P. marneffei was rare prior to the AIDS epidemic in this area, but the number of cases has since risen dramatically. In Thailand, penicilliosis is a major opportunistic infection in AIDS patients, particularly in the north of the country (4, 16). Infection with P. marneffei usually presents as a disseminated and progressive illness in patients with CD4 counts below 100, with fever, anemia, leukopenia, weight loss, diarrhea, cough, massive hepatosplenomegaly, generalized lymphadenopathy, and characteristic multiple umbilicated skin lesions particularly affecting the face and upper body (18, 19). Penicilliosis is fatal if it is left untreated but responds well to antifungal agents such as amphotericin B and itraconazole (19). A presumptive diagnosis of P. marneffei infection is made on clinical grounds in conjunction with microscopic identification of characteristic septate yeast cells in smears of skin lesions, blood, or bone marrow. Definitive diagnosis relies on the isolation of P. marneffei by culture from clinical specimens. However, cultures often require at least 3 days of incubation before definitive identification of the fungus is possible. Identification of P. marneffei by means of histological (15) or immunohistochemical (1, 12) techniques and exoantigen tests (11, 17) has been reported. These tests are specific, but time-consuming and laborious. Serological methods have also been developed. Detection of antibodies to P. marneffei by use of an immunodiffusion test (9, 13), an indirect fluorescent-antibody technique (23), and an enzyme-linked immunosorbent assay (ELISA) antibody detection test (3) have each been described, but these tests, while specific, are less sensitive than culture.
Antigen detection tests have also been studied (6, 13). We have shown previously that P. marneffei antigen can be detected at high titers in urine by ELISA with a fluorescein isothiocyanate (FITC)-labeled purified rabbit polyclonal hyperimmune immunoglobulin G (IgG) (6). This test is highly sensitive and specific. We now report on the development of a simplified dot blot ELISA and a latex agglutination (LA) test for detecting P. marneffei antigenuria by using the polyclonal anti-P. marneffei antibody used previously in the urinary antigen ELISA. The dot blot ELISA and LA tests were clinically evaluated in an area of northeast Thailand where penicilliosis is endemic and were compared with the urinary antigen ELISA for the diagnosis of penicilliosis.
MATERIALS AND METHODS
Patients and urine samples.
The study was conducted between June 1995 and July 2000. Adult patients with suspected or confirmed penicilliosis admitted to Sappasitprasong Hospital, Ubon Ratchathani, northeast Thailand, were included in the study. All patients were seen by a member of the study team, and full clinical details were recorded on a standard form. Routine hematological and biochemical tests were performed, and the patients were evaluated for the presence of human immunodeficiency virus (HIV) antibody when indicated. The diagnosis of penicilliosis was confirmed by isolation of P. marneffei from blood, urine, lymph node biopsy, and liver aspirate specimens or throat or skin lesion swab specimens; a presumptive diagnosis was made by direct identification of septate yeast cells in stained smears made from appropriate clinical specimens.
A total of 37 urine samples were obtained on admission from HIV-positive patients confirmed to have penicilliosis. Control urine samples were obtained from healthy individuals (n = 52); HIV-seropositive patients with other fungal infections (total, n = 34; cryptococcosis, n = 31; histoplasmosis, n = 1; candidiasis, n = 2); patients with melioidosis (n = 168), other bacterial causes of septicemia (n = 12), and other bacterial infections (n = 7); and inpatients for whom all cultures were negative (n = 27). All urine samples were stored at −30°C and thawed only at the time of testing.
Antigen preparation.
Antigen was prepared as described previously (6). Briefly, killed fission-form arthroconidia of a clinical isolate of P. marneffei (grown in brain heart infusion broth) were used to immunize rabbits and as the control antigen for the ELISA, dot blot ELISA, and LA test.
Anti-P. marneffei IgG preparation.
Hyperimmune rabbit sera with antibodies against P. marneffei were prepared as described previously (6). The purified IgG fraction was used in the ELISA, dot blot ELISA, and LA test.
ELISA.
Urinary P. marneffei antigen was detected by a sandwich ELISA, as described previously (6). The ELISA uses a polyclonal anti-P. marneffei antibody conjugated to FITC and an anti-FITC antibody amplification system. A titer of 1:40 or greater was considered a positive result for P. marneffei antigen.
Dot blot ELISA.
Optimal incubation conditions and reagent concentrations for the dot blot ELISA were determined by checkerboard titration. Urine samples were boiled for 6 min and centrifuged at 5,000 × g for 6 min to remove any precipitate before testing. A 2-μl aliquot of each urine sample was then dotted onto an 11.5-cm2 nitrocellulose (NC) membrane sheet (Bio-Rad Laboratories, Richmond, Calif.) at 1.3-cm intervals. The blotted NC sheet was dried at 37°C for 30 min and subsequently blocked with blocking buffer consisting of 2% bovine serum albumin (Sigma Chemical Company, St. Louis, Mo.) in phosphate-buffered saline (PBS) containing 0.02% Tween 20 (PBS-T) for 20 min at room temperature. After the NC sheet was washed with PBS-T, it was incubated with purified rabbit anti-P. marneffei IgG diluted 1:500 in blocking buffer for 1 h at room temperature. The sheet was washed once and was incubated for 1 h at room temperature with alkaline phosphatase-conjugated swine anti-rabbit immunoglobulin (Dakopatt, Copenhagen, Denmark) diluted 1:500 in blocking buffer. After further washing, the NC sheet was immersed in substrate solution (freshly prepared by mixing 330 μl of nitro blue tetrazolium [10 mg/ml] with 33 μl of 5-bromo-4-chloro-3-indolyl phosphate p-toluine salt [50 mg/ml of 100% dimethyl formamide; Sigma] and diluted to 10 ml with substrate buffer [0.1 M Tris buffer {pH 9.5} containing 100 mM sodium chloride and 5 mM magnesium chloride]) and then incubated for 10 min at room temperature to allow color development before it was rinsed with stopping buffer (0.02 M Tris buffer containing 0.2 M disodium EDTA). The NC sheet was then washed with distilled water and air dried. A purple dot indicated a positive reaction, and a pale brown dot or a clear area was read as a negative reaction. The same controls used in the ELISA were included in this assay. All urine samples and controls were tested in duplicate.
Preparation of LA test.
A purified IgG fraction of the pooled rabbit immune serum with antibodies against P. marneffei was adjusted to a concentration of 15 to 20 mg/ml and used to coat the test latex particles. A 1% suspension of latex polystyrene beads (diameter, 0.6 μm; Sigma) in glycine-buffered saline (pH 8.2) was sensitized by mixing of the beads with purified rabbit anti-P. marneffei IgG at 37°C for 2 h before the addition of bovine serum albumin to a final concentration of 0.5% (wt/vol). The optimal quantity of immunoglobulin used was the highest dilution that produced a clear agglutination with the highest reactive dilution of P. marneffei culture suspension (starting at a concentration of 106 yeast cells/ml in a box titration).
Control latex particles were coated with purified, pooled normal rabbit IgG. The suspensions were stored at 4°C and brought to room temperature before use.
LA method.
All urine samples were boiled for 6 min, cooled, and centrifuged at 5,000 × g for 6 min to remove any precipitate before testing. The LA test was performed by placing 15 μl of test and control latex suspensions on a clean glass slide. Five microliters of urine was added to the latex particles. After the components were mixed, the slide was rocked gently for 3 min. Agglutination with the test latex but not the control latex indicated a positive result, while a lack of agglutination with either latex indicated a negative result. Agglutination with both the test and the control latexes was interpreted as a nonspecific (negative) reaction.
Fungal and bacterial strains.
To evaluate the specificities of the dot blot ELISA and the LA method, Merthiolate-killed brain heart infusion broth cultures (20-ml broth volumes) of various fungal species (Penicillium griseofulvum ATCC 48166, Penicillium chrysogenum ATCC 9480, Penicillium notatum ATCC 9478, Aspergillus terreus, Aspergillus fumigatus BCC 123, Aspergillus flavus BCC 235, Histoplasma capsulatum var. capsulatum, Candida albicans [two strains], Candida kefyr, Cryptococcus neoformans var. neoformans, Cryptococcus neoformans var. gattii, Sporothrix schenkii, and Trichosporon beigelii) were tested at various concentrations, together with seven clinical strains of P. marneffei (three hyphal forms and four yeast-like forms). The cultures were incubated for 48 h at 37°C (Candida, Cryptococcus, and Trichosporon), 4 days at 37°C (P. marneffei yeast phase), or 4 days at room temperature (P. marneffei hyphal form and all other species). The final yeast cell or spore counts in broth were determined with a counting chamber. Following subculture to ensure purity, the organisms were killed by overnight incubation with Merthiolate (final Merthiolate concentration, 0.2%). Appropriate test dilutions were then made in sterile urine (boiled) from a healthy human volunteer. Strains of Burkholderia pseudomallei (strain 203a), Salmonella enterica serovar Enteritidis, and Staphylococcus aureus, which are common causes of systemic infection in Ubon Ratchathani, were also tested, in addition to a strain of S. enterica serovar Typhi. These fungal and bacterial control strains were all as described previously (6). All samples were tested in duplicate, and each test was repeated three times.
Statistical analysis.
Data were analyzed with SPSS for Windows (version 9.0; SPSS Inc., Chicago, Ill.) computer software. Quantitative variables with a nonnormal distribution were expressed as medians and ranges. Qualitative variables were expressed as frequencies and percentages. The sensitivities (proportions of positive samples correctly identified by the test) and the specificities (proportions of negative samples correctly identified by the test) of the dot blot ELISA and LA test were compared with those of the ELISA and evaluated by using the method of Galen (8). The sensitivities and specificities of the dot blot ELISA and LA test were compared with those of the ELISA by using the McNemar test. The measures of agreement between any pair of these three tests were assessed by use of the kappa statistic, which was interpreted as the chance corrected proportional agreement between the methods. A P value of <0.05 was considered significant.
RESULTS
Specificities of tests for P. marneffei.
Specificity results for the dot blot ELISA, the LA test, and the ELISA are presented in Table 1. All strains of P. marneffei culture supernatant, from both yeast and hyphal forms, gave positive results by the dot blot ELISA as well as by the LA test. In the LA test, the time for the appearance of agglutination for yeast-form whole-cell culture supernatants of P. marneffei (median, 4 s [range, 3 to 6 s]; n = 4) was significantly shorter than that for culture supernatants of the hyphal form (median, 7 s [range, 7 to 15 s]; n = 3) (P = 0.031). The lowest detection limits for P. marneffei antigen in both the dot blot ELISA and the LA test were 103 yeast cells/ml. Similar titration results were found when either PBS or urine was used as the diluent. These findings were similar to those of the established ELISA. However, the ELISA was more sensitive; it gave positive results at 102 yeast cells/ml. All the other fungal culture supernatants gave negative results in the dot blot ELISA, the LA test, and the ELISA up to concentrations of 108 cells/ml. All bacterial strains except S. aureus were nonreactive in these tests; S. aureus gave positive results in the dot blot ELISA and ELISA (but not the LA test), but only at a concentration of 108 cells/ml.
TABLE 1.
Reactivity of purified rabbit anti-P. marneffei IgG against control organisms in dot blot ELISA, LA test, and ELISA
| Species (no. of strains) | Forma | Reactivity with rabbit anti-P. marneffei IgG by:
|
||
|---|---|---|---|---|
| Dot blot ELISA | LA test | ELISA | ||
| Penicillium marneffei (4) | Y | + (103/ml)b | + (103/ml) | + (102/ml) |
| Penicillium marneffei (3) | H | + | + | + |
| Penicillium griseofulvum (1) | H | − | − | − |
| Penicillium chrysogenum (1) | H | − | − | − |
| Penicillium notatum (1) | H | − | − | − |
| Cryptococcus neoformans var. neoformans (1) | Y | − | − | − |
| Cryptococcus neoformans var. gatti (1) | Y | − | − | − |
| Histoplasma capsulatum var. capsulatum (1) | Y | − | − | − |
| Candida albicans (2) | Y | − | − | − |
| Candida kefyr (1) | Y | − | − | − |
| Aspergillus terreus (1) | H | − | − | − |
| Aspergillus fumigatus (1) | H | − | − | − |
| Trichosporon beigelii (1) | Y | − | − | − |
| Burkholderia pseudomallei (1) | − | − | − | |
| Salmonella enterica serovar Enteritidis (1) | − | − | − | |
| Salmonella enterica serovar Typhi (1) | − | − | − | |
| Staphylococcus aureus (1) | + (108/ml) | − | + (108/ml) | |
Y, yeast or yeast-like form; H, hyphal form.
Values in parentheses are the lowest detection limits (numbers of cells per milliliter).
Detection of P. marneffei antigen in urine by dot blot ELISA.
The results of the dot blot ELISA with urine from patients with penicilliosis and the control groups are given in Table 2. The results were compared with those of the established ELISA. Of the 37 HIV-positive patients with culture-confirmed P. marneffei infection, 35 (94.6%) were positive for P. marneffei urinary antigen by the dot blot ELISA and 36 (97.3%) were positive by the ELISA at a cutoff titer of ≥1:40. All 52 samples from healthy volunteers were negative by the dot blot ELISA. Of the 34 samples from patients with other fungal infections, 2 (5.9%) were positive by the dot blot ELISA. False-positive results were also found for 2 (1.2%) of 168 urine samples from patients with melioidosis, 1 (8.3%) of 12 urine samples from patients with other bacterial septicemias, 1 (14.3%) of 7 urine samples from patients with other bacterial infections, and 2 (7.4%) of 27 subjects who were culture negative. Thus, false-positive results were found for 8 (2.7%) of 300 control urine samples overall by the dot blot ELISA. This compares with 6 (2%) of 300 control urine samples that were positive by ELISA.
TABLE 2.
Comparison of dot blot ELISA, LA test, and ELISA for detection of P. marneffei antigen in urine specimens
| Patient group (no. of patients) | No. (%) of patients positive by:
|
||
|---|---|---|---|
| Dot blot ELISA | LA test | ELISAa | |
| Penicilliosis (37) | 35 (94.6) | 37 (100) | 36 (97.3) |
| Other fungal infection (34) | 2 (5.9) | 2 (5.9) | 4 (11.8) |
| Melioidosis (168) | 2 (1.2) | 0 (0) | 0 (0) |
| Other septicemia (12) | 1 (8.3) | 0 (0) | 1 (8.3) |
| Other bacterial infection (7) | 1 (14.3) | 0 (0) | 1 (14.3) |
| Culture negative (27) | 2 (7.4) | 0 (0) | 0 (0) |
| Normal healthy controls (52) | 0 (0) | 0 (0) | 0 (0) |
A titer in urine ≥1:40 was considered a positive reaction for P. marneffei antigen.
Detection of urinary antigen by the LA test.
All 37 samples from confirmed penicilliosis patients were positive by the LA test. The results are shown in Table 2. The median time to agglutination was 30 s (range, 15 to 180 s); i.e., the time to agglutination was longer than that observed with whole-cell cultures of P. marneffei. All except 2 of the 300 control urine samples were negative; these 2 (5.9%) samples were from among the 34 patients with other fungal infections (the 2 samples were both reactive by the dot blot ELISA and the ELISA, and both patients were HIV antibody positive). Thus, false-positive results were found for only 2 (0.7%) of 300 control urine samples overall by the LA test. Only one urine sample (from a patient in the “other septicemia” group) gave a nonspecific reaction.
Diagnostic sensitivity and specificity.
The sensitivity and specificity of the dot blot ELISA, the ELISA, and the LA test are summarized in Table 3. By using a cutoff titer of 1:40 in the ELISA, the overall sensitivity for penicilliosis was 97.3% and the specificity, using data for all control groups, was 98% (positive predictive value [PPV], 85.7%; negative predictive value [NPV], 99.7%). The sensitivity of the dot blot ELISA was lower, at 94.6%, with a specificity of 97.3% (PPV, 81.4%; NPV, 99.3%). The sensitivity and specificity of the LA test were 100 and 99.3%, respectively (PPV, 94.9%; NPV, 100%). There were no significant differences between the three tests (dot blot ELISA versus ELISA, P = 0.99; LA test versus ELISA, P = 0.45; and dot blot ELISA versus LA test, P = 0.29). In addition, there was significant agreement between each pair of tests (dot blot ELISA versus ELISA, κ = 0.825 and P < 0.001, LA test versus ELISA, κ = 0.902 and P < 0.001, and dot blot ELISA versus LA test, κ = 0.889 and P < 0.001).
TABLE 3.
Sensitivity and specificity of P. marneffei urinary antigen detection by dot blot ELISA, LA test, and ELISA
| P. marneffei antigen detec- tion test | % Sensi- tivity | % Specificity
|
||
|---|---|---|---|---|
| All patients (n = 300) | Other inpatients (n = 248) | Healthy controls (n = 52) | ||
| Dot blot ELISA | 94.6 | 97.3 | 96.8 | 100 |
| LA test | 100 | 99.3 | 99.2 | 100 |
| ELISA | 97.3 | 98 | 97.6 | 100 |
These results indicate that although the LA test achieved the highest sensitivity and PPV for detection of P. marneffei antigen in urine, there is little in terms of performance from which to choose among the three tests for the diagnosis of penicilliosis.
DISCUSSION
Penicilliosis is now recognized as one of the most important opportunistic infections in AIDS patients in Thailand, affecting 6.8% of AIDS patients in the north of the country (4). At present, the diagnosis of P. marneffei infection relies on the identification of yeast cells in smears of skin lesion, blood, or bone marrow specimens, but there may be confusion with histoplasmosis or disseminated cryptococcosis. Culture is often the only way to confirm the diagnosis, particularly if characteristic skin lesions are absent, but this may take several days. Thus, a rapid diagnostic or confirmatory test would be of clinical value, and its results could be used to direct the appropriate antifungal therapy. Rapid identification of fungal pathogens, once isolated in pure culture, can be achieved with specific DNA probes (14), but these tests have not yet been applied to clinical specimens for rapid diagnosis.
There have been few advances in diagnostic tests for penicilliosis since a previous report (6). Several specific protein antigens of P. marneffei have been identified to be immunologically reactive in Western blot assays for approximately 50% of serum specimens from patients with penicilliosis and 20% of serum specimens from patients with cryptococcosis or candidiasis (5, 10, 20). A mannoprotein antibody ELISA has been reported to be positive for 80% of HIV-positive penicilliosis patients (3), but serum specimens from patients with other fungal infections were not included in that study, and there have been no reports of prospective clinical evaluations. Thus, the usefulness of antibody tests for diagnosis of penicilliosis in areas where penicilliosis is endemic remains uncertain.
Antigen detection tests may be more appropriate than antibody detection tests for the diagnosis of penicilliosis in immunocompromised patients, in whom antigen loads are high and antibody responses may be muted (22). Detection of P. marneffei antigen also suggests active rather than past infection, which is particularly relevant in an area of endemicity. A fluorescent-antibody test for the tissue form of P. marneffei that uses a rabbit polyclonal antibody raised against whole P. marneffei yeast cells has been described (12), as has an LA test for detection of P. marneffei antigen in serum and urine (this was evaluated with a small number of patient and control serum samples, and only two urine samples from penicilliosis patients were tested) (13). Prospective clinical evaluations of these tests have not been reported. A cell wall mannoprotein antigen has been demonstrated in the serum of 65% of 26 penicilliosis patients; the sensitivity was increased to 88% if this test was used in conjunction with an antibody detection test (2). A urinary antigen detection test with a rabbit IgG antibody developed for diagnosis of H. capsulatum var. capsulatum infection has been reported to give positive results for 17 of 18 confirmed penicilliosis patients (21). More recently, we reported that a rabbit polyclonal antibody raised against killed whole-fission-form arthroconidia of P. marneffei could be used to quantify urinary antigen by ELISA (6). At a cutoff titer of 1:40, this urinary antigen ELISA had a diagnostic sensitivity of 97% and a specificity of 98%. The polyclonal rabbit antibody used previously in the ELISA was used in the present study for the development of the dot blot ELISA and the LA test.
Each of our tests, the dot blot ELISA, the ELISA, and the LA test, appears to be highly sensitive and specific. The LA test detected antigen in all 37 urine specimens from patients with culture-confirmed penicilliosis, whereas the dot blot ELISA and ELISA detected antigen in 35 and 36 urine specimens, respectively. The two specimens with false-negative dot blot ELISA results had ELISA titers of only 1:40 (the cutoff titer). Of the remaining 35 dot blot ELISA-positive specimens, 34 had ELISA titers of 1:320 or greater. One dot blot ELISA-positive undiluted urine specimen had antigen detectable only by ELISA (the patient presented initially with disseminated cryptococcal infection). The LA test was also rapid; agglutination occurred in all 37 positive specimens by 180 s (median, 30 s). Only two positive results were found among 300 control specimens; these specimens were positive by all three tests. Both specimens were from HIV-positive patients with cryptococcosis, and we cannot exclude the possibility that they were also infected with P. marneffei, although we were unable to culture it from suitable specimens. False-positive results by the dot blot ELISA (eight patients) were slightly more common than false-positive results by the ELISA (six patients). Two patients gave false-positive results by both the dot blot ELISA and the ELISA; one of these patients was also HIV positive and presented with a Salmonella septicemia, while a coliform organism was cultured from the urine of the other patient (whose HIV infection status was unknown); again, we are unable to exclude penicilliosis in these patients.
The antigens detected by the polyclonal rabbit antibody remain to be characterized. However, it is evident from our studies that detection of antigen in urine with this antibody is highly specific and sensitive whether the antibody is used in an ELISA, a dot blot ELISA, or an LA test. There is little in terms of performance from which to choose among the three tests. However, the LA test is by far the more preferable for routine diagnostic use: it is a simple, convenient, rapid, and reliable method for detecting P. marneffei antigenuria, which should allow its widespread use even where laboratory facilities are very limited. The ELISA and dot blot ELISA do not offer any advantages over the LA test and are considerably more laborious and time-consuming. The LA test reagent can be readily produced by sensitizing latex particles with our rabbit polyclonal antibody, and it should prove useful as a diagnostic test for P. marneffei infection. It should therefore be developed further. We also hope to evaluate this test for the detection of antigenemia and use it to assess responses to antifungal therapy in a study of serial urinary antigen testing during treatment and subsequent follow-up.
Acknowledgments
We thank Wipada Chaowagul, Yupin Supputamongkol, Wirongrong Chierakul, Brian Angus, Boongong Pimsa-ad, and Sayan Langla for assistance and Sornchai Looareesuwan (dean of the Faculty of Tropical Medicine, Mahidol University) and Wichai Supanaranond (head of the Department of Clinical Tropical Medicine) for support and encouragement. We also thank Mandy Walsh for original work, plus the director of Sappasitprasong Hospital and the medical and nursing staff of the Department of Medicine for continued support.
This study was part of the Wellcome-Mahidol University, Oxford Tropical Medicine Research Programme, funded by the Wellcome Trust of Great Britain.
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