To the best of our knowledge, studies on fungi associated with the healthy conjunctiva using NGS are scarce, but reviews exist that enumerate all fungi associated with the conjunctiva of keratitis individuals by using the conventional culturable approach.
36,39,40 Karsten et al.
36 studied all publications reporting microorganisms implicated in keratitis between 1950 and 2012 and identified 92 genera, and subsequently, Thomas and Kaliamurthy
39 reported 65 genera of fungi as associated with mycotic keratitis. In most of these publications, only single fungal genera or at the most three genera have been reported. In comparison, the present study identified 65 genera out of which only 18 and 21 genera were common with the study of Thomas and Kaliamurthy
39 and Karsten et al.,
36 respectively. The three studies shared 26 genera that included
Acremonium,
Candida,
Rhodotorula,
Aspergillus,
Penicillium,
Paecilomyces,
Phoma,
Cladosporium,
Aureobasidium,
Phialophora,
Colletotrichum,
Myrothecium,
Alternaria,
Saccharomyces,
Pyrenochaeta,
Torula,
Trichoderma,
Rhizopus,
Cercospora,
Stachybotrys,
Chrysosporium,
Malassezia,
Fusarium,
Cryptococcus,
Ustilago, and
Phaeosphaeriopsis, which were common to this study and one or two of the above studies.
36,39 These findings may imply that the above 26 genera are potential opportunistic pathogens. The reason why the remaining 39 genera in this study did not match the findings of Karsten et al.
36 and Thomas and Kaliamurthy
39 is surprising because some of them are known to cause keratitis, such as fungi affiliated to the genera
Pichia,
Nectria,
Arthrinium,
Pestalotiopsis,
Khuskia, and
Nigrospora. In a recent review, Maharana et al.
40 reported that fungi such as
Fonsecaea pedrosoi,
Lasiodiplodia theobromae,
Cylindrocarpon species,
Scedosporium prolificans,
Metarhizium anisopliae,
Paecilomyces species, and
Pythium insidiosum are rare in mycotic keratitis. In addition, the observed variation in the above studies may also be attributed to the fact that the age of the individuals sampled, the region of their origin, and the pathology were not identical.
24,41–43 It is also possible that some of them are not pathogenic and are associated only with healthy conjunctiva. In addition, the discrepancy in the detection of microorganisms by the culturable method versus the NGS method could be attributed to several other factors. For instance, organisms present in low numbers (even one) if amenable to culture would get enriched following culture but getting sufficient DNA from a single organism may not be possible. Other factors that contribute to the discrepancy include ease in DNA extraction, sequencing artifacts, taxonomic misidentification, choice of reagents and kits, and PCR bias.
44,45 Nevertheless, the culture-independent method reveals a greater degree of diversity, for instance 12 to 24 different genera per sample as observed in this study versus 1 to 2 culturable fungi.
20,46