Assessing the incubation time in antifungal susceptibility testing using the agar-based disk diffusion method

Abstract

Background

The use of agar-based disk diffusion (ABDD) for susceptibility testing of fungal pathogens is well-established. However, the incubation time may affect the result. Therefore, this study aims to determine the optimal incubation period for assessing the inhibition zone against Trichophyton spp., one of the most common pathogens among patients with dermatophytosis.

Methods

This in-vitro study involved 129 cryopreserved strains of Trichophyton spp., including T. rubrum complex (67 strains) and T. mentagrophytes complex (62 strains). The strains were recovered in Sabouraud’s dextrose agar, and conidia were harvested after 5 to 7 days of culture. The ABDD method was performed in Müeller-Hinton agar with five antifungals: itraconazole (ITC), griseofulvin (GRI), clotrimazole (CLO), miconazole (MCL), and voriconazole (VOR). The inhibition diameter zone (IZD) was measured consecutively on days 3, 5, and 7 by the caliper. Data analysis was performed by SPSS 25.

Results

The mean IZD of all antifungals to T. rubrum was highest on day 5. The results were similar in T. mentagrophytes, except VOR presented the highest IZD on day 3. We noticed significant differences in the IZD of ITC, CLO, MCL, and VOR of T. rubrum. In T. mentagrophytes, only VOR showed a marked difference between periods.

Conclusion

The study’s findings recommend the incubation time in dermatophyte susceptibility testing. Specifically, the incubation time should be 5 days for ITC, GRI, CLO, and MCL for both T. rubrum and T. mentagrophytes. Regarding VOR, it should be 5 days for T. rubrum and 3 days for T. mentagrophytes.

Introduction

Trichophyton spp. is one of the three main genera causing cutaneous mycoses with high prevalence.^1–3 Antifungal resistance rate is rising globally and becoming a concern in the clinical and mycological field.^1,4 Antifungal susceptibility testing of dermatophytes has been performed drastically, and the agar-based disk diffusion (ABDD) method is simple, applicable, and interpretable, leading to its wide use.^5,6 The Clinical and Laboratory Standard Institute (CLSI) guidelines have not mentioned a standard dermatophyte protocol for the ABDD method.

Several factors strongly affect the interpretation of the ABDD result, ⁷ including the incubation time. Regularly, the appropriate incubation time in the ABDD method is when the diameter of the inhibition zone presents the most visible margin^6,8 Many CLSI guidelines have proposed proper incubation times for bacteria, yeasts, and non-dermatophyte molds.^9,10 Prolonged incubation time leads to a higher rate of false resistance, which has been observed in bacteria. ¹¹ The issue is likely to happen in yeast and filamentous fungi. Hence, for dermatophytes, there needs to be a recommendation for the incubation time in the ABDD method. There are two concurrent phenomena: in-vitro degradation of antifungals ¹² and dermatophyte growth. ¹³ The two phenomena could lead to an objective error while interpreting the ABDD result. Many papers have published the IZD results of dermatophytes with inconsistent incubation times.^7,14–19 In the context of no specific guideline, the proper incubation time of the ABDD method should be determined, depending on dermatophyte complexes and antifungals. Therefore, the study aims to assess the optimal incubation time of Trichophyton spp. to perform antifungal susceptibility testing.

Methods

Study design

The in-vitro study involved 129 strains of Trichophyton spp., including 67 strains of T. rubrum complex and 62 strains of T. mentagrophytes complex. All strains were cryopreserved in glycerol 50% (v/v) at −20°C at the Department of Microbiology and Parasitology, School of Medicine, Vietnam National University Ho Chi Minh City. The main exclusion criterion was contaminated strains during recovery;

Laboratory procedures

The study of 129 strains assessed the antifungal susceptibility by the agar-based disk diffusion method according to the standardized protocol of Nweze E. I. et al. (2010). ⁷ The antifungals used were itraconazole (ITC) 8 µg, griseofulvin (GRI) 10 µg, clotrimazole (CLO) 50 µg, miconazole (MCL) 10 µg and voriconazole (VOR) 1 µg; all the antifungal disks were from Liofilchem®, Italy.

The strains were recovered on potato dextrose agar to harvest conidia, following the CLSI M38-A2 guideline. ²⁰ After 5 to 7 days of incubation, a sterile cotton swab wetted in a sterile solution of 0.9% saline and Tween 20 was used to gently scrape the surface of the colony. Then, we formed conidia suspension by stirring the swab in saline and adjusting the conidia density at 10⁶ – 5.10⁶ conidia/milliliter by a hemocytometer. ²¹

The suspension was spread on a Müeller-Hinton agar plate (4 millimeters thick ²² ) and let dry for 1-2 minutes. Antifungal disks were then placed on the surface: for each strain, two Müeller-Hinton agar plates were used to test five antifungals (ITC and GRI on one plate, CLO, MCL, and VOR on the other). The plates were invertedly incubated at 30°C.

On days 3, 5, and 7, the largest IZD of every antifungal of every strain was measured. The IZD was defined as the largest diameter of non-growing fungus in the area around the antifungal disc; pinpoint microcolonies at the zone edge were ignored.

Statistical analysis

Data were analyzed by SPSS 25 and presented as mean ± standard deviation. Differences of IZD by three periods were tested based on the null hypothesis (H₀) as no difference; Bayes factor (BF) was used to interpret the level of hypothesis acceptance (Table 1):

Table 1.

Bayes factor’s interpretation.

Bayes factor	Interpretation
>100	Decisive evidence for hypothesis Ha
30 to 100	Very strong evidence for hypothesis Ha
10 to 30	Strong evidence for hypothesis Ha
3 to 10	Substantial evidence for hypothesis Ha
1 to 3	Not worth more than a bare mention
1/3 to 1	Not worth more than a bare mention
1/10 to 1/3	Substantial evidence for hypothesis H0
1/30 to 1/10	Strong evidence for hypothesis H0
1/100 to 1/30	Very strong evidence for hypothesis H0
<1/100	Decisive evidence for hypothesis H0

H_a: null hypothesis; H₀: alternative hypothesis.

Results

The highest mean IZD yielded in 67 strains of T. rubrum complex after 5 days of incubation Figure 1:

Figure 1.

Mean inhibition zone diameter of Trichophyton rubrum complex at three incubation times (days 3, 5, and 7).

In 62 strains of T. mentagrophytes complex, ITC, GRI, CLO, and MCL yielded the highest mean IZDs on day 5, except VOR reached the highest mean IZD on day 3 Figure 2:

Figure 2.

Mean inhibition zone diameter of Trichophyton mentagrophytes complex at three incubation times (days 3, 5, and 7).

In the T. rubrum complex, GRI was the only case that expressed no statistical difference in the IZDs by time. Other tested antifungals showed significant differences in IZDs by periods (from “strong evidence” to “decisive evidence”). Regarding T. mentagrophytes complex, IZDs by VOR significantly differed among periods (BF = 105.7; decisive evidence). Other antifungals showed no differences in IZD (Table 2).

Table 2.

Comparison of the inhibition zone diameter of five antifungals between three reading times.

Strains (N = 129)	Antifungal agent	Incubation time	Mean ± SD (mm)	Range (mm)<	Bayes factor
Trichophyton rubrum complex (n = 67)	ITC	3rd	37.84 ± 6.69	31.12 - 44.15	12.83
		5th	38.4 ± 8.21	30.12 - 46.19
		7th	36.88 ± 8.25	28.62 - 45.63
	GRI	3rd	26.49 ± 8.21	18.22 - 34.28	2.53
		5th	27.55 ± 8.84	18.72 - 36.71
		7th	26.02 ± 8.61	17.42 - 34.41
	CLO	3rd	39.21 ± 10.37	28.82 - 49.84	35.05
		5th	40.9 ± 10.03	30.82 - 50.87
		7th	39.45 ± 10.79	28.62 - 50.66
	MCL	3rd	23.97 ± 6.3	17.62 - 30.67	43.58
		5th	24.28 ± 6.22	18.02 - 30.06
		7th	22.63 ± 6.25	16.32 - 28.38
	VOR	3rd	43.57 ± 13.12	30.42 - 56.45	233.4
		5th	44.21 ± 13.51	30.72 - 57.7
		7th	41.22 ± 13.51	27.72 - 54.71
Trichophyton mentagrophytes complex (n = 62)	ITC	3rd	35.15 ± 4.5	30.62 - 39.65	1.63
		5th	35.34 ± 6.84	28.52 - 42.5
		7th	33.58 ± 8.05	25.52 - 41.53
	GRI	3rd	26.29 ± 5.69	20.62 - 31.6	0.37
		5th	26.45 ± 5.95	20.52 - 32.5
		7th	25.03 ± 5.73	19.32 - 30.3
	CLO	3rd	34.11 ± 9.69	24.42 - 43.42	0.09
		5th	34.47 ± 10	24.42 - 44.47
		7th	33.66 ± 9.31	24.32 - 42.35
	MCL	3rd	18.71 ± 4.82	13.82 - 23.89	1.22
		5th	20.08 ± 4.94	15.12 - 25.14
		7th	19.32 ± 5.12	14.22 - 24.2
	VOR	3rd	34.77 ± 14.35	20.42 - 49.42	105.7
		5th	32.52 ± 16.06	16.42 - 48.46
		7th	30.03 ± 14.75	15.22 - 44.28

SD: Standard deviation.

Discussion

In the present study, the dermatophytes belonging to the genus Trichophyton were investigated due to the difference in Trichophyton conidia compared to other genera. Trichophyton produces predominantly microconidia, while other genera form large amount of macroconidia. ²² The use of macroconidia to test susceptibility is still in debate. B. Fernández-Torres et al. (2003) recorded different susceptibility patterns when performing on conidia suspension compared to mycelial suspension. ²³ The results indicated the impact of conidia wall thickness on antifungal susceptibility; from these, the susceptibility patterns between microconidia and macroconidia suspensions might differ. ²³ Therefore, to ensure the consistency of the results in this study, we assessed the susceptibility patterns on microconidia suspension of two commonest clinical complexes: T. rubrum and T. mentagrophytes.

The incubation time recorded in the study was day 3, day 5, and day 7. Previous studies preferred using one of three periods to read the antifungal susceptibility results. J. Singh et al. (2007) measured the IZD from day 4 to day 7 without any specific indication for antifungals or dermatophyte species. ²⁴ E. I. Nweze et al. (2010) proposed that the optimal incubation time was 7 days. ⁷ However, there was no explanation for each specific incubation time.

A study by A. B. Alió et al. (2005) has demonstrated that after 192 hours (approximately 8 days), the growth rate of dermatophytes increased exponentially in broth microdilution assay. ¹³ Other findings from the study were that T. rubrum required at least 105.96 hours to reach a biomass of 0.00,654 grams, while T. mentagrophytes needed 81.62 hours to form 0.01 grams of biomass. ¹³ These findings seemed to be under what we observed: after 5 days of incubation, T. rubrum and T. mentagrophytes formed enough colonies to cover the agar surface, which were enough to detect the IZD of most antifungals, compared to day 3. On day 7, we noted the reduction of IZD, possibly because of a tremendous increase in dermatophyte mycelium. Previously, the same phenomenon has been observed by M. E. Mulligan et al. (1987) on Staphylococcus aureus antimicrobial susceptibility testing. ¹¹ The phenomenon also occurs in fungi, so the optimal incubation time is needed to interpret the results as accurately as possible. In terms of VOR, we noticed the highest mean IZDs of dermatophyte strains after 3 days of incubation, which is consistent with the results of C. C. Méndez et al. (2008) ¹⁹ ; the IZDs tended to decrease gradually following the incubation time. However, we have not yet found an explanation for VOR.

When we compared the IZDs of T. rubrum complex on different periods, we observed that the IZDs caused by ITC, CLO, MCL, and VOR were significantly higher on day 5 (BF >10), except for GRI, which showed no statistical difference. This result led us to a conclusion: when performing ABDD, the antifungal susceptibility of ITC, CLO, MCL, and VOR to T. rubrum complex SHOULD be interpreted on day 5, and the IZD of GRI could be measured from day 3 to 7. In the T. mentagrophytes complex, only VOR showed a significantly higher IZD on day 3 (BF = 105.7); other antifungals showed no difference in IZDs from day 3 to 7, with day 5 being the highest. These results were evident in an optimal incubation time in ABDD. Our results address the importance of interpreting AFST by disk diffusion method: proper incubation time should be an issue to obtain accurate results. The results also serve as fundamental values for a standard protocol of AFST.

As with other studies, we focused solely on the incubation time of the ABDD method, leaving many other factors aside. Another limitation is that we have yet to investigate other genera of dermatophytes (Microsporum and Epidermophyton) due to macroconidia-forming features (thicker cell wall may lead to low penetration of the drug) and their relatively low frequencies. Other upcoming studies could resolve these limitations and standardize the procedure of dermatophytes antifungal susceptibility testing by using the agar method.

Conclusions

The results proposed that the Trichophyton susceptibility patterns to five antifungals (ITC, GRI, CLO, MCL, and VOR) by the ABDD method should be interpreted on day 5, except for the IZD of VOR should be measured on day 3 in the case of T. mentagrophytes complex. These findings could serve as one of the first steps to standardize the antifungal susceptibility testing of dermatophytes by the ABDD method.