*7.4.1 Polymerase chain reaction (PCR)*

PCR is a highly sensitive technique that allows rapid amplification of tiny samples of DNA. In the context of infection, it may be used to detect the presence of pathogenic DNA of specific microorganisms [96]. The 16S and 18S rRNA are the most frequently used marker genes for assessing bacterial and fungal profiles, respectively. They are found in all respective microorganisms and have enough variation for phylogenetic analysis and sequence conservation for accurate alignment [97]. The 16S rRNA gene sequence is 1,550 bp long, and it is composed of nine variable regions (V1-V9) interspaced in more conserved regions. By amplifying the 16S rRNA region with PCR, the background host contamination encountered in routing culturing techniques is reduced significantly [98].

Kim et al. compared the detection rate of PCR compared with traditional cultures in patients with infectious corneal ulcers [99]. Of 108 samples taken from ulcers, 52% were culture-positive and 89% PCR-positive for fungal primers (18S rRNA), bacterial primers (16s rRNA), or both. Of note, other nonpathogenic organisms (i.e., *Ralstonia, Oerksovia,* and *Leclercia* species) were also identified in 60% and 52% of the PCR samples and control swabs, respectively. Airborne contamination and false-positive results for pan-fungal and pan-bacterial PCR constitute a significant limitation of the technique [100]. Moreover, when analyzing culture-positive samples, 24% and 6.5% were PCR-negative for bacteria and fungi, respectively, suggesting that PCR does not replace traditional culturing. PCR, however, accurately distinguishes fungal from bacterial pathogens [99]. In patients with suspected *Acanthamoeba* keratitis, PCR and *in-vivo* confocal microscopy (IVCM, see Section 7.5) are preferred over conventional cultures since the latter has a low sensitivity and requires special media and extended incubation periods [101]. Goh et al. compared traditional cultures, PCR, and IVCM in the early diagnosis of *Acanthamoeba* keratitis. All methods exhibited a specificity and positive predictive value of 100%. However, the diagnostic sensitivities were 100% for IVCM, 71.4% for PCR, and 33.3% for traditional cultures. Since IVCM is an expensive device and requires an experienced operator, PCR is considered as a valuable adjunct to cultures when *Acanthamoeba* is suspected [101].
