Laboratory confirmation is hindered by delay in culturing, prior therapy, quality and quantity of specimen, inexperienced personnel, and microbial growth rate [1, 3, 7]. Lack of simple, rapid, and accurate methods also adds to the delay in laboratory identification and confirmation.

Organisms

Among the more than 100 mycobacterial species, less than 20 have been associated with microbial keratitis (Table 1.1) [8, 9]. A minimal of 300 cases of mycobacterial keratitis have been reported since the first case by Turner and Stinson in 1965. Nontuberculous species (NTM), also referred to as “atypical mycobacteria” or mycobacteria other than tuberculosis (MOTT) are the most common. The majority of these continue to be rapidly growing, saprophytic species with diverse environmental reservoirs including fresh, salt, and recreational waters, soil, animals and healthy colonized human. Members of the Mycobacteria chelonae complex (Runyoun Group IV) have been the most frequent pathogens, constituting 63% and 60% of LASIK and traumatic inoculation cases, respectively (Table 1.1).

True pathogens M. tuberculosis and M. leprae can directly invade corneal tissue but disease usually results via systemic dissemination or accidental direct inoculation. Less than 5% of patients with M. tuberculosis will develop keratitis [10–12]. The clinical presentation is usually an allergic reaction. The numbers are a little higher for patients suffering from leprosy due to direct corneal invasion and ulceration associated with corneal anesthesia [13]. Although rare, these pathogens cause significant morbidity and mortality in endemic areas and are increasingly encountered in the rising population of HIV patients [14].

Nontuberculous species are ubiquitous in nature and resistant to traditional mycobacterial drugs as well as chlorine and other disinfectants. Increasingly, sporadic single cases or outbreaks are associated with unusually or slow growing mycobacteria such as M. skulgai, M. immunogenum, and/or M. terrae. The Runyon classification with modifications is still used to characterize these infections. The classification of isolates is based on the time it takes for colonies to appear on solid media from a subculture rather than growth from clinical samples. Common rapid growers (M. abscessus, M. chelonae, M. fortuitum) usually grow from subculture within 3–7 days while growth after subculture for the slower growers taking up to 8 weeks. All four Runyon groups have been associated with microbial keratitis.

Detection

Conventional techniques for the identification of mycobacterial species employ a battery of phenotypic (growth rate, colony morphology) characteristics and biochemical tests. These are time consuming, labor intensive, expensive, and often inconclusive. The delay in laboratory recovery and identification may impede clinical diagnosis [2, 15–17]. An updated algorithm using molecular and traditional

stains (Kinyoun (cold) and Ziehl Neelsen (hot)) are important tools in the rapid and direct detection of acid fast bacilli in corneal scrapings, biopsies, and material collected from under the LASIK flap. The basic stain includes flooding a heatfixed slide with carbol fuschin for 3–5 min, decolorization with acid alcohol, wash step, and application of a 1-min counterstain. A modified Kinyoun stain using a weaker decolorizer may be more sensitive for confirming the rapidly growing mycobacteria due to their wearker or inconsistent staining with the first two preparations (<10%) [9, 18].

Although fluorescent stains (auramine or auramine–rhodamine) are generally more rapid and sensitive than the carbol fuschin stains, many of the rapid growers may not stain with these fluorochromes [9, 18]. In positive smears, acid-fast organisms will appear orange-yellow in a black background or yellow-green in the absence of the counterstain. In the review of mycobacteria keratitis cases by Huang, only 50% of culture positive cases were detected by smear [5]. Limitations of these studies is the requirement for the presence of a high number of organisms (³103–4 CFU/ml) required for positivity. Initial corneal scrapings/smears contain numbers below this threshold and are often acid fast negative. Correlation between smears and culture is poor.

A recent, new modification of the auramine stain, Rapid-Auramine O, (Scientific Device, Inc, Des Plaines, IL) provides for quicker (2 min vs. 22 min) and more sensitive (brighter, less debris) screening of mycobacteria from clinical samples including M. fortuitum (100%) and M. chelonae (80%) [19]. This might be useful in evaluating or screening ocular samples collected from LASIK flaps or corneal biopsies where the infecting microorganism may be above the stain’s detection threshold.

Culture Media

Both liquid and solid media are recommended for optimal recovery and quicker identification of mycobacteria species. The most frequently involved pathogens (the rapid growers) are recovered from corneal scrapings, tissues, or biopsy within 3 days on routine solid media (chocolate, blood agar, Sabouraud agars) and special media (Lowenstein-Jensen and Middlebrook agars) (Figs. 1.5 and 1.6). Dependent on the quality and quantity of sample, initial recovery of mycobacterial species can take up to 10 days.

Slow growers (Runyoun Groups I-III) and Mycobacterium tuberculosis grow poorly or not at all on routine laboratory media. Scrapings should be inoculated on to Lowenstein-Jensen, Middlebrook, or Ogawa media for recovery. Recovery rates range from 2 to 8 weeks. Broth media (MGIT, Middlebrook, and Bactec media) have been used to recover mycobacteria from corneal samples. Organisms were recovered on average within 3 days from inoculation. M. leprae has not been grown on artificial laboratory media.

A new type of solid media has been developed for the recovery, identification, and susceptibility of mycobacteria species. TK Medium (M. tuberculosis complex)