A Closer Look at Onychomycosis and Terbinafine Resistance

Onychomycosis is one of the most common medical conditions encountered by podiatrists and, in our experience, the treatment of fungal nail infections often takes 12 months or longer, regardless of if oral antifungals, topical antifungals, or a combination of different treatments are utilized. We have observed patients and podiatrists frequently expressing frustration with toenail pathology, as determining the exact cause and specific treatments for the successful eradication of the fungal infection for each patient can be challenging. We believe the recent emergence of terbinafine-resistant dermatophytes as an etiologic agent in onychomycosis warrants clinical vigilance. Patients presenting with onychomycosis, even if diagnosed and treated appropriately with evidence-based medicine, may fail to respond to traditional prescription medicines commonly used to treat fungal nails.

The vast majority of superficial mycosis cases, both cutaneous and involving the nail unit, result from infection by members of the Trichophyton genera, with Trichophyton rubrum being the primary infectious organism, followed by members of the T mentagrophytes complex. The treatment of choice for onychomycosis has traditionally been oral terbinafine as this provides the highest rates of mycological and clinical cure rates.¹

However, terbinafine resistance in clinical patient samples has been identified in several studies and mycology laboratories and is on the rise worldwide.^4–6 In our estimation, this resistance is likely a product of the broad and often inappropriate use of antifungal agents, whether prescription or over-the-counter, resulting in selection pressure for the emergence of resistant strains.

The fungicidal activity of terbinafine is via the inhibition of squalene epoxidase (SE), leading to interference in the production of an essential cell membrane component, ergosterol, and an accumulation of squalene, which is toxic to the fungus.^2–8 Mutations, primarily point mutations, in the SE gene result in an elevation of the minimum inhibitory concentration (MIC) for both T rubrum and T interdigitale (T mentagrophytes complex) with the degree of elevation in the MIC being dependent on the specific single nucleotide polymorphism (SNP) present.⁶

Although terbinafine resistance associated with SNPs in the SE gene has been identified worldwide, the high rates of occurrence in particular regions have made it a recognized public health issue, whereas in those regions where rates are low, the issue has yet to gain the attention necessary. At this time, India shows some of the highest rates of terbinafine resistance reported in patients with onychomycosis.⁹ However, with patterns of world migration and increased travel, there is no reason to believe this is, or will remain, an isolated phenomenon.

Insights on Onychomycosis Tests

When a patient presents to a podiatrist with signs and symptoms of onychomycosis, a sample of nail and subungual debris of the affected nail submitted to a diagnostic laboratory can objectively determine if there is an infectious (dermatophyte, saprophyte, yeast, bacteria, etc.) or a noninfectious (microtrauma, psoriasis, neoplasm, lichen planus, etc.) etiology causing the nail deformity. It is critical that podiatrists sample the most proximal area of the affected nail and subungual debris where the highest probability of capturing the infectious organism lies, as submitting distal nail clippings alone will result in misleading pathology reports.¹⁰ Additionally, podiatrists who experience a perceived unexpected low positivity for onychomycosis from their patient samples sent to a laboratory may benefit from review of patient sample collection protocol for onychomycosis to assure best practices in tissue collection.

Histochemical stains such as periodic acid-Schiff (PAS), Gomori methenamine silver (GMS) and Fontana-Masson (FM) are sensitive tests designed to detect fungi and melanin and allow for a diagnosis of non-infectious nail dystrophy like microtrauma, but do not provide speciation of an infectious organism. Molecular diagnostic testing such as polymerase chain reaction (PCR assay) analysis is a specific DNA test designed to identify the genus and species of causative organisms in cases of nail dystrophy due to fungal infections. In our experience, combination testing (PAS, GMS, FM, PCR) provides the most comprehensive evaluation of nail unit dystrophy, incorporates both the highest sensitivity and highest specificity, and enables precise targeted therapy for the underlying etiology, infectious or non-infectious.

Although antibiotic sensitivity testing is routinely used to help physicians determine the most appropriate treatment for a bacterial infection, antifungal drug susceptibility testing is not routinely performed with dermatophytes due technical difficulties, required expertise, and suboptimal reproducibility.¹¹ Minimum inhibitory concentration (MIC) can occasionally be used for testing the susceptibility of fungi to antifungal drugs, as the MIC test involves growing a culture of the fungus in the presence of increasing concentrations of the antifungal drug in an attempt to determining the lowest concentration that prevents growth. MIC50 is the minimum inhibitory concentration at which 50% of the isolates tested are inhibited (susceptibility) and MIC90 is the minimum inhibitory concentration at which 90% of the isolates tested are inhibited (efficacy of the agent). However, this test method is not 100% reliable and has some limitations.¹¹

The limitations associated of antimicrobial drug sensitivity testing are compounded when dealing with fungal organisms due to the requirement of fungal growth, which in some cases may be challenging even in the presence of a viable organism and the length of time required for mycologic culture. Additionally, there is no clear correlation between clinical outcome and in-vitro dermatophytes drug susceptibility patterns and MICs, and all species of dermatophytes may not have the same pattern of drug susceptibility.¹²

A Closer Look at Innovative PCR Testing

Bako Diagnostics has recently developed a novel and specific real-time TaqMan PCR test for the detection of 12 mutations known to confer terbinafine resistance in both T rubrum and T mentagrophytes (Figure 1 and Table 1). In addition, the company aimed to establish a frequency of resistance in T rubrum and T mentagrophytes in United States onychomycosis samples for the first time. All cases of terbinafine resistance identified were further defined as to which SNP or other mutation was present within the SE gene.

The BakoDx Terbinafine (TRB) Drug Resistance (DR) mutation detection panel is a real-time PCR (rPCR) assay intended for the detection of mutations within the squalene epoxidase gene, which is involved in ergosterol biosynthesis in Trichophyton sp., the fungicidal target for terbinafine. The assay is designed to detect 12 SPNs in squalene epoxidase gene through 4 reactions.
Clinical validation is a laboratory process using control samples to test the PCR machines and is a laboratory operating protocol process to make sure the machines are accurate. This was performed using fungal cultures with antifungal sensitivity and the company’s terbinafine assay with a total of 281 samples, including 259 clinical tinea unguium nail specimens (T rubrum 89.8% and T mentagrophytes 10.2%) plus 22 terbinafine drug resistant isolates. Thirteen samples of the 259 clinical nail specimens and the 22 terbinafine drug resistant isolates were culture-positive on fresh terbinafine SDA plate. The results of this comparison study are shown in Table 2.

Performance of the company’s TRBDR assay against the fungal culture results had a sensitivity of 35/35 (100%) and specificity of 246/246 (100%). Moreover, full length squalene epoxidase (SQLE) gene Sanger sequencing was performed for all of the terbinafine culture positive clinical samples and isolates. Drug resistance mutations were detected by Sanger sequencing methodology in all the 35 samples that were positive on TRBDR assay and terbinafine culture.

Clinical testing of the company’s Terbinafine PCR Assay using 1301 US clinical nail samples confirmed as Trichophyton species by OIAD Dermatophytic Fungi Reflex Assay were tested using the BakoDx TRBDR assay. Of the 1,301 clinical samples collected in US patients, 87.2% were speciated as T rubrum and 12.8% as T mentagrophytes. Fifty-one clinical samples (3.92%) carried a mutation of conferring terbinafine resistance: 47 (4.14%) were T rubrum and 4 (2.4%) were T mentagrophytes. The distribution of the mutations is shown in Table 3.

Further Insights on Terbinafine Resistance

The difficulties and challenges in providing effective treatment strategies for onychomycosis are well known. Recalcitrant cases and treatment failures may result from mechanical and structural issues inhibiting penetration of topical therapies, lower efficacy of some antifungal therapies, and antifungal resistance. Terbinafine resistance is an emerging public health issue that may result in at least a portion of the recalcitrant cases and treatment failures noted in patients with onychomycosis.

In our experience, clinical misdiagnosis and widespread over-the-counter home treatment in many regions of the world are likely significant contributors to resistance development and propagation through inappropriate antifungal utilization. In addition, there has been speculation that combinations of antifungal medications and steroids may also play a role in resistance development.²

While traditional treatment strategies fail to consider fungal resistance, this standard may be insufficient in the current and evolving world. Due to this current treatment paradigm, fungal sensitivity testing is rarely performed and when it is, the inherent difficulty with mycologic culture can inhibit accurate results. Potential issues encountered in fungal culture may be associated with the extended incubation times required for fungal isolation and identification, as well as other issues including antifungal treatment prior to nail sampling. Traditional mycologic culture requires anywhere from 2 weeks for a positive fungal culture to 6 weeks for negative confirmation depending on the growth rate of the specific organism. During this incubation period, samples are subject to potential contamination by bacteria and/or non-dermatophytic fungi, which may be from possible sample contaminations. These contaminants may lead to overgrowth of the primary culture and thus making accurate interpretation impossible, a contributing factor in the lower sensitivity observed in fungal culture.¹³

Terbinafine resistance associated with point mutations in the SE gene are primarily isolated to SNPs at 4 amino acid loci: Leu393, Phe397, Phe415 and His440.2-8 The degree of resistance is directly related to the amino acid substitution present, as evidenced by differences in MICs present in different isolates possessing alternate substitutions.⁶ The patient data we collected confirms the presence of terbinafine resistance associated SNPs within currently submitted clinical samples at an overall rate of 3.92% (T rubrum, 4.14% and T mentagrophytes, 2.4%). Traditional mycologic culture using terbinafine infused agar was utilized to further support these findings. Comparison of these samples demonstrated full concordance between the culture positive resistant isolates and PCR-identified resistance-associated SNPs. No false positives were identified. An additional difficulty which may be encountered posing an obstacle for traditional culture is the potential for mixed mutant/wild type cultures. Given the lower sensitivity of traditional mycologic culture, it is unlikely that both genetic variants would be identified, especially if the mutant form comprises a minority constituent. Our experience has shown that this issue is essentially nonexistent with the use of molecular analysis. Although the company designed a very sensitive method for the detection of resistance associated point mutations within the SE gene, other mechanisms of resistance may also be emerging, and continued monitoring of this issue is essential.

While approximately 4% of clinical onychomycosis samples showing T rubrum and T mentagrophytes harbor the point mutations in the SE gene that confer terbinafine resistance in the current data set, other parts of the world have shown significantly higher degrees of resistance.^4–6 Given the fact that the current level of resistance is likely an underestimate because of the high rate of empiric therapy without laboratory confirmation and the lack of fungal sensitivity testing in general, we believe it is reasonable to assume that this rate will only escalate. Due to the relatively limited options available for antifungal agents, it is recommended that cases of clinically suspected onychomycosis have laboratory confirmation prior to the initiation of oral and/or topical therapy. In addition, when the infectious agent is confirmed to be dermatophytic, T rubrum and T mentagrophytes complex, molecular identification of potential resistance associated gene mutations would be prudent.

There are several strategies that can be used to address the problem of treating terbinafine resistance in onychomycosis. One strategy is to prescribe alternative antifungal medications, such as itraconazole (200mg daily) or off-label fluconazole (150–300mg once weekly). These medications have a different mechanism of action than terbinafine and may be effective in patients who have developed resistance to terbinafine. Additionally, topical antifungal treatments, such as efinaconazole 10% solution, tavaborole 5% solution, and ciclopirox 8% lacquer can be effective in mild-to-moderate cases of onychomycosis. Combination therapy consisting of oral mediations and topical medications used together are beginning to show tremendous success. However, there is a need for further study to determine the most effective protocol.

Finally, lifestyle modifications, the use of copper or silver embedded socks, ultraviolet shoe sterilizers, and regular visits to podiatry to address nail pathology along with potential undiagnosed tinea pedis will also improve patient outcomes.

Dr. William P. Scherer is board certified by the American Board of Foot and Ankle Surgery, served 17 years as an Adjunct Professor at Barry University School of Podiatric Medicine. He is the Senior Podiatric Medical Advisor to Bako Diagnostics, and is in Delray Beach, FL.

Dr. Wayne L. Bakotic is board certified in anatomic pathology, clinical pathology, and dermatopathology. He is the co-founder and chief medical officer at Bako Diagnostics, Alpharetta, GA.

References
1.    Gupta AK, Ryder JE, Chow M, Cooper EA. Dermatophytosis: the management of fungal infections. Skinmed. 2005 SepOct;4(5):305-10.
2.    Gupta AK, Renaud HJ, Quinlan EM, Shear NH, Piguet V. The growing problem of antifungal resistance in onychomycosis and other superficial mycoses. Am J Clin Dermatol. 2021 Mar;22(2):149-157.
3.    Fattahi A, Shirvani F, Ayatollahi A, et al. Multidrugresistant Trichophyton mentagrophytes genotype VIII in an Iranian family with generalized dermatophytosis: report of four cases and review of literature. Int J Dermatol. 2021 Jun;60(6):686-692.
4.    Saunte DML, Hare RK, Jørgensen KM, et al. Emerging terbinafine resistance in Trichophyton: clinical characteristics, squalene epoxidase gene mutations, and a reliable EUCAST method for detection. Antimicrob Agents Chemother. 2019 Sep 23;63(10):e01126-19.
5.    Singh A, Masih A, Khurana A, et al. High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene. Mycoses. 2018 Jul;61(7):477-484.
6.    Yamada T, Maeda M, Alshahni MM, et al. Terbinafine resistance of Trichophyton clinical isolates caused by specific point mutations in the squalene epoxidase gene. Antimicrob Agents Chemother. 2017 Jun 27;61(7):e00115-17.
7.    Ghelardi E, Celandroni F, Gueye SA, et al. Potential of ergosterol synthesis inhibitors to cause resistance or cross-resistance in Trichophyton rubrum. Antimicrob Agents Chemother. 2014 May;58(5):2825-9.
8.    Nowosielski M, Hoffmann M, Wyrwicz LS, et al. Detailed mechanism of squalene epoxidase inhibition by terbinafine. J Chem Inf Model. 2011 Feb 28;51(2):455-62.
9.    Ebert A, Monod M, Salamin K, et al. Alarming India-wide phenomenon of antifungal resistance in dermatophytes: A multicentre study. Mycoses. 2020 Jul;63(7):717-728.
10.    Leelavathi M, Tzar M. Brief report: nail sampling technique and its interpretation. Malays Fam Physician. 2011 Aug 31;6(2-3):58-9. PMID: 25606224; PMCID: PMC4170424.
11.    Dogra S, Shaw D, Rudramurthy SM. Antifungal drug susceptibility testing of dermatophytes: laboratory findings to clinical implications. Indian Dermatol Online J. 2019 May-Jun;10(3):225-233. doi: 10.4103/idoj.IDOJ_146_19. PMID: 31149563; PMCID: PMC6536077.
12.    Ghannoum MA, Arthington-Skaggs B, Chaturvedi V, et al. An interlaboratory study of quality control isolates for a broth antifungal susceptibility method for the testing of dermatophytes. J Clin Microbiol. 2006;44:4353–6.
13.    Falotico JM, Lipner SR. Updated perspectives on the diagnosis and management of onychomycosis. Clin Cosmet Investig Dermatol. 2022 Sep 15;15:1933-1957. doi: 10.2147/CCID.S362635. PMID: 36133401; PMCID: PMC9484770.