Abstract
We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H . capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens.
Introduction
Histoplasmosis caused by the dimorphic fungus Histoplasma capsulatum is a common endemic mycosis of humans and animals. 1 , 2 Some infections may cause acute pulmonary histoplasmosis (APH), and immunocompromised patients such as those infected with human immunodeficiency virus 3 and transplant recipients can develop progressive disseminated histoplasmosis (PDH) with a poor prognosis if not treated. 4 Chronic pulmonary histoplasmosis (CPH) is classically described as a cavitary disease in smokers with underlying chronic obstructive pulmonary disease. 5 Histoplasmosis is diagnosed according to the results of microbiological culture, histopathology, and antibody and antigen assays; however, these techniques have limited value, because H. capsulatum must be cultured for as long as 4 weeks. 6 Moreover, because of its high virulence, culturing H. capsulatum is generally restricted to biosafety level 3 laboratories. Antigen detection in urine and serum is useful for the diagnosis of PDH, 7 , 8 but this test is not available in the majority of non-endemic areas. 9
Recently, polymerase chain reaction (PCR)-based assays to detect H. capsulatum DNA in clinical specimens were described as important tools to facilitate the diagnosis of cultures, 9,-13 although they are not widely used for routine diagnosis in human histoplasmosis. In the present study, we developed a new real-time PCR and a nested real-time PCR based on cycling probe technology 14 for detecting the gene encoding the H. capsulatum antigen N-acetylated α-linked acidic dipeptidase (NAALADase). 15
Materials and methods
Fungal strains
H. capsulatum clinical strains (n = 19) and other fungal strains (n = 43) were provided by the Medical Mycology Research Center (MMRC), Chiba University, Chiba, Japan; Hospital das Clínicas, Faculdade de Medicina, University of São Paulo, Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto), University of São Paulo, and Julio Miller Universitary Hospital, Faculty of Medicine, Federal University of Mato Grosso, Brazil.
Clinical specimens
Sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissues (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1) were collected from five patients of histoplasmosis (proven, n = 4; probable, n = 1). The diagnosis is based on the EORTC/MSG ciriteria. 16 All specimens were collected from various hospitals in Japan between 2011 and 2015 and sent to MMRC for laboratory testing. Sera of healthy volunteers were collected and used as normal control (n = 4).
DNA extraction
Fungal genomic DNA was extracted using PrepMan TM Ultra sample preparation reagent (Life Technologies, CA, USA) according to the manufacturer's instructions. DNA extraction from 240 μl of serum was performed using the NucleoSpin ® Plasma XS (Macherey Nagel, Düren, Germany) according to the manufacturer's instructions. DNA extraction from FFPE tissue was performed using the NucleoSpin ® FFPE DNA (Macherey Nagel) according to the manufacturer's instructions, with a slight modification. In brief, 10 μl of 50 mg/ml fungal cell wall-digesting enzyme (Yatalase™; Takara Bio, Shiga, Japan) was used instead of Proteinase K. DNA extraction from cell pellets prepared from BALF was performed using a High Pure PCR template preparation kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer instructions. DNA extraction efficiency was evaluated by performing PCR amplification of partial fungal 28s ribosomal RNA gene with a primer pair NL1/NL4 17 and partial human beta-globin gene with a primer pair G1/G2. 18
Primers and probe design
DNA sequences of the gene encoding NAALADase from 4 H. capsulatum strains isolated from different geographic origins (North America, WU24; Latin America, G186AR; and Africa, H143 and H88), and 6 other fungi ( Blastomyces dermatitidis SLH 14081, Coccidioides immitis RS, Talaromyces marneffei ATCC 18224, Aspergillus fumigatus Af293, Fusarium graminearum PH-1, and Arthroderma otae CBS 113480) were obtained from the Broad Institute of Harvard and MIT. The primer pairs HcN4F/HcN4R and HcN2F/HcN1R and the cycling probe Hist1probe3 were designed from the conserved regions of the NAALADase genes between 4 H. capsulatum strains using the CycleavePCR ® Assay Designer program (Takara Bio). A BLAST search was performed on the primers and probe sequences to confirm the specificity.
Standard plasmid DNA
A 279-bp PCR product of the NAALADase gene was amplified from H. capsulatum IFM 46159 using the primer pair HcN4F/HcN4R. The PCR product was cloned into the pCR2.1 TA cloning vector using the TOPO ® TA Cloning kit (Life Technologies). Plasmid DNA digested with HindIII (Takara Bio) was used as the DNA standard.
Real-time PCR assay
The assays were conducted using the primer pair HcN2F/HcN1R and the cycling probe Hist1probe3 in a 20-μl reaction mixture containing the following components: 10 μl of 2× Cycleave PCR ® Reaction Mix (Takara Bio), 0.2 μM of each primer, 0.08 μM of cycling probe, and 1 μl of template DNA. When testing clinical specimens, internal positive control (IPC) primer pair (Lambda-primerF1/Lambda-primerR2), IPC cycling probe (Lambda-probe1), and IPC plasmid DNA were added to the PCR reaction mixture according to Muraosa et al. 19 to detect PCR inhibition. Reactions were performed using an Applied Biosystems 7300 real-time PCR system (Life Technologies) under the following conditions: 95°C for 10 s, followed by 40 cycles at 95°C for 5 s, 55°C for 10 s, and 72°C for 30 s. For the no-template control (NTC), sterile distilled water was used instead of the template DNA. For the positive control (PC), 50 copies of the standard plasmid DNA were used as template DNA.
Nested real-time PCR assay
For the NAALADase gene was initially amplified using conventional PCR with the primer pair HcN4F/HcN4R in a total volume of 25 μl of a solution containing the following components: 2.5 μl of 10× EX Taq buffer (Takara Bio), 2 μl of 2.5 mM dNTPs (Takara Bio), 0.5 μM of each primer, 0.625 U of Takara EX Taq Hot Start Version (Takara Bio), and 1 μl of template DNA. Reactions were performed using an iCycler (Bio-Rad, Berkeley, CA) as follows: 30 cycles at 98ºC for 10 s, 55ºC for 30 s, and 72°C for 30 s. Second-round real-time PCR was performed using the primer pair HcN2F/HcN1R and the cycling probe Hist1probe3. For analysis of specificity, a 1,000-fold dilution of first-round PCR products (1 μl) was used as a template. To detect the DNA extracted from clinical specimens, undiluted first-round PCR products (1 μl) were used as templates. Second-round real-time PCR was performed for 30 cycles using the conditions described above. For the NTC, sterile distilled water was used instead of the template DNA. For the PC, 50 copies of the standard plasmid DNA were used as template DNA. Real-time and nested real-time PCR products from clinical specimens were confirmed by DNA sequence analysis.
Analytical specificity and sensitivity
The specificities of real-time and nested real-time PCR assays were determined by testing duplicate samples of DNA from H. capsulatum clinical strains (n = 19), other fungal strains (n = 43), and human DNA (n = 1).
The limit of detection (LOD) of the real-time PCR assay was determined by testing 10-fold serial dilutions (1 to 1 × 10 5 DNA copies per PCR reaction) of the standard plasmid DNA using Easy dilution (Takara Bio). For the NTC, Easy dilution (Takara Bio) was used instead of the template DNA. Six independent experiments were performed. To compare the analytical sensitivity of the two assays, human serum spiked with 10-fold serial dilutions of the standard plasmid DNA (5 to 500 DNA copies per 240 μl of serum) was tested. Serum without the spiked standard plasmid DNA was used as a NTC. Six independent experiments were performed. The LOD of the assays was defined as the lowest concentration at which all replicates tested positive.
Results
The DNA sequences of NAALADase genes amplified by the primer pair HcN4F/HcN4R were highly conserved between H. capsulatum strains (similarity, 98.2–100%), showing low similarity (51.0–85.3%) between H. capsulatum and non- H. capsulatum fungi (Supplementary Fig. 1). The sequences and characteristics of the primers and cycling probe are shown in Supplementary Table 1.
The real-time and nested real-time PCR assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi (Supplementary Table 2) or human DNA. The LOD of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA (Fig. 1A ) and 50 DNA copies when using human serum spiked with the standard plasmid DNA (Table 1 ). A standard curve was created by plotting copy number of standard DNA logarithm values on X-axis against Ct values on Y-axis (Fig. 1B ). The plot showed good linearity with R 2 value of 0.9855 (Fig. 1B ). No amplification signals were detected in the NTC. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. (Table 1 ). No amplification signals were detected in the NTC.
(A) Analytical sensitivity of real-time PCR assay evaluated using 10-fold serial dilutions (1 to 1 × 10 5 DNA copies per PCR reaction) of the standard plasmid DNA. (B) A standard curve of the real-time PCR assay. Delta Rn, the magnitude of the signal generated during the PCR at each time point; NTC, no template control; Ct, cycle threshold.
Comparison of the limit of detection (LOD) of the real-time PCR and nested real-time PCR assays evaluated using serum spiked with the standard plasmid DNA.
| Concentration of the standard plasmid DNA | Real-time PCR | Nested real-time PCR | ||
|---|---|---|---|---|
| (copies/240 μl of serum) | Number of positive reactions | Ct value (mean ± SD) | Number of positive reactions | Ct value (mean ± SD) |
| 500 | 6/6 | 33.7 ± 1.2 | 6/6 | 6.5 ± 1.3 |
| 50 | 6/6 | 36.3 ± 2.3 | 6/6 | 8.3 ± 2.2 |
| 5 | 3/6 | 37.1 ± 0.5 | 6/6 | 9.5 ± 1.9 |
| NTC | 0/6 | – | 0/6 | – |
| Concentration of the standard plasmid DNA | Real-time PCR | Nested real-time PCR | ||
|---|---|---|---|---|
| (copies/240 μl of serum) | Number of positive reactions | Ct value (mean ± SD) | Number of positive reactions | Ct value (mean ± SD) |
| 500 | 6/6 | 33.7 ± 1.2 | 6/6 | 6.5 ± 1.3 |
| 50 | 6/6 | 36.3 ± 2.3 | 6/6 | 8.3 ± 2.2 |
| 5 | 3/6 | 37.1 ± 0.5 | 6/6 | 9.5 ± 1.9 |
| NTC | 0/6 | – | 0/6 | – |
Note : The limit of detection for each assay is highlighted in bold. NTC, no template control; SD, standard deviation; Ct, cycle threshold.
Comparison of the limit of detection (LOD) of the real-time PCR and nested real-time PCR assays evaluated using serum spiked with the standard plasmid DNA.
| Concentration of the standard plasmid DNA | Real-time PCR | Nested real-time PCR | ||
|---|---|---|---|---|
| (copies/240 μl of serum) | Number of positive reactions | Ct value (mean ± SD) | Number of positive reactions | Ct value (mean ± SD) |
| 500 | 6/6 | 33.7 ± 1.2 | 6/6 | 6.5 ± 1.3 |
| 50 | 6/6 | 36.3 ± 2.3 | 6/6 | 8.3 ± 2.2 |
| 5 | 3/6 | 37.1 ± 0.5 | 6/6 | 9.5 ± 1.9 |
| NTC | 0/6 | – | 0/6 | – |
| Concentration of the standard plasmid DNA | Real-time PCR | Nested real-time PCR | ||
|---|---|---|---|---|
| (copies/240 μl of serum) | Number of positive reactions | Ct value (mean ± SD) | Number of positive reactions | Ct value (mean ± SD) |
| 500 | 6/6 | 33.7 ± 1.2 | 6/6 | 6.5 ± 1.3 |
| 50 | 6/6 | 36.3 ± 2.3 | 6/6 | 8.3 ± 2.2 |
| 5 | 3/6 | 37.1 ± 0.5 | 6/6 | 9.5 ± 1.9 |
| NTC | 0/6 | – | 0/6 | – |
Note : The limit of detection for each assay is highlighted in bold. NTC, no template control; SD, standard deviation; Ct, cycle threshold.
The clinical sensitivity of the nested real-time PCR assay was higher than that of the real-time PCR assay (nested real-time PCR assay, 77%; real-time PCR assay, 33%) (Table 2 ). DNA sequence analysis identified the amplicons generated from clinical specimens by each assay as those of the H. capsulatum NAALADase gene. No amplification signals were detected in the normal control sera. PCR inhibition was not observed in any of the clinical samples and the normal control sera.
Examinations summary of patients with histoplasmosis.
| Patient | Travel to | Serum antibody | Presence of fungal yeast forms | Sample | Real-time | Nested real-time | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| No. | Age | Sex | endemic area | HIV | (ELISA a index value) | in the histopathological specimen b | Diagnosis | type | (Ct value) | PCR (Ct value) |
| 1. | 48 | M | Brazil | Pos. | Neg. (0.812) | Pos. | Proven PDH | Serum | Pos. (34.8) | Pos. (14.8) |
| FFPE (Oral mucosal tissue) | Pos. (32.3) | Pos. (8.0) | ||||||||
| 2. | 33 | M | Mexico | Neg. | Pos. (1.362) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (14.2) | ||||||||
| 3. | 49 | M | Panama, USA (Ohio) | ND | Pos. (1.958) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (12.6) | ||||||||
| 4. | 65 | M | Mexico | Neg. | Pos. (1.706) | ND | Probable APH | Serum | Neg. | Pos. (15.1) |
| BALF | Neg. | Pos. (12.1) | ||||||||
| 5. | 55 | M | Indonesia | ND | Pos. (4.360) | Pos. | Proven CPH | FFPE (Lung tissue) | Pos. (37.2) | Pos. (9.9) |
| Patient | Travel to | Serum antibody | Presence of fungal yeast forms | Sample | Real-time | Nested real-time | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| No. | Age | Sex | endemic area | HIV | (ELISA a index value) | in the histopathological specimen b | Diagnosis | type | (Ct value) | PCR (Ct value) |
| 1. | 48 | M | Brazil | Pos. | Neg. (0.812) | Pos. | Proven PDH | Serum | Pos. (34.8) | Pos. (14.8) |
| FFPE (Oral mucosal tissue) | Pos. (32.3) | Pos. (8.0) | ||||||||
| 2. | 33 | M | Mexico | Neg. | Pos. (1.362) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (14.2) | ||||||||
| 3. | 49 | M | Panama, USA (Ohio) | ND | Pos. (1.958) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (12.6) | ||||||||
| 4. | 65 | M | Mexico | Neg. | Pos. (1.706) | ND | Probable APH | Serum | Neg. | Pos. (15.1) |
| BALF | Neg. | Pos. (12.1) | ||||||||
| 5. | 55 | M | Indonesia | ND | Pos. (4.360) | Pos. | Proven CPH | FFPE (Lung tissue) | Pos. (37.2) | Pos. (9.9) |
a Using Histoplasma DxSelect ™ (Focus Diagnostics, CA, USA).
b Using Gomori's methenamine silver stain method.
Pos., positive; Neg., negative; M, male; PDH, progressive disseminated histoplasmosis; APH, acute pulmonary histoplasmosis; CPH, chronic pulmonary histoplasmosis; FFPE, formalin-fixed paraffin-embedded; BALF, bronchoalveolar lavage fluid; HIV, human immunodeficiency virus; ELISA, enzyme-linked immunosorbent assay; ND, not determined; Ct, cycle threshold.
Examinations summary of patients with histoplasmosis.
| Patient | Travel to | Serum antibody | Presence of fungal yeast forms | Sample | Real-time | Nested real-time | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| No. | Age | Sex | endemic area | HIV | (ELISA a index value) | in the histopathological specimen b | Diagnosis | type | (Ct value) | PCR (Ct value) |
| 1. | 48 | M | Brazil | Pos. | Neg. (0.812) | Pos. | Proven PDH | Serum | Pos. (34.8) | Pos. (14.8) |
| FFPE (Oral mucosal tissue) | Pos. (32.3) | Pos. (8.0) | ||||||||
| 2. | 33 | M | Mexico | Neg. | Pos. (1.362) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (14.2) | ||||||||
| 3. | 49 | M | Panama, USA (Ohio) | ND | Pos. (1.958) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (12.6) | ||||||||
| 4. | 65 | M | Mexico | Neg. | Pos. (1.706) | ND | Probable APH | Serum | Neg. | Pos. (15.1) |
| BALF | Neg. | Pos. (12.1) | ||||||||
| 5. | 55 | M | Indonesia | ND | Pos. (4.360) | Pos. | Proven CPH | FFPE (Lung tissue) | Pos. (37.2) | Pos. (9.9) |
| Patient | Travel to | Serum antibody | Presence of fungal yeast forms | Sample | Real-time | Nested real-time | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| No. | Age | Sex | endemic area | HIV | (ELISA a index value) | in the histopathological specimen b | Diagnosis | type | (Ct value) | PCR (Ct value) |
| 1. | 48 | M | Brazil | Pos. | Neg. (0.812) | Pos. | Proven PDH | Serum | Pos. (34.8) | Pos. (14.8) |
| FFPE (Oral mucosal tissue) | Pos. (32.3) | Pos. (8.0) | ||||||||
| 2. | 33 | M | Mexico | Neg. | Pos. (1.362) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (14.2) | ||||||||
| 3. | 49 | M | Panama, USA (Ohio) | ND | Pos. (1.958) | Pos. | Proven APH | Serum | Neg. | Neg. |
| FFPE (Lung tissue) | Neg. | Pos. (12.6) | ||||||||
| 4. | 65 | M | Mexico | Neg. | Pos. (1.706) | ND | Probable APH | Serum | Neg. | Pos. (15.1) |
| BALF | Neg. | Pos. (12.1) | ||||||||
| 5. | 55 | M | Indonesia | ND | Pos. (4.360) | Pos. | Proven CPH | FFPE (Lung tissue) | Pos. (37.2) | Pos. (9.9) |
a Using Histoplasma DxSelect ™ (Focus Diagnostics, CA, USA).
b Using Gomori's methenamine silver stain method.
Pos., positive; Neg., negative; M, male; PDH, progressive disseminated histoplasmosis; APH, acute pulmonary histoplasmosis; CPH, chronic pulmonary histoplasmosis; FFPE, formalin-fixed paraffin-embedded; BALF, bronchoalveolar lavage fluid; HIV, human immunodeficiency virus; ELISA, enzyme-linked immunosorbent assay; ND, not determined; Ct, cycle threshold.
Discussion
In the present study, we describe the development of new cycling probe-based real-time polymerase chain reaction (PCR) and nested real-time PCR assays for the detection of H. capsulatum that was designed to detect the gene encoding NAALADase, which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. NAALADase gene sequences were highly conserved between H. capsulatum strains (similarity, 98.2–100%). However, there was low similarity between H. capsulatum strains and genetically related fungi such as B. dermatitidis (similarity, 85.3%).
The real-time PCR assay using the H. capsulatum -specific primer pair HcN2F/HcN1R and the cycling probe Hist1probe3 was highly specific for H. capsulatum strains and sensitive (analytical sensitivity: 10 DNA copies per PCR reaction). However, in clinical specimens, this assay was not sufficiently sensitive to detect H. capsulatum DNA (clinical sensitivity = 33%). Therefore, we developed a nested real-time PCR assay using the primer pair HcN4F/HcN4R, which was designed from a region outside of that represented by the primer pair HcN2F/HcN1R. The nested real-time PCR assay detected 5 copies of the standard plasmid DNA per 240 μl of human serum, which represents a 10-fold higher LOD than that observed with the real-time PCR. Further, the nested real-time PCR assay increased the clinical sensitivity (77%).
Although the number of clinical specimens tested here was very limited, the results suggest that the nested real-time PCR assay has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens. The nested real-time PCR assay described here may provide a rapid and precise diagnostic tool for clinical specimens, however nested PCR method in general has shortcomings such as the higher risk of cross-contamination, longer procedure, and higher cost compare to the real-time PCR. Although we have not experienced any of these, further examination is warranted for the reliability of this method, and validation is required for its use in routine clinical practice in the diagnosis of histoplasmosis.
This study was supported by a Health Science Research Grant from the Ministry of Health, Labor, and Welfare of Japan (Research on Emerging and Re-emerging Infectious Diseases, H25-shinko-ippan-006) and in part by the National BioResource Project—Pathogenic Microbes funded by the Ministry of Education, Culture, Sports, Science, and Technology, Japan ( http://www.nbrp.jp/ ). The authors thank Rio Seki (MMRC) for technical assistance and Drs. Roberto Martinez (Faculdade Medicina Ribeirão Preto) and Cor Jesus (Faculty of Medicine, Federal University of Mato Grosso) for the DNA of Histoplasma isolates.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
Supplementary Material
Supplementary material is available at Medical Mycology online ( http://www.mmy.oxfordjournals.org/ ).
References
