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Onychomycosis: An evaluation of three sampling methods
Journal of the American Academy of Dermatology Volume 39, Number 6
Brief communications 1015
Onychomycosis: An evaluation of three sampling methods P. R. Hull, MD, PhD(Med), FFDerm(SA), FRCPC,a A. K. Gupta, MD, MA, FRCPC,b and R. C. Summerbell, PhDc,d Saskatchewan and Ontario, Canada
From the Division of Dermatology, Department of Medicine, Royal University Hospital and the University of Saskatchewan, Saskatoona; the Division of Dermatology, Department of Medicine, Sunnybrook Health Science Centre and the University of Toronto, Ontariob; the Ontario Ministry of Health, Laboratory Services Branch, Torontoc; and the Department of Laboratory Medicine and Pathology, University of Toronto, Ontario.d Supported in part by Janssen-Ortho Inc, Canada. Reprint requests: P. R. Hull, Head, Division of Dermatology, Department of Medicine, Royal University Hospital, Saskatoon, Saskatchewan, S7N OW8, Canada. J Am Acad Dermatol 1998;39:1015-7. Copyright © 1998 by the American Academy of Dermatology, Inc. 0190-9622/98/$5.00 + 0 16/54/92154
The availability of more effective systemic antifungals has resulted in increasing numbers of patients receiving treatment for clinically suspected onychomycosis. Although positive microscopy is frequently used as an indication for starting treatment, cultural identification of the responsible fungus has important implications when treatment options are considered. Frequently, however, the fungus fails to grow in culture. Reasons advanced to explain this include the presence of nonviable fungi in the distal nail, sampling error,
Journal of the American Academy of Dermatology December 1998
1016 Brief communications
Table I. Culture results from the three sampling methods in 75 nails
NaOH n = 75 Culture n = 75 Pathogenic fungi D n = 61
Clipping
Curetting
Plate
Cl + Cu
Cl + Pl
Cu + Pl
Cl + Cu + Pl
56 (75%) 42 (56%)
61 (81%) 49 (65%)
54 (72%) 47 (63%)
66 (88%) 58 (77%)*
67 (89%) 56 (75%)
70 (93%) 62 (83%)†
75 (100%) 66 (88%)
38 (61%)‡
45 (73%)
43 (79%)
54 (89%)*
52 (85%)
57 (95%)†
61 (100%)
Cl, Nail clipping; Cu, subungual curetting; D, dermatophytes; NaOH, direct microscopy with sodium hydroxide; Pl, nail plate sample. *P < .01 when compared with curetting method. †P < .001 when compared with curetting method. ‡As a percentage of the nails with dermatophytes.
and possible interfering substances or microorganisms.1,2 There is little available literature evaluating which routine sampling method is the most reliable. We compared three practical methods of nail sampling: clipping of the free end of the nail plate, nail plate sampling by means of a No. 15 surgical blade, and with the use of a 1-mm curette to obtain subungual debris. PATIENTS AND METHODS The study included 75 patients with toenail onychomycosis. Two centers were involved in the study. The inclusion criteria were a previous positive microscopy or culture; exclusion criteria were the use of antifungal treatment, either topical or systemic, within the preceding 3 months. A single infected toenail was selected and all 3 methods of sampling applied to that nail. The methods used were (1) nail clipping—the nail plate was clipped off as far proximally as possible with standard nail clippers; (2) subungual material was curetted from below the nail plate by means of a sterile 1-mm curette; and (3) a sterile, new, No. 15 scalpel blade was used to pare down the nail plate overlying the discolored nail. Outer and uninvolved shavings were discarded, and the sample was only collected when it was apparent that the shavings were from the diseased portion of the nail plate. The nail specimens were all processed in the same reference laboratory. A comparison between the 3 sampling methods was made by means of McNemar test.3 RESULTS
Seventy-five patients (55 male and 20 female, average age 56 years, range 17 to 91 years) were entered into this study. Dermatophytes were isolated from 61 nails (81%) and in 54 (72%) were the only organisms cultured. The yield of fungal organisms from the 3 sampling methods and combinations of methods are
shown in Table I. Positive cultures were obtained from 56% of the nail clippings, 65% of subungual samples obtained by curetting, and 63% of the nail plate samplings. These differences are not statistically significant. However, the combination of subungual curetting with any other sampling method yields a significantly higher yield of fungi than any single sampling method. Thus, when the results of clippings are combined with curettage (a total of 58 positive cultures) and compared with curettage samplings alone (49 positive cultures) the result is significant (χ2 = 8.1 and P < .01). When dermatophyte infections are considered separately, there is no significant difference among the sampling methods, but a combination of two methods that includes subungual curetting again yielded a significantly larger number of positive cultures. DISCUSSION
In the most common form of onychomycosis, the distal and lateral subungual type, the fungal organism gains entry to the subungual region by invading through the hyponychium or lateral nail sulci. The fungus invades proximally in this plane and invades the ventral and middle portions of the nail plate. The dorsal nail plate is usually not invaded except when the infection has spread proximally to invade the nail matrix. Clippings taken from the distal nail plate will contain fungal elements carried distally by a combination of nail invasion and nail growth. It is also usual for these clippings to include some adherent subungual debris. Subungual curettings are expected to contain a rich harvest of invading fungi but are more difficult to perform and are best obtained with a 1-mm curette. Both the clippings and the subungual curettings may include fungi that are saprophytic or contaminants, particularly
Journal of the American Academy of Dermatology Volume 39, Number 6
if significant onycholysis exists. Fungi growing in the middle third or ventral portion of the nail plate are less likely to be contaminated with air-borne fungi. Direct sampling of the nail plate with drills and fraises has been advocated, but these methods lack easy applicability in the clinical setting and have not achieved widespread use.4-7 The method used in this study requires no special skill or instrumentation. The difficulty of obtaining positive cultures is well recognized.7 Culture failure rates in nails having a positive direct microscopy by using only clippings taken from the distal end of the nail range from 19.5% to 35.5%.8-10 Nail drilling specimens from the proximal and presumably most viable area failed to grow fungi in 14%.5 The present study has shown that none of the 3 sampling methods used was consistently superior. When only a single sampling method was used, negative cultures were found in more than 35% of the nails examined. Of practical importance is the fact that it is possible to improve significantly the yield of positive cultures by combining either a nail clipping or nail plate sampling with a curetting sample taken from beneath the nail plate. When the results of all 3 methods are combined,
Brief communications 1017 88% of the sodium hydroxide–positive nails were culture positive. These results suggest that sampling different sites in the affected nail improves culture positivity. REFERENCES 1. Zaias N. Onychomycosis. Arch Dermatol 1972;105:26374. 2. Midgley G, Moore MK, Cook JC, Phan QG. Mycology of nail disorders. J Am Acad Dermatol 1994;31:S68-S74. 3. Woolson RF. Statistical methods for analysis of biomedical data. New York: John Wiley & Sons; 1987. p. 205-9. 4. Epstein S. Examination of nails for fungi. Arch Dermatol Syphilol 1945;57:209. 5. English MP, Atkinson R. An improved method for the isolation of fungi in onychomycosis. Br J Dermatol 1973;88:237-41. 6. Blecher P, Korting HC. A new combined diagnostic approach to clinically and microscopically suspected onychomycosis unproven by culture. Mycoses 1993;36: 321-4. 7. Heikkila H. Isolation of fungi from onychomycosis-suspected nails by two methods: clipping and drilling. Mycoses 1996;39:479-82. 8. English MP, Lewis L. Ringworm in the South-West of England, 1960-1970, with special reference to onychomycosis. Br J Dermatol 1974;90:67-75. 9. Walshe MM, English M. Fungi in nails. Br J Dermatol 1966;78:198-207. 10. Gentles JC. Laboratory investigation of dermatophyte infections of nails. Sabouraudia 1971;9:149-52.
Brief communications 1015
Onychomycosis: An evaluation of three sampling methods P. R. Hull, MD, PhD(Med), FFDerm(SA), FRCPC,a A. K. Gupta, MD, MA, FRCPC,b and R. C. Summerbell, PhDc,d Saskatchewan and Ontario, Canada
From the Division of Dermatology, Department of Medicine, Royal University Hospital and the University of Saskatchewan, Saskatoona; the Division of Dermatology, Department of Medicine, Sunnybrook Health Science Centre and the University of Toronto, Ontariob; the Ontario Ministry of Health, Laboratory Services Branch, Torontoc; and the Department of Laboratory Medicine and Pathology, University of Toronto, Ontario.d Supported in part by Janssen-Ortho Inc, Canada. Reprint requests: P. R. Hull, Head, Division of Dermatology, Department of Medicine, Royal University Hospital, Saskatoon, Saskatchewan, S7N OW8, Canada. J Am Acad Dermatol 1998;39:1015-7. Copyright © 1998 by the American Academy of Dermatology, Inc. 0190-9622/98/$5.00 + 0 16/54/92154
The availability of more effective systemic antifungals has resulted in increasing numbers of patients receiving treatment for clinically suspected onychomycosis. Although positive microscopy is frequently used as an indication for starting treatment, cultural identification of the responsible fungus has important implications when treatment options are considered. Frequently, however, the fungus fails to grow in culture. Reasons advanced to explain this include the presence of nonviable fungi in the distal nail, sampling error,
Journal of the American Academy of Dermatology December 1998
1016 Brief communications
Table I. Culture results from the three sampling methods in 75 nails
NaOH n = 75 Culture n = 75 Pathogenic fungi D n = 61
Clipping
Curetting
Plate
Cl + Cu
Cl + Pl
Cu + Pl
Cl + Cu + Pl
56 (75%) 42 (56%)
61 (81%) 49 (65%)
54 (72%) 47 (63%)
66 (88%) 58 (77%)*
67 (89%) 56 (75%)
70 (93%) 62 (83%)†
75 (100%) 66 (88%)
38 (61%)‡
45 (73%)
43 (79%)
54 (89%)*
52 (85%)
57 (95%)†
61 (100%)
Cl, Nail clipping; Cu, subungual curetting; D, dermatophytes; NaOH, direct microscopy with sodium hydroxide; Pl, nail plate sample. *P < .01 when compared with curetting method. †P < .001 when compared with curetting method. ‡As a percentage of the nails with dermatophytes.
and possible interfering substances or microorganisms.1,2 There is little available literature evaluating which routine sampling method is the most reliable. We compared three practical methods of nail sampling: clipping of the free end of the nail plate, nail plate sampling by means of a No. 15 surgical blade, and with the use of a 1-mm curette to obtain subungual debris. PATIENTS AND METHODS The study included 75 patients with toenail onychomycosis. Two centers were involved in the study. The inclusion criteria were a previous positive microscopy or culture; exclusion criteria were the use of antifungal treatment, either topical or systemic, within the preceding 3 months. A single infected toenail was selected and all 3 methods of sampling applied to that nail. The methods used were (1) nail clipping—the nail plate was clipped off as far proximally as possible with standard nail clippers; (2) subungual material was curetted from below the nail plate by means of a sterile 1-mm curette; and (3) a sterile, new, No. 15 scalpel blade was used to pare down the nail plate overlying the discolored nail. Outer and uninvolved shavings were discarded, and the sample was only collected when it was apparent that the shavings were from the diseased portion of the nail plate. The nail specimens were all processed in the same reference laboratory. A comparison between the 3 sampling methods was made by means of McNemar test.3 RESULTS
Seventy-five patients (55 male and 20 female, average age 56 years, range 17 to 91 years) were entered into this study. Dermatophytes were isolated from 61 nails (81%) and in 54 (72%) were the only organisms cultured. The yield of fungal organisms from the 3 sampling methods and combinations of methods are
shown in Table I. Positive cultures were obtained from 56% of the nail clippings, 65% of subungual samples obtained by curetting, and 63% of the nail plate samplings. These differences are not statistically significant. However, the combination of subungual curetting with any other sampling method yields a significantly higher yield of fungi than any single sampling method. Thus, when the results of clippings are combined with curettage (a total of 58 positive cultures) and compared with curettage samplings alone (49 positive cultures) the result is significant (χ2 = 8.1 and P < .01). When dermatophyte infections are considered separately, there is no significant difference among the sampling methods, but a combination of two methods that includes subungual curetting again yielded a significantly larger number of positive cultures. DISCUSSION
In the most common form of onychomycosis, the distal and lateral subungual type, the fungal organism gains entry to the subungual region by invading through the hyponychium or lateral nail sulci. The fungus invades proximally in this plane and invades the ventral and middle portions of the nail plate. The dorsal nail plate is usually not invaded except when the infection has spread proximally to invade the nail matrix. Clippings taken from the distal nail plate will contain fungal elements carried distally by a combination of nail invasion and nail growth. It is also usual for these clippings to include some adherent subungual debris. Subungual curettings are expected to contain a rich harvest of invading fungi but are more difficult to perform and are best obtained with a 1-mm curette. Both the clippings and the subungual curettings may include fungi that are saprophytic or contaminants, particularly
Journal of the American Academy of Dermatology Volume 39, Number 6
if significant onycholysis exists. Fungi growing in the middle third or ventral portion of the nail plate are less likely to be contaminated with air-borne fungi. Direct sampling of the nail plate with drills and fraises has been advocated, but these methods lack easy applicability in the clinical setting and have not achieved widespread use.4-7 The method used in this study requires no special skill or instrumentation. The difficulty of obtaining positive cultures is well recognized.7 Culture failure rates in nails having a positive direct microscopy by using only clippings taken from the distal end of the nail range from 19.5% to 35.5%.8-10 Nail drilling specimens from the proximal and presumably most viable area failed to grow fungi in 14%.5 The present study has shown that none of the 3 sampling methods used was consistently superior. When only a single sampling method was used, negative cultures were found in more than 35% of the nails examined. Of practical importance is the fact that it is possible to improve significantly the yield of positive cultures by combining either a nail clipping or nail plate sampling with a curetting sample taken from beneath the nail plate. When the results of all 3 methods are combined,
Brief communications 1017 88% of the sodium hydroxide–positive nails were culture positive. These results suggest that sampling different sites in the affected nail improves culture positivity. REFERENCES 1. Zaias N. Onychomycosis. Arch Dermatol 1972;105:26374. 2. Midgley G, Moore MK, Cook JC, Phan QG. Mycology of nail disorders. J Am Acad Dermatol 1994;31:S68-S74. 3. Woolson RF. Statistical methods for analysis of biomedical data. New York: John Wiley & Sons; 1987. p. 205-9. 4. Epstein S. Examination of nails for fungi. Arch Dermatol Syphilol 1945;57:209. 5. English MP, Atkinson R. An improved method for the isolation of fungi in onychomycosis. Br J Dermatol 1973;88:237-41. 6. Blecher P, Korting HC. A new combined diagnostic approach to clinically and microscopically suspected onychomycosis unproven by culture. Mycoses 1993;36: 321-4. 7. Heikkila H. Isolation of fungi from onychomycosis-suspected nails by two methods: clipping and drilling. Mycoses 1996;39:479-82. 8. English MP, Lewis L. Ringworm in the South-West of England, 1960-1970, with special reference to onychomycosis. Br J Dermatol 1974;90:67-75. 9. Walshe MM, English M. Fungi in nails. Br J Dermatol 1966;78:198-207. 10. Gentles JC. Laboratory investigation of dermatophyte infections of nails. Sabouraudia 1971;9:149-52.