**6. Polymerase chain reaction techniques and other novel methodologies for the diagnosis of acute postoperative endophthalmitis**

Since the advent of PCR, molecular laboratory techniques have increased rapidly. Its use is part of many routine processing of clinical samples in microbiology laboratories, establishing a new era for diagnosing infectious diseases [17]. The use of PCR for APIE diagnosis increases significantly bacterial detection sensitivity and speed for etiologic diagnosis in vitreous and aqueous humors. The bacteria detection rate in aqueous and vitreous samples increased from approximately 48% to up to 95% using PCR techniques [17–21].

PCR and nested PCR protocols followed by post-PCR techniques such as RFLP (restriction fragment length polymorphism), DNA sequencing, and DNAprobe hybridization have all been utilized to improve the etiologic diagnosis of APIE [22, 23].

Reported sensitivities for bacterial identification in vitreous samples for several PCR techniques like nested PCR and real-time multiplex PCR are 84%, and 90–95%, respectively, although reported sensitivities vary among different publications [17].

Real-time PCR technology (RT-PCR) is a modification and enhancement of the PCR technique. It is a homogeneous technique in which DNA amplification and detection of the target sequence co-occur, decreasing PCR products' handling and

#### *Acute Postoperative Infectious Endophthalmitis: Advances in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.97545*

risks of carryover contamination. Simultaneous DNA amplification and detection allow higher reliability as compared to other traditional multi-step techniques. One of the main advantages of real-time PCR technology is the rapid access to results, with microbial detection times of 30 to 50 minutes, compared to 1–14 days for previous PCR methods [18–21].

Green 16S rDNA–Based Universal PCR (SGRU-PCR) and a Multiplex Gram-Specific TaqMan–Based PCR (MGST-PCR) are useful for microorganism detection in many culture-negative samples. In one study, 90% were PCR positive among ten microbiologically negative samples, and five gave interpretable sequence data [17]. The pathogens identified included one coagulase-negative *Staphylococcus*, one *Moraxella spp*., and two *Streptococcus mitis* group, all APIE causative bacteria. Sequencing of PCR-positive/culture-negative samples also included the identification of a *Proteus spp*. causing APIE, which is a rare causative microorganism in postoperative endophthalmitis [17].

Albeit being useful in APIE diagnosis, molecular identification of pathogen microorganisms remains an expensive technique. It is not available in many small cities, underdeveloped countries, laboratory settings, and in many instances, it requires a high workload that makes it inadequate for routine use. Furthermore, clinical definitions of some species do not match those used for 16S rRNA identification [17, 18].

Among another novel microbial detection methods that could eventually be used in the expedite diagnosis of APIE cases are Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) [24, 25] and the utilization of magneto-DNA nanoparticle Dovepress system [26]. The latter method was reported to simultaneously identify up to thirteen species of bacteria in under two hours [26].

Bacterial recognition directly from samples and colonies using MALDI-TOF MS has been described as a revolutionary method that better adapts to the clinical microbiology laboratory. MALDI-TOF MS is used to identify bacterial species and to detect microorganisms previously misidentified [24]. Moreover, detection of antimicrobial resistance using MALDI-TOF MS has been reported for many bacteria, including *Escherichia coli* and *Staphylococcus aureus*. Another advantage of MALDI-TOF MS is that the time required for pathogen identification declines by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with culture and molecular gene sequencing, respectively [24, 25].

The Magneto-DNA nanoparticle Dovepress system is an assay that utilizes magnetic nanoparticles and oligonucleotide probes to detect target nucleic acids from the pathogen. Rather than sequencing the whole RNA strand, a series of primers and probes were established to amplify and detect specific regions of interest within common bacterial types. They used a miniaturized micro-nuclear magnetic resonance system that requires only small volumes of sample for processing for signal readout [26].

It is hypothesized that ribosomal RNA sequence information from microorganisms such as bacteria could be used in a robust magneto-DNA assay. Because this magnetic detection strategy allows near background-free sensing, the assay steps are greatly simplified, and detection is much faster [26].
