Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

The incidence of invasive fungal infections has increased in recent years and has been important cause of morbidity and mortality in immunocompromised patients such as recipients of bone marrow transplants, patients with hematological malignancies (with or without chemotherapy) and AIDS patients (1)(2)(3).
The conventional methods for the identification of fungemia agents are often based on the examination of phenotypic characteristics.This approach can be timeconsuming and its dependence on the variable expression of phenotypic characteristics can lead to inconsistent results (4)(5)(6).
The polymerase chain reaction (PCR) is being increasingly applied within hospital laboratories for the routine detection of human genes and pathogenic microorganisms (7).Modern PCR techniques such as multiplex PCR and multiplex real-time PCR have been used for the detection and identification of pathogenic fungi (8)(9)(10)(11).However, these methods have some weaknesses such as: a) the multiplex techniques are still limited to the identification of only a few fungal species per reaction (8)(9), b) the multiplex PCR is still presenting problems of low sensitivity and specificity for fungal detection in biological samples (8)(9)(10), and c) the multiplex real-time PCR is not a well-disseminated method because of the necessity of specific and expensive equipment (11).
The ribosomal genes are popular targets for PCRbased systems for the detection and identification of fungal pathogens (15)(16)(17).The internal transcribed space regions (ITS1 and ITS2) are highly variable sequences that have been used for the identification of fungi at the species level in different formats (17)(18)(19)(20).
A number of procedures using PCR/RFLP amplification techniques have been suggested for the identification of Candida species.However, few identification studies included fungemia agents.
The aim of the present study was to evaluate three PCR primers and three restriction enzymes for the identification of fungemia agents on the basis of size and variation of the rDNA internal space regions.

Material and Methods
Seven reference strains of fungemia agent species (obtained from the FMTAM fungal collection) were included: Candida albicans FMT170, Candida parapsilosis FMT72, Candida tropicalis FMT44, Candida glabrata FMT66, Cryptococcus neoformans FMT1420, Cryptococcus gattii FMT1170, and Histoplasma capsulatum FMT1400.These microorganisms were identified by conventional methods (20) and their identities were confirmed by the sequencing of their ITS1 gene from ribosomal DNA.

Preparation of template DNA
Strains were cultivated on solid Sabouraud agar plates for 3-15 days at 30°C.Sabouraud broth medium (10 mL) was inoculated and the cells were grown in 50-mL tubes (Falcon, Becton Dickinson, USA) at room temperature with shaking overnight.After centrifugation at 2268 g for 10 min, the supernatant was removed and the pellet was washed by adding 20 mL washing buffer (150 mM NaCl, 10 mM Tris-HCl, 0.5 mM EDTA, pH 8.0) at room temperature.Fungal DNA was extracted with the QIAamp tissue kit (Qiagen, Germany) using the silica column technology according to manufacturer instructions for DNA extraction from yeast, including digestion with Lyticase and RNAase.

Restriction digest
The PCR product was digested individually with 10 U of the restriction enzymes HaeIII, BfaI, and DdeI (New England Biolabs, UK) during an overnight incubation at 37°C.

Electrophoresis analysis
The PCR products were analyzed by 2% agarose gel electrophoresis and the gels were stained with ethidium bromide.Gene Ruler DNA ladder Mix (SM0331, MBI Fermentas, Germany) was used as the DNA marker.The PCR products were purified using the QIAquick PCR Purification kit (Qiagen) according to manufacturer instructions.The PCR products were diluted in 40 µL of the buffer included in the kit.

Selection of the rDNA region
The regions of ribosomal DNA described for the three different pairs of primers were successfully amplified from all the reference strains tested (Figure 1).
As expected, a PCR product was consistently obtained for each microorganism.Using the pair of primers ITS1/ITS4, the reference strains identified as C. glabrata, C. gattii and C. neoformans yielded unique product sizes of approximately 400, 600, and 650 bp, respectively, and a product of approximately 550 bp was obtained for the remaining species.Using the pair of primers NL4/ITS5, only C. glabrata DNA strain yielded unique product sizes of approximately 800 bp and a product of approximately 1100 bp was obtained for the remaining species.Using the pair of primers Primer1/ Primer3, C. glabrata, C. gattii, and C. neoformans strains yielded product sizes of approximately 200, 300 and 300 bp, respectively, and a product of approximately 350 bp was obtained for the remaining species.

Selection of the restriction enzyme
The PCR products were treated with the restriction enzymes Bfal, Ddel and HaeIII for further differentiation.Figure 2 shows a typical gel electrophoresis of the patterns obtained after digestion of the PCR products.
The PCR products obtained using the pair of primers ITS1/ITS4 after treatment with HaeIII, Ddel and Bfal pro-duced 5, 6, and 5 RFLP patterns, respectively.The products obtained using the pair of primers NL4/ITS5 after treatment with HaeIII, Ddel, and Bfal produced 6, 7, and 0 RFLP patterns, respectively.The products obtained using the pair of primers Primer1/Primer3 after treatment with HaeIII, Ddel, and Bfal produced 6, 0, and 0 RFLP patterns, respectively.Among the combinations of pairs of primers and restriction

Evaluation of the detection limit and the time required for microorganism identification by PCR/RFLP
In order to study the detection limit of the PCR/RFLP, different concentrations (10 6 , 10 5 , 10 4 , 10 3 , and 10 2 /mL) of cells of the fungemia agents were submitted to the DNA extraction and PCR amplification using the pair of primers ITS1/ITS4.The minimum concentration of cells necessary for the amplification of C. neoformans and H. capsulatum was 10 4 cells/mL; however, for C. albicans amplification a concentration of 10 5 cells/mL was necessary.The genotyping identification proved to be more adequate than phenotyping methods (gold standard) for Candida species identification.Cryptococcus sp (40 strains) and H. capsulatum (4 strains) were identified identically using both conventional methods and PCR/RFLP.The time necessary for the identification of a fungemia agent using PCR/RFLP was approximately 2 days in a routine laboratory: 1 day for extraction and amplification and 1 additional day for enzyme restriction and electrophoresis assay.

Discussion
In the present study, we investigated three of the most important methodologies described for the identification of Candida species by PCR/RFLP (12)(13)(14) in order to evaluate them for the identification of fungemia agents.The results obtained demonstrated that it is possible to use a single RLFP/PCR procedure, on the basis of size and structural differences in the rDNA space regions, for correct identification.The present study was also the first to describe the differentiation of Cryptococcus species using the primer pair NL4/ITS5 and the restriction enzyme Ddel.
Although PCR/RFLP can be considered to be an "old tool" for PCR product analysis, it is still being used for studies of microorganism characterization because of its simplicity, reliability, easy adaptation for identifying several genera or species, and because it does not require expensive materials or equipment (12)(13)(14).These characteristics are important in cost-effective studies and indicate that the use of this technique will probably become routinely accepted in mycology laboratories before the DNA sequencing or real-time PCR methods are used.
In the present study, some species could be identified by evaluating the size of the PCR products with different primers.However, the enzyme digestion of the PCR products was necessary for the identification of most species.The combination of the PCR products using the pair of primer NL4/ITS5 and digestion with the restriction enzyme Ddel was the only one able to produce a single PCR/RFLP pattern for each one of the reference species.
The PCR/RFLP presented differs from those previously reported by Irobi et al. ( 13) that identified 7 medically important Candida species.In this new study, we included three different species: C. neoformans, C. gattii and H. capsulatum.It is important to note that the new method proposed here was able to effectively differentiate between C. neoformans and C. gattii.Both the reference and patient strains gave exactly the same RFLP patterns and the results obtained in the present study were reproducible and consistent for the isolates of the fungemia cultures tested.
The traditional methods for identifying fungemia agents are often based on the examination of phenotypic characteristics.This approach can be time-consuming and the reliance on the variable expression of phenotypic characteristics can lead to inconsistent results (3)(4)(5)(6) and consequently genotype-based approaches may be preferable.The phenotypic identification of Histoplasma capsulatum, Cryptococcus sp and Candida sp cultures takes approximately 30, 10, and 5 days, respectively.The method described, PCR/RFLP, was able to identify cultures containing more than 1 x 10 5 cells within about 48 h.This detection limit is sufficient for the investigation of blood cultures or small/ young cultures from agar plates.
The present methodology was developed and evaluated in order to be applied in the public health system of developing countries.The information presented in this manuscript could be useful for hospitals of medium and high complexity that need to identify fungemia agents and already have the standard materials for PCR.
In summary, three PCR amplification sites and three different cutter restriction enzymes were evaluated for the identification of fungemia species.Seven reference strains and 60 clinical isolates were successfully analyzed.The reliability of the technique was demonstrated by the examination of a large number of isolates.