Abstract
In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.
The diagnosis of histoplasmosis is based on culture, histopathology, and antibody and antigen detection. A biosafety level 3 laboratory is required to grow the etiological agent, Histoplasma capsulatum, and the sensitivity of culture varies depending on the kind of specimen examined (9). Only a few laboratories perform antigen assays, which have a high sensitivity in cases of disseminated disease but limited value for localized infections (16). The sensitivity of serological assays also depends on the patient's immunity as well as the stage and type of disease (9). Staining of histological sections reveals fungal elements, but identification is sometimes difficult because of similarities in the appearances of yeast cells belonging to several dimorphic fungal species and the inability to grow the fungus from formalin-fixed biopsy specimens. Immunocytochemistry has not improved the identification of H. capsulatum to the species level in fixed tissue samples due to cross-reacting antibodies (14).
PCR assays amplifying sequences of fungal genes have been introduced successfully into the armamentarium for diagnosis of invasive fungal infections (11). They could be useful in the diagnosis of histoplasmosis in regions with inadequate culture facilities and inadequate experience in isolating H. capsulatum. In order to achieve high sensitivity, target sequences within rRNA genes are often used for diagnostic PCR, because multiple gene copies are usually present within a single genome. Accordingly, we have described a sensitive 18S ribosomal DNA (rDNA) nested PCR assay to monitor murine histoplasmosis (4). However, ribosomal genes are conserved regions bearing the risk of nonspecific amplifications (3). Therefore, by using our previous experience with paracoccidioidomycosis (2), a distinctive target gene of H. capsulatum was sought in order to develop a diagnostic PCR assay with high specificity. Recently, a 100-kDa-like protein was described as being essential for the survival of H. capsulatum in human cells (13). We developed a nested PCR assay targeting the gene coding for this unique protein. In order to evaluate this novel assay and the previously described 18S rDNA nested PCR assay in human tissue samples, paraffin-embedded biopsy specimens were examined. Use of this kind of specimen provides the possibility of repeated examinations, but the requirement for formalin fixation inhibits control by culture. Additionally, the quality and the amount of extractable DNA may vary (1, 12). A sensitive PCR targeting a human gene is therefore necessary as a control for DNA extraction. This is essential in judging the diagnostic value of our PCR assays for detection of H. capsulatum DNA in formalin-fixed human tissue samples.
(The data presented here are part of the doctoral thesis of Antje Feucht.)
MATERIALS AND METHODS
In the computerized register of the Department of Histopathology, University of Zimbabwe, 50 cases of histoplasmosis and 50 biopsy specimens negative for histoplasmosis were identified. The tissue samples had been routinely embedded in paraffin and stained with hematoxylin-eosin and periodic acid-Schiff stain. For the 50 cases of histoplasmosis, infection with Histoplasma capsulatum var. capsulatum was confirmed by microscopy only. The biopsy specimens included 25 skin samples (50%), 16 samples of ulcers or tumors of the oropharynx (32%), 7 lymph node samples (14%), and 1 rectal and 1 parotid gland sample (4%). For comparative purposes, a similar distribution of tissues was chosen for 50 negative biopsy specimens in which no H. capsulatum could be detected. These included 32 (64%) skin, 11 (22%) oropharyngeal, 6 (12%) lymph node, and 1 (2%) rectal biopsy specimen. Further examinations and analysis unrelated to the data of the 100 patients were carried out. Three 5-μm-thick sections of each biopsy specimen were placed in Eppendorf tubes with coded labels and sent to Tübingen for DNA extraction and PCR assays.
DNA extraction.
One thousand microliters of xylene was added to one Eppendorf tube containing one 5-μm section, which was then incubated on a shaker for 5 min at room temperature and subsequently centrifuged at 10,000 × g for 2 min. The supernatant was removed, and 1,000 μl of absolute ethanol was added, followed by centrifugation at 10,000 × g for 3 min. After removal of the supernatant and repetition of the ethanol and centrifugation steps, the supernatant was removed and samples were air dried. As the next step, 180 μl of ATL buffer from the QIAamp tissue kit (Qiagen, Hilden, Germany) and proteinase K (Qiagen) to a final concentration of 2 mg/ml were added. After incubation at 55°C for at least 2 h or overnight, samples were boiled for 5 min and then exposed to three cycles of freezing in liquid nitrogen for 1 min and boiling for 2 min to disrupt the fungal cells. After cooling to room temperature, DNA was extracted by use of the QIAamp tissue kit (Qiagen), based on binding of the DNA to silica columns, in accordance with the manufacturer's instructions.
Primer design of the H. capsulatum 18S rDNA PCR.
The outer primer set fungus I (5′-GTT AAA AAG CTC GTA GTT G-3′) and fungus II (5′-TCC CTA GTC GGC ATA GTT TA-3′) is complementary to a highly conserved region of the small-subunit rRNA gene of H. capsulatum (GenBank accession number X58572), amplifying a 429-bp sequence of several fungi pathogenic for humans. The inner primer set histo I (5′-GCC GGA CCT TTC CTC CTG GGG AGC-3′) and histo II (5′-CAA GAA TTT CAC CTC TGA CAG CCG A-3′), complementary to positions 643 to 666 and 873 to 849 of the small-subunit rDNA, respectively, delimits a specific 231-nucleotide sequence.
Primer design of the H. capsulatum 100-kDa PCR (Hc100 PCR)
The outer primer set Hc I (5′-GCG TTC CGA GCC TTC CAC CTC AAC-3′) and Hc II (5′-ATG TCC CAT CGG GCG CCG TGT AGT-3′) delimits a 391-nucleotide sequence of a gene coding for a 100-kDa-like protein unique to H. capsulatum (accession number AJ005963). The inner primers Hc III (5′-GAG ATC TAG TCG CGG CCA GGT TCA-3′) and Hc IV (5′-AGG AGA GAA CTG TAT CGG TGG CTT G-3′) are complementary to positions 2291 to 2314 and 2500 to 2476, respectively. The nested PCR product is 210 bp long.
H. capsulatum PCR assays.
The reaction mixture for the primary PCRs consisted of 10 μl of DNA extract in a total volume of 50 μl with final concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 2.5 mM MgCl2 (10× Perkin-Elmer buffer II plus MgCl2 solution [Roche Molecular Systems, Branchburg, N.J.]); a 1 μM concentration of each primer of the outer primer sets (Roth, Karlsruhe, Germany); 1.5 U of AmpliTaq DNA polymerase (Roche); and a 100 μM concentration of each deoxynucleoside triphosphate (Promega, Madison, Wis.). The reaction mixture for the nested PCRs was identical, except that 1 μl of the first reaction product, a 50 μM concentration of each deoxynucleoside triphosphate, and a 1 μM concentration of each primer of the inner primer sets were used. Reaction mixtures with the outer primer sets were thermally cycled once at 94°C for 5 min; 35 times at 94°C for 30 s, 50°C (65°C in the Hc100 PCR) for 30 s, and 72°C for 1 min; and then once at 72°C for 5 min. For the nested PCR products, reaction mixtures were thermally cycled once at 94°C for 5 min, 30 times at 94°C for 30 s and at 72°C for 1 min, and then once at 72°C for 5 min. The high melting temperatures of the inner primer sets allowed two-step nested PCRs with high stringency.
GAPDH PCR.
In order to prove the presence of amplifiable DNA, a nested PCR with a target sequence within the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (GenBank accession number J04038.1) was carried out. The outer primers gap 1 (5′-GAC AAC AGC CTC AAG ATC ATC-3′) and gap 2 (5′-GAC GGC AGG TCA GGT CCA CCA-3′) amplify a 610-nucleotide sequence of genes (positions 3816 to 4425) and a 441-nucleotide sequence of pseudogenes, respectively. The inner primers gap 3 (5′-AAT GCC TCC TGC ACC ACC-3′) and gap 4 (5′-ATG CCA GTG AGC TTC CCG-3′) amplify 325- and 248-bp products (positions 3932 to 4372).
The reaction mixture was identical to that for the PCR assays described above, except that all primers were used at a concentration of 0.3 μM and that 2 μl of the first-reaction product was used for the nested PCR. Reaction mixtures with the outer primer sets were thermally cycled once at 94°C for 5 min; 35 times at 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s; and then once at 72°C for 5 min. The reaction with the inner primer set was identical, except that 40 cycles were carried out. All PCRs were run in a Primus PCR thermocycler Tc 9600 (MWG Biotech, Ebersberg, Germany). The nested PCR products were analyzed by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.
Cloning.
The amplicons of the primary PCRs targeting the 18S rDNA and the unique H. capsulatum 100-kDa-like-protein gene, respectively, using template DNA extracted from a laboratory strain of H. capsulatum (strain 93/255; M. G. Rinaldi, Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio), were purified by Qiagen spin columns. The amplicons were inserted into the pCR2.1-TOPO cloning vector by using the Original TA Cloning Kit in accordance with the manufacturer"s instructions (Invitrogen, Groningen, The Netherlands). After culturing and harvesting of the bacteria, plasmid DNA was purified by using the Qiagen Plasmid Maxi Kit, consisting of alkaline lysis of bacteria, separation, binding of plasmid DNA to anion-exchange resin, wash steps, and final elution. The DNA concentration was measured by absorption at 260 nm. Serial dilutions were used in order to determine the detection limit of the nested PCR assays. The amplified products were sequenced to prove homology to the sequences in the GenBank database.
Controls.
Ten microliters containing 100 fg of purified plasmid DNA with an insert of either the H. capsulatum 18S rDNA partial sequence or the H. capsulatum 100-kDa-like partial gene sequence was used in every corresponding PCR assay as a positive control. In order to monitor crossover contamination, sterile water was included in the DNA extraction and was used as a negative control after every fifth sample in the nested PCR assay. Reaction mixtures without DNA were run in the first and nested PCRs to detect contamination. To exclude PCR inhibitors, 20 fg of plasmid DNA was added to 8 μl of DNA extract from a PCR-negative sample, and the corresponding PCR assay was repeated.
Sequencing.
Nested PCR products were purified by using the QIAquick PCR purification kit (Qiagen), based on DNA binding to a silica membrane. Automated sequencing was done with the BigDye terminator cycle sequencing kit and primers of the nested PCR in accordance with the manufacturer's instructions, and PCR products were analyzed on an ABI 373 automated DNA sequencer (Applied Biosystems Division, Perkin-Elmer, Foster City, Calif.). Sequences were generated from both strands, edited and aligned with Sequence Navigator software (Applied Biosystems), and used for a BLAST search (National Center for Biotechnology Information, Washington, D.C.) of the GenBank database.
RESULTS
Several genes and pseudogenes coding for GAPDH are usually present in each human cell (8). Failure to amplify the target sequence within the GAPDH gene by nested PCR is an unequivocal sign of the absence of amplifiable human DNA. This may be due to failure to extract DNA or destruction of DNA by formalin fixation. As shown in Table 1, a GAPDH nested PCR product was obtained for 29 of 50 histoplasma-positive and 33 of 50 histoplasma-negative biopsy specimens. In order to rule out any failure in extracting DNA, extraction was repeated for all samples initially negative by the GAPDH nested PCR, but without additional success.
TABLE 1.
Results of PCR assays and sequencing of 100 biopsy specimens examined
| Method | No. of positive results for specimens with the following histopathology result:
|
|
|---|---|---|
| Positive (n = 50) | Negative (n = 50) | |
| GAPDH nested PCR product (amplifiable human DNA) | 29 | 33 |
| 18S rDNA nested PCR product | 26 | 18 |
| H. capsulatum identification by sequencing | 20 | 0 |
| 100-kDa-like-protein nested PCR product | 20 | 0 |
| H. capsulatum identification by sequencing | 20 | 0 |
Detection limits.
Serially diluted cloned plasmid DNA was repeatedly used as a template in the two H. capsulatum PCR assays. A product was detected by using a minimum amount of 0.1 to 1 fg of plasmid DNA. Depending on the number of gene copies per genome, this corresponds to a detection limit of 1 to 5 genome equivalents per sample.
Results for specimens found histoplasma positive by histopathology.
Twenty out of 29 biopsy specimens with amplifiable human DNA revealed a product in the PCR assay targeting the 100-kDa-like-protein gene. Sequencing disclosed 100% homology of all 20 PCR products to the gene sequence coding for the 100-kDa-like protein in GenBank. The 18S rDNA PCR revealed a DNA band in the agarose gel in 26 of the 29 GAPDH-positive biopsy specimens. Sequencing confirmed only 20 of these as identical to the 18S rDNA sequence of H. capsulatum, whereas the remaining 6 were 95 to 99% homologous to sequences of several fungi belonging to the class of Euascomycetes. Identity of the 18S rDNA PCR product with the H. capsulatum gene sequence in the GenBank database was found exclusively in samples which were also positive with the Hc100 PCR assay.
In summary, histoplasmosis was diagnosed by a nested PCR for 11 skin biopsy specimens (55% of all positive specimens), 6 oropharyngeal specimens (30%), and 3 lymph node (15%) specimens. The pattern of PCR-positive tissue samples in percentage terms is similar to the distribution of tissues examined. Thus, detection of H. capsulatum-specific DNA by any of the two PCR assays was unrelated to the kind of tissue examined.
In order to verify the nine negative results of the Hc100 PCR assay, DNA extraction was repeated. For a second time, inhibitors were excluded and amplifiable human DNA was detected. Nonetheless, the specific H. capsulatum nested PCR remained negative for all nine samples.
Results for specimens found histoplasma negative by histopathology.
The GAPDH nested PCR assay amplified human DNA in 33 of 50 microscopically histoplasma-negative biopsy specimens. All 50 tissue samples were negative by the nested PCR assay targeting the unique H. capsulatum 100-kDa-like-protein gene, whereas a product of the 18S rDNA PCR assay was found for 18 samples. Sequencing revealed that these products were 95 to 99% homologous to 18S rDNA sequences of several species belonging to Euascomycetes, i.e., none of them were identical to the H. capsulatum 18S rDNA sequence. Amplifiable human DNA was found by the GAPDH-PCR for only 12 of the 18 biopsy specimens.
Discordant results of the two H. capsulatum PCR assays were obtained only for biopsy specimens from the skin, oropharynx, or rectum, i.e., tissue samples with possible contamination or colonization with nonpathogenic fungi. Due to the universal primers, the 18S rDNA PCR amplifies DNA from related pathogenic fungi and from nonpathogenic fungal species, which can be distinguished from H. capsulatum by sequencing only. A constant contamination during paraffin embedding or DNA extraction seems unlikely, because the sequences obtained were neither identical nor homologous to a single fungal species.
Controls.
All DNA extraction controls were proven negative in all PCR assays carried out. Contamination during the extraction procedure can therefore be excluded. No crossover contamination was found in any of the PCR assays, because all reaction mixture controls of the first and the nested PCRs remained negative. PCR inhibitors were ruled out in all 80 samples found negative by the PCR assay targeting the gene of the 100 kDa-like protein, because all samples turned positive after addition of 20 fg of cloned plasmid DNA to the sample DNA.
DISCUSSION
The two nested PCR assays described in this study are capable of identifying H. capsulatum in formalin-fixed, paraffin-embedded human tissue samples. The detection limits and sensitivities of the two assays turned out to be identical. No false-positive results were obtained by the PCR assay targeting the gene coding for the specific 100-kDa-like protein. In contrast, the 18S rDNA nested PCR assay revealed products in 24 additional samples. These required sequencing to distinguish them from H. capsulatum, identifying them as nonspecific reaction products. These findings confirm earlier observations on diagnostic PCR assays of the closely related fungus Paracoccidioides brasiliensis (2). A PCR assay targeting the rRNA genes might achieve higher sensitivity in detecting low numbers of a variety of human fungal pathogens by universal primers. However, the ubiquitous occurrence of these conserved genes is a disadvantage in clinical specimens from nonsterile body sites, because nonpathogenic contaminating or colonizing fungi can cause considerable nonspecific amplifications. Even the high stringency of our nested PCR using a high annealing temperature of 72°C cannot avoid nonspecific amplifications, which may not even be revealed by subsequent sequencing (5).
Reid and Schafer have described a nested PCR for detection of H. capsulatum DNA in soil samples using primers of the internal transcribed spacer (ITS) region (15). Our detection limits are in accordance with that of 10 yeast cells per sample reported by these authors. Universal primers complementary to sequences of the ITS region have been used for identification and phylogenetic placement of fungal species (17). However, their diagnostic relevance in clinical specimens has not been proven so far. According to our unpublished findings, a nested PCR based on primers of the ITS region amplifies several products per sample, even when sterile human specimens are examined. Panfungal PCR assays, described as being able to detect DNA of H. capsulatum, will face difficulties similar to those of the 18S rDNA PCR assay described above if biopsy specimens from skin, mucous membranes, or bronchoalveolar lavage fluid are examined.
The failure of a sensitive nested PCR targeting the multicopy GAPDH gene to amplify human DNA in 38% of our samples reveals the well-known disastrous effect of formalin fixation on DNA (1, 12). No information concerning the fixation time and the usage of buffered or unbuffered formalin was available for our specimens.
For nine microscopically positive samples, both H. capsulatum PCR assays failed to amplify specific DNA. This may be due to small amounts of fungal DNA in the total volume extracted. The significantly higher sensitivity of the GAPDH nested PCR assay compared to that of the H. capsulatum nested PCR assay is due to the presence of more than 10 copies of the GAPDH gene and pseudogenes per human cell, in contrast to (presumably) a single copy of the specific gene per yeast cell in samples consisting mainly of human cells with only a few fungal cells. Thus, traces of amplifiable human DNA detected by the GAPDH nested PCR do not necessarily indicate the presence of sufficient amounts of amplifiable fungal DNA. However, mutations in the targeted H. capsulatum gene may also be responsible for the failure to detect specific DNA. Another possibility could be incorrect identification of the 2- to 4-μm intracellular organisms in tissue sections. This seems unlikely in an area in which visceral leishmaniasis is not known to be endemic, but it cannot be formally excluded as another cause of failure.
Recently, an in situ hybridization protocol for the identification of yeast-like organisms in tissue sections by using specific and panfungal oligonucleotides complementary to 18S and 28S rRNA genes was published (10). A sensitivity of 50% and a specificity of 100% were reported when 20 positive H. capsulatum samples confirmed by culture were examined. Compared to PCR assays requiring intact DNA strands of as many as 500 bp, the binding of specific oligonucleotides is less sensitive to destruction of DNA by formalin. Thus, in situ hybridization may be superior to PCR assays for detection of H. capsulatum DNA in formalin-fixed tissues. However, in this study paraffin-embedded samples were used for the evaluation of the diagnostic PCR assays, which are intended for use with various unfixed clinical specimens such as bronchoalveolar lavage fluid, cerebrospinal fluid, organ biopsy specimens, and blood samples.
H. capsulatum var. capsulatum has been isolated in Zimbabwe (7). For the first time our data reveal that the gene coding for a protein which seems to be essential for the intracellular survival of the fungal pathogen (6) is widespread among H. capsulatum strains from human specimens in this African country. All 210-bp PCR products were 100% identical to the sequence in the GenBank database.
There were no cases of African histoplasmosis in our study. According to the data in GenBank, the 18S rDNA sequence of Histoplasma capsulatum var. duboisii is identical to the sequences of the other two varieties of this species. Thus, the 18S rDNA PCR described here should detect DNA of the varietas duboisii. It is not known whether the gene coding for the 100-kDa-like protein is present in the genome of H. capsulatum var. duboisii, or of any other dimorphic fungi; this needs to be elucidated in further studies.
In conclusion, we have developed a nested PCR assay specific for the detection and identification of H. capsulatum DNA in human tissue samples by targeting a gene coding for a unique fungal 100-kDa-like protein. In contrast, products of the equally sensitive PCR assay targeting the 18S rRNA genes have to be confirmed by sequencing in order to avoid a high rate of false-positive results.
Acknowledgments
This study was supported by Förderverein AIDS im Kindesalter e.V., Bonn, Germany.
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