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Letter
Amphotericin B and natamycin are not synergistic in vitro against Fusarium and Aspergillus spp. isolated from keratitis
  1. Prajna Lalitha1,
  2. Brett L Shapiro2,
  3. Allison R Loh2,
  4. Annette W Fothergill3,
  5. N Venkatesh Prajna1,
  6. M Srinivasan1,
  7. Catherine E Oldenburg2,
  8. David A Quigley2,
  9. Jaya D Chidambaram2,
  10. Stephen D McLeod2,
  11. Nisha R Acharya2,
  12. Thomas M Lietman2
  1. 1Aravind Eye Care System, Madurai, India
  2. 2University of California San Francisco (UCSF), San Francisco, California, USA
  3. 3University of Texas Health Sciences Center San Antonio (UTHSCSA), San Antonio, Texas, USA
  1. Correspondence to Professor Thomas M Lietman, F.I. Proctor Foundation, Room S309, 513 Parnassus Ave, UCSF, San Francisco, CA 94143-0412, USA; tom.lietman{at}ucsf.edu

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Introduction

Infectious keratitis is a major cause of monocular blindness worldwide.1 Although fungal ulcers are relatively uncommon in developed nations, they occur frequently in developing countries, especially those in tropical regions. In South India, up to half of infectious ulcers are fungal.2 Fungal ulcers are notoriously difficult to treat compared with bacterial ulcers, and physicians frequently use combination therapy.3 Amphotericin B and natamycin are two commonly used topical agents to treat filamentous fungal keratitis.3 However, combination therapy may increase the risk of potential drug toxicity as well as the cost of therapy. Currently, there are no data on whether natamycin and amphotericin B interact synergistically in vitro against filamentous fungi.

Here, we investigate whether amphotericin B and natamycin interact synergistically in vitro when tested against Fusarium and Aspergillus spp. isolated from patients with fungal keratitis.

Methods

Ten Fusarium spp. and ten Aspergillus spp. isolates were randomly selected from 98 consecutive cases of culture-positive fungal keratitis treated at Aravind Eye Hospital in South India. All patients underwent cornea culture and were treated with natamycin, the local standard of care. Stored fungal isolates were sent to the University of Texas Health Sciences Center San Antonio (Texas, USA) for minimal inhibitory concentration (MIC) susceptibility testing to natamycin alone and amphotericin B alone using methods described previously.4 5

Drug interactions were tested using the checkerboard microdilution method. Interaction was determined by calculating the fractional inhibitory concentration index (FICI) with standard definitions: synergy FICI ≤0.5, indifference FICI >0.5 and ≤4, and antagonism FICI >4.

Results

The median MIC (MIC50) and 90th percentile (MIC90) of natamycin and amphotericin B were individually calculated for both Fusarium and Aspergillus spp. (table 1). These are comparable to previously published work.4 Drug interactions showed indifference for all 20 isolates; none of the isolates demonstrated synergy or antagonism. The geometric mean FICI was 1.02 (95% CI 1.00 to 1.05) for Fusarium spp. and 0.93 (95% CI 0.79 to 1.11) for Aspergillus spp. (with FICI=1.00 indicating indifference). These data are summarised and represented graphically in figure 1.

Table 1

Minimum inhibitory concentration median (MIC50) and 90th percentile (MIC90) (μg/ml) of all 20 isolates from filamentous fungal keratitis

Figure 1

Summation of fractional inhibitory concentration (FIC) index endpoints for combination testing of amphotericin B and natamycin against Fusarium spp. (n=10) and Aspergillus spp. (n=10). The FIC of a drug is the fraction of the minimum inhibitory concentration (MIC) required to inhibit fungal growth when used in combination with another drug. This is a way of normalising the respective MICs of each drug so they can be compared when used in combination in the checkerboard. If the drugs do not interact, the FIC remains at the normalised value of 1.00 (underlined in the figure). If the drugs interact synergistically, the FIC decreases. If the drugs interact antagonistically, the FIC increases. The FIC index (FICI) is the sum of the two FIC values from the x- and y-axes for the checkerboard square corresponding to the visual endpoint. The endpoint is the checkerboard square with the smallest FICI that inhibits growth. There is one endpoint (one FICI) for each isolate (each checkerboard). The figure is a graphical overlay of the 20 checkerboards with a numeric summation of the endpoint locations (white text) with corresponding FICI values (black text). All 20 isolates showed indifference to combination treatment. No isolate showed synergy or antagonism.

Discussion

Amphotericin B and natamycin are often combined in the treatment of fungal keratitis. We were unable to demonstrate synergy between amphotericin B and natamycin in any of the 20 filamentous fungal isolates. These results suggest that synergy is not a rationale for treating fungal ulcers with amphotericin and natamycin simultaneously. This is not surprising since both drugs are polyenes that are thought to work by the same mechanism.

Synergy is not the only reason to use multiple drugs. Combination therapy can broaden coverage, decrease toxicity, shorten therapy, and prevent the emergence of de novo resistance. Drugs with different toxicities can be combined to levels that could not be attained with either agent alone, a common practice in oncology. Another example is in toxoplasmosis, where the dose of sulfa drugs was originally limited by crystallisation in the kidney causing crystalluria; triple sulfas lowered the dosage of individual components to reduce the risk of crystalluria.6 Multiple drugs can prevent the emergence of resistance, as in the treatment of tuberculosis and HIV/AIDS. It is also possible that synergy could be realised in vivo despite the lack of in vitro evidence. It has been suggested that the increased toxicity of amphotericin may break down the corneal epithelium and allow better penetration of natamycin. We are not suggesting that combination therapy be avoided, but rather that treating physicians have a rationale for using amphotericin and natamycin together. In this case, synergy is not a rationale.

References

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Footnotes

  • Funding Supported in part by an unrestricted grant from That Man May See, Inc., San Francisco, California; an unrestricted grant from Research to Prevent Blindness, New York; and the National Institutes of Health (NIH) grant U10 EY015114.

  • Competing interests None.

  • Ethics approval Obtained from the University of California, San Francisco Committee on Human Research and the Aravind Eye Care System, Madurai Institutional Review Board.

  • Provenance and peer review Not commissioned; not externally peer reviewed.

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