Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Serological Survey of Paracoccidioidomycosis in Sheep

The objective of this study was to evaluate the prevalence of antibodies against Paracoccidioides brasiliensis in sheep from Guarapuava, Paraná State, Brazil. The seroepidemiological study was carried out in 262 sheep. The samples were analyzed by ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. Initially, two sheep were immunized with P. brasiliensis to evaluate whether contact with the fungal cells could induce a humoral immune response against gp43 and exoantigen from P. brasiliensis. Both animals produced antibodies against gp43 and exoantigen, the main antigens used for diagnosis and seroepidemiology of paracoccidioidomycosis. A reactivity of 37% was observed to the P. brasiliensis gp43 antigen by ELISA although no reactivity had been observed by the immunodiffusion test. Sheep under extensive grazing system showed higher frequency of positivity to P. brasiliensis (P ≤ 0.05) than those under intensive and semi-intensive systems. These data suggest that sheep may be a useful epidemiological marker of P. brasiliensis presence in the environment and reinforce that contact with soil is an important risk factor for infection.     

Phenotypic differences among single-oospore cultures of Phytophthora palmivora and P. botryosa from Heavea brasiliensis

Oospores ofPhytophthora palmivora andP. botryosa fromHevea brasiliensis were produced when complementary strains of the same species were incubated on V-8 juice agar in continuous darkness, with or without a subsequent period of continuous light. The oospores germinated at a rate of 15–30 % in demineralised water at 26 °C in normal daylight conditions. Other substrates did not improve the germination rate. Single-zoospore colonies derived from sporangia formed by a single oospore were similar to each other in morphology and in pathogenicity toHevea leaves. Over 400 single-oospore isolates from four intraspecific matings ofP. palmivora, and 102 from one pairing ofP. botryosa, were examined. The progeny differed in morphological appearance, mating behaviour, temperature-growth relations, pathogenicity toHevea leaf petioles and cacao pods, rate of production, shape and size of sporangia and in the abundance of chlamydospores. The progeny from an intraspecific cross ofP. botryosa was more variable, with a few isolates being similar in appearance toP. palmivora, having permanently lost their parental characteristic of producing small oval sporangia in clumps. One isolate in particular was indistinguishable fromP. palmivora in morphology and in its ability to produce functional oospores when mated withP. palmivora. Oospores formed by interspecific crosses could not be germinated. With both species, many progeny was less pathogenic than the parents, and many completely non-infective isolates with respect toHevea, cacao and other host plants were produced. Sexual reproduction resulted in a diversity of phenotypes, and both parental types and recombinants were recovered.     

Serological detection of antibodies against Paracoccidioides brasiliensis in dogs with leishmaniasis

The aim of this study was to detect antibodies against Paracoccidioides brasiliensis in dogs seropositive and seronegative for leishmaniasis. Sera from 836 dogs (449 positive and 387 negative to leishmaniasis) were analysed by ELISA and the immunodiffusion test using gp43 and exoantigen, respectively. The analysis of the 836 serum samples by ELISA and the immunodiffusion test showed a positivity of 67.8 % and 7.3%, respectively, for P. brasiliensis infection. The dogs positive to leishmaniasis showed a higher reactivity to gp43 (79.9%) and exoantigen (12.7%) than the negative ones (54.0% and 1.0%, respectively). The higher reactivity to P. brasiliensis antigens may be due to cross-reactivity or a co-infection of dogs by Leishmania and P. brasiliensis. The lower correlation (0.187) observed between reactivity to gp43 and Leishmania antigen reinforces the latter hypothesis.     

Antigenic relationships between Aracoccidioides loboi and other pathogenic fungi determined by immunofluorescence

Summary The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.Deceased. Last address: Fundacão Gonçalo Moniz, Salvador, Bahia, Brazil. Requests for reprints should be sent to Dr.William Kaplan.Dr.Miranda is in private practice in Rio de Janeiro, Brazil.     

Adenosine triphosphatase in yeast phaseParacoccidioides brasiliensis

We have previously detected various enzymatic activities inP. brasiliensis. In the present study we have examined Adenosine Triphosphatase (ATPase) activity in yeast phase cultures ofP. brasiliensis of increasing age. We employed Wachstein and Meisel's method and electron microscopy and found specific electron-dense deposits indicating ATPase activity to be present in the cytoplasm around vacuoles. Their distribution varied according to age. Deposits decreased or became absent in old cultures. We assume that inP. brasiliensis, ATPase is involved (as in other systems) with transport of Na⁺ and K⁺.     

Immunoelectrophoretic characteristics ofOphiobolus graminis Sacc. as an aid in classification and determination

Antigens from four cultures ofO. graminis were compared immunoelectro-phoretically. Each culture produced a characteristic immunogram. More common antigens were found between the two cultures isolated from wheat or the two cultures isolated from oat than between a wheat and an oat isolate. Cell-wall antigens were the best reference antigens for serologic analysis of strain relationship. O. graminis antisera were cross-reacted with antigens from a number of other species of fungi. Relatively few of these cross-reacted with antisera to cell-wall antigens whereas more cross-reacted with antisera to whole-cell antigens.Immunoelectrophoretic analysis of antigens from a range of isolates ofO. graminis indicates specific immunograms which can be determined and separated from the immunograms developed by all other fungi when tested againstO. graminis antiserum. Immunoelectrophoresis can therefore be used as an aid in determiningO. graminis.     

The effect of ethanol on cell wall antigens ofSaccharomyces cerevisiae and specific isolation of high ethanol producing strains of this yeast,making use of a serological technique

Generally, natural isolates of high ethanol producingSaccharomyces cerevisiae obtained by screening are used in alcoholic industries. The methods involved in their isolation and identification are elaborate. Antigenic analysis using antibodies raised against wholeSaccharomyces cells indicated species specificity of cell wall surface thermostable antigens. By affinity purification, the specific antibodies could be obtained and used for specific isolation ofS. cerevisiae. Antigenic studies using antibodies raised against isolated cell walls of fermentatively grownS. cerevisiae indicated the occurrence of thermolabile antigens common toSaccharomyces species. Higher concentrations of these antigens could be detected in thoseS. cerevisiae that had the ability for high ethanol production. The concentrations of these cell wall common antigens increased with increasing culture age and ethanol accumulation in culture broths. In younger yeast cells, the concentration could be increased by growing the cells in a medium containing added ethanol. Using dilutions of cross absorbed antibody specific for common antigens and Ouchterlony test, high ethanol producingS. cerevisiae could be identified.     

Immuno-identification of cultures of fungi pathogenic to man

Because isolates of the fungal pathogensBlastomyces dermatitidis. Coccidioides immitis, Histoplasma capsulatum, andParacoccidioides brasiliensis frequently vary widely in gross and microscopic features and are often difficult or impossible to convert to their tissue forms, a simple diagnostic procedure not dependent upon sporulation is needed to identify them specifically and rapidly. The exoantigen technique has been found to meet this need effectively. On the basis of studies with 166Histoplasma spp. isolates, 128C. immitis isolates, 59B. dermatitidis isolates, 30P. brasiliensis isolates, and 181 saprophytes, we determined that the exoantigen test is valuable for the presumptive identification of the four fungal pathogens studied. All of the positive reactions have correlated with the cultural, histologic, or other available laboratory data, and we are unaware of any false positive or flase negative reactions.     

Application of PCR in Serum Samples for Diagnosis of Paracoccidioidomycosis in the Southern Bahia-Brazil

Paracoccidioidomycosis (PCM) cannot always be diagnosed by conventional means such as direct examination of histopathology or clinical samples, and serological methods, used as an alternative, still have many cases of cross-reactivity. In this scenario, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of this mycosis. In this study we analyzed 76 serum samples from patients in southern Bahia suspected of having paracoccidioidomycosis using a conventional PCR with primers for the ITS1 ribosomal DNA of P. brasiliensis. Of these 76 patients, 5 were positive for PCM by double immunodiffusion and/or direct examination and histopathology. To test specificity of PCR, we used human DNA and three isolates of P. lutzii (1578, 01 and ED01). Additionally, we analyzed by serial dilutions of DNA the limit of detection of the assay. The test of PCR proved specific, as only a 144 bp fragment of the three isolates of P. lutzii and no human DNA was amplified. Detection limit was 1.1 pg/µL of DNA. Despite the high detection limit and specificity of PCR none of the 76 serum samples were found positive by PCR, but a biopsy specimen obtained from one of the patients with PCM was positive. These results, albeit limited, show that PCR is not effective in detecting DNA of P. brasiliensis or P. lutzii in serum, but could perhaps be used with other types of clinical samples, especially in those instances in which conventional methods fail.     

Serology of Paracoccidioidomycosis Due to Paracoccidioides lutzii

Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. In western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.     

Ultrastructural changes produced by ketoconazole in the yeast-like phase of Paracoccidioides brasiliensis and Histoplasma capsulatum

The ultrastructural changes produced by ketoconazole on the yeast-phase ofH. capsulatum andP. brasiliensis were studied by means of scanning and transmission electron microscopy.The observed alterations on both fungi were very similar to those induced by the same drug on the ultrastructure ofC. albicans. These alterations include surface changes, abnormal membrane proliferation, fatty degeneration of the cytoplasm and lysis of subcellular organelles. P. brasiliensis seems to be more sensitive to ketoconazole thanH. capsulatum, since the necrosis of most of the cells was obtained in the former at a concentration of 0.1 gmg/ml and in the latter at 1 g/ml.     

Virulence of Paracoccidioides brasiliensis: The influence of in vitro passage and storage

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidioidomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated from patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.Abbreviations ATCC American Type Culture Collection - CFU colony-forming units - LD Lethal dose - MMv-M modified McVeigh Morton - PMN polymorphonuclear neutrophil     

Pathogenicity of Nocardia caviae,N. asteroides and N. brasiliensis

The pathogenicity ofNocardia caviae, N. asteroides andN. brasiliensis has been tested for white mice, guinea pigs and rabbits and chorio-allantoic membrane of the developing chick embryo. Altogether, 14 strains belonging to the 3Nocardia species originating from soil, human and animal sources in India or abroad were tested. All of them proved pathogenic though the degree of virulence varied from strain to strain. Incorporation of hog gastric mucin in the inoculum enhanced the virulence of all the 3Nocardia species for white mice.N. caviae strains were uniformly more virulent than those ofN. asteroides andN. brasiliensis.In the white mice inoculated intraperitoneally, a greater dissemination of the disease was apparent withN. caviae than withN. asteroides. Of the 6 strains ofN. caviae tested, 5 disseminated to the lung, 3 to the heart and 2 to the brain. InN. asteroides dissemination of the disease to the brain was observed with 2 of its 3 strains.N. brasiliensis showed no dissemination.N. caviae was found to be equally virulent for white mice, guinea pigs and rabbits. On the other hand,N. asteroides andN. brasiliensis were more virulent for white mice than for guinea pigs and rabbits. The lesions caused byN. caviae in mice, guinea pigs and rabbits persisted up to 4 weeks. In strong contrast to this the lesions due toN. asteroides andN. brasiliensis found in the guinea pigs and rabbits showed a strong tendency towards spontaneous clearance.Histologically, the lesions caused byN. caviae, N. asteroides andN. brasiliensis in mice, guinea pigs and rabbits were in the form of abscesses which showed an acute or chronic reaction. In the case ofN. caviae these abscesses showed both granules and freely dispersed cocco-bacillary bodies or filaments. As forN. asteroides it occurred in the form of cocco-bacillary bodies or filaments whereasN. brasiliensis consistently produced granules in the lesions.The lesions caused by the 3Nocardia species on the chorio-allantoic membrane of the developing chick embryo were in the form of abscesses which contained cocco-bacillary bodies and branching filaments but no granules.This forms a part of the thesis submitted by P.V.K. for Ph. D. degree, of the University of Delhi.     

Exoantigens of Trypanosoma cruzi. I. Conditions for Their Detection and Immunogenic Properties in Experimental Infections1

ABSTRACT Exoantigens of Trypanosoma cruzi were produced in experimentally infected BALB/c mice. The exoantigens were detected by the counterimmunoelectrophoresis method (CIE), with antisera raised in rabbits by immunization with total homogenates of culture forms of ***T. cruzi in plasma from ***field animals obtained by centrifugation and filtration. Control experiments indicated that exoantigens are not somatic components of T. cruzi leaked during the preparative procedure. Exoantigens were detected in male and female mice, 11-90 days old, between 6 and 60 days of infection, and in all mice with patent parasitemia. After 13 days of infection, mice developed antibodies to exoantigens; by CIE up to three populations of antibodies were revealed in different groups of animals. In mice between 13 and 60 days of infection, the coexistence of exoantigens and homologous antibodies was also observed. The exoantigens are not strain specific since a cross reactivity between antigens from three strains of T. cruzi (Tulahuén, Higueras, and Alejandro) was seen. Finally, the presence of antibodies to exoantigens in humans with chronic Chagas’ disease was demonstrated.     

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (imunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment #2, the reference system for P. brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

Penicillosis marneffei: Serological and exoantigen studies

An immunodiffusion (ID) test has been developed to diagnose infections caused byPenicillium marneffei. A 20 X concentrated culture-filtrate of six-week-old shake cultures (25 C) ofP. marneffei was employed as an antigen. This preparation was found to be better in quality than that from still cultures of the same age (30 C). Anti-P. marneffei rabbit sera were produced by injecting rabbits with increasing dosages of the inoculum for at least six weeks. These sera demonstrated two to three precipitin lines following their reaction with their antigens for 24–48 h at 25 C. TheP. marneffei antigenic preparations did not react with rabbit antisera to five species ofAspergillus, which commonly cause aspergillosis, or to antisera for four dimorphic systemic fungi. Similarly, the antigens of these other fungi did not react against the anti-P. marneffei rabbit serum. However, the anti-P. marneffei rabbit sera demonstrated antibody titres (132 to 164) to histoplasmin, blastomycin and coccidioidin in the complement fixation test. Cross-reactions were not observed with any of the human sera in the suspected or proven cases of opportunistic or systemic mycotic infections. Therefore, the ID test for penicillosis marneffei is considered to be highly specific. Exoantigen studies demonstrated that, of a total of 34 isolates of ten species ofPenicillium tested, only the extracts ofP. marneffei (6 isolates) and one isolate (PLM 771) of aP. species reacted positively with the anti-P. marneffei rabbit serum, giving at least two lines of identity with reference reagents. Based on this analysis, theP. species (PLM 771) was identified as P. marneffei. The exoantigen test is considered to be a specific and rapid method for the identification and confirmation ofP. marneffei isolates.

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Zusammenfassung Eine Immun-Verbreitung (ID) Test war ausgearbeitet fur die Diagnose der Infektionen diePenicillium marneffei verursachen. Die 20 X conzentrierte Kultur-Fieltrier von der sechs Wochen alten schuttelten Kulturen (25 C) vonP. Marneffei war gebraucht fur Antigen. Diese Preparation war besser in Qualitat denn die stillen Kulturen von dem selben Alter (30 C). Die Anti-P. Marneffei Kaninchen Sera wurde produziert durch die Injektion der Kaninchen mit immer vergrosserden Dosen von dem Imfstoff-mindestebs wahrend sechs Wochen. Diese Sera zeigte zwei — drei Ubere sturzung Linien durch die Reaktion von ihrer Antigenen wahrend 24–48 Stunden im 25 C. Die Antigen-Preparationen vonP. Marneffei gaben keine Reaktionen mit Kaninchen Anti-sera fur funf Species vonAspergillus, die stellen an gewohnlich Aspergillosis, oder zu der Antisera fur fier dimorpische systemische Pilze. Gleichweis, die Antigenen von diese Plize gaben keine Reaktionen gegen die Kaninchen Antisera vonP. Marneffei. Jedoch die Kaninchen Antiserum vonP. marneffei zeigte Anti-Korper Titer (132 bis 164) zu Histoplasmin, Blastomycin und Coccidioidin in der Complement (Erganzung) Fixing Test. Kreuz-Reaktionen wurden nicht observiert mit keine menschlichen Sera in verdachtigen oder erwiesenen Krankengeschichten von passenden oder systematischen mycotischen Infectionen. Deswegen die ID Test fur Penicillosis marneffei ist sehr spezifisch. Die Untersuchungen haben demonstriert, dass 34 isolationen von zehn Species vonPenicillium — die untersucht waren — nur der Auszug vonP. Marneffei (6 Isolationen) und ein Isolation (PLM 771) vonP. Species eine positive Reaktion mit der Kaninchen Anti-Serum vonP. Marneffei, und es gab mindestens zwei identische Linen mit die Referenz Reagenten. An diese Analyse grunden wir dass, dieP. Species (PLM 771) war identifiziert wieP. Marneffei. Die Exo-Antigen Test ist anbetrachtet wie eine spezifische und schnelle Methode fur die Identifikation und Bestatigung der Isolierten vonP. Marneffei.

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Portion of a thesis submitted by the second author to the Canadian Society of Laboratory Technologists, Hamilton, Ontario, Canada, in partial fulfilment of the requirements for Advanced registered Technologist, Certification in Immunology.     

Usefulness of a microimmunodiffusion test for the detection of Penicillium marneffei antigenemia,antibodies, and exoantigens

Eight sera from culturally-proven cases of penicilliosis marneffei and their corresponding isolates were examined for circulating antibody(ies) and antigen, and exoantigens, respectively, using a microimmunodiffusion (MID) test. Two of the 8 sera produced strong precipitins (1-2) when reacted against control Penicillium marneffei antigen (5-week-old shaken cultures at 25 C) in the presence of control rabbit anti-P. marneffei serum. Five of the 8 sera produced a strong precipitin line when reacted against control hyperimmune serum to P. marneffei. These five sera, and one additional serum, which tested negative for antibody to P. marneffei, demonstrated the presence of antigen by reacting only against the anti-P. marneffei serum. Serological evaluations of the sera revealed that the MID test is capable of detecting antibody and antigen in AIDS patients having penicilliosis marneffei infections. Exoantigen analysis of the 8 P. marneffei isolates, which were previously identified using this conventional and time-consuming macro- and micro-morphological characteristics, showed the presence of 1 to 4 specific exoantigens in MID. With the exoantigen analysis, the identity of all of the isolates was confirmed as P. marneffei. Our studies indicated that the serological tests are useful for detecting circulating antibody and/or antigen in patients' sera, and that the exoantigen test is reliable for confirming the identity of P. marneffei cultures. This revised version was published online in June 2006 with corrections to the Cover Date.