**Diagnostics Methods in Ocular Infections−From Microorganism Culture to Molecular Methods**

Victor Manuel Bautista-de Lucio, Mariana Ortiz-Casas, Luis Antonio Bautista-Hernández, Nadia Luz López-Espinosa, Carolina Gaona-Juárez, Ángel Gustavo Salas-Lais, Dulce Aurora Frausto-del Río and Herlinda Mejía-López

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52468

**1. Introduction**

#### **1.1. Conventional methods of microbiological diagnosis: Culture, isolation and phenotypic identification**

Despite advances in the medical field, 4 of the 10 leading causes of death worldwide are due to infectious diseases [1]. At the eye, infections are one of the most common diseases, and bacterias are the first causative agent, followed by fungus and virus. Between these bacte‐ rias, *Staphylococcus* genus, *Streptococcus genus, Corinebacterium sp, Chlamydia sp, Pseudomonas aeruginosa, Escherichia coli, Enterococcus sp, Serratia sp*are frequent in keratitis, conjunctivitis, endophthalmitis and cornea ulcer [2]; *Fusarium sp, Aspergillus sp* and *Candida sp,* are the com‐ monly fungus found in keratitis infections [3]; *Adenovirus, HSV-1 (Herpes Simplex Virus-1), HSV-2 (Herpes Simplex Virus-2), VZV (Varicella Zoster Virus), HPV (Human Papilloma Virus)* are important in conjunctivitis and keratitis [4].

In order to reduce complications from ocular infectious diseases is very important to pro‐ vide appropriate early treatment. To make this possible, is essential microbiological identifi‐ cation of the causative agent of infection in the shortest time possible. However, the

microbiological diagnosis by conventional methods considered as gold standards, based on the culture followed by phenotypic identification of the microorganism once isolated, taken between 48 and 72 hours, depending on the requirements of the microorganism, and in the case of fungal infections, identifying and obtaining the antifungal susceptibility profile, come to take over a week. Identification time may be reduced by using automated equip‐ ment whose bases are the same as those used for manual identification, through biochemical profile of microorganisms. These tests are based on the ability to ferment, oxidize, degrade or hydrolyze different substrates or to grow on different carbon sources producing changes in pH that may be monitored using compounds that turn color depending on the pH. Auto‐ mated systems work with cards containing dehydrated culture media with suitable sub‐ strates. Culture time elapses while the cards are automatically read and data are collected by a system confronts the data collected with a database through the microorganism is identi‐ fied. Among the available automated identification systems are the VITEK 2 (bioMérieux, France) and BD Phoenix (USA), these Systems reduce time of identification to 6-12 h.

Although the main disadvantage is the time it takes the identification, cultivation allows the discovery of new or atypical strains, conservation of strains for further characterization and the ability to determine the antimicrobial susceptibility directly [5].

Given the urgency with which requires identification of microorganisms other strategies have been designed which further reduce the periods of time. These include the identifica‐ tion by endpoint PCR, real time PCR, microarrays and mass spectrometry directed to the de‐ tection of proteins or nucleic acids.

#### *1.1.1. Endpoint PCR and real time PCR*

Although the identification of microorganisms through culture is the gold standard this methodology presents some complications that are resolved using molecular biology techni‐ ques such as endpoint PCR and real time PCR, significantly reducing the outcome of days to a couple of hours. The identification of microorganisms using traditional microbiology is limited by slow growing organisms or poorly viable, besides giving false negative results due to treatment of patients with pre antimicrobial sampling [5]. Identification by culturing are required pure colonies, because in mixtures of microorganisms is impossible to identify the components of the mixture. All these constraints are solved by PCR. Using real time PCR is possible the detection of several microorganisms in the same assay [6]. This requires as‐ sembling multiple reactions in which the detection of microorganisms is carried out at the end of the amplification when by increasing the temperature gradually build dissociation curve (melt curve). Thus, if we know the temperature to which the DNA strands of ampli‐ cons are separated from each microorganism, then microorganisms present in the specimen can be identified [6,7].

Some of the limitations to the identification by PCR are that this reaction requires the use of specific oligonucleotides for each microorganism, then each PCR reaction is performed for one or a particular group of organisms suspected. The design of specific oligonucleotides re‐ quires knowledge of the genome of the microorganism as well as genomics variants, some‐ times new strains or mutations cause that oligonucleotides designed for the identification might not align correctly. Due to test sensitivity is possible to detect even one copy of the target sequence [6], so that contamination is one of the main problems, one reason for this is the inadequate ways of taking the samples and the presence of microorganisms from normal flora cause false positives [5].

Even with the disadvantages mentioned, there are reasons that justify the use of PCR for di‐ agnosis of ocular infections, due to the importance of receiving prompt treatment to stop the infectious process because otherwise compromises the functionality of the eye. Ocular infec‐ tions caused by fungi are those with more advantages in the detection by PCR, because fun‐ gi growth is very slowly, and detection by molecular means is very quickly, and it allows giving a specific treatment [8].

Sensitivity of culture in cases of endophthalmitis is less than 70% and keratitis is not more than 80% due to the small inoculum [8]. If you have a sample that will uncover the presence fungal crop procedure and identification can take over a week delaying treatment. There‐ fore, the PCR for fungi provides the sensitivity needed in case of poor sample, even on the day of the sample obtaining [8]. Very few are currently available for clinical diagnostic kits for PCR. Roche provides kits for detection of *Chlamydia trachomatis*, CMV, EBV, Hepatitis A, B and C, HSV 1 and 2, VZV, HIV and *Neisseria gonorrhoeae*, meanwhile Bio-Rad have kits on‐ ly for *Chlamydia trachomatis* and Mycoplasma. None company offers multiple trials or gener‐ ic that can detect the presence of fungi and/or bacteria. Generic detection of bacteria is performed using oligonucleotides designed to bind to the conserved region of 16S ribosomal RNA gene, whereas for the detection of fungi, target is the 18S gene. While it is important to know the identity of the organism for appropriate treatment, discrimination between bacte‐ ria or fungi as causal agents of infection allows the introduction of generic treatment as early identification of the organism is carried out. The absence of commercial testing kitsallow that diagnostic laboratories use "home" methods at endpoint PCR and real time PCR de‐ signed and validated by themselves [8].

#### *1.1.2. Identification by full genome sequencing*

microbiological diagnosis by conventional methods considered as gold standards, based on the culture followed by phenotypic identification of the microorganism once isolated, taken between 48 and 72 hours, depending on the requirements of the microorganism, and in the case of fungal infections, identifying and obtaining the antifungal susceptibility profile, come to take over a week. Identification time may be reduced by using automated equip‐ ment whose bases are the same as those used for manual identification, through biochemical profile of microorganisms. These tests are based on the ability to ferment, oxidize, degrade or hydrolyze different substrates or to grow on different carbon sources producing changes in pH that may be monitored using compounds that turn color depending on the pH. Auto‐ mated systems work with cards containing dehydrated culture media with suitable sub‐ strates. Culture time elapses while the cards are automatically read and data are collected by a system confronts the data collected with a database through the microorganism is identi‐ fied. Among the available automated identification systems are the VITEK 2 (bioMérieux,

France) and BD Phoenix (USA), these Systems reduce time of identification to 6-12 h.

the ability to determine the antimicrobial susceptibility directly [5].

tection of proteins or nucleic acids.

2 Common Eye Infections

*1.1.1. Endpoint PCR and real time PCR*

can be identified [6,7].

Although the main disadvantage is the time it takes the identification, cultivation allows the discovery of new or atypical strains, conservation of strains for further characterization and

Given the urgency with which requires identification of microorganisms other strategies have been designed which further reduce the periods of time. These include the identifica‐ tion by endpoint PCR, real time PCR, microarrays and mass spectrometry directed to the de‐

Although the identification of microorganisms through culture is the gold standard this methodology presents some complications that are resolved using molecular biology techni‐ ques such as endpoint PCR and real time PCR, significantly reducing the outcome of days to a couple of hours. The identification of microorganisms using traditional microbiology is limited by slow growing organisms or poorly viable, besides giving false negative results due to treatment of patients with pre antimicrobial sampling [5]. Identification by culturing are required pure colonies, because in mixtures of microorganisms is impossible to identify the components of the mixture. All these constraints are solved by PCR. Using real time PCR is possible the detection of several microorganisms in the same assay [6]. This requires as‐ sembling multiple reactions in which the detection of microorganisms is carried out at the end of the amplification when by increasing the temperature gradually build dissociation curve (melt curve). Thus, if we know the temperature to which the DNA strands of ampli‐ cons are separated from each microorganism, then microorganisms present in the specimen

Some of the limitations to the identification by PCR are that this reaction requires the use of specific oligonucleotides for each microorganism, then each PCR reaction is performed for one or a particular group of organisms suspected. The design of specific oligonucleotides re‐ quires knowledge of the genome of the microorganism as well as genomics variants, some‐ times new strains or mutations cause that oligonucleotides designed for the identification Time spent in genome sequencing has decreased. The first genome sequenced in 1995 (*Hae‐ mophilus influenzae*) took more than a year [9], whereas today technology is able to sequence hundreds of thousands of times faster. There is currently information around 3,144 complete genome deposited at the GenBank database [10]. All this information makes it possible to implement identification techniques based on sequencing. Through the comparison of se‐ quences obtained from the analysis of clinical samples with the sequences contained in the databases, microbiological identification takes only a few hours with high certainty. These new technologies use PCR to amplify DNA, coupled to a parallel sequencing system using methods such as pyrosequencing, sequencing by ligation and sequencing by synthesis.

Although this technology is available from Roche 454 platforms, SOLID platform from Life Technologies and Illumina platform, plus the costs of technology, interpretation of results is perhaps the greatest barrier to the implementation of the sequencing genome as a routine identification technique in clinical laboratories [10].

In 2005, Yeo *et al.,* reported an outbreak of acute hemorrhagic conjunctivitis in Singapore [11]. Patients were diagnosed clinically with acute hemorrhagic conjunctivitis and it was identified by PCR the presence of an enterovirus and molecular typing confirmed a variant of coxsackievirus A24 (CA24v). Full-length genome sequencing results showed that CA24v virus was responsible for the outbreak and it was evolved from virus emerged 40 years ago.

#### *1.1.3. PCR coupled to mass spectrometry using electrospray ionization (PCR / ESI-MS)*

For identification by PCR/ESI-MS using oligonucleotides specific for bacterial groups rather than to a particular species, although variable regions are amplified between species and strains. Additionally, there are species-specific oligonucleotides used as primers that target genes for antibiotic resistance or some pathogenic characteristics [12]. Subsequent to amplifi‐ cation, amplicons are subjected to mass spectrometry and the pattern obtained is compared with those in the databases. The ability to identify an organism without prior knowledge of the Gram, or group of microorganisms is another advantage [12], since the stains are not re‐ quired or previous isolates that provides fast trial and will always be possible to identify the microorganisms. This technology will be improving the identification of microorganism in ocular infections, it takes some advantages as certainty and specificity, and however the cost is the major disadvantage.

Kaleta *et al.,* designed a study to evaluate the feasibility of the use of PCR/ESI-MS to identify microorganisms directly from blood culture bottles in the clinical microbiology laboratory [12]. The high concordance of the results of this technique with those of standard methods, particularly at the genus level, demonstrates that PCR/ESI-MS technique is capable of rapid‐ ly evaluating clinically complex specimens providing information as to the selection and ad‐ ministration of targeted antibiotics.

About eye microorganisms, Pedreira *et al.,*evaluated the efficacy of a prophylactic regimen of daily topical 0.5% moxifloxacin and 5% povidone-iodine in patients with Boston type I. The patients with the prophylactic regimen were sampled and analyzed by standard culture methods and by PCR/ESI-MS [13]. The molecular diagnostic approach using PCR/ESI-MS yielded data comparable with those obtained using standard microbiologic techniques. Be‐ cause of the high throughput nature and rapid results, the method might be a useful surveil‐ lance tool in patients with Boston type I.
