Comparison of diagnostic methods to detect Histoplasma capsulatum in serum and blood samples from AIDS patients
Comparison of diagnostic methods to detect Histoplasma capsulatum in serum and blood samples from AIDS patients
Katia Cristina Dantas 1
Roseli Santos de Freitas
Marcos Vinicius da Silva 0
Paulo Ricardo Criado
Olinda do Carmo Luiz
Adriana Pardini Vicentini
Etsuro Ito, Waseda University, JAPAN
0 Emilio Ribas Institute of Infectious Diseases , Consultant , Ministry of Health, Department of Medicine, Catholic University of Sao Paulo, and Professor, Program in Postgraduate Sciences and Coordination of Disease Control, Department of State Health , Sao Paulo , Brazil , 4 Full Researcher at ABC Medical School , Sao Paulo , Brazil , 5 Preventive Medicine Department, Sao Paulo University Medical School , Sao Paulo , Brazil , 6 Immunology Center, Adolfo Lutz Institute , São Paulo , Brazil
1 Department of Pathology, Sao Paulo University Medical School , Sao Paulo, Brazil, 2 Medical Mycology Laboratory-LIM 53/HCFMUSP and Institute of Tropical Medicine, University of Sao Paulo , Sao Paulo , Brazil
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was supported by the State of
São Paulo Research Foundation (FAPESP) under
grant n. 2009/50362-0. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Although early and rapid detection of histoplasmosis is essential to prevent morbidity and
mortality, few diagnostic tools are available in resource-limited areas, especially where it is
endemic and HIV/AIDS is also epidemic. Thus, we compared conventional and molecular
methods to detect Histoplasma capsulatum in sera and blood from HIV/AIDS patients.
We collected a total of 40 samples from control volunteers and patients suspected of
histoplasmosis, some of whom were also infected with other pathogens. Samples were then
analyzed by mycological, serological, and molecular methods, and stratified as histoplasmostic
with (group I) or without AIDS (group II), uninfected (group III), and infected with HIV and
other pathogens only (group IV). All patients were receiving treatment for histoplasmosis
and other infections at the time of sample collection.
Comparison of conventional methods with nested PCR using primers against H. capsulatum
18S rRNA (HC18S), 5.8S rRNA ITS (HC5.8S-ITS), and a 100 kDa protein (HC100) revealed
that sensitivity against sera was highest for PCR with HC5.8S-ITS, followed by
immunoblotting, double immunodiffusion, PCR with HC18S, and PCR with HC100. Specificity was
equally high for double immunodiffusion, immunoblotting and PCR with HC100, followed for
PCR with HC18S and HC5.8-ITS. Against blood, sensitivity was highest for PCR with
HC5.8S-ITS, followed by PCR with HC18S, Giemsa staining, and PCR with HC100.
Specificity was highest for Giemsa staining and PCR with HC100, followed by PCR with
HC18S and HC5.8S-ITS. PCR was less efficient in patients with immunodeficiency due to
HIV/AIDS and/or related diseases.
Molecular techniques may detect histoplasmosis even in cases with negative serology and
mycology, potentially enabling early diagnosis.
Ajellomyces capsulatus (anamorph Histoplasma capsulatum), a dimorphic fungus that takes a
saprophytic mycelial form in the soil but a pathogenic yeast form in the host lung [
histoplasmosis, a widely distributed systemic mycotic infection. However, histoplasmosis is
significantly more prevalent in immunocompromised individuals, especially among HIV or
AIDS patients who have limited access to antiretroviral therapy. In addition, the mortality rate
among HIV/AIDS patients diagnosed with histoplasmosis is 30% in Latin America, but only
4±8% in the United States [
]. Histoplasmosis is particularly common in Brazil, where it is
the second most frequent invasive fungal infection in HIV/AIDS patients and results in high
]. According to Ostrosky-Zeichner , early diagnosis of invasive fungal
infections is critical, as delays often render antifungal therapy ineffective or even cause death.
Histoplasmosis is traditionally and directly diagnosed by histopathology using specific
stains, as well as by isolation of the fungus in culture, which is considered the gold standard
]. Indirect immunological assays to detect antibodies and/or antigens are also valuable [
In any case, both direct and indirect assays vary in sensitivity and specificity depending upon
the method, clinical form of the disease, and immune status of the host [
]. More recently,
molecular techniques have gained prominence due to greater speed, sensitivity, and specificity
]. Although perhaps not yet routinely employed, these methods include double
immunodiffusion, counterimmunoelectrophoresis , and PCR [
]. Indeed, PCR methods
were recently developed based on blood samples spiked with H. capsulatum DNA , as well
as on sera and whole blood from histoplasmosis patients [
]. The aim of this study was to
compare conventional, i.e., mycology and serology, and molecular methods to detect H.
capsulatum in sera and blood from patients with AIDS, with a view to assist clinicians in early
diagnosis and choice of therapy.
The study was approved by institutional ethics committees at University of São Paulo Medical
School Hospital (no. 0372/09), Institute Adolfo Lutz (no. 007/2010), and Emilio Ribas Institute
of Infectious Diseases (no. 348/2009).
Blood samples (n = 40) were collected between January 2009 and December 2011 from
patients admitted with suspected histoplasmosis to the emergency units at Emilio Ribas
Institute of Infectious Diseases and Clinical Medical Hospital at University of Sao Paulo
Medical School. The samples were tested at both institutions for HIV, hepatitis, syphilis,
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Paracoccidioides brasiliensis, Cryptococcus neoformans, Aspergillus fumigatus, and Histoplasma
sp. In addition, samples were analyzed by culture and direct microscopy on Sabouraud glucose
agar. After results were obtained from mycological (positive or negative) and serological
(reactive or nonreactive) assays, patients were requested to participate in this study, and those who
agreed were asked to fill out a relevant questionnaire and sign a form indicating informed
consent. Patients who were pregnant or younger than 18 years were excluded. After diagnosis,
samples were classified as histoplasmotic with AIDS (group I, n = 12) or without AIDS (group
II, n = 8), uninfected (group III, n = 10), or infected with HIV and other pathogens only
(group IV), including P. brasiliensis (n = 2), C. neoformans (n = 2), Aspergillus spp. (n = 2),
Leishmania (n = 2), and rheumatoid factor (n = 2). In cases where Histoplasma was not isolated
from patient samples, diagnosis was confirmed by histopathology or, in some cases, by
To establish diagnostic sensitivity and specificity, heterologous control strains were selected
based on clinical similarity to H. capsulatum, and consisted of P. brasiliensis 18 and B-339
(ATCC 32069), Candida albicans, C. parapsilosis, C. neoformans ATCC 24067, and Aspergillus
spp., all of which were obtained from Micoteca do Instituto de Medicina Tropical de Sao
Paulo. Positive control strains consisted of H. capsulatum ATCC 28308 (CDC: B973), ATCC
12700 (CDC: A811), and HC200 (GenBank: DQ239887).
Giemsa-stained smears were observed by direct microscopy for oval elements in phagocytes
that are 3±4 μm in diameter with typical cap coloration (nuclear chromatin at poles) and
small, surrounding light halos (false capsules). Smears were prepared from serial blood
samples collected and maintained under sterile conditions and inoculated on Sabouraud-Dextrose
agar (Difco Laboratories, Detroit, MI), Brain-Heart Infusion agar (Difco Laboratories, Detroit,
MI), and tryptone soya broth (Oxoid, London, England). Cultures were incubated at 35ÊC,
and pathogen growth was assessed for 60 days.
Double immunodiffusion and immunoblotting were performed according to Freitas et al. [
and Passos et al. [
], respectively, with some modifications.
To extract DNA from cell cultures, 200 μL samples were mixed with 40 μL of 60 mg/mL lysing
enzymes from Trichoderma harzianum (cat. no. L1412, Sigma Chemical Co., St. Louis, MO,
USA) in 1 M sorbitol, 100 mM EDTA, and 14 mM β-mercaptoethanol. Samples were then
incubated for 30 min at 30ÊC and centrifuged at 5,000 ×g (Eppendorf, Hamburg, Germany) at
room temperature. Precipitated cells were resuspended in 180 μL of ATL buffer (QIAamp
DNA Mini Kit, Qiagen, Hilden, Germany), and lysed for 3 h at 56ÊC with 100 mg/mL
proteinase K. DNA was then extracted using QIAamp DNA Mini Kit. To extract DNA from serum
and blood, 200 μL samples were lysed for 3 h at 56ÊC with 100 mg/mL proteinase K, and DNA
was then extracted using QIAamp Blood DNA Mini Kit (Qiagen).
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The presence of amplifiable DNA was confirmed by nested PCR of a fragment of human
glyceraldehyde-3-phosphate dehydrogenase (GADPH; GenBank: J04038.1), as described
]. Outer primers 5' GAC AAC AGC CTC AAG ATC ATC 3' and 5' GAC GGC
AGG TCA GGT CCA CCA 3' were used to amplify a 610 bp fragment, and inner primers 5'
AAT GCC TCC TGC ACC ACC 3' and 5' ATG CCA GTG AGC TTC CCG 3' were then
used to amplify an internal 248 bp product. In the first round, targets were amplified from
2 μL DNA extract in 25 μL of 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.3 μM
each of outer primers, 1.5 U AmpliTaq DNA polymerase, and 100 μM of each dNTP, over one
cycle at 94ÊC for 5 min, 35 cycles at 94ÊC for 30 s, 56ÊC for 30 s, and 72ÊC for 45 s, and final
extension at 72ÊC for 5 min. In the second round, targets were amplified from 2 μL of the
initial amplification product in 50 μM of each dNTP and 0.3 μM of each inner primer, over the
same thermal profile as the first reaction, except that 40 cycles were carried out. Positive and
negative controls without DNA were included in all assays.
H. capsulatum 18S rRNA gene (HC18S) was amplified according to reaction conditions
adapted from Bialek et al. [
]. Briefly, outer primers 5' GTT AAA AAG CTC GTA GTT G
3' and 5' TCC CTA GTC GGC ATA GTT TA 3' were used to amplify a 429 bp sequence
from several fungi that are pathogenic to humans. Inner primers 5' GCC GGA CCT TTC
CTC CTG GGG AGC 3' and 5' CAA GAA TTT CAC CTC TGA CAG CCG A 3' were
then used to amplify a 231 bp sequence specific to Histoplasma spp. Reaction conditions for a
100 kDa H. capsulatum protein (HC100) were similarly adapted from Bialek et al. [
particular, outer primers 5' GCG TTC CGA GCC TTC CAC CTC AAC 3' and 5' ATG TCC
CAT CGG GCG CCG TGT AGT 3' were used to amplify a 391 bp sequence, and inner
primers 5' GAG ATC TAG TCG CGG CCA GGT TCA 3' and 5' AGG AGA GAA CTG TAT
CGG TGG CTT G3' were then used to amplify a 210 bp sequence specific to Histoplasma were
amplified in 25 μL reactions as described previously [
]. In the first round, reactions
consisted of 2 μL DNA extract, 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 1 μM of
each outer primer, 1.5 U of Platinum Taq DNA poly Brazil, and 100 μM of each dNTP. The
thermal profile consisted of one cycle at 94ÊC for 5 min, 35 cycles at 94ÊC for 30 s, 50ÊC
(HC18S) or 65ÊC (HC100) for 30 s, and 70ÊC for 1 min, and one cycle at 72ÊC for 5 min.
Reaction mixtures for the second round were identical, except that 1 μL of the first reaction
product, 50 μM dNTP, and 1 μM of each outer primer were used. The thermal profile in this round
consisted of one cycle at 94ÊC for 5 min, 30 cycles at 94ÊC for 30 s and 72ÊC for 1 min, and
then one cycle at 72ÊC for 5 min. High annealing temperatures were used in this round to
For nested PCR of H. capsulatum 5.8S rDNA ITS (HC5.8S-ITS), all strains were first
sequenced with primers 5' TCC GTA GGT GGA CCT GCG 3', 5' GCA TCG ATG AAG
AAC GCA GC 3', and 5' TCC TCC GCT TAT TGA TAT GC 3', to target the conserved
18S, 5.8S, and 28S regions of the rRNA gene . ITS1 and ITS4 were then used to amplify the
intervening HC5.8S-ITS sequence in 25 μL as described previously , using conditions
described in Fujita et al. [
]. In the primary round, reactions consisted of 2 μL DNA extract
in 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 1 μM outer primers, 1.5 U Platinum
Taq DNA poly Brazil, and 100 μM each of dNTP. Targets were amplified over one cycle at
94ÊC for 5 min, 35 cycles at 94ÊC for 30 s, 65ÊC for 30 s, and 72ÊC for 1 min, and one cycle at
72ÊC for 5 min. Reaction mixtures in the second round were identical, except that 2 μL of the
first reaction product, 50 μM dNTP, and 1 μM each of inner primers were used.
All PCR reagents were obtained from Invitrogen (Carlsbad, CA, USA), and samples were
processed and amplified three times on a Veriti 96 thermocycler (Applied Biosystems, Life
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Technologies Corporation, Carlsbad, CA, USA). To avoid contamination of components,
preparation of reaction mixtures and addition of template DNA were performed in separate
rooms. All assays included negative controls without DNA and positive controls with DNA
from H. capsulatum ATCC A811 and B923, C. neoformans ATCC 24067, and P. brasiliensis 18
and 339. Products were electrophoresed on 1.5% agarose, stained with ethidium bromide, and
visualized on a UV transilluminator.
PCR products were purified with PureLink Kit (Invitrogen), and sequenced according to
manufacturer protocols on a MegaBACE 1000, a system with 96 capillaries, using DYEnamic
ET Dye Terminator Kit with Thermo Sequence II DNA polymerase (GE Healthcare formerly
Amersham Biosciences, Marlborough, MA, USA). Pathogens were identified by BLAST
(http://www.ncbi.nlm.nih.gov/Blast) against GenBank.
Direct observation was used to identify tests that would provide greater sensitivity when
performed in parallel. A combined test was considered positive when either of the tests performed
in parallel were positive. Fungal isolation was considered the gold standard for diagnosis failed,
histopathology was used to confirm histoplasmosis.
Data obtained from conventional (mycology and serology) and molecular (nested PCR) assays
were analyzed according to Fletcher et al. [
] to determine sensitivity, specificity, positive
predictive value, negative predictive value, and accuracy. Results from H. capsulatum cultures,
histopathology or, in some cases, by autopsy were used as reference. Agreement between the
reference method and other methods was assessed by inter-rater agreement (Cohen's Kappa)
], as interpreted using Landis and Koch-Kappa Benchmark Scale [
homogeneity was assessed using McNemar's test.
The study population consisted of biological samples from 20 individuals suspected to have
histoplasmosis and 10 uninfected individuals. Patients with disseminated histoplasmosis
(group I) presented high fever, diarrhea, weight loss, generalized lymphadenopathy,
hepatosplenomegaly, neurological symptoms, acute renal failure, respiratory failure, and skin lesions,
and 100% of these patients had CD4 lymphocytes fewer than 200 cells/mm3. Other infections
detected in some group I patients are described in Table 1. Of the 8 patients in Group II, 2
presented disseminated diseases, 2 presented acute pulmonary histoplasmosis, 1 presented
subacute pulmonary histoplasmosis, 2 presented chronic pulmonary histoplasmosis, and 1
presented supra renal histoplasmosis. These patients also presented respiratory tract infections,
fever, headache, cough, night sweats, weight loss, chest pain, neurological symptoms, and
abdominal pain. Patients with histoplasmosis were often mistaken as having tuberculosis, as
well as having other associated diseases (Table 1).
Histoplasmosis was confirmed by mycology in 83.33% and 12.5% of patients with and
without HIV, respectively (Table 2, Fig 1), with H. capsulatum isolated from 50% and 25% of blood
samples. However, one isolate suggestive of histoplasmosis based on Giemsa staining was
subsequently identified as Candida glabrata. Control strains were also characterized by
conventional and molecular methods, and were found by sequencing of HC5.8S-ITS to be at least
98% identical to reference species.
Serology by double immunodiffusion revealed that 25% and 12.5% of histoplasmotic
patients with and without HIV, respectively, had circulating H. capsulatum antibodies. All
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other patients did not react against H. capsulatum antigens. On the other hand, 66.7% and
25.0% of samples from histoplasmotic patients with or without HIV reacted with H.
capsulatum H and M fractions on immunoblots, while samples from all other patients did not
(Table 2, Fig 1).
PLOS ONE | https://doi.org/10.1371/journal.pone.0190408
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Fig 1. Mycological and serological tests for histoplasmosis. A, Mycological tests suggestive of histoplasmosis in a sample from a histoplasmotic patient with HIV. a,
Pleural fluid stained by Giemsa, 1,500×; b, intracellular yeast in alveolar macrophages with cytoplasmic retraction by direct exams, 1,500×; c and d, blood smear stained
with Giemsa, showing basophil nuclei and intracellular yeasts with cytoplasmic retraction. B, Immunoblotting for circulating H. capsulatum antibodies in sera from
histoplasmotic patients with (GI) and without HIV (GII), and from patients with HIV or other infections only (GIV). C+, polyclonal H. capsulatum antibody (positive
control); 1±10, sera from patients with suspected histoplasmosis. Fractions H (108±120 kDa) and M (70 and 94 kDa) are indicated.
Nested PCR against the housekeeping gene GADPH was used to test for the presence or
absence of amplifiable DNA, as well as for the presence of PCR inhibitors (Table 2). Against
sera from histoplasmotic patients with HIV, the sensitivity of nested PCR was highest using
HC5.8S-ITS primers (92%), followed by HC18S primers (34%), and HC100 primers (25%).
However, but was still highest for HC5.8S-ITS primers (25%), followed by HC18S (12.5%)
primers. H. capsulatum DNA was undetectable in these patients using HC100 primers. Against
blood from histoplasmotic patients with HIV, sensitivity was also highest for HC5.8S-ITS
primers (91.66%), followed by HC18S primers (66.6%), and HC100 primers (33.3%). The
sensitivity profile was again different in histoplasmotic patients without HIV, and was highest for
HC18S primers (50%) but comparable (37.5%) for HC100 and HC5.8S-ITS primers (Table 2).
All three-primer pairs exhibited 100% specificity when tested against uninfected sera and
blood samples. Among patients without histoplasmosis but with HIV and other infections,
100% specificity was achieved only with HC100 primers. On the other hand, specificity was
60% in sera and blood samples tested with HC5.8S-ITS primers, and 70% in sera and 80% in
blood samples tested with HC18S primers.
All samples from histoplasmotic patients with or without HIV were analyzed by
sequencing, and results confirmed the presence of H. capsulatum with 98% identity to reference
strains. A strain of C. glabrata was isolated in culture from a histoplasmotic patient without
HIV (sample 1), along with a strain of H. capsulatum from another patient in the same group
(sample 4). Sequences from the latter strain were 97% identical to those of Pichia kudriavzevii
and 99% identical to those of H. capsulatum. No fungal cells were isolated from samples 7 and
8 of histoplasmotic patients without HIV; however, sequences derived from these blood
samples were 98% identical to H. capsulatum and 99% identical to Rhodotorula mucilaginosa,
respectively. Sequencing was not possible for strains 2, 3, 5, and 6.
Among all methods, sensitivity for H. capsulatum in sera was highest for PCR with
HC5.8S-ITS, followed by immunoblotting, by double immunodiffusion and PCR with HC18S,
which have equal sensitivity, and, finally, by PCR with HC100. Against blood, sensitivity was
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also highest for PCR with HC5.8S-ITS, followed by PCR with HC18S, Giemsa staining,
and PCR with HC100. In contrast, specificity was equally high against sera for double
immunodiffusion, immunoblotting, and PCR with HC100, followed by PCR with HC18S and
HC5.8S-ITS. On the other hand, specificity against blood was highest for Giemsa staining and
PCR with HC100, followed by PCR with HC18S and HC5.8S-ITS (Table 2). Positive predictive
value against sera was equally high for double immunodiffusion, immunoblotting, and PCR
with HC100, followed by PCR with HC5.8S-ITS and then by PCR with HC18S. Negative
predictive value was highest for PCR with HC5.8S-ITS, followed by immunoblotting, PCR with
HC100, PCR with HC18S, and double immunodiffusion (Table 2). Against blood, positive
predictive value was highest for both Giemsa staining and PCR with HC100, followed by PCR
with HC18S, and then by PCR with HC5.8S-ITS, while negative predictive value was highest
for PCR with HC5.8S-ITS, followed by PCR with HC18S, Giemsa staining, and PCR with
HC100 (Table 2).
Direct observation of data was used to verify that sensitivity was greater for the following
combination of tests: fungal isolation and PCR with HC5.8S-ITS, and fungal isolation and
PCR with HC18S against blood. For these combined tests, the sensitivity, specificity, and
negative and positive predictive values were 90%.
Kappa analysis confirmed substantial agreement of the results of HC18 against blood,
HC5.8 against blood, HC100 against blood, and HC5.8 against serum with the results of the
gold standard, while HC18 and HC100 against serum showed moderate agreement and the
other tests showed slight agreement (Table 2).
Analysis using McNemar's test indicated that results of HC5.8-ITS against blood and
serum, HC18S against blood, and the combined test did not differ significantly from those of
the gold standard.
Bahr et al. [
] argued that, as a consequence of HIV pandemicity, progressive disseminated
histoplasmosis has grown more prevalent not only in known endemic regions, but also in
areas not considered endemic. The increasingly expanding suite of immunosuppressive
medications and biologics has also compounded this trend, which appears to be independent of
geographic location or patient travel. However, histoplasmosis remains challenging to
diagnose, as the turnaround time for a positive culture, the current gold standard of diagnosis, can
be significant [
]. Hence, we compared various diagnostic methods against blood and sera
collected from infected patients.
Direct microscopy and other mycological assays may only be suggestive but not conclusive
of histoplasmosis, owing to the similarity in structure between H. capsulatum yeast and other
pathogens, which can lead to false positives [
]. Indeed, H. capsulatum is difficult to
differentiate by histopathology and microscopy from other yeasts such as C. glabrata and other
Candida species, as well as from diminutive forms of other pathogens such as Cryptococcus, P.
brasiliensis, Pneumocystis jirovecii, and even from protozoa such as Leishmania donovani and
Toxoplasma gondii . In addition, Guimarães et al. [
] reported that the sensitivity of
microscopy (Giemsa staining) and histopathology in histoplasmostic patients with limited,
acute/subacute, chronic, disseminated pulmonary, or mediastinal HIV/AIDS are 9%, 10%, 17±40%,
43%, and < 25%, respectively, indicating low sensitivity in HIV/AIDS and possibly in other
H. capsulatum isolated in vitro may also exhibit similar morphology as non-pathogenic
species like Chrysosporium, Corynascus, Renispora, and Sepedonium. There are also atypical H.
capsulatum isolates that may prevent accurate identification [
]. In this study, fungi
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suggestive of H. capsulatum were isolated from 50% and 25% of histoplasmotic patients with
and without HIV, respectively, and confirmed by morphology in 27% and 12.5%, respectively.
Of note, sample 3 among patients with HIV revealed co-infection with C. albicans, which was
found to be predominant based on sequencing. Collectively, the data confirm that cultures
have low sensitivity in histoplasmotic patients with or without HIV [
]. Moreover, in vitro
isolation of H. capsulatum from patients with HIV/AIDS may be inhibited by administration
of sulfamethazole-trimethoprim to treat lung infections, primarily those caused by P. jirovecii
]. Indeed, we found that all isolates were inhibited by sulfamethazole-trimethoprim.
Nevertheless, this drug is effective against paracoccidioidomycosis, and is often the treatment of
choice depending on socioeconomic conditions. We note, however, that the drug is not used
in Brazil to treat histoplasmosis.
Of existing serological assays, double immunodiffusion is most often used in the clinic.
This technique is inexpensive, but has variable sensitivity and specificity, with predictive values
86±100% depending on the antigen used. It also enables evaluation of therapeutic effectiveness
based on titers of specific fungal antibodies [
]. However, double immunodiffusion has
low sensitivity in immunocompromised patients who produce immunoglobulins at reduced
]. We found that 25% of sera from histoplasmotic patients with HIV tested positive
for H. capsulatum antibodies on double immunodiffusion, with titers ranging from 1:4 to 1:16.
However, 66.7% (8/12) of samples testing negative on double immunodiffusion subsequently
tested positive on immunoblots. Of these samples, five reacted more strongly with the M
fraction than with the H fraction, indicating active disease. On the other hand, results from both
assays were consistent for four samples. Among histoplasmotic patients without HIV, H.
capsulatum antibodies were detected on double immunodiffusion in 12.5% of sera, with titers 1:4,
while 25% tested positive on immunoblots. Results were consistent between methods for
12.5% of these samples. Accordingly, sensitivity was 25% for double immunodiffusion and
50% for immunoblotting against histoplasmotic patients with or without HIV. These low
percentages are due to the general inability of patients with AIDS and other severe diseases to
mount an adequate antibody response to circulating antigens [
]. Moreover, the potential
for false-positives and cross-reactivity with other pathogens such as cutaneous leishmaniasis is
a serious limitation. Non-specific reactivity has been attributed to carbohydrate C, a
thermostable galactomannan found in most systemic dimorphic fungi .
Blood was collected from histoplasmotic patients during hospitalization and antifungal
therapy. Although the double immunodiffusion methodology presents a high degree of
specificity, its sensitivity is moderate. It should also be noted that some of these patients had
circulating H. capsulatum titers that were below the detection limit for the methodology. Therefore,
we propose that immunoblotting and/or PCR be included in the methodology as confirmatory
In contrast, several studies using specific PCR primers have demonstrated high sensitivity
and specificity for histoplasmosis. Samples evaluated using these primers have included
isolated fungal cultures [
], whole blood [
], and paraffin-embedded tissues [
However, DNA-based detection of H. capsulatum has not yet been validated as a diagnostic tool,
and is not commercially available [
]. Results using HC100 primers demonstrated 100%
specificity and reliability in total blood and serum, confirming previous results [
Indeed, Ohno et al.  demonstrated that these primers have great potential in initial
diagnosis, with high sensitivity, 90% specificity against blood, and 85% specificity against sera, but
emphasized the need for concurrent use of conventional methods. HC18S primers performed
better against blood, and had higher specificity and positive predictive value than HC5.8S-ITS.
The specificity of the former was similarly higher against sera, although the positive predictive
value was higher for the latter. Nevertheless, both primers exhibited relatively lower specificity
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due to genomic similarity between H. capsulatum and other species such as P. brasiliensis and
Aspergillus fumigatus. This explains false positives observed in patients who only have HIV,
aspergillosis, cryptococcosis, or paracoccidioidomycosis.
The generally low sensitivity of all methods tested against blood and sera from patients with
HIV/AIDS confirms previous findings by Buitrago et al. [
], Toranzo et al. [
FrÂõasDe-LeoÂn et al. [
]. In particular, PCR-based methods exhibited lower efficacy against patients
with immunodeficiency, presumably because of ongoing treatments for histoplasmosis and/or
The association of histoplasmosis with other pathologies such as pulmonary or
disseminated tuberculosis, neurological disorders, cancer, diabetes mellitus, hypertension, and
infection with other fungi, viruses, or parasites (e.g., toxoplasmosis, leishmaniasis) significantly
complicates the detection of the histoplasmosis. Nevertheless, we recommend the use of PCR
with rDNA primers in conjunction with conventional methods, especially since PCR is faster
than culture, and does not require handling of infectious fungi. In addition, these tools may
enable early diagnosis, even in cases of negative serology and mycology. We note, however,
these methods remain in-house, with limited availability and without independent validation.
Although further studies are needed, our results indicate that using a combination of tests
may increase diagnostic capacity. Sensitivity may be increased by simultaneously performing
HC18 against blood, HC5.8 against serum, and fungal isolation to identify histoplasmosis. For
such combinations, the sensitivity, specificity, and negative and positive predictive value were
90%. However, to resolve the occurrence of 10% false negatives, we suggest further
confirmatory analysis of negative results with a more specific combined test, such as HC100 primers
(blood and serum), Giemsa staining, DI, and IB, which presented 100% specificity.
This study was supported by the State of São Paulo Research Foundation under grant no.
2009/50362-0. We thank Mrs. Suely Campos Cardoso, a librarian at the Faculty of Medicine,
University of Sao Paulo, for her support in editing the figure and revision of references.
Conceptualization: Katia Cristina Dantas, Roseli Santos de Freitas, Adriana Pardini
Formal analysis: Katia Cristina Dantas, Roseli Santos de Freitas, Olinda do Carmo Luiz,
Adriana Pardini Vicentini.
Funding acquisition: Paulo Ricardo Criado.
Investigation: Katia Cristina Dantas, Roseli Santos de Freitas.
Methodology: Katia Cristina Dantas, Roseli Santos de Freitas, Adriana Pardini Vicentini.
Project administration: Katia Cristina Dantas, Roseli Santos de Freitas, Marcos Vinicius da
Silva, Adriana Pardini Vicentini.
Validation: Roseli Santos de Freitas.
Writing ± original draft: Katia Cristina Dantas, Roseli Santos de Freitas, Adriana Pardini
Writing ± review & editing: Katia Cristina Dantas, Roseli Santos de Freitas, Adriana Pardini
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