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To test the hypothesis that the coexistence of Acanthamoeba with other forms of microbial keratitis, especially fungal keratitis (FK), is more prevalent than suspected.A prospective diagnostic study whereby patients presenting with stromal keratitis were additionally tested for Acanthamoeba, irrespective of the initial diagnosis. In addition to the routine workup with Gram stain, KOH mount, and cultures on blood agar and potato dextrose agar, nonnutrient agar was included. Confocal microscopy was performed where feasible. Samples for polymerase chain reaction studies were also obtained. We present the preliminary report of the first 100 culture-positive cases. The primary outcome measured was the number of coexistent Acanthamoeba and FK. The secondary outcomes were the total number of Acanthamoeba cases detected and the correlation between clinical diagnosis and microbiological observations.Of the first 100 cases, 22 were culture positive for Acanthamoeba, of which 9 were associated with concurrent FK, 5 with bacterial keratitis, and 8 in isolation. However, only 2 cases were diagnosed clinically as Acanthamoeba, whereas 5 were Acanthamoeba suspects. An additional 4 cases of fungal/Acanthamoeba coexistence in keratitis were revealed purely by confocal microscopy.Acanthamoeba can coexist with other forms of microbial keratitis. The frequency of infection coexistent or otherwise is higher than reported, and the possibility of coinfection must be considered especially in unresponsive cases. Including nonnutrient agar and confocal microscopy in all cases of keratitis would perhaps translate into better treatment strategies and outcomes.