*3.2.1 Polymerase chain reaction*

Polymerase chain reaction (PCR) involves repeated cycles of denaturation, amplification, and replication, in which segments of deoxyribonucleic acid (DNA) are continuously multiplied. Specific DNA primers are employed to indicate the presence of the microorganism in question [64]. PCR has emerged as a sensitive and specific test for the diagnosis of fungal keratitis. Several techniques of PCR have been evolved and currently used for identification of fungi.

Traditional PCR by using single pair of primer to amplify the target genomic sequence is simple and efficient technique, but generation of nonspecific products can affect the results. In Nested PCR, two pairs of primers are used; one set of primer is an amplified sequence, and the other is complementary to the sequence amplified by the first one. It is more specific than traditional PCR; amplifies only the specific sequences looked for; but identify a set of fungal pathogens, not a single specific species.

In multiplex PCR Multiple primer, pairs are used. Advantage is Rapid amplification of multiple sequences, conserves template DNA, and minimizes expense; recognizes many pathogens at once. In real time PCR, one set of primers is used; amplified sequence is linked with a fluorescent probe, which emits light when bound to the amplified product. It is more specific, sensitive, and reproducible but not ideal for multiplexing [65–67].

PCR reported higher sensitivity in comparison to culture and stains for both bacteria and fungi [68, 69]. Zhao et al. reported significantly higher positive detection rate of PCR for fungal keratitis (84.5%) as compare to the positivity rate for culture (35.3%) and stain (64.7%) [69]. A higher sensitivity of PCR for infectious keratitis compared to culture (98% versus 47%), but a slightly lower specificity (83% versus 100%) is reported in this study [69].

The PCR is rapid test, it takes 4–8 h, and only a small clinical sample is needed for diagnosis **[**7]. The limitation of PCR is that it is expensive, not readily available and specificity is lower than culture. Extraction of artifacts and amplification of

non-pathogenic DNA can lead to over diagnosis [66]. However, it can be used to detect fungal DNA in corneal scrape material, to start antifungal therapy at an early stage of the keratitis.

#### *3.2.2 Metagenomic deep sequencing*

Metagenomic deep sequencing (MDS) is a new technique for the diagnosis of FK; with next generation sequencing rapid and accurate diagnosis is possible. Next generation sequencing is high-throughput sequencing methods where billions of nucleic acid fragments can be sequenced simultaneously and independently. MDS is an unbiased approach that interrogates all genomes in a clinical sample and identify any organism whereas PCR is a targeted test the clinician must know the suspected causative organism.

It has been shown to enhance detection of common and unusual pathogens from the intraocular fluid of patients with infectious uveitis and other systemic infections [70–72]. A study by Seitzman et al. in a case series of nine patients of infectious keratitis diagnosed by conventional methods reported that MDS detected all the microorganisms identified by culture or PCR. MDS was able to identify parasitic, fungal, bacterial, and viral infections as a single assay. The pathogenic organisms ranged in size from smaller genomes (*herpes simplex virus-1* and *adenovirus*) to larger genomes (*Acanthamoeba* and *Aspergillus*). In one case, the MDS identified the organism not supposed to be a cause of infectious keratitis. The case was culture positive for Purpureocillium lilacinum was identified as the second most abundant organism and, the most abundant organism in the sample was Auricoccus indicus, which is not known to cause ocular infections and not even listed in the University of California San Francisco's mass spectrometry's database for identifiable organisms [73].

### **4. Recent advances in medical treatment**

Polyenes (Amphotericin B and Natamycin) and azoles (fluconazole, itraconazole, ketoconazole, miconazole, voriconazole, and posaconazole) constitute two major classes of antifungal drugs used to treat ocular fungal infections including fungal keratitis. In Comparison to antibacterial agents, antifungals have a lower efficacy due to their mechanism of action (usually fungistatic, with dose dependent fungicidal action), lower tissue penetration, and the indolent nature of the fungal infection [74]. Still for the management of fungal keratitis, the traditional anti-fungal drugs like natamycin and fluconazole in topical and oral form are used most commonly. In recent years, other new drugs and drug delivery system to increase bioavailability of drugs have been evaluated. Anti-fungal agents are summarized in **Table 1**.

#### **4.1 Natamycin**

Natamycin is first antifungal agent approved for FK by Food and Drug Administration in the 1960s. After that, many antifungal agents have been evaluated, no single agent has emerged as the best and most cost effective agent [7]. Cochrane systematic review in 2008 and 2012, found no evidence that any single drug, or combination of drugs, is more effective in the management of fungal keratitis. The trials included in this review were of variable quality and were generally underpowered [75, 76].



#### **Table 1.**

*Summary of antifungal agents used in Fungal Keratitis*

Natamycin is a polyene antifungal drug, it binds preferentially to ergosterol on the fungal plasma membrane and causes localized membrane disruptions by altering membrane permeability. Natamycin is currently considered the most effective medication against Fusarium and Aspergillus [7]. Cochrane systematic review in 2015 found that there is evidence that natamycin is more effective than voriconazole in the treatment of fungal ulcers. However, the trials included in this review were of variable quality and were generally underpowered. Future research should evaluate treatment effects according to fungus species [77].

Several studies reported that fungal keratitis due to fusarium responded better to Natamycin as compare to itraconazole and voriconazole [77]. NTM is the treatment of choice for filamentous keratitis, especially that due to Fusarium species. However its poor penetration into corneal stroma, limits its use in deep stromal keratitis. In deep keratitis or with involvement of intraocular structures, natamycin should be associated with other antifungal agents using a different route of administration.

#### **4.2 Amphotericin B**

Amphotericin B is the first broad-spectrum antifungal agent, produced by the actinomycetes, *Streptomyces nodosus*. It acts by binding to ergosterol and by promoting oxidative action on cells, thus altering their metabolic functions. This binding also results in formation of pores or channels in the fungal cell membrane and increasing cell permeability. Its binding to cholesterol in human cells is responsible for its side effects. It is effective against *Aspergillus* and *Candida* species but less effective against *Fusarium* species [74]. It is administered as a topical solution in concentration of 1.5 to 5 mg/ml.

Amphotericin B has poor ocular penetration after intravenous administration and is toxic to human cells at a higher dose. Due to systemic (nephrotoxicity) and ocular toxicity (punctate epithelial erosions and greenish discoloration of the cornea), amphotericin B is not currently a first line agent in treating fungal keratitis.

In a study, Morand K, et al. compared the commercial 0.15% Amphotericin B with a liposomal formulation and found that the liposomal form was more stable and less toxic. The liposomal formulation also increased the potential amount of loaded drug by 3-fold compared with the conventional form [78].
