بررسی ضایعات کاندیدایی ناخن و تعیین هویت گونه‌های جدا شده با روش‌های قارچ‌شناسی و مولکولی
محورهای موضوعی : میکروب شناسی
مژگان سقازاه
1
(استادیار،گروه میکروبیولوژی، واحد قم، دانشگاه آزاد اسلامی، قم، ایران)
الهام نجفی
2
(دانشجوی کارشناسی ارشد، میکروبیولوژی، واحد قم، دانشگاه آزاد اسلامی، قم، ایران)
کلید واژه: PCR-RFLP, کاندیدا, آزمایش مستقیم, کشت, اونیکومایکوزیس,
چکیده مقاله :
هدف:اونیکومایکوزیس شایع‌ترین بیماری ناخن محسوب می‌شود که مخمرها از عوامل مهم بروز آن می‌باشند. هدف مطالعه حاضر بررسی ضایعات کاندیدیایی ناخن و تعیین هویت گونه‌های جدا شده PCR-RFLP می‌باشد. روش‌شناسی: در این مطالعه مقطعی از 280 بیمار دچار دیستروفی ناخن مراجعه‌کننده به آزمایشگاه تخصصی قارچ‌شناسی در تهران نمونه تهیه شد. آزمایش مستقیم و کشت در محیط‌های سابورودکستروز آگار، کروم آگار کاندیدا، کورن میل آگار و تست لوله زایا بر روی مخمرهای جدا شده از ضایعات ناخن انجام گرفت و در نهایت برای تشخیص نهایی گونه‌های کاندیدایی که تعیین هویت نشده، روش مولکولی PCR-RFLP با استفاده از آنزیم Msp1 انجام شد. یافته‌ها:از 280 بیمار، 87 نفر انیکومایکوزیس کاندیدایی داشتند که 54 مورد کاندیدا آلبیکنس و پس از آن به ترتیب کاندیدا پاراپسیلوزیس 16 مورد، کاندیدا تروپیکالیس7 مورد، کاندیدا گلابراتا 6 مورد و کاندیدا کروزئی 3 مورد و یک مورد کاندیدا گیلرموندی بود. به منظور تعیین هویت گونه‌های مجهول و همچنین تائید نتایج حاصل از روش‌های تشخیصی، تست ژنوتیپی بر روی 20 نمونه انجام گردید و نتایج حاصل از کشت و آزمایش مستقیم و همچنین تعیین هویت گونه‌های مجهول را تایید کرد. نتیجه‌گیری:به دلیل زمان‌بر بودن مراحل تشخیصی معمول در آزمایشگاه‌ها از جمله کشت، استفاده از روش‌های ژنوتیپی سریع و مطمئن مانند PCR-RFLP که شناسایی را در حد گونه انجام می‌دهد، بسیار کار‌آمد خواهد بود و روش‌های نوین مولکولی می‌تواند در تشخیص و شناسایی گونه‌ها بسیار کمک کننده باشد.
Background:Onychomycosis consider as the most common nail diseases and yeasts are the important causative agents in onychomycosis. The aim of current study is the investigation of candidial onychomycosis and investigation of isolated species using direct examination.culturing and PCR-RFLP techniques. Methods: In this cross- sectional study nail sampling has been examined in 280 patients with dystrophic nail problem who were the applicant of Medical mycology laboratory in Tehran. Direct examination test, Culturing on SDA, candida Chrome agar, Corn meal agar and germ tube test were used for identification of isolated yeasts from onychomycosis and finally PCR-RFLP techniques using MSP1 enzyme were performed for definitive diagnosis of Candida species which had not been identified by conventional methods. Results:Out of 280 patients with onychomycosis 54 cases (candida albicanse), 16 cases (candida parapsilosis), 7 cases (candida tropicalis), 6 cases (candida glabrata), 3 cases (candida krusei) and 1 case (candida guilliermondii) have been identified respectively.In order to identification of unknown species and also confirmation of the results of conventional methods PCR-RFLP genotyping methods have been performed on 20 samples. The results obtained from Culturing and direct examination and the identification of unknown species have been confirmed using PCR-RFLP methods. Conclusion:As conventional diagnostic laboratory methods such as culturing, are time-consuming, it is better to use fast and safe genotyping techniques (PCR-RFLP) due to the possibility of better identification in species level. Therefore using modern molecular methods for diagnosis and identification in species level would be useful.
Agarwalla A, Agrawal S, Khanal B. Onychomycosis in eastern Nepal. Nepal Med Coll J. 2006; 8(4): 215-9.
Midgley G, Moore MK. Nail infections. Dermatol Clin.1996; 14(1): 41-49.
Anaisse E, Mecinnis MR, Pfaller MA. Clinical Mycology. Churchill Livingstone. 2003:95-239.
Lange M, Roszkiewicz J, Sczerknowska-Dobosz A, Jasiel-Walikowska E, Bykowska B. Onychomycosis is no longer a rare finding in children. Mycoses. 2006; 49:55-59.
Foster KW, Ghannoum MA, Elewski BE. Epidemiologic surveillance of cutaneous fungal infection in the United states from 1999 to 2002. J Am Acad Dermatol .2004; 50:
748-752.
Zaini F, Mehbod ASA, Emami M. Comprehensive Medical mycology. Tehran: Tehran University Press. 2009: 133. [In Persian].
Dalle F, Franco N, Lopez J. Comparative genotyping of Candida albicans bloodstream and non-bloodstream isolates at a polymorphic microsatellite locus. J Clin Microbial. 2000; 38: 4554-4559.
Karahan ZC, Guriz H, Agirbasli H, Balaban N, Gocmen JS, Aysev D & et al. Genotype distribution of Candida albicans isolates by 25S intron analysis with regard to invasiveness. Mycoses. 2004; 47: 465-469.
Elewski BE. Onychomycosis: pathogenesis, diagnosis, and management. Clin Microbiol Rev. 1998; 11(3): 415-29.
Borkowski P, Williams M, Holewinski J, Bakotic B. Onychomycosis: an analysis of 50 cases and a comparison of diagnostic techniques. J Am Podiatr Med Assoc. 2001; 91(7): 351-5.
Surjushe A, Kamath R, Oberai C, Saple D, Thakre M, Dharmshale S, Gohil A. A clinical and mycological study of onychomycosis in HIV infection. Indian J Dermatol Venereol Leprol. 2007; 73(6): 397- 401.
Midgley G, Moore MK, Cook JC, Phan QG. Mycology of nail disorders. J Am Acad Dermatol. 1994; 31(3 pt 2): S68-74.
Alvarez MI, González LA, Castro LA. Onychomycosis in Cali, Colombia. Mycopathologia. 2004; 158(2): 181-6.
Vélez A, Linares MJ, Fenández-Roldán JC, Casal M. Study of onychomycosis in Córdoba, Spain: prevailing fungi and pattern of infection. Mycopathologia. 1997; 137(1): 1-8.
Khosravi AR, Shokri H, Mansouri P, Katiraee F, Ziglari T. Candida species isolated from nails and their in vitro susceptibility to antifungal drugs in the department of Dermatology (University of Tehran, Iran). J Myc Med. 2008; 18(4): 210-5.
Anane S, Chtourou O, Chedi A, Triki S, Belhaj S, Kaouech E, Kallel K, Chaker E. Onychomycosis in the elderly. Ann Dermatol Venereol. 2007; 134 (10 Pt 1): 743-7.
Gerami Shoar M, Zomorodian K, Emami M, Tarazoei B, Saadat F. Study and identification of the etiological agents of onychomycosis in Tehran, Capital of Iran. Iranian J Publ Health.2002; 31(3-4): 100-4.
Fidel PL Jr, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev. 1999; 12: 80-96.
Gauzit R, Cohen Y, Dupont H, Hennequin C, Montravers P, Timsit JF & et al. Infections by Candida sp. in intensive care. Survey of French practices. Press Med. 2003; 32: 440-9.
Staats CC, Korstanje MJ. Fungi causing onychomycoses in The Netherlands. Ned Tijdschr Geneeskd. 1994;138(47): 2340-3
Del Castillo L, Bikandi J, Nieto A, Quindos G, Sentandreu R, Ponton J. Comparison Of morphotypic and genotypic methods for strain delineation in Canada. Mycoses. 1997; 40: 445-450.
Freydie’re AM, Parant F, Chaux C, Gille Y. Candida ID, a new chromagenic medium compared to Albicans ID2. Clin Microbial Infect. 2000; 6(supp1.1): 181.
Lott TJ, Burns BM, Zancope-Oliveira R, Elie DM, Reiss E. Sequence analysis of the internal transcribed spacer 2 (ITS2) from yeast species within genus Candida. Current Microbiol. 1998; 36: 63-69.
Chen YC, Eisner JD, Kattar MM, Rassoulian- Barrett SL, LaFe K, Yarfitz SL, Limaye P, Cookson BT. Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes. J Clin Mirobiol. 2000; 38: 2302-2310.
Ajello L, Hay RJ . Medical mycology. Ninth edition. Arnold Publication Unicelluar Ascomycetous Candida species. 1999: 423-450.
Ahmad S, Khan Z, Mustafa AS, Khan ZU. Seminested PCR for diagnosis of candidemia: Comparison with culture, antigen detection, and biochemical methods for species identification. J Clin Microbiol. 2002; 40: 2483-2489.
Shadzi Sh. Pathogenic yeasts. In: Medical Mycology. Isfahan University: Jihad Press; 2007:43-59. [In Persian]
Kurtzman CP, Fell JW. The Yeasts: A Taxonomic Study. 4th ed. Amesterdam: Elsevier. 1998.
Odds FC, Bernaerts R. CHROM agar candida, a new differential isolation medium for presumptive identiation of clinically important Candida species. J Clin Microbiol. 1994; 32(8): 1923-9.
Rezaee Matekalaee A. Quantitative assessment of Bead-beating, Boiling, and freez and thaw for Genomic DNA extraction from some medically important yeasts. Presented for the M.Sc. Tehran: Tehran University of Medical Sciences, 2005. [In Persian]
Mirhendi H, Makimura K, Khoramizadeh M, Yamaguchi H. A one-enzyme PCR-RFLP assay for identification of six medically important Candida species. Nippon Ishinkin Gakkai Zasshi. 2006; 47(3): 225-9.
Mirhendi SH, Kordbacheh P, Kazemi B, Samiei S, Pezeshki M, Khoramizadeh MR. A PCR-RFLP Method to Identification of the Important Opportunistic Fungi: Candida Species, Cryptococcus neoformans, Aspergillus fumigatus and Fusarium solani. Iranian J Publ Health. 2001; 30(3-4): 103-6.
Mosca CO, Moragues MD, Llovo J, Al Mosaid A, Coleman DC, Pontón J. Casein agar: a useful medium for differentiating Candida dubliniensis from Candida albicans. J Clin Microbiol. 2003; 41(3):1259-62.
Heikkla H, Stubb S. The prevalence of onychomycosis in Finland. Br. J. Dermatol. 1995; 133: 699-703.
Piraccini BM & Alessandrini A. Onychomycosis: A Review. J Fungi (Basel). 2015; 1(1): 30-43. Doi: 10.3390/jof1010030.
Roseeuw D. Achilles foot screening project: Preliminary results of patients screened by dermatologists. J. Eur. Acad. Dermatol. Venereol. 1999; 12: S6-S9.
Scher RK, Rich P, Pariser D, Elewski B. The epidemiology, etiology, and pathophysiology of onychomycosis. Semin. Cutan. Med. Surg. 2013; 32: S2-S4.
Gupta AK, Drummond-Main C, Cooper EA, Brintnell W, Piraccini BM, Tosti A. Systematic review of nondermatophyte mold onychomycosis: Diagnosis, clinical types, epidemiology, and treatment. J. Am. Acad. Dermatol. 2012; 66: 494-502.
Jayatilake JA, Tilakatatne WM, Panagoda GJ. Candidal onychomycosis: A mini-review. Mycopathologia. 2009; 168: 165-173.
Tulumoglu S, Kariptas`E, Erdem B. Identification and antifungal susceptibility of Candida isolates from various clinical specimens in Doctor Behcet Uz hospital. Anatol J Clin Investig. 2009; 3(3): 170-3.
Ghahri M, MirhendiSH, Yadegari MH, Hajizadeh E, Shidfar MR. Identification of pathogenic yeasts isolated from onychomycosis in Tehran, using polymerase chain Reaction Modares. Journal of Medical Sciences. 2020; 13(1):79-91. [In Persian].
Ghasemi Z, Falahati M, Nami S, Nozari Sh, Ahmadi F, Ghaffarpour GhH. Investigation of prevalence of onychomycosis due to yeast fungi in dystrophic nails of patients who referred to Razi ospital (2010-2011). Razi Journal of Medical Sciences. 2012; 19(96):
27-33. [In Persian].
Shokohi T, Heidari Z, Haqhani I, Khalilian A, Aghili R, Miahi S. Study 101 Case onychomycosis in patients refers o Boo ali sina hospital and toba clinic in Sari. J Mazandaran Uni Med Sci. 2009; 33-4. [In Persian].
Mohammadi R, Mirhendi, H, Yadegari MH, Shadzi Sh, Jalalizand N. Identification and Frequency of Candida Species in Patients with Different Forms of Candidiasis in Isfahan, Using PCR-RFLP Method. Journal of Isfahan Medical School. 2011; 29(133): 1-8.
Hashemi SJ, Zaini F, Charsizadeh A & et al. Prevalence of candida and non-candida yeasts isolated from patients with yeast fungal infections in Tehran labs. Tehran University Medical Journal. 2011; 69(1): 55-62.
Zaini F, Mahmoudi M, Mehbod ASA, Kordbacheh P, Safara M. Fungal Nail Infections in Tehran. Iran Iranian J Publ Health. 2009; 38(3): 46-53.
Reisberger EM, Abels C, Landthaler M, Szeimies RM. Histopathologic diagnosis of onychomycosis by periodic acid- Schiffstained nail clippings. Br J Dermatol. 2003; 148: 749-754.
Mitchel TG, Sandin RL, Bowman BH, Meyer W, Merz WG. Molecular mycology: DNA probes and application of PCR technology. J. Med. Vet.Mycol.1994; 32(1): 351-366.
Reiss E, Tanaka K, Bruker G, Chazalet V, Coleman D, Debeaupuis JP & et al. Molecular diagnosis and epidemiology of fungal infections. Med. Mycol.1998; 36(1): 249- 257.
Morace G, Pagano L, Sanghinetti M, Posteraro B, Mele L, Equitani F & et al. PCR-restriction enzyme analysis for detection of Candida DNA in blood from febrible patiens with hematological malignancies. J.Clin. Microbiol. 1999; 37: 1871-1875.
Williams DW, Wilson MJ, Lewis MA, Potts AJ. Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J. Clin. Microbiol.1995; 33: 2476-2479.
_||_Agarwalla A, Agrawal S, Khanal B. Onychomycosis in eastern Nepal. Nepal Med Coll J. 2006; 8(4): 215-9.
Midgley G, Moore MK. Nail infections. Dermatol Clin.1996; 14(1): 41-49.
Anaisse E, Mecinnis MR, Pfaller MA. Clinical Mycology. Churchill Livingstone. 2003:95-239.
Lange M, Roszkiewicz J, Sczerknowska-Dobosz A, Jasiel-Walikowska E, Bykowska B. Onychomycosis is no longer a rare finding in children. Mycoses. 2006; 49:55-59.
Foster KW, Ghannoum MA, Elewski BE. Epidemiologic surveillance of cutaneous fungal infection in the United states from 1999 to 2002. J Am Acad Dermatol .2004; 50:
748-752.
Zaini F, Mehbod ASA, Emami M. Comprehensive Medical mycology. Tehran: Tehran University Press. 2009: 133. [In Persian].
Dalle F, Franco N, Lopez J. Comparative genotyping of Candida albicans bloodstream and non-bloodstream isolates at a polymorphic microsatellite locus. J Clin Microbial. 2000; 38: 4554-4559.
Karahan ZC, Guriz H, Agirbasli H, Balaban N, Gocmen JS, Aysev D & et al. Genotype distribution of Candida albicans isolates by 25S intron analysis with regard to invasiveness. Mycoses. 2004; 47: 465-469.
Elewski BE. Onychomycosis: pathogenesis, diagnosis, and management. Clin Microbiol Rev. 1998; 11(3): 415-29.
Borkowski P, Williams M, Holewinski J, Bakotic B. Onychomycosis: an analysis of 50 cases and a comparison of diagnostic techniques. J Am Podiatr Med Assoc. 2001; 91(7): 351-5.
Surjushe A, Kamath R, Oberai C, Saple D, Thakre M, Dharmshale S, Gohil A. A clinical and mycological study of onychomycosis in HIV infection. Indian J Dermatol Venereol Leprol. 2007; 73(6): 397- 401.
Midgley G, Moore MK, Cook JC, Phan QG. Mycology of nail disorders. J Am Acad Dermatol. 1994; 31(3 pt 2): S68-74.
Alvarez MI, González LA, Castro LA. Onychomycosis in Cali, Colombia. Mycopathologia. 2004; 158(2): 181-6.
Vélez A, Linares MJ, Fenández-Roldán JC, Casal M. Study of onychomycosis in Córdoba, Spain: prevailing fungi and pattern of infection. Mycopathologia. 1997; 137(1): 1-8.
Khosravi AR, Shokri H, Mansouri P, Katiraee F, Ziglari T. Candida species isolated from nails and their in vitro susceptibility to antifungal drugs in the department of Dermatology (University of Tehran, Iran). J Myc Med. 2008; 18(4): 210-5.
Anane S, Chtourou O, Chedi A, Triki S, Belhaj S, Kaouech E, Kallel K, Chaker E. Onychomycosis in the elderly. Ann Dermatol Venereol. 2007; 134 (10 Pt 1): 743-7.
Gerami Shoar M, Zomorodian K, Emami M, Tarazoei B, Saadat F. Study and identification of the etiological agents of onychomycosis in Tehran, Capital of Iran. Iranian J Publ Health.2002; 31(3-4): 100-4.
Fidel PL Jr, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev. 1999; 12: 80-96.
Gauzit R, Cohen Y, Dupont H, Hennequin C, Montravers P, Timsit JF & et al. Infections by Candida sp. in intensive care. Survey of French practices. Press Med. 2003; 32: 440-9.
Staats CC, Korstanje MJ. Fungi causing onychomycoses in The Netherlands. Ned Tijdschr Geneeskd. 1994;138(47): 2340-3
Del Castillo L, Bikandi J, Nieto A, Quindos G, Sentandreu R, Ponton J. Comparison Of morphotypic and genotypic methods for strain delineation in Canada. Mycoses. 1997; 40: 445-450.
Freydie’re AM, Parant F, Chaux C, Gille Y. Candida ID, a new chromagenic medium compared to Albicans ID2. Clin Microbial Infect. 2000; 6(supp1.1): 181.
Lott TJ, Burns BM, Zancope-Oliveira R, Elie DM, Reiss E. Sequence analysis of the internal transcribed spacer 2 (ITS2) from yeast species within genus Candida. Current Microbiol. 1998; 36: 63-69.
Chen YC, Eisner JD, Kattar MM, Rassoulian- Barrett SL, LaFe K, Yarfitz SL, Limaye P, Cookson BT. Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes. J Clin Mirobiol. 2000; 38: 2302-2310.
Ajello L, Hay RJ . Medical mycology. Ninth edition. Arnold Publication Unicelluar Ascomycetous Candida species. 1999: 423-450.
Ahmad S, Khan Z, Mustafa AS, Khan ZU. Seminested PCR for diagnosis of candidemia: Comparison with culture, antigen detection, and biochemical methods for species identification. J Clin Microbiol. 2002; 40: 2483-2489.
Shadzi Sh. Pathogenic yeasts. In: Medical Mycology. Isfahan University: Jihad Press; 2007:43-59. [In Persian]
Kurtzman CP, Fell JW. The Yeasts: A Taxonomic Study. 4th ed. Amesterdam: Elsevier. 1998.
Odds FC, Bernaerts R. CHROM agar candida, a new differential isolation medium for presumptive identiation of clinically important Candida species. J Clin Microbiol. 1994; 32(8): 1923-9.
Rezaee Matekalaee A. Quantitative assessment of Bead-beating, Boiling, and freez and thaw for Genomic DNA extraction from some medically important yeasts. Presented for the M.Sc. Tehran: Tehran University of Medical Sciences, 2005. [In Persian]
Mirhendi H, Makimura K, Khoramizadeh M, Yamaguchi H. A one-enzyme PCR-RFLP assay for identification of six medically important Candida species. Nippon Ishinkin Gakkai Zasshi. 2006; 47(3): 225-9.
Mirhendi SH, Kordbacheh P, Kazemi B, Samiei S, Pezeshki M, Khoramizadeh MR. A PCR-RFLP Method to Identification of the Important Opportunistic Fungi: Candida Species, Cryptococcus neoformans, Aspergillus fumigatus and Fusarium solani. Iranian J Publ Health. 2001; 30(3-4): 103-6.
Mosca CO, Moragues MD, Llovo J, Al Mosaid A, Coleman DC, Pontón J. Casein agar: a useful medium for differentiating Candida dubliniensis from Candida albicans. J Clin Microbiol. 2003; 41(3):1259-62.
Heikkla H, Stubb S. The prevalence of onychomycosis in Finland. Br. J. Dermatol. 1995; 133: 699-703.
Piraccini BM & Alessandrini A. Onychomycosis: A Review. J Fungi (Basel). 2015; 1(1): 30-43. Doi: 10.3390/jof1010030.
Roseeuw D. Achilles foot screening project: Preliminary results of patients screened by dermatologists. J. Eur. Acad. Dermatol. Venereol. 1999; 12: S6-S9.
Scher RK, Rich P, Pariser D, Elewski B. The epidemiology, etiology, and pathophysiology of onychomycosis. Semin. Cutan. Med. Surg. 2013; 32: S2-S4.
Gupta AK, Drummond-Main C, Cooper EA, Brintnell W, Piraccini BM, Tosti A. Systematic review of nondermatophyte mold onychomycosis: Diagnosis, clinical types, epidemiology, and treatment. J. Am. Acad. Dermatol. 2012; 66: 494-502.
Jayatilake JA, Tilakatatne WM, Panagoda GJ. Candidal onychomycosis: A mini-review. Mycopathologia. 2009; 168: 165-173.
Tulumoglu S, Kariptas`E, Erdem B. Identification and antifungal susceptibility of Candida isolates from various clinical specimens in Doctor Behcet Uz hospital. Anatol J Clin Investig. 2009; 3(3): 170-3.
Ghahri M, MirhendiSH, Yadegari MH, Hajizadeh E, Shidfar MR. Identification of pathogenic yeasts isolated from onychomycosis in Tehran, using polymerase chain Reaction Modares. Journal of Medical Sciences. 2020; 13(1):79-91. [In Persian].
Ghasemi Z, Falahati M, Nami S, Nozari Sh, Ahmadi F, Ghaffarpour GhH. Investigation of prevalence of onychomycosis due to yeast fungi in dystrophic nails of patients who referred to Razi ospital (2010-2011). Razi Journal of Medical Sciences. 2012; 19(96):
27-33. [In Persian].
Shokohi T, Heidari Z, Haqhani I, Khalilian A, Aghili R, Miahi S. Study 101 Case onychomycosis in patients refers o Boo ali sina hospital and toba clinic in Sari. J Mazandaran Uni Med Sci. 2009; 33-4. [In Persian].
Mohammadi R, Mirhendi, H, Yadegari MH, Shadzi Sh, Jalalizand N. Identification and Frequency of Candida Species in Patients with Different Forms of Candidiasis in Isfahan, Using PCR-RFLP Method. Journal of Isfahan Medical School. 2011; 29(133): 1-8.
Hashemi SJ, Zaini F, Charsizadeh A & et al. Prevalence of candida and non-candida yeasts isolated from patients with yeast fungal infections in Tehran labs. Tehran University Medical Journal. 2011; 69(1): 55-62.
Zaini F, Mahmoudi M, Mehbod ASA, Kordbacheh P, Safara M. Fungal Nail Infections in Tehran. Iran Iranian J Publ Health. 2009; 38(3): 46-53.
Reisberger EM, Abels C, Landthaler M, Szeimies RM. Histopathologic diagnosis of onychomycosis by periodic acid- Schiffstained nail clippings. Br J Dermatol. 2003; 148: 749-754.
Mitchel TG, Sandin RL, Bowman BH, Meyer W, Merz WG. Molecular mycology: DNA probes and application of PCR technology. J. Med. Vet.Mycol.1994; 32(1): 351-366.
Reiss E, Tanaka K, Bruker G, Chazalet V, Coleman D, Debeaupuis JP & et al. Molecular diagnosis and epidemiology of fungal infections. Med. Mycol.1998; 36(1): 249- 257.
Morace G, Pagano L, Sanghinetti M, Posteraro B, Mele L, Equitani F & et al. PCR-restriction enzyme analysis for detection of Candida DNA in blood from febrible patiens with hematological malignancies. J.Clin. Microbiol. 1999; 37: 1871-1875.
Williams DW, Wilson MJ, Lewis MA, Potts AJ. Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J. Clin. Microbiol.1995; 33: 2476-2479.
