Is inclusion of Sabouraud dextrose agar essential for the laboratory diagnosis of fungal keratitis?

Purpose:

To determine whether the inclusion of Sabouraud dextrose agar (SDA) is essential in the diagnosis of fungal keratitis.

Materials and Methods:

Corneal scrapings of 141 patients with microbial keratitis were smeared and cultured. Sheep blood agar (BA), chocolate agar (CA), SDA, non-nutrient agar (NNA) with Escherichia coli overlay, and brain heart infusion broth (BHI) were evaluated for time taken for growth and cost. The media were also evaluated experimentally for rate of growth and time taken for identification.

Results:

Twenty-six of 39 patients positive for fungus in corneal scrapings by microscopy were culture-positive. Fungus grew on BA in 22/39, on CA in 18/39, on SDA in 17/39, on NNA in 17/39, and on BHI in 13/39 cases. Growth on SDA was higher in ulcers with larger infiltrate (6/18 versus 9/13, P = 0.04). Estimated saving with inclusion of only BA/CA was Rs. 600 per patient. Performance of all media was similar in in vitro experiment although the characteristic spores and color were seen earlier on SDA.

Conclusion:

Fungal keratitis can be reliably confirmed on BA or CA, which support growth of both bacteria and fungus.     

【In Press】Mincle is involved in the innate immune response of mice fungal keratitis

AIM: To investigate how macrophage inducible C-type lectin (Mincle) influences inflammation in mice fungal keratitis induced by A. fumigatus. METHODS: C57BL/6 mice were infected with A. fumigatus after pretreated with Mincle agonist TDB or Mincle neutralizing antibody (mincleAb), taking DMSO or IgG as control group respectively. The cornea lesions were monitored with slit-lamp microscope and evaluated by clinical score. Mincle expression was assessed using RT-PCR and immunostaining. The expression of cytokines (IL-1β, TNF-α and IL-6) chemokines (CXCL-1 and MIP-2) was determined by RT-PCR and ELISA. Neutrophil infiltration was observed by immunostaining. The levels of nitric oxide (NO) generated by corneas were tested by Griess reaction. RESULTS: Mincle mRNA and protein levels were higher in infected corneas than normal corneas of C57BL/6 mice, saving clinical scores revealed differences. When pretreated with Mincle agonist TDB, the mRNA and protein levels of IL-1β, TNF-α and IL-6 in infected corneas were significantly increased compared with the control group (P<0.01). Results of the counterpart in corneas pretreated with Mincle neutralizing antibody was decreased consistently (P<0.01). Expression of CXCL1 and MIP-2 mRNA levels were up-regulated in TDB group and down-regulated in MincleAb group (P<0.01), coincide with neutrophil aggregation degree in corneas showed by immunostaining. As for the concentration of NO, it was promoted in TDB group compared with DMSO control group, and decreased in mincleAb group compared with IgG control group. CONCLUSION: Mincle plays a dual role in mice fungal keratitis. It participates in the innate immune system by enhancing inflammation. What’s more, Mincle can mediate cytotoxic effects by regulating the formation of nitric oxide.     

【In Press】 Modified ulcer debridement in the treatment of the superficial fungal infection of the cornea

AIM: To evaluate the efficacy of modified corneal ulcer debridement in superficial fungal keratitis unresponsive to medications. METHODS: A total of 209 patients (209 eyes) with fungal keratitis, involving no more than 50% of the stromal depth and not responding to antifungal agents for 2wk, were recruited in this retrospective, noncomparative study. The patients were treated with modified corneal ulcer debridement. All visible corneal infiltrates were removed under an operating microscope to obtain a clean stromal bed and smooth incised edges. Antifungal drugs were used immediately after surgery. Healing time of the ulcers was recorded. Fungal recurrence, visual acuity, corneal thickness and risk factors for treatment failure were monitored. RESULTS: The follow-up was 13.6±5.8mo. The corneal ulcers healed in 195 of 209 eyes (93.3%), with a mean healing time of 8.4±6.8d. The other 14 eyes were further treated by penetrating keratoplasty (1 eye), anterior lamellar keratoplasty (7 eyes), conjunctival flap covering (4 eyes) or amniotic membrane transplantation (2 eyes). The best corrected visual acuity was ≥20/70 in 80.3% of the eyes, ≥20/40 in 56.9% of the eyes, and ≥20/25 in 27.3% of the eyes. The corneas at the lesions became thinner, but all in the safe range. No fungal recurrence or corneal ectasis developed during the follow-up. The risk of treatment failure was higher in patients with preoperative hypopyon (P=0.036) and ever using steroid (P=0.025). CONCLUSION: Modified surgical debridement is a simple and effective method for the treatment of superficial fungal infection of the cornea, with improved visual acuity and no recurrence. Such an intervention in time can rapidly control fungal infection and largely shorten corneal ulcer healing time.     

Two-step strategy—conjunctival flap covering surgery combined with secondary deep anterior lamellar keratoplasty for the treatment of high-risk fungal keratitis

AIM: To investigate whether the two-step strategy [conjunctival flap covering surgery (CFCS) combined with secondary deep anterior lamellar keratoplasty (DALK)] is effective for patients with high-risk fungal keratitis (FK). METHODS: In this noncomparative, retrospective case series, 10 subjects (6 males, 4 females) with a mean age of 56.5±7.1 (range 47-72)y with high-risk FK undergone the two-step strategy were included. Reported outcome measures were healing of the corneal ulcer, recurrence of FK, reject reaction, improvement in best corrected visual acuity (BCVA) and relevant complications. RESULTS: The average diameter of corneal infiltrates was 7.50±0.39 mm, ranging from 6.94 to 8.13 mm. The mean depth of corneal infiltrates was 422.4±77.1 μm, ranging from 350 to 535 μm. The mean corneal thickness was 597.4±117.3 μm, ranging from 458 to 851 μm. Hypopyon and endothelial plaques were presented in all patients. The period between the two steps was 3.65±0.9 (ranging from 3 to 5)mo. The graft diameter was 7.75±0.39 mm. At the last follow-up (average 9.25±3.39, ranging from 5.5 to 17mo), no fungal recurrence or graft rejection appeared, and all patients showed improvement of BCVA. One patient suffered from liver function impairment due to oral voriconazole for 4wk and recovered spontaneously after 1wk of drug withdrawal. CONCLUSION: The two-step strategy is safe and effective in the treatment of high-risk FK by transforming intentional therapeutic penetrating keratoplasty during acute infection to later optical DALK. It is a practical strategy, especially in areas lacking fresh donor corneas and eye bank services.