|Year : 2013 | Volume
| Issue : 2 | Page : 83-86
In vitro susceptibilities of fungal isolates against amphotericin B and voriconazole in Aspergillus keratitis: A comparative study
Niranjan Nayak1, Gita Satpathy1, Sujata Prasad1, Ravinder Mohan Pandey2, Namrata Sharma3, Bhavna Chawla3, Jeewan S Tityal3, Radhika Tandon3
1 Dr Rajendra Prasad Centre for Ophthalmic Sciences, Ocular Microbiology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India
3 Cornea and Refractive Surgery, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||01-Oct-2012|
|Date of Acceptance||13-Feb-2013|
|Date of Web Publication||20-May-2013|
Professor of Ocular Microbiology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
Objectives: To study the susceptibility patterns of clinical isolates of Aspergillus species to amphotericin B and voriconazole. Materials and Methods: Fifty isolates of Aspergillus species (21 A. flavus, 14 A. fumigatus and 15 A. niger) from keratitis cases were tested for their susceptibilities to amphotericin B and voriconazole by a broth microdilution method, standardized in our laboratory. The relative MIC 50 units were calculated from the absolute MIC 50 (μg/ml) upon topical prescription dose in gm/100 ml. Results: The MIC values of amphotericin B for A. fumigatus and A. niger ranged between 0.2 mg/L to 6.25 mg/L (MIC 50; 1.56 mg/L and MIC 90; 6.25 mg/L) and for A. flavus between 0.78-6.25 mg/L (MIC 50 and MIC 90; 6.25 mg/L). MICs of voriconazole for A. fumigatus were in the ranges of 0.1 to 0.2 mg/L, for A. niger 0.05 to 0.2 mg/L (MIC 50 and MIC 90; 0.2 mg/L for both fungi) and against A. flavus 0.05 to 0.39 mg/L (MIC 50 ; 0.2 mg/L and MIC 90 ; 0.39 mg/L). The relative MIC 50 of voriconazole against all the three Aspergillus species was 0.002, compared to those of amphotericin B which were 0.104 against A. fumigatus and A. niger and 0.416 against A. flavus. Conclusion: Voriconazole had lower MIC values and relative MICs than amphotericin B against Aspergillus species, the commonest fungi causing keratitis in this part of the country. Combined with its reported higher bio-availability, it appears far superior to amphotericin B for the treatment of fungal keratitis.
Keywords: Antifungal agents, amphotericin B, MIC, sensitivity testing, voriconazole
|How to cite this article:|
Nayak N, Satpathy G, Prasad S, Pandey RM, Sharma N, Chawla B, Tityal JS, Tandon R. In vitro susceptibilities of fungal isolates against amphotericin B and voriconazole in Aspergillus keratitis: A comparative study. J Clin Ophthalmol Res 2013;1:83-6
|How to cite this URL:|
Nayak N, Satpathy G, Prasad S, Pandey RM, Sharma N, Chawla B, Tityal JS, Tandon R. In vitro susceptibilities of fungal isolates against amphotericin B and voriconazole in Aspergillus keratitis: A comparative study. J Clin Ophthalmol Res [serial online] 2013 [cited 2019 Oct 22];1:83-6. Available from: http://www.jcor.in/text.asp?2013/1/2/83/112175
Infective keratitis of fungal origin represents around 35-40% of all culture proven cases.  Corneal opacity leading to blindness is one of the serious complications of this condition and represents a major public health problem in India. , The choice of the antifungal agents to manage such cases is often difficult in view of the increasingly developing resistance amongst the pathogenic fungi to one or more antifungal drugs. , Besides, many Aspergillus species are intrinsically resistant to polyenes such as amphotericin B.  In addition, lack of proper standards of antifungal drug susceptibility testing in many laboratories, especially for filamentous fungi, has led to the situation that the treatment of fungal corneal ulcer, even today, is still most commonly empirical. ,,,
Thus, optimization of antifungal therapy is of great concern amongst the microbiologists and the clinicians and in vitro susceptibility testing has been found to be an important tool in resolving this issue. Voriconazole, a triazole antifungal, was recently documented to have had potent in vitro activity against clinical yeast isolates. 
Thus, considering the limitations of the many commonly used antifungals in ophthalmic practice as mentioned above, ,,,, we planned this study to focus on the in vitro susceptibility testing for determining and comparing the MICs of amphotericin B and voriconazole, the commonly used topical antifungals in our setup, towards the clinical isolates of Aspergillus species in patients with keratitis.
| Materials and Methods|| |
This is a retrospective study in which we included fifty clinical isolates of Aspergillus species from equal number of eyes of 50 patients, who reported to the out patients department of our center between the years 2008 to 2010 and were diagnosed as cases of infectious keratitis. The Institute ethics committee clearance waiver was obtained in this retrospective analysis. The isolates comprised of 21 species of Aspergillus flavus, 14 of A. fumigatus and 15 A. niger. The organisms were identified by the recommended procedures  and were stored at -20 o C on Sabouraud's Dextrose Agar (SDA) slants until used.
There were 28 males and 22 females. The mean age at presentation was 43.6 ± 17.4 years (range 14-68 years). The right eye was affected in 29 cases and the left in 21 cases. None of the subjects had bilateral keratitis. The most common presenting symptoms included redness (96% cases) and diminution of vision (88% cases). A definite history of trauma was noted in 34 (64%) cases.
The method adopted for inoculum preparation was that described elsewhere  with some alterations. Prior to the study, fungi were sub-cultured onto fresh SDA slants, which were incubated at 25 o C for 3-5 days or until such time as there was confluent mycelial growth. The inoculum was prepared by flooding the surface of the agar slant with sterile distilled water and scraping the sporulated aerial mycelia with a sterilized loop. The suspension, thus, obtained was filtered through sterile gauze folded three times in order to remove hyphae as far as possible. The filtrate was then allowed to settle down and the supernatant was collected. The number of conidia in the suspension was counted by a Neubauer chamber and diluted in 2x yeast nitrogen glucose broth (YNGB), (Difco Laboratories, and 5% glucose) to produce four inoculum sizes in serial tenfold dilutions starting from 10 6 spore forming units (SFU) / ml to 10 3 SFU/ml.
All the isolates were tested against amphotericin B and voriconazole. The drugs were diluted in dimethyl sulfoxide (DMSO) to give doubling dilutions in the range of 100 mg - 0.05 mg/L.
For susceptibility testing, the method described earlier  was followed with minor modifications. Briefly, 100 μl portions of each drug concentration were added to the wells of a microtiter plate. Then 100 μl of spore suspension of each concentration were added to all the 12 wells of the respective rows for each spore inoculum size. Thus, the final antibiotic concentration ranged between 50 mg - 0.025 mg/L and the final inoculum sizes were between 5 × 10 5 SFU/ml to 5 × 10 2 SFU/ml. Control wells constituted (a) growth control i.e. 100 μl of 2 × YNGB and 100 μl of each spore concentration (b) solvent control i.e. 100 μl of the solvent for the drug and 100 μl inoculum of each concentration to see if the diluent used for the drug had any inhibitory effect on fungal growth and (c) a negative control i.e. 200μl of only the spore diluent i.e. YNGB. Observations were noted, when there was visible growth in the growth control wells. The lowest concentration of the drug that prevented visible growth was considered as the MIC. Candida parapsilosis ATCC 90018 reference strain was tested simultaneously with each set of experiment as control.
The tests were repeated at least thrice to check the reproducibility. Since our method showed very good reproducibility; for all our subsequent studies such as the statistical evaluation of the MIC 50 and MIC 90 values, we used the data obtained against the inoculum size of 5 × 10 4 CFU/ml, which is supposed to be the ideal sample size to give optimum reproducible results for filamentous fungi like Aspergillus, as recommended by CLSI. , MIC 90 and MIC 50 values of the drug were calculated for all ocular isolates, using the software STATA 10.0.
The relative MIC 50 was calculated as the percentage of the prescription dose of the topical antifungal agent (in mg/L) taking the absolute MIC 50 as the numerator. 
Paired t-test was employed for comparison of the geometric mean MIC s of amphotericin B and voriconazole against all the three Aspergillus species.
| Results|| |
The MIC values of amphotericin B ranged between 0.2 mg/L to 6.25 mg/L both for A. fumigatus and A. niger and between 0.78 - 6.25 mg/L for A. flavus. In contrast to this the MICs of voriconazole for A. fumigatus were in the range of 0.1 - 0.2 mg/L; for A. niger , 0.05 - 0.2 mg/L; and for A. flavus, 0.05 - 0.39 mg/L [Table 1]. The geometric mean MICs of amphotericin B for all the three Aspergillus species were higher than those recorded for voriconazole and these differences were found to be statistically significant [Table 1]. It was interesting to note that voriconazole was inhibitory against A. fumigatus and A. niger at a concentration 1/10 the concentration of amphotericin B and against the A. flavus at <1/20 the concentration of amphotericin B as evidenced by the Geometric Mean MICs of both the drugs [Table 1].
|Table 1: Antifungal activities of amphotericin B and voriconazole against ocular isolates of Aspergillus species|
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As depicted in [Table 2], the MIC 50 and MIC 90 values of voriconazole were remarkably low as compared to those noted for amphotericin B against all the Aspergillus species.
|Table 2: MIC50s and MIC90s of amphotericin B and voriconazole against ocular fungal isolates|
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The relative MIC 50 of voriconazole against all three Aspergillus species was 0.002, which was much lower than those of amphotericin B (0.104 both for A. fumigatus and A. niger and 0.416 for A. flavus [Figure 1].
|Figure 1: Relative MICs of voriconazole vs. amphotericin B against all three Aspergillus species, AF = Aspergillus flavus, A FU = Aspergillus fumigatus, AN = Aspergillus niger|
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| Discussion|| |
We studied the in vitro efficacy of voriconazole and amphotericin B against isolates from fungal keratitis using a microbroth dilution test. Very little information is available on the activity of voriconazole against Aspergilli in systemic fungal infections, and more so in fungal keratitis. In one of the earlier studies,  it was shown that voriconazole had the lowest MICs for Aspergillus species isolated from keratitis and endophthalmitis and that voriconazole and amphotericin B had the lowest MICs against Fusarium species. In a large series involving isolates from systemic diseases, Johnson et al., documented that voriconazole and amphotericin B were more effective than itraconazole against Fusarium species, and voriconazole was found to be the most effective agent against Aspergillus species. The above mentioned studies , also documented that no single agent was universally most effective. However, voriconazole had superiority over the rest of the antifungals.
The findings in our study were in good agreement with those reported earlier. ,, We showed that voriconazole was much more effective in vitro against all three Aspergillus species compared to amphotericin B, which is often used in our referral hospital as a topical antifungal empirically in clinically suspected cases of fungal keratitis. In a similar study  the researchers compared the in vitro efficacies of voriconazole and amphotericin B against Aspergillus and Fusarium species and found that voriconazole was far superior to amphotericin B. Our data on the geometric mean MICs and MIC ranges of amphotericin B were comparable to those noted by others.  However, as mentioned above, while comparing the efficacies of both the drugs, we noticed the superiority of voriconazole over amphotericin B, not only with respect to the geometric mean MICs and the MIC ranges, but also in terms of the MIC 50 and MIC 90 values. This is in total agreement with the findings documented elsewhere. , In yet another similar study, Suttan et al., demonstrated that the geometric mean MIC of amphotericin B towards clinical isolates of A terreus was 15 times higher as compared to that of voriconazole. Contrary to this, it was observed that non-A. fumigatus Aspergilli such as A. terreus, A. niger and A. flavus showed higher MICs towards both amphotericin B and voriconazole , and A. lentulus exhibited reduced susceptibilities not only towards amphotericin B, but also to many azoles and echinocandins. ,
Though the MIC 50 values in our study were comparable to those observed by others,  the high MIC 90s amongst our isolates towards amphotericin B is of great concern. This might have been due to the prolonged empirical administration of the drug, long before referring the patients to a tertiary care hospital like ours. Nevertheless, high MIC 90 values amongst the isolates in the present study would mean that majority i.e. 90% of the organisms would respond to very high dosage of antifungals so as to produce the tissue concentration at the site of infection equivalent to the MIC 90 . In addition, the remaining 10% would not even respond to that high dosage regimen. 
Therefore, an index of suspicion should always be there for the isolates of Aspergilli from cases of infectious keratitis regarding their resistance to amphotericin B. In such a situation, therapeutic modalities may need to be changed, keeping in view the higher and broad spectrum antifungal values of voriconazole. The superiority of voriconazole over amphotericin B is further substantiated by our observations on the relative MIC data, which clearly indicated that voriconazole had much lower relative MICs (0.002 for all three Aspergillus species) than amphotericin B (0.416 for A. flavus and 0.104 each for A. niger and A. fumigatus). In other words, even if only a minimal 0.002 percent of the topical voriconazole preparation was retained in the cornea after the instillation of the scheduled prescribed drug, it would be effective, as opposed to a much higher proportion of retention (0.416 percent or 0.104 percent) in case of amphotericin B. The higher the relative MIC, a comparatively larger proportion of drug retention would be needed to produce the desired effect suggesting thereby that the agent having the lower relative MIC would be more effective than that with higher relative MIC. Moreover, preliminary pharmacokinetic data on experimental animals indicated that after a single oral dose of voriconazole (4 mg/kg), the mean ± SD voriconazole concentration in aqueous humor was 0.86 ± 0.22 mg/L. At the same time, the mean ± SD voriconazole concentration in aqueous humor after topical administration of 1.0 % solution was 2.35 ± 0.78 mg/L and the drug was detected in the plasma for one hour after the final topically administered dose without any signs of ocular toxicity.  Our data showed that the MIC 90s and MIC 50s of voriconazole were much below the above mentioned pharmacokinetic values [Table 2]. Although interpretative breakpoints for the non-A. fumigatus Aspergillus have not yet been elucidated clearly, Verweij et al, 18] applying EUCAST susceptibility testing, proposed the following breakpoints for voriconazole against A. fumigatus: <2 mg/L (susceptible), 2 mg/L (intermediate susceptible) and >2 mg/L (resistant). The authors even put forth the view that additional work would be required to confirm these breakpoints, including in vivo and clinical responses. 
In notwithstanding the above lapses in our knowledge, it is universally agreeable that amphotericin B has widely been used as a topical and systemic drug for ocular infections. Preparation of a 0.15% suspension of this drug, reconstituted from a 50 mg vial powder (for IV formulation) is universally adopted for topical use for candida keratitis, as well as for keratitis due to mycelial fungi. After topical application, this drug can penetrate deep into the corneal stroma and 0.15% suspension is well tolerated, when instilled round the clock every two hourly.  Once healing starts, the dosage schedule can be changed to four times a day for a minimum period of two weeks.
Voriconazole, on the other hand, has a broad spectrum of activity against fungi causing keratitis. In a recent study,  in vitro susceptibility of various fungal isolates in infectious keratitis towards voriconazole was 100%, whereas towards other azoles it ranged in between 60% to 82.4%. It was also noted that voriconazole MIC 90 for Candida species was as low as 0.016 μg/ml. This drug, therefore, was advocated to be a better alternative for therapeutic management of Candida and Aspergillus ocular infections, as compared to other antifungals. For topical application, a one percent suspension is made from the commercially available powder form and is instilled every two hourly for the first 48 h and thereafter every four hourly till the ulcer heals.
Thus, to conclude with, our observations certainly emphasize that voriconazole can be used with promising therapeutic value in fungal keratitis due to Aspergillus species, which are the commonest etiological agents of fungal keratitis in this part of our country.
| Acknowledgement|| |
The work was supported by internal funding from the Center. No financial implication from any other government agency or granting body is involved.
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[Table 1], [Table 2]