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Journal of Clinical Microbiology, April 1998, p. 949-954, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification and Purification of Specific
Penicillium marneffei Antigens and Their Recognition by
Human Immune Sera
L.
Jeavons,1,*
A. J.
Hamilton,1
N.
Vanittanakom,2
R.
Ungpakorn,3
E. G. V.
Evans,4
T.
Sirisanthana,5 and
R. J.
Hay1
Dermatology Laboratory, St. John's Institute of
Dermatology, Thomas Guy House, Guy's Hospital, London
SE1-9RT,1 and
Department of
Microbiology, University of Leeds, Leeds, LS2,4
United Kingdom, and
Department of
Microbiology2 and
Department of
Medicine,5 Chiang Mai University Hospital,
Chiang Mai 50200, and
Institute of Dermatology,
Bangkok,3 Thailand
Received 27 October 1997/Returned for modification 2 December
1997/Accepted 10 January 1998
 |
ABSTRACT |
Disseminated infection with the dimorphic pathogenic fungus
Penicillium marneffei is increasingly seen among patients
with AIDS in southeast Asian countries. Previous studies have
demonstrated the presence of humoral immune responses to this fungus in
patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop
Western blots of P. marneffei cytoplasmic yeast antigen
(CYA). P. marneffei CYA was then partially purified by
liquid isoelectric focusing, and fractions were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
Western blotting. Immunoenzyme development of the Western blots with
pooled sera from patients with P. marneffei infection and
with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis
(n = 9), and histoplasmosis (n = 11)
revealed three antigens with relative molecular masses of 61, 54, and
50 kDa. These antigens were specifically recognized by the pooled sera
from the P. marneffei-infected patients. The 61- and 54-kDa
antigens were subsequently purified to homogeneity by preparative gel
electrophoresis, and the 50-kDa antigen was partially purified by the
same technique. N-terminal amino acid sequencing revealed that the
61-kDa antigen had a strong homology (87% identity) with the
antioxidant enzyme catalase. The three antigens were then subjected to
SDS-PAGE and Western blotting and to immunoenzyme development with
individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera
from 71% recognized the 54-kDa antigen, and sera from 48% recognized
the 50-kDa antigen. These specifically recognized antigens are the
first to be purified from P. marneffei and can be used
either singly or in combination to detect antibody responses in a large
percentage of individuals infected with P. marneffei.
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INTRODUCTION |
Penicillium marneffei was
first described in 1956 after its isolation from a bamboo rat
(Rhizomys sinensis) (1), although the organism's
pathogenicity in humans was not established until the infection of a
laboratory worker in 1959 (17). It is the only known
Penicillium species which displays dimorphism, and it is
thought to be free-living in the mycelial form but exists in tissue as
yeast or fission arthroconidia which divide by transverse fission
(5, 11). Until recently, human infection with P. marneffei was only rarely observed, although it has been suggested that this disease may frequently be misdiagnosed as tuberculosis (19). The majority of cases reported in the literature have occurred in individuals with some form of immunosuppression
(6), and there has been an increase in the rate of infection
with this fungus as the human immunodeficiency virus (HIV) pandemic has penetrated areas where P. marneffei is endemic. For example,
between 1991 and 1994 in Chiang Mai University Hospital in Chiang Mai, which is in northern Thailand, there were 550 cases of infection with
P. marneffei (2).
A presumptive diagnosis of P. marneffei infection is made on
the basis of clinical symptoms in conjunction with the identification of yeast dividing by transverse fission on microscopic examination of
bone marrow aspirate and/or touch smears of skin or lymph node biopsy
specimens stained with Wright's stain (18). Diagnosis is
confirmed by direct culture of the organism (21). There is, however, a need for the development of serologically based diagnostic tests which would enable the identification of either those individuals with initial asymptomatic forms of disease or those demonstrating nonspecific symptoms of P. marneffei infection. Until
relatively recently little was known of the antigenic composition of
P. marneffei, making the development of such diagnostic
systems problematic. Several studies have now, however, identified a
number of antigenic determinants in secreted antigen preparations from
P. marneffei (3, 20). These include antigen with
relative molecular masses of 54 and 50 kDa which were recognized by
sera from 60.6 and 57.6% of 28 P. marneffei-infected
patients, respectively, and more recently a 38-kDa molecule recognized
by 45% of sera from a sample of AIDS patients with culture-confirmed
P. marneffei infection (3).
However, it is noteworthy that to date there have been no reports of
the purification of P. marneffei antigens and their direct use in the detection of specific serological responses in patients with
P. marneffei infection. In this report we describe the
identification of three specifically recognized P. marneffei
antigens, two of which have been purified to homogeneity, and the
recognition of these antigens by the sera of individuals with P. marneffei infection.
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MATERIALS AND METHODS |
Fungal isolates.
The clinical isolate P. marneffei NCPF 4160 was provided by the National Collection of
Pathogenic Fungi, Mycological Reference Laboratory, Bristol, United
Kingdom. The clinical isolate P. marneffei F1620 was
provided by Chiang Mai University, Chiang Mai, Thailand.
Antigen preparation.
P. marneffei NCPF 4160 was
cultured for antigen preparation in the yeast phase at 37°C on brain
heart infusion medium (BHIM; Difco Laboratories Ltd., Surrey, United
Kingdom) agar slopes. For the preculture a 10-µl inoculating loop was
used to add yeast cells from a 7-day-old slope to 50 ml of BHIM broth
in a 250-ml conical flask, which was then incubated in a shaking
incubator at 120 rpm for 48 h at 37°C. Subsequently, 10 ml of
the preculture was used to inoculate 200 ml of BHIM broth in a 1-liter
conical flask which was incubated under the same conditions for 5 to 7 days (approximately mid-log phase). After treatment with 0.02 g of
thimerosal per liter at room temperature for 24 h, the yeasts were
harvested by centrifugation at 2,000 × g for 20 min.
P. marneffei F1620 was grown at Chiang Mai University under
the same conditions except that incubation was achieved with a shaking
heated water bath. P. marneffei cytoplasmic yeast antigen
(CYA) was prepared by mixing packed yeast cells with an equal volume of
0.5-mm glass Ballotini beads in phosphate-buffered saline (PBS). The
mixture was added to the chamber of a Bead Beater homogenizer (Biospec Products, Bartlesville, Okla.), packed in ice, and homogenized 15 times
in 1-min bursts with an interval of 10 min between each homogenization.
The homogenate was transferred to 40-ml centrifuge tubes, the insoluble
debris was removed by centrifugation at 10,000 × g for
15 min, and the cytoplasmic antigen solution was decanted (10). The Coomassie blue method was used to determine the
protein content of each preparation (16).
Liquid IEF of CYA.
Approximately 15 to 20 mg of CYA from
P. marneffei NCPF 4160 was dialyzed overnight at 4°C with
several changes of distilled water to remove the buffer salts present
from the PBS used in the antigen preparation protocol and was then made
up to 50 ml by the addition of 1 ml of ampholytes (Biolyte; pH range, 3 to 10; Bio-Rad Laboratories Ltd., Hemel Hempstead, United Kingdom) and
distilled water. The sample was loaded onto a liquid isoelectric focusing (IEF) system (Rotofor; Bio-Rad), and separation was achieved with a constant power input of 12 W. The 20 fractions were harvested once the voltage reading had stabilized for 30 min or more. The pH of
the fractions was measured, and the protein content was determined by
the Coomassie blue method (16). IEF fractions were then
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and Western blotting, followed by immunoenzyme development
with human immune sera.
SDS-PAGE, Western blotting, and immunoenzyme development.
SDS-PAGE was performed with 10% polyacrylamide gels as described
previously (8). The gels were stained with either Coomassie brilliant blue (16) or silver stain (14). The
relative molecular masses of the protein bands were determined by a
standard curve method with kaleidoscope markers (Bio-Rad). For protein
transfer onto polyvinylidene fluoride membranes (Millipore UK Ltd.,
Hertfordshire, United Kingdom), the gels were subjected to Western
blotting as described previously (8). The blots that were to
be used for the N-terminal amino acid sequencing of purified protein
were prepared by using the appropriate modification as described
previously (10). The blots that were to be used for
immunoenzyme development were incubated overnight at 4°C in 2%
casein with sodium azide (0.02%) in PBS and were then air dried and
stored at 4°C until required.
Immunoenzyme development of the blots was carried out as described
previously (8) with pooled human sera at a dilution of
1:1,000 in 1% casein-PBS-Tween (0.05% Tween 20; Sigma Aldrich Ltd.,
Dorset, United Kingdom) or individual serum specimens at a dilution of
1:100 in 1% casein-PBS-Tween. Goat anti-human immunoglobulin G (IgG)
peroxidase-linked conjugate (Jackson, West Grove, Pa.) was used at a
dilution of 1:1,000 in 1% casein-PBS-Tween to recognize bound human
IgG, and the blots were developed with tablets of 10 mg of
3,3'-diaminobenzidine tetrahydrochloride (Sigma Aldrich Ltd.) and 30 mg
of 4-chloro-1-naphthol (Sigma Aldrich Ltd.). Each tablet was dissolved
by mixing in 2 ml of absolute ethanol, and the mixture was added to 100 ml of PBS before the addition of 30 µl of
H2O2. After development in this solution, the
blots were washed in 0.1 M H2SO4 and, finally,
in distilled water before being allowed to air dry.
Human sera.
Sera from patients with a variety of fungal
infections, as listed in Table 1, were
used in immunoblot assays. Those from the patients with P. marneffei infections were used either as pools of sera or as
individual serum samples. Those from the patients with other mycoses
were used as pools of sera. Control sera from healthy human controls
were taken from an area of low endemicity for P. marneffei
(Bangkok, Thailand). Two pools of sera from P. marneffei-infected patients were used for the immunoblot assays: Pool Pm1 was pooled sera from 28 P. marneffei-infected
patients taken in 1995 at Chiang Mai University Hospital and had
previously been used in a study by Vanittanakom et al. (20).
The individual sera from the study by Vanittanakom et al.
(20) were no longer available and could not be used in the
present study. The second pool of sera (pool Pm2) was a mixture of 14 serum samples taken from P. marneffei-infected patients
diagnosed by positive histology and culture at Chiang Mai University
Hospital in 1996 plus a further 7 samples from patients diagnosed on
the same basis at the Institute of Dermatology, Bangkok, Thailand. All
serum samples were taken at the time of diagnosis, when patients had
active P. marneffei infection. The individual serum samples
which made up pool Pm2 were those used in the single-antigen immunoblot
studies.
Purification of antigens by Prep-cell preparative gel
electrophoresis.
IEF fractions 10 and 11 were pooled and subjected
to separation on the basis of size by using the Prep-cell preparative
gel electrophoresis system (Bio-Rad). Samples were reduced with
2-mercaptoethanol (Sigma Aldrich Ltd.) and were loaded onto a gel
column consisting of a 1.75-cm stacking gel and an 8-cm, 7.5%
resolving gel (acrylamide/bisacrylamide ratio, 35:1). Electrophoresis
was performed at a constant power of 10 W, and once the dye front was
eluted from the column, 2.5-ml volumes were collected. Fractions were
concentrated either by the use of Microsep centrifugal concentrators
(Flowgen Instruments Ltd., Kent, United Kingdom) or by acetone
precipitation (15) and were analyzed by SDS-PAGE.
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RESULTS |
Immunoenzyme development of Western blots of P. marneffei CYA with sera from patients with P. marneffei infection.
Pooled immune sera from P. marneffei-infected patients reacted with a large number of
P. marneffei antigenic determinants in CYA with relative
molecular masses of between 10 and 200 kDa. Of particular note were
highly reactive determinants at approximately 40, 61, 88, 135, and 190 kDa. Individual human sera demonstrated a wide variety of patterns of
reactivity (Fig. 1, lanes 3 to 23) varying from a small degree of recognition (lanes 3, 9, and 21) to the
recognition of a large number of determinants (lanes 6, 8, and 18).
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FIG. 1.
Reactivities of pooled and individual serum samples from
P. marneffei-infected patients to P. marneffei
CYA by Western blotting. Lanes 1 and 2, reactivities of sera in pools
Pm1 and Pm2, respectively (pooled sera were used at 1:100, and
peroxidase-conjugated goat anti-human IgG polyclonal sera were used at
1:1,000); lanes 3 to 23, reactivities of individual sera from P. marneffei-infected patients (sera were used at 1:100, and
peroxidase-conjugated goat anti-human IgG polyclonal sera were used at
1:1,000) to P. marneffei CYA; lane 24, negative control (no
primary antibody). Molecular masses (on the left) are in kilodaltons.
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|
Separation of P. marneffei CYA by IEF and
identification of specific P. marneffei antigens.
P.
marneffei CYA was separated into 20 fractions with the Rotofor
liquid IEF system (Fig. 2). Large amounts
of precipitated proteins (which could not be measured) were found at
the anodic and cathodic ends of the Rotofor system. Of the remaining
fractions, fractions 4 and 9 contained the most detectable protein.
Individual fractions obtained with the Rotofor system were subjected to
SDS-PAGE, Western blotting, and immunoenzyme development with pooled
sera from the groups described in Table 1.
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FIG. 2.
Separation of P. marneffei CYA by liquid IEF.
Bars, protein concentration (in micrograms per milliliter); diamonds,
pH units.
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|
Fractions 10 and 11 obtained with the Rotofor system contained a number
of antigens which were strongly reactive with pooled
sera from
P. marneffei-infected patients (Fig.
3, lane D and E;
data are
presented for IEF fraction 11, but those for fraction
10 were
identical). The Pm2 serum pool recognized bands of 40,
50, 54, 61, 70, 78, 90, 135, and 190 kDa, although not all of
these were visualized
when the bands were stained with Coomassie
blue (Fig.
3). Three of
these antigens with relative molecular
masses of 61, 54, and 50 kDa
demonstrated no cross-reactivity
with pooled sera from healthy
individuals or with sera from patients
with histoplasmosis, candidosis,
aspergillosis, and cryptococcosis
(Fig.
3). On the basis of the pH of fractions
10 and 11 obtained
with the Rotofor system, these antigens would appear
to have pIs
of between 6.5 and 7.0.
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FIG. 3.
Immunoenzyme development of Western blots of fraction 11 obtained by IEF with various pooled sera. Lanes A and B, reactivities
of pooled normal human sera (n = 30); lanes D and E,
pooled sera from P. marneffei-infected patients (pool Pm2;
n = 21); lanes G and H, pooled sera from histoplasmosis
patients (n = 11); lanes K and L, pooled sera from
candidiasis patients (n = 9); lanes N and O, pooled
sera from aspergillosis patients (n = 20); lanes Q and
R, pooled sera from cryptococcosis patients (n = 10);
lanes C, F, I, M, P, and S, molecular mass markers; lanes J and T,
duplicates of Coomassie blue-stained fraction 11 obtained by IEF.
Pooled sera were used at a dilution of 1:1,000, and the
peroxidase-conjugated goat anti-human IgG peroxidase conjugate was used
at 1:1,000. Molecular masses (on the left) are in kilodaltons.
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Purification of the 61-, 54-, and 50-kDa antigens.
The 61- and
54-kDa antigens were purified to homogeneity from IEF fractions 10 and
11 by Prep-cell fractionation. Purification of each antigen was
monitored by Coomassie blue staining (data not shown) and silver
staining analysis of SDS-polyacrylamide gels (Fig.
4). A 39-kDa band was sometimes observed
to copurify with the 61-kDa molecule after Prep-cell preparative
electrophoresis (data not shown). It proved to be impossible to purify
the 50-kDa antigen to homogeneity because of the presence of a
contaminating antigen at 46 kDa in all Prep-cell fractions containing
the 50-kDa antigen (data not shown).
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FIG. 4.
Purification of the 61-kDa (A) and 54-kDa (B) P. marneffei antigens as monitored by silver staining. Lane A, CYA;
lane B, fractions 10 and 11 (pooled) obtained with the Rotofor system;
lane C, purified antigen after preparative SDS-PAGE (61- or 54-kDa
antigen as annotated on the right). Molecular masses (on the left) are
in kilodaltons.
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N-terminal amino acid sequencing of the 61-kDa antigen revealed a
sequence with 87% identity with
Escherichia coli catalase
(Table
2). Despite attempts at N-terminal
amino acid analysis
for the 54-kDa antigen, we obtained no data
suggesting that the
N terminus of this protein is blocked.
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TABLE 2.
N-terminal amino acid sequence of 61-kDa antigen and its
homology with other proteins in the GENEMBL database
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Recognition of the 61-, 54-, and 50-kDa antigens by sera from
patients infected with P. marneffei.
The purified 61-kDa
antigen was recognized by 86% (18 of 21) of the serum samples from
P. marneffei-infected patients (Fig. 5A) when the samples were used in the
immunoenzyme development of Western blots. A 39-kDa molecule, which had
been seen to sometimes copurify with the 61-kDa antigen, was reactive
with 48% (10 of 21) of the serum samples from P. marneffei-infected patients (Fig. 5A). The 54-kDa antigen was
recognized by 71% of the sera from P. marneffei-infected
patients (Fig. 5B), while the 50-kDa antigen (Fig. 5C) was reactive
with 48% (10 of 21) of the patient sera. In total, 18 of 21 (86%) of
all serum samples from P. marneffei-infected patients tested
recognized at least one of these antigens. Other faint immunoreactive
bands were observed on the immunoblots, although these proteins were
not visible by Coomassie blue or silver staining techniques (Fig. 5B).
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FIG. 5.
Reactivities of 21 immune serum samples from P. marneffei-infected patients to the 61-kDa (A), 54-kDa (B), and
50-kDa (C) antigens as determined by immunoenzyme development of
Western blots. Lanes 1 to 21, individual sera from P. marneffei-infected patients (sera were used at 1:100).
Peroxidase-conjugated goat anti-human IgG conjugate was used at
1:1,000. Molecular masses (on the left) are in kilodaltons.
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DISCUSSION |
Three antigens which may be of use in the diagnosis of
asymptomatic P. marneffei infection have been identified and
purified either partially or to homogeneity from P. marneffei CYA. The first of these, a 61-kDa antigen, may be the
same as that previously reported as being present in P. marneffei culture medium (20). However, there is a
disparity between the number of patient sera recognizing this antigen
in the earlier study compared to the number recognizing this antigen in
the present study. Thus, in our study the purified 61-kDa antigen from
P. marneffei CYA was recognized by 86% of 21 serum samples
by Western blotting, whereas only 24% of 28 serum samples were
positive in the previous study (20). N-terminal amino acid
sequencing revealed that the 61-kDa antigen was probably a catalase;
similar data would have to be obtained for the previously identified
extracellular 61-kDa antigen to establish the relationship between the
two antigens. Catalase antigens are known to be produced by a number of
pathogenic fungi including Histoplasma capsulatum
(7) and Aspergillus fumigatus (13) and
may play a role in virulence (12). It is of note that while
both H. capsulatum and A. fumigatus possess
catalases which induce immune responses (7, 13), sera from
patients with these infections do not appear to recognize the putative P. marneffei catalase, indicating antigenic differences
between these enzymes.
A 39-kDa molecule was often observed to copurify with the 61-kDa
antigen. It is very likely that this is a breakdown product of the
61-kDa antigen since this molecule typically appeared in fractions
containing the 61-kDa antigen which had been separated by Prep-cell
fractionation. The latter procedure is capable of separating molecules
with a size difference of as little as 2 kDa, and therefore, the
copurification of two unrelated molecules with relative molecular
masses of 61 and 39 kDa by this method is a highly unlikely event. The
reactivity of the 39-kDa molecule with sera from patients infected with
P. marneffei supports the supposition that this molecule is
a breakdown product of the 61-kDa antigen: in this study the 39-kDa
molecule was reactive with 48% of the human immune serum samples. This
was lower than the percentage of sera which recognized the 61-kDa
antigen, but the result could be accounted for by the small amount of
this antigen on the blot; the 39-kDa band was often invisible when
either gels or blots were stained by conventional techniques. It is
also possible that some reactivity is lost in the process of
degradation. It is noteworthy that Vanittanakom et al. (20)
also mentioned the reactivity of a 39-kDa extracellular protein which
gave a positive reaction with 27% of immune sera in their study.
Furthermore, recently published data have suggested that a 38-kDa
molecule secreted from P. marneffei is specific to this
fungus, and antibody to a 38-kDa molecule has been detected in 45% of
AIDS patients with culture-confirmed P. marneffei infections
(3).
The second antigen identified in this study had a reduced relative
molecular mass of 54 kDa and was purified to homogeneity by a method
similar to that described for the 61-kDa antigen. When used in a
Western blot system with patient sera, this antigen demonstrated 100%
specificity for P. marneffei, with no cross-reactivity with
pooled sera from patients with other mycoses. It is quite possible that
this molecule corresponds to a protein of 54 kDa identified by
Vanittanakom et al. (20) in P. marneffei culture filtrate. Thus, of the 21 serum samples from patients used in this
study, 71% recognized the 54-kDa molecule, whereas 60% of the serum
samples from 28 patients in the culture filtrate antigen study
recognized this molecule (20). This similarity in the pattern of recognition by patient sera suggests that the 54-kDa antigens described in the two studies are probably the cytoplasmic and
extracellular forms of the same molecule. However, in contrast to the
situation with the 61-kDa antigen, we were unable to obtain any
N-terminal amino acid data for the 54-kDa antigen (as a result of
N-terminal blockage), and as such, the identity of the 54-kDa antigen
remains unknown.
Purification of the third antigen, the 50-kDa molecule, was difficult
since it was present only in very small amounts in the IEF fractions
used. However, this antigen was specific for P. marneffei
and it was recognized by sera from 48% of the patients. The latter
value equates broadly with the rate of recognition (58%) of a
previously described 50-kDa antigen present in P. marneffei culture filtrate, which is again suggestive of a relationship between
the two antigens (20). In common with the other two antigens
described in this study, the 50-kDa antigen has a pI of approximately
6.5 to 7.0 on the basis of the results of liquid IEF analysis.
The specificity and relatively high sensitivity of the 61-, 54-, 50-, and 39-kDa molecules when used in the detection of humoral immune
responses to P. marneffei make these antigens directly applicable to the serological diagnosis of P. marneffei
infections. It is highly likely that many patients harbor asymptomatic
P. marneffei infection for many months before developing
disseminated disease. This hypothesis is supported by the number of
individuals who develop this infection many months after leaving an
area where the organism is endemic (4, 9). These patients
are likely to develop disseminated P. marneffei infection
because their immunity is eroded by the progression to full-blown AIDS,
and these are the patients who may benefit from targeted antifungal
therapy. Antibody reactivity against the 38-kDa molecule described by
Chongtrakool et al. (3) was found in 17% of the sera from
apparently P. marneffei-negative, HIV-positive patients
living in areas where P. marneffei is endemic, whereas it
was found in the sera of only 2% of those living in areas where the
organism is not endemic, which the authors suggested was due to
asymptomatic infection. Since there is evidence to suggest that the
38-kDa molecule of Chongtrakool et al. (3) may be recognized
by asymptomatic P. marneffei-infected individuals, it could
be of high diagnostic value if tests with this molecule were applied to
sera from at-risk populations such as HIV-positive patients living in
areas where the organism is endemic. The theory that many individuals
may suffer asymptomatic P. marneffei infection for a
considerable time before clinical symptoms become obvious is given
further support by the results obtained by Vanittanakom et al.
(20), since serum from one of the AIDS patients used as a
negative control repeatedly gave a positive reaction against the 54- and 50-kDa antigens present in P. marneffei yeast-phase
culture filtrate. This patient finally developed clinical symptoms of
P. marneffei infection 2 months after initial testing,
indicating that antibody responses can be present before clinical
symptoms become apparent. The utility of the use of the antigens
described in this study in tests with sera from an at-risk population
without obvious symptoms of P. marneffei infection merits
further investigation. Especially useful in this regard would be the
elucidation of any relationship between the cytoplasmic antigens
described in this report with the secretory antigens of Vanittanakom et
al. (20), of which the 54- and 50-kDa antigens have already
been demonstrated to be useful indicators of early P. marneffei infection in at least one patient.
Our initial studies with sera from AIDS patients with disseminated
P. marneffei infection demonstrate that these patients do
mount detectable humoral immune responses, despite having low CD4
counts. However, any approach involving the detection of antibody responses as a marker of P. marneffei infection in this
group of patients should involve a number of antigens in order to
identify as many asymptomatic sufferers as possible. The 61-, 54-, 50-, and 39-kDa antigens are prime candidates for this purpose since they
appear to be identified only by the antibody responses of individuals
with P. marneffei infections and are not recognized by sera
from patients with other mycoses, which may, like mycosis caused by
H. capsulatum, occur in the same area where P. marneffei is endemic.
As yet, the relationship (if any) of the 61-, 54-, and 50-kDa antigens
described in this study to the secretory antigens described by
Vanittanakom et al. (20) has not been ascertained because a
direct comparison of one group of sera to both secretory and cytoplasmic antigens has not been possible (the individual sera which
comprise the Pm1 pool are no longer available). It is hoped that
further studies will elucidate any such relationship, and efforts are
under way to purify the secretory antigens for such a study.
Future studies will involve an examination of the 39-kDa molecule in an
attempt to determine its identity by N-terminal amino acid analysis,
which would reveal its precise relationship with the 61-kDa antigen.
Work already under way involves the development of an enzyme-linked
immunosorbent assay incorporating various combinations of the 61-, 54-, and 50-kDa antigens for the identification of individuals with both
symptomatic and nonsymptomatic P. marneffei infection.
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ACKNOWLEDGMENT |
This work was supported by a grant from the Wellcome Trust.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dermatology
Laboratory, St. John's Institute of Dermatology, 5th Floor, Thomas Guy House, Guy's Hospital, London SE1-9RT, United Kingdom. Phone: 0171 955 4663. Fax: 0171 407 6689. E-mail:
l.jeavons{at}umds.ac.uk.
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Journal of Clinical Microbiology, April 1998, p. 949-954, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
