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Fermentation Technology Division, Central Drug Research Institute, Lucknow, India
ABSTRACT
The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus-positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. Immunological methods of diagnosis have not been found to be satisfactory, and recent research has been diverted to the use of PCR for the sensitive and early diagnosis at the molecular level. In the present study we targeted different regions of the rRNA gene to diagnose cases of mycotic keratitis and identify the causal agents. Six fungus-specific primers (primers ITS1, ITS2, ITS3, ITS4, invSR1R, and LR12R) were used, and the amplified products were analyzed by single-stranded conformation polymorphism (SSCP) analysis. Dendrograms of these SSCP patterns, prepared on the basis of Jaccard's coefficient, indicated that the PCR products obtained with primer pair ITS1 and ITS2 were the best for the identification of fungi. The results were confirmed by sequencing of the PCR products, and the approach was successfully tested experimentally for the detection of mycotic keratitis caused by Aspergillus fumigatus and was used for the diagnosis of fungal corneal ulcers in patients.
INTRODUCTION
Mycotic keratitis or fungal corneal infections have a worldwide distribution, and the incidence is higher in tropical and subtropical countries (8). More than 105 species of fungi belonging to 56 genera have been reported to cause oculomycosis. However, species of Fusarium, Aspergillus, Candida, and other hyaline and dematiaceous hyphomycetes are the usual isolates from patients with mycotic keratitis (20). The management of keratomycosis depends on rapid identification of the causal agent. The diagnosis is often delayed because of the poor availability of infected material from the cornea and the slow growth of a large number of fungi in routinely used culture media, and therefore, early intervention is not always possible and the patient's vision is often lost. In general, the diagnosis of fungal corneal ulcer is dependent on Gram and Giemsa staining, which have low sensitivities of about 50 to 80% (4). Recent advances in molecular biology techniques have opened the door for culture-independent diagnostic methods. Immunological detection (13) and identification by use of distinctive metabolites (2) and nucleic acid probes (7, 25) are the tools most often used for diagnosis. One such technique is PCR, which has been shown to be useful for the culture-independent diagnosis of various microbial infections, including mycoses (10, 11, 22). To date, a few cases of mycotic keratitis have successfully been diagnosed by PCR (5, 6).
Ribosomal DNA is the most conserved region in the genome, with capabilities of phylogenetic divergence (14). The whole rRNA gene contains a small subunit (SSU) 18S rRNA, 5.8S rRNA, and a large subunit (LSU) 28S rRNA. Internal transcribed spacer (ITS) region I (ITSI) and ITSII are more variable than the rest of the ribosomal gene subunits and are found between SSU rRNA and 5.8S rRNA and between 5.8S rRNA and LSU rRNA, respectively. Besides this, intergenic spacer (IGS) region I (IGSI) and IGSII are found between the end of the LSU and start of the next SSU sequence (24). Many workers (9, 15-17) have used the single-stranded conformation polymorphism (SSCP) technique to identify sequence variations in a single strand of DNA due to its adoption to a unique conformation under nondenaturing conditions (12). Here we report on the experimental proof and the clinical laboratory diagnosis of three cases of corneal ulcer by PCR by the ITS SSCP technique, in which useful vision could be restored due to prompt diagnosis and specific antifungal therapy.
MATERIALS AND METHODS
Cultures. Aspergillus fumigatus, Aspergillus flavus, Candida albicans, Candida krusei, Candida parapsilosis, Cryptococcus neoformans, Fusarium spp., Sporothrix schenckii, Trichophyton mentagrophytes, and three patient isolates (responsible for mycotic keratitis) were maintained on Sabouraud dextrose agar (SDA). Pseudomonas aeruginosa was maintained on nutrient agar.
Extraction of fungal DNA. All the fungal strains were inoculated in 100 ml of Sabouraud dextrose broth under shaking conditions at 37°C to obtain log-phase cultures. Microscopic examination was done to test the purity of the cultures, and the cells were harvested by centrifugation at 6,000 x g for 15 min at 4°C. The pellets were washed twice with 0.8% physiological saline and transferred to 200 μl of extraction buffer (0.2 M Tris-HCl [pH 7.6], 0.5 M NaCl, 0.1% sodium dodecyl sulfate, 0.01 M EDTA). Glass beads were added to this mixture at a 1:1 ratio and vortexed vigorously in a bead beater (HamiltonBeach/Proctor-Silex, Inc., Southern Pines, N.C.) to achieve 60% lysis of the cell mass. Fungal DNA from this lysate was recovered with a DNeasy plant mini kit (Qiagen, Hilden, Germany), electrophoresed on a 1% agarose gel with 1x TBE buffer (8.9 mM Tris-borate, 0.2 mM EDTA), and analyzed after staining with ethidium bromide. The purity of the extracted DNA was checked at 260 and 280 nm (UV/VIS 911A; GBC Scientific Equipment, Dandenong, Australia) and stored at –20°C for further analysis.
Sample collection. (i) Experimental mycotic keratitis. Experimental A. fumigatus keratitis was produced in bred albino New Zealand rabbits by the method of Agrawal et al. (1). A spore suspension of A. fumigatus (103 CFU/ml) was prepared in physiological saline from a 5-day-old SDA slant culture incubated at 28°C. It was inoculated intracorneally into the right eye of each rabbit while the rabbit was under local anesthesia by using a 26-gauge needle. Prior clearance from the local animal ethics committee was obtained. Corneal lesions started to develop within 24 to 48 h of inoculation. Corneal scrapings were collected aseptically at 2, 3, 4, and 5 days postinfection for DNA extraction in lysis buffer (QIAamp DNA mini kit; Qiagen) and recovery by culture on SDA slants.
(ii) Patients with keratitis. Four patients suspected of having mycotic keratitis visited an ophthalmologist with general complaints of pain, lacrimation, swollen eyelids, and diminution of vision in their affected eyes. All four patients had undergone some treatment before reporting to the ophthalmologist. Corneal scrapings were collected from all of the patients while they were under local anesthesia by use of a slit-lamp microscope and a flame-sterilized Kimura spatula. One part of each scraping was directly transferred to lysis buffer for DNA extraction, and the other part was inoculated aseptically on SDA slants, which were incubated at 28°C in a biological oxygen demand incubator. The scraping in lysis buffer was immediately processed for extraction of DNA, according to the instructions of the manufacturer. The fungal growth on the SDA slants with the corneal scrapings from three positive patients was also processed for DNA isolation, as described above.
Negative controls. (i) Corneal tissue. Corneal tissue was taken from the other eye of each experimental rabbit, and the DNA extraction procedure was carried out with a QIAamp DNA mini kit, as described above.
(ii) Bacteria. P. aeruginosa, one of the causal agents of bacterial keratitis, was grown overnight in nutrient broth at 37°C on an orbital shaker. Cells were harvested by centrifugation at 5,000 x g for 10 min at 4°C and lysed by sonication. The lysate was mixed with a mixture of phenol-chloroform-isoamyl alcohol (25:24:1). The aqueous layer was mixed with 100 μl each of 3 M sodium acetate and 1 M sodium chloride, and the mixture was incubated at 4°C for 30 min. Then, an equal volume of isopropanol was added and the mixture was centrifuged at 12,000 x g for 15 min. The pellet was washed with 70% ethanol, air dried, and dissolved in TE buffer (100 mM Tris-HCl, 10 mM EDTA).
PCR. The extracted DNA was subjected to amplification with a thermal cycler (Helena Biosciences, Sunderland, United Kingdom) and the primers listed in Table 1. All the primers, synthesized by Sigma Aldrich House, Suffolk, United Kingdom, were used in four sets of PCRs, as follows. The first set of PCRs was standardized to amplify ITSI by using primers ITS1 and ITS2. The 25-μl reaction mixture contained 100 μM deoxynucleoside triphosphates, 0.1 μM each primer, 1x PCR buffer with 2.0 mM MgCl2, 2 μl of template DNA sample, and 1 U of Taq polymerase (Qiagen). The reaction involved initial denaturation at 96°C for 10 min, followed by 30 cycles in series of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, with a final step of one cycle at 72°C for 10 min to final extension. The second set of PCRs was done to amplify ITSII with primer pair ITS3 and ITS4. The reaction mixture and conditions were the same as those used for the first set of PCRs, with the exception that the annealing temperature was 56°C. The third set of PCRs was performed to amplify both ITSI and ITSII along with the 5.8S rRNA gene by using primer pair ITS1 and ITS4. The reaction mixture was of the same composition as described above. The first cycle of initial denaturation was performed at 95°C for 10 min, followed by 30 cycles in series of 95°C for 1 min, 55°C for 1 min, and 72°C for 90 s, with a final cycle at 72°C for 10 min. The fourth and final set of PCRs involved primers invSR1R and LR12R, specific for the IGS region along with the 5S rRNA gene. The reaction mixture contained all of the components at the same concentrations described above, except that MgCl2 was used at 3.0 mM and the condition for annealing was fixed at 55°C for 1 min and extension was at 72°C for 150 s. For all the PCR protocols, a reaction mixture without sample DNA was used as a negative control and the products were analyzed and stored as described above.
SSCP analysis. The amplified products were denatured at 95°C for 10 min and snap-cooled on ice before they were mixed with denaturing buffer (80% [wt/vol] deionized formamide, 10 mM EDTA [pH 8.0], 1 mg of xylene cynol per ml, 1 mg of bromophenol blue per ml). The samples were then electrophoresed on a 5% acrylamide gel. The gels were then silver stained and analyzed (3).
Similarity-dissimilarity analysis. SSCP profiles were generated by using all of the samples from the PCRs and the four sets of primers. These profiles were analyzed by the presence or the absence (which were given values of 0 and 1, respectively) of bands to prepare a dendrogram for these fungal strains on the basis of Jaccard's coefficient.
Sequencing of PCR products. To determine the complete sequences of ITSI and ITSII, the amplified products obtained with primer pair ITS1 and ITS4 were sequenced with an ABI Prism automated DNA sequencer (model 3100, version 3.0; Applied Biosystems, Warrington, United Kingdom) with the single primer ITS1. These sequences were used to identify the fungi with the help of the BLASTn program (www.ncbi.nlm.nih.gov/BLAST), and multiple-sequence alignments were determined with the Clustal W program.
RESULTS
The 260 nm/280 nm ratios for the DNAs extracted from all the fungal strains were found to be between 1.7 and 1.8. These DNA samples gave positive results in all sets of reactions. Amplification of DNA samples with primer pairs ITS1-ITS2, ITS3-ITS4, ITS1-ITS4, and invSR1R-LR12R resulted in fragments of approximately 200 bp, 350 bp, 550 bp, and 2.0 kb, respectively. None of these primers amplified bacterial DNA.
Experimental mycotic keratitis caused by A. fumigatus was successfully produced in the right eyes of the albino rabbits (Fig. 1a). The progression of the corneal infection was monitored daily for up to 7 days, and the scrapings from the infection sites were found to be positive by PCRs with all the primer pairs described above. Also, A. fumigatus was isolated on SDA at 2, 3, 4, and 5 days postinoculation. We could achieve amplification of fungal DNA from the corneal scrapings by PCR as early as 48 h postinoculation (Fig. 2). These primer pairs did not amplify fragments from the corneal tissue from the control eye of any of the rabbits. Scrapings from three of the four patients suspected of having mycotic keratitis produced amplicons of approximately the same size as those produced from the standard fungal strains.
PCR products were first checked on a 1% agarose gel in 1x TBE buffer (Fig. 3a to c) and were then further diluted to 1:10 for SSCP analysis to reduce the background effects. The optimum temperature and voltage for SSCP analysis were found to be 22°C and 5 V/cm, respectively. Overall, four SSCP patterns specific for different regions of the ribosomal gene were obtained from a total of 15 DNA samples (9 from standard cultures, 3 from isolates from the patients with mycotic keratitis, and 3 from the corneal scrapings from these patients). Variations in the sequences of the different regions of the ribosomal gene were clearly evident in the SSCP patterns (Fig. 4a to c).
Agarose gel electrophoresis of the PCR products showed that ITSII, amplified by primer pair ITS3 and ITS4, was almost of the same size in all the fungi tested, whereas the PCR products of ITSI, which was amplified with primers ITS1 and ITS2, varied in size, thereby indicating that ITSII is the more conserved region in the 18S and 28S rRNA genes (Fig. 3a and b). However, a considerable difference in the band patterns of the amplified products of both the ITSI and the ITSII regions was observed by SSCP analysis (Fig. 4a and b). Similarly, the products amplified from the ITSI and the ITSII regions, including the 5.8S rRNA region (amplified with primer pair ITS1 and ITS4), showed little variation in size, as detected by agarose gel electrophoresis (Fig. 3c), whereas a significant difference in the band patterns of these amplified products (from all 15 DNA samples) was obtained by SSCP analysis (Fig. 4c). All the PCR products obtained with primer pair ITS1 and ITS4 were sequenced with primer ITS1 and were identified by using the BLASTn program. These sequences were found to be 90 to 100% similar to the sequences of the ITSI, 5.8S rRNA gene, and ITSII regions of the respective fungi. On the basis of the results of these studies, the three patient isolates were identified as the Colletotrichum state of Glomerella cingulata, Curvularia inaequalis, and Epidermophyton floccosum by PCR of the corneal scrapings as well as by conventional identification methods. The multiple-sequence alignment obtained with the Clustal W program is presented in Fig. 5. The fourth patient had a case of bacterial keratitis and did not give positive results for fungi by PCR analysis.
Primers invSR1R and LR12R, used in the present study for amplification of the IGS region of the fungi, were inferior, as they failed to amplify the DNA of C. albicans and T. mentagrophytes under normal conditions. These primers also did not give positive results with DNA from the corneal scrapings. However, variation of the MgCl2 concentration in the reaction mixtures resulted in positive results. SSCP analysis of the amplified products obtained with this primer pair was not satisfactory, as the product size was found to be about 2.0 kb (Fig. 2) for all the fungal isolates; therefore, almost similar patterns were obtained (data not shown).
The SSCP patterns of the ITS regions were found to differentiate the fungal strains tested to the species level. The SSCP patterns of the Candida and Aspergillus strains differed significantly (Fig. 4a to c, lanes 1, 2, 3, 5, and 6), but no such difference was evident in the products amplified from the DNA from the corneal scrapings or in those from their subsequent cultures (Fig. 4a to c, lanes 10, 11, 12, 13, 14, and 15). Dendrograms were prepared from the three SSCP patterns described above on the basis of Jaccard's coefficient. Analysis of these dendrograms also confirmed the findings indicated above (Fig. 6).
DISCUSSION
The identities of the fungi determined by PCR in the present study matched 100% the conventional identities of the respective strains. Three of four patients with mycotic keratitis were positive for fungi by this technique, while the fourth patient, who was negative for a fungus in this study, was found to be negative for fungus in culture as well, thereby indicating the specificity of the ITS PCR-SSCP technique.
The high purity of the fungal DNA isolated in the present study was indicated by the 260 nm/280 nm ratios, which ranged from 1.7 to 1.8 for all fungi. We achieved the same high grade of purity of DNA obtained by the extraction methods used earlier, including those with commercial kits, by use of the QIAamp tissue mini kit (Qiagen). We also used the DNeasy plant mini kit (Qiagen) for DNA extraction, but this kit was not found to be suitable for use with corneal scrapings, in which it is supposed that only a few fungal elements may be present. The added advantage to our extraction method was that we could isolate the pure form of DNA in significantly less time (<1 h) than is required for other methods (11, 15). In addition, the kit that we used apparently had a capacity of removing all PCR-inhibiting substances (5) from DNA extracts of the corneal scrapings.
We were interested in the section of the genome that includes the 18S, 5.8S, and 28S genes, which code for rRNA and whose nucleotide sequence is also relatively conserved among fungi. This section also includes the intervening ITS regions, called ITSI and ITSII, whose DNA sequences vary. Although the ITS-coding regions are not translated into proteins, they have a critical role in the development of functional rRNA; and because of the sequence variations of these regions among species, these regions show promise for use as signatures for molecular biology-based assays (25). A number of probes have been designed by many workers for the identification of fungal DNA by the hybridization procedure, but PCR is the most sensitive and widely used technique for the identification of fungi and is also best suited for use with clinical samples in which DNA is poorly available. Ferrer et al. (5) used primers ITS1, ITS4, and ITS86 to identify fungal strains by nested PCR, and Kumeda and Asao (15) also used PCR with primers ITS1 and ITS4 followed by SSCP analysis to identify fungi pathogenic for plants (15). Besides these, other primer pairs have been designed for the identification of fungi (21). On the basis of these facts, the primers described by White et al. (24) and Vilgalys et al. (23), which anneal to different regions of the ribosomal gene, were selected and the sequence variations in each segment were determined simultaneously. An IGS region-specific primer pair was also used to compare the sequence variations within the ITS region. To analyze the variations in nucleotide sequences, direct sequencing of the amplified product, restriction fragment length polymorphism analysis (21), temperature gradient gel electrophoresis, denaturing gradient gel electrophoresis, amplified rRNA gene restriction analysis for the ribosomal gene, and SSCP analysis (9, 15-17) are being used by various workers. Among all these techniques, SSCP analysis is one of the most accurate and is often used for mutational studies and the detection of single nucleotide polymorphisms (12). In the present study the ITS PCR-SSCP technique was used for the successful identification of fungi.
We tried to assess these techniques for their abilities to diagnose experimental keratomycosis in rabbits. The prognosis of the disease depends on an early and a prompt diagnosis. In the case of mycotic keratitis, the delay in diagnosis is due to the lack of sensitive techniques and the continued dependence on classical methods, such as direct microscopic examination, culture of material obtained from deep corneal scrapings, corneal biopsy, and anterior chamber paracentesis. The availability of less clinical material and the slow growth of fungi often lead to delays in therapy and ultimately result in corneal damage. Using all four primer pairs, we succeeded in diagnosing experimental fungal keratitis due to A. fumigatus within 8 h from the time of collection of the corneal scrapings from the rabbits (Fig. 2), which was faster than the time to the recovery of fungi by conventional culture on SDA. Encouraged by this we applied our technique to four patients who were suspected of having fungal corneal ulcers and who consulted a private ophthalmic practitioner (N. K. Mishra).
The fungi isolated from the human cornea were identified in the laboratory on the basis of micro- and macromorphological characteristics. One of the isolates was identified as the Colletotrichum state of G. cingulata. Interestingly, this is the second case of mycotic keratitis caused by this fungus (Fig. 1b) in the vicinity of our city (Lucknow, India) and has been encountered after a gap of more than 20 years (18). The other two isolates were identified as a Curvularia sp. and an Epidermophyton sp. Species of the genus Curvularia are frequently isolated, while an Epidermophyton sp. has rarely been reported as a cause of mycotic keratitis (19, 26). The PCR product sequencing studies resulted in the identification of these isolates as the Colletotrichum state of G. cingulata (100% similarity), C. inaequalis (99% similarity), and E. floccosum (98% similarity). This indicates the authenticity and superiority of PCR-based identification of fungi (Fig. 5).
The amplified products obtained with primer pair invSR1R and LR12R were almost similar; therefore, the SSCP patterns derived from this IGS region did not differentiate all the fungi, whereas the ITS region-specific primer pair ITS1 and ITS4 resulted in amplified products of various sizes that could be differentiated on SSCP gels due to sequence variations (Fig. 4c). ITSI has a more variable segment than ITSII, and ITS1 was amplified by primers ITS1 and ITS2 (Fig. 4a and b). This was also confirmed by the use of Jaccard's coefficient, as represented in the dendrogram in Fig. 6. The dendrogram prepared with the ITSII sequences showed no difference between A. flavus and Fusarium; similarly, the dendrogram prepared with the complete sequences of ITSI, 5.8S, and ITSII failed to differentiate S. schenckii and the Colletotrichum state of G. cingulata. On the other hand, the dendrogram prepared with the ITSI sequences differentiated all the fungal strains to the species level (Fig. 6). However, the patient isolates of the Colletotrichum state of G. cingulata, C. inaequalis, and E. floccosum were found to be closely related to A. fumigatus, A. flavus, and T. mentagrophytes, respectively. This satisfies our sequencing results for the DNA sample amplified with primers ITS1 and ITS4. In conclusion, the successful diagnosis of three cases of mycotic keratitis within 8 h of collection of corneal scrapings, identification of the fungi isolated by sequencing of the PCR products and dendrogram analysis, and confirmation of the identities by conventional methods are satisfying.
ACKNOWLEDGMENTS
We thank the director of CDRI and the head of the Fermentation Technology Division, CDRI, Lucknow, India, for providing facilities. We are also thankful to N. K. Mishra, the ophthalmologist who provided patient material for this study.
M.K. thanks the Council of Scientific and Industrial Research, New Delhi, India, for a junior research fellowship.
CDRI communication no. 6620.
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