**6.1 Microbiology evaluation**

The microbiological evaluation consists of smear examination and culture of corneal scrapings into several media to grow organisms for identification [32]. Culture is the only way to determine, which antibiotics the pathogenetic agent is susceptible to. In the case of sight-threatening keratitis, the culture is an indispensable diagnostic tool. The results of culture allow to shorten the duration of treatment and avoid unnecessary drug use. The efficiency of corneal cultures and smears is much higher if done before antibiotic treatment is initiated. However,

when a patient previously used antibiotics, antibiotic therapy should be discontinued and scraping can be delayed for 12–24 hours to improve test performance. Lately, calcium alginate swabs with trypticase soy broth have been employed to obtain corneal specimens for obtaining a higher yield of bacteria [33]. When obtaining specimens, we should be very careful in the case of descemetocele, deep stromal keratitis, or corneal melting. The corneal ulcer samples are performed under topical anesthesia (i.e., 1% lignocaine, 0.5% proparacaine, or proxymetacaine 0.5%). There should be preferred preservative-free formulation because preservative may lower the bacterial viability for culture. Before performing scraping, dead and necrotic tissue and loose mucus are removed from the ulcer surface. Then, the corneal ulcer samples are collected from the area of corneal infiltration (the margin and the base of the ulcer) using a disposable number 11 or 15 Bard-Parker blade or typically 25-gauge or 26 G bent hypodermic needle or sterile kimura or platinum spatula. The first samples are placed on glass slides for staining, and then onto the media for culture [12, 33, 34]. The obtained material is smeared onto one or two glass slides for microscopic evaluation along with a gram stain. Gram staining detects the type of organism in 60–75% of bacterial cases, and it is beneficial providing results in 5 minutes [12, 31]. Repeat scraping is performed, and the sample is placed on various culture media that should be taken from the fridge and left for 1 h to reach room temperature. Various stains used for bacteria and various culture media for bacteria are shown in **Tables 1** and **2** [35].

According to literature data from around the world, the positive culture rate from corneal scrapes ranges from 38 to 66% [36–42]. When smear and culture results are negative two times, and there is a clinical progression of ulcer despite the best antibacterial therapy a corneal biopsy can be performed. It is obtained with the help of a dermal trephine or freehand dissection, and the specimen is divided into two halves to


#### **Table 1.**

*Various stains used for bacteria.*


#### **Table 2.**

*Various culture media for bacteria.*

#### *Bacterial Keratitis DOI: http://dx.doi.org/10.5772/intechopen.113365*

allow histopathological and microbiological analysis [9, 43]. Conjunctival swab culture (calcium alginate swabs give the best results) may be another important additional diagnostic method in severe cases when culture growth is negative [44]. Anterior chamber paracentesis is another needed diagnostic method, which is performed in the case of negative scraping culture, or there is a progression of ulcer despite the best antibacterial therapy. A 0.1 to 0.2 ml sample is obtained with the help of a 25 G needle by a side port [45]. In addition to corneal scrapping, it is worth to obtain culture from contact lenses, liquids, and containers for lenses and from other potential sources of infection, for example, from inflamed eyelids. A relationship has been demonstrated between cultures of the abovementioned sources and corneal scraping [46]. We should keep in mind, that as interpreting results caution is needed because most eyelid and ocular surface commensal organisms are gram-positive and likely to contaminate the sample [47].

#### **6.2 Polymerase chain reaction test**

The polymerase chain reaction (PCR) test is another test used in the diagnosis of microbial keratitis. This is a molecular technique for the detection and analysis of specific DNA sequences, consisting of repeated cycles of denaturation, amplification, and replication, in which segments of DNA are continuously multiplied to enable their detection [48]. The advantages of PCR, including sensitivity, speed, and cost-effectiveness relative to culture and staining. It also gives an ability to quickly differentiate bacterial and fungal ulcers. It also gives a possibility to detect of slow-growing bacteria and organisms that are difficult to cultivate or identify with traditional microbiological methods [49–51]. This technology also has an important role in diagnosing rare organisms, such as atypical mycobacteria and Nocardia species [52]. There are also some disadvantages, including the high rate of false positive errors from commensal contaminants or dead bacteria, lower specificity compared with culture and staining, difficulty to interpret results and treating by clinician, more expensive procedure, and less cost-effective when performed with a multi-organisms PCR approach, and is not readily available at all sites [49–51].

### **6.3** *In vivo* **confocal microscopy (IVCM)**

*In vivo* confocal microscopy (IVMC) is a noninvasive examination that shows real-time visualization of corneal layers and structures and pathological agents within the corneal tissue. The advantages of IVCM are repeatability, rapidity, and noninvasiveness, thus also being useful in monitoring the therapy. The high sensitivity and specificity of IVCM is a valuable adjuvant to the other diagnostic assays. Thanks to immediate results obtained after conducted rapid in vivo corneal examination, it allows the prompt beginning of appropriate treatment, and some authors recommend its use as a very good diagnostic tool early in the course of the disease [53]. IVCM is also useful appreciate the depth of the infection in the corneal stroma, what is an important prognostic factor of IK [54]. However, there are some disadvantages that include patient collaboration and patience are required during testing, the high price of the device, lack of availability at all sites, and difficulty in both acquiring and interpreting images by non-experienced clinician [54]. When we have access to IVCM, we should always consider to perform this examination in the following clinical situations: deeply situated infiltrates, where corneal scrapes do not have access to avoid invasive corneal biopsy, MK occurring after corneal surgery (i.e., intracorneal implants, refractive surgery), lack of the positive results in current treatment with antifungal or anti-*Acanthamoeba* spp. therapy, when actively proliferating microorganisms are found in the profound, inaccessible corneal stroma [53, 54]. IVCM is highly sensitive and specific, and thus is very useful in cases of fungal or *Acanthamoeba keratitis*. As for nowadays, IVCM should be used alongside cultures and smears. Other new diagnostic modalities, such as immunohistochemistry, enzyme immunoassay, and radioimmunoassay, are recent upcoming modalities but still have a limited role in diagnosing BK [55].
