Dr. H N MadhavanApplication of nucleic acid based amplification techniques in bacterial...

Dr. H N Madhavan
Application of nucleic acid based amplification techniques in bacterial endophthalmitis – A review

Dr. H N Madhavan President, Vision Research Foundation Director & Prof of Microbiology, vSankara Nethralaya, Chennai, India

Dr. H N Madhavan
President, Vision Research Foundation
Director & Prof of Microbiology,
vSankara Nethralaya, Chennai, India

 

Title of the paper : Application of nucleic acid based amplification techniques in bacterial endophthalmitis – A review
Authors : Madhavan HN*, MBBS, MD, PhD, FAMS, FIC Path
Bagyalakshmi R MSMLT, PhD
Affiliation : ALarsen and Toubro Microbiology Research Centre
Kamal Nayan Bajaj Research Centre
Vision Research Foundation,
No.41, College Road, Chennai -600006
Title of the paper : Dr H N Madhavan MBBS, MD, PhD, FAMS, FIC Path
President –Vision Research Foundation
Larsen and Toubro Microbiology Research Centre
Kamal Nayan Bajaj Research Centre
Vision Research Foundation
No.41, College Road, Chennai -600006
Phone No: 91-044- 28270229, Fax No: 91 44 2825 4180
E mail ID: drhnm@snmail.org

 

ABSTRACT

Bacterial endophthalmitis is a serious intraocular infection which frequently results in vision loss. Conventional microbiological methods for microbiological characterization of eyes with suspected endophthalmitis lack sensitivity and are laborious and time-consuming . The application of molecular methods which are rapid, specific and sensitive enhances significantly laboratory confirmation of bacterial endophthalmitis PCR-based techniques, including post-PCR methods such as Restriction Fragment Length Polymorphism (RFLP), DNA probe hybridization and PCR based dHPLC and DNA sequencing have been successfully used for the diagnostic elucidation of clinically suspected bacterial endophthalmitis cases, showing promising application in the routine practice of ocular microbiology laboratories. This review focuses on the application of various nucleic acid based amplification techniques which proved useful in aetiological diagnosis of bacterial endophtahlmitis Endophthalmitis is an inflammation of intraocular tissues, which can result from the introduction of a bacterial agent in the posterior segment of the eye.1 It requires urgent medical attention because it is a potentially destructive condition for the eye, and even with therapeutic and surgical intervention, it can lead to partial or complete vision loss after a few days of inoculation. Regarding the acquisition mechanism, endophthalmitis is
classified as:
(1) postoperative (acute or delayed-onset), (2) posttraumatic, (3) endogenous and (4) miscellaneous (e.g., secondary to keratitis) . Postoperative endophthalmitis is the most common presentation and it is frequently associated with cataract surgery. Causative pathogens generally originate from the normal conjunctival and eyelid flora. 2,3 In the Endophthalmitis Vitrectomy Study (EVS), the recovery of gram-positive bacteria (94.2%) was far greater than gram-negative (6.5%) in acute postoperative endophthalmitis cases, where staphylococci, streptococci and enterococci were more frequently isolated. 4 Posttraumatic endophthalmitis is mainly caused by normal ocular flora and environmental isolates. Staphylococci and B. cereus are the most common isolates.5,6
Endogenous endophthalmitis is a less common category, corresponding to 2 to 8% of all endoph-thalmitis cases. As a consequence of hematogenous dissemination, specially in compromised hosts or intravenous drug abusers, any pathogen causing bacteremia/sepsis could gain access to the posterior segment of the eye resulting in infection. However, it has been shown that the most common bacterial isolates are Staphylococcus aureus, group B streptococci, Streptococcus pneumoniae, Listeria monocytogenes, Klebsiella spp., Escherichia coli, Pseudomonas aeruginosa, and Neisseria meningitidis.7 Use of molecular techniques for the diagnosis of infectious diseases. The introduction of molecular biology techniques in diagnostic medicine, such as the polymerase chain reaction (PCR), and its application in clinical microbiology, established a new era in the detection and characterization of microorganisms. PCR makes it possible to detect microorganisms that are difficult to detect using traditional microbiological methods and to reduce the time necessary for a confirmatory laboratory report, which is an important improvement in the characterization of microorganisms involved in serious and rapidly developing infections. Due to the high sensitivity of detection, even in cases that the microorganisms have already been killed, the utilization of PCR increases results for patients undergoing treatment should be rigorously evaluated.8 Although direct microscopy is the easiest and most rapid method to detect bacterial etiologies of endophthalmitis, its sensitivity is very low, with positive result varying from 4.2% to 46.5% for vitreous samples, which decreases further in aqueous fluid.9,10 More sensitive than microscopy, culture is considered “ the gold standard”. However, there have been no significant improvements in the yield of culture methods.11 Postoperative endophthalmitis is a vision threatening complication of cataract surgery and presents even further diagnostic challenges. The organisms are frequently present in low numbers, and they can be difficult to culture. Yields from diagnostic vitreous biopsies in this condition are less than 50%. The Endophthalmitis Vitrectomy Study reported culture yields of only 70% . Culture results are also slow to return, thus requiring patients be treated with broad-spectrum antibiotics for several days, even for relatively indolent bacteria. In cases where conventional techniques have low sensitivity, PCR, characterized by its high sensitivity and specificity, would be an ideal technique to detect bacterial pathogens in the eye. 12 All bacteria share common, highly repetitive DNA sequences for their 16S ribosomal RNA. By designing primers to these conserved 16S sequences, PCR can be performed on biopsy material from eyes with suspected endophthalmitis, with the results available within 6-8 hours. Therese et al. 10demonstrated the utility of this approach for culture-negative endophthalmitis Bacterial aetiology was established in 100% of culture-positive and 44% of culture-negative cases.. Lohmann et al 13 used 16S ribosomal primers as well as fungal PCR primers, along with culture and stain for 25 eyes with delayed-onset endophthalmitis. Aqueous culture and microscopy each had a 0% yield, but vitreous culture had a 24% yield in these patients. PCR of the aqueous yielded a diagnosis in 84% of the cases and PCR of a vitreous biopsy yielded a diagnosis in 92%. PCR thus has clear superiority to any other conventional techniques.

The need for PCR in ophthalmology

The rapid identification of the causal agent and early institution of a specific antibiotic treatment are associated with better visual outcomes in endophthalmitis. Microbiological endophthalmitis diagnosis is performed routinely by culture and microscopic examination of the vitreous (VH) and aqueous (AH) humors. However, conventional microbiological techniques are frequently insufficient to confirm clinical cases suspected of endophthalmitis, and the time necessary to obtain culture results can vary from 2 to 12 days. The consequences for eyes not treated can be dreadful, the lack or delay of a microbiological laboratory confirmation can lead to the wrong use of some ophthalmic pharmacological therapy with potential ocular toxicity, as well as the use of broad-spectrum antibiotics  for several days, being the correct antibiotic therapy
initiated only after the definitive identification of the causative microorganism.14,15

In clinical cases suspected of endophthalmitis, cultures show a positivity of about 25% to 56% . The low sensitivity of microbiological culture is due to various factors such as small quantity of specimen, fixation of microorganisms in solid surfaces (intraocular lens, lens fragments, capsule) and consequent decrease of cells in the vitreous/aqueous humor, use of antibiotics before the collection of clinical material and the presence of fastidious microorganisms such as agents causing endophthalmitis.16

Molecular detection and identification in bacterial endophthalmitis

Usually, the detection of bacteria of clinical interest by molecular biology laboratories is completed by the amplification of the 16S rRNA gene which codes for the small subunit of ribosomal RNA. Post-PCR analysis, utilizing hybridization with DNA probes, restriction analysis of PCR products by RFLP (restriction fragment length polymorphism), or direct sequencing of these products allow the species identification of the pathogen. A platform that utilizes mass spectrometry of ionized PCR products (including products of the 16S rRNA gene) was developed which is capable of integrating in the same system, identification of the microorganism, genotyping, and detection of virulence and antibiotic resistance genes.17
The 16S rRNA gene shows regions that are highly conserved among all bacterial species, and highly variable regions that permit the differentiation between species. Nine hypervariable regions are present while the 16S rRNA, which demonstrates the phylogenetic diversity among different bacterial species, allows their identification in most cases. By the utilization of universal primers that are complementary to the conserved regions flanking the variable regions of the gene, it is possible to carry out PCR directly on the vitreous and/or aqueous humors followed by sequencing of the amplified product. This allows the identification of the bacterial species causing the infection by alignment with sequences obtained from available databanks, usually BLAST/Genbank(http://www.ncbi.nlm.nih.gov/blast/). 18

The first study applied the PCR technique for the diagnosis of delayed postoperative endophthalmitis. Utilizing the nested PCR technique with universal primers and a combination of species-specific primers for Propionibacterium acnes, the method was capable of detecting bacterial DNA in 17 (74%) of 23 specimens of vitreous humor, while the culture was positive in 10 (43.5%) of these specimens. Bacterial DNA was detected by PCR in 8 specimens with a negative culture and in all cases where there was bacterial growth in culture.19

Soon after, various authors showed how the utilization of PCR for the direct detection of pathogens from aqueous and vitreous humor samples could impact effectively the diagnosis of bacterial endophthalmitis, mainly by increasing significantly the number of cases that were characterized microbiologically with the use of PCR but that were shown to be negative by microscopy or culture. 10,11,13,15,19,The nested PCR method was later used in specimens of vitreous (n=30) and aqueous (n=28) humor collected from 55 patients with clinical diagnosis of endophthalmitis(21). This study utilized universal primers for the bacterial 16S rRNA gene and a set of species-specific primers for P. acnes. Among the 58 samples included, 27 (46.5%) were positive by culture, 20
(34.5%) were positive for bacteria and 7 (12%) for fungi and 31 (53.5%) were negative after culture. On the other hand, PCR was capable of detecting bacterial DNA in 37 (63.8%) of the specimens tested, demonstrated 100% concordance with cases characterized microbiologically by culture, and was capable of detecting bacterial DNA in 44.7% of the specimens found negative for bacterial culture.10  Later studies were able to observe a sensitivity of detection of pathogens by PCR varying from 57% to 100% while sensitivity of the culture varied between 24% and 56%.11,13,14,15,20

A multicenter study published recently where PCR was applied for the diagnosis of bacterial endophthalmitis, included 100 patients with acute endophthalmitis following cataract surgery who were treated in 4 academic hospitals in France.21
The specimens were divided into two groups, those collected at the time of admission and before the initiation of antibiotic therapy (n=76 for AH and n=38 for VH) and those collected at a second time (n=53 for AH and n=57 for VH) only in patients who required another intravitreal injection of antibiotic or were submitted to vitrectomy via pars plana. The sensitivity of the culture and PCR for the specimens collected before the initiation of antibiotic therapy was 38.2% and 34.6% for aqueous humor and 54.0% and 56.7% for vitreous humor, respectively. In this group, PCR was positive in specimens of aqueous humor in 9.7% and vitreous humor in 40.0% of cases showing negative cultures. For the specimens of aqueous humor collected after the administration of antibiotics, the sensitivity of PCR decreased to 23.4% and culture to 13.6%. However, for the specimens of vitreous humor included in this second group, PCR was positive in 70.1% and culture in 8.8% of the cases, where PCR was positive in 73.0% of the cases with negative cultures. The detection of pathogens by PCR depends only on the presence of bacterial DNA in the specimen, whether or not there are viable microorganisms, characterizing one of the advantages of this technique in relation to culture, mostly in fluids where normal flora is not present.21

The use of PCR for bacterial detection in aqueous and vitreous humor from patients with suspected endophthalmitis has been described by several authors and the same is  represented in Table 1.

Table 1: Review of literature on infectious bacterial endophtahlmitis

Endophthalmitis literature Key points of the study References
Infectious endophthalmitis due to retention of foreign body Risk factors and prognostic indicators
of infectious endophthalmitis in eyes
with penetrating injury and retained
intraocular foreign body.
Thompson et al5 1993
Delayed post operative endophthalmitis 16SrRNA PCR targeting Propionibacterium acnes Hykin et al 19 1994
Rapid laboratory diagnosis of infectious endophthalmitis Molecular methods which identify conserved sequences from common causative microbes of endophthalmitis, or which can specify whether the organism is a bacterium or a fungus is an important diagnostic tool Kinnear et al2 1995
Endophthalmitis vitrectomy study Spectrum of aetiological agents causing endophthalmitis Han et al4 1996, Anand et al 37 2000 b
Molecular diagnosis of post operative endophthalmitis Application of 16SrRNA PCR to detect bacterial genome in intraocular specimens   Therese et al10    1998,Lohmann et al 131998, Knox et al22 1999, Van Gelder15 et al, 2001,Ugahary et al 23 2004, Taban et al 24 2005,Chiquet et al 21, 2008
Speciation of bacteria causing endophthalmitis 100% concordance between PCR and culture results. Application of PCR-RFLP to speciate bacteria  causing endophthalmitis Okhravi et al 14 2000
Molecular epidemiology of Staphylococcus aureus and
Enterococcus faecalis in endophthalmitis
Genomic DNA fingerprinting which identified five clonal types of Staphylococcus aureus and   clonally diverse population of Enterococcus faecalis causing endophthalmitis Booth et al 24 , 1998
Relationship between clinical presentation and visual outcome in infectious endophthalmitis Risk factors for poor visual outcome in post operative and post-traumatic endophthalmitis in South Central India Das et al 6 2005
Gram reaction of bacteria causing endophthalmitis PCR based hybridization using specific Gram positive and Gram negative probes Anand et al 11  2000a
Endophthalmitis literature Key points of the study References
Discrimination of Gram positive and Gram negative bacteria causing endophthalmitis Nested PCR protocol has been developed for the detection of and discrimination between14 species of gram-positive and -negative bacteria in samples of ocular fluids Carroll et al 25  2000
Propionibacterium acnes endophthalmitis Application of PCR on intraocular specimens for specific detectionof Propionibacterium acnes in intraocular specimens Therese et al10 1998, Buggage et al26  2003, Lai et al27 2006, Bispo et al 28 2009, Maalouf et al 29 2012
Neisseria meningitidis endogenous endophthalmitis Application of 16SrRNA PCR to establish the aetiology of endogenous endophthalmitis Kerkhoff et al 30 2004
Frelich et al 31 2003
Acute endogenous endophthalmitis caused by Bartonella henselae Application of 16SrRNA PCR to establish the aetiology of endogenous endophthalmitis Goldstein et al 32 2001
Endogenous endophthalmitis caused bySerratia marcescens Application of 16SrRNA PCR to establish the aetiology of endogenous endophthalmitis Sanchez et al33,2003
Simultaneous detection of eubacterial, P. acnes and panfungal genomes in intraocular specimens A novel multiplex PCR assay developed targeting the 16SrRNA ofbacterial and P. acnes genome and 28SrrNA of panfungal genome Bagyalakshmi et al34 2006
Review of endophthalmitis in India Conventional methods positivity ranges from 25 to 60% highlighting the increased sensitivity of  PCR Sharma et al 35 2010
Development of Real Time PCR to discriminate Gram positive and Gram negative bacteria causing intraocular infections       Multiplex Gram specific Taqman based PCR detected 31 clinicallyimportant pathogens   (20 Gram positive and 11 Gram negative bacteria) Bispo et al36 2011
Identification of bacteria in culture negative endophtahlmitis PCR based dHPLC to separate mixed bacterial genomes followedby DNA sequencing revealed novel bacteria(soil and environmental origin ) causing endophthalmitis Aarthi et al 39 2011
Viability of bacteria causing endophthalmitis Development of Reverse Transcriptase PCR to determine bacterial viability in intraocular specimens Aarthi et al40 2012

PCR for differentiation of Gram positive and Gram negative bacteria

A nested PCR protocol has been developed by Carroll et al 25  for the detection and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. The nested PCR consisted of  first-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). The authors reported a sensitivity between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative.

In an other  study by Anand et al11 PCR combined with DNA probe hybridization was evaluated to determine the Gram reaction of the bacterium in intraocular specimens from patients with infectious endophthalmitis. In this study 57 intraocular specimens – 17 aqueous humor and 40 vitreous fluid from 55 patients with clinically diagnosed infectious endophthalmitis and 25 control intraocular specimens from non-infectious ocular disorders (10 aqueous humor and 15 vitreous fluid) were evaluated by microscopy, culture and PCR-DNA probe hybridization to detect the Gram reaction of the bacterium.
PCR-DNA probe hybridization was specific and sensitive to detect 30 fg of both gram-positive and gram-negative bacterial DNA. None of the controls showed bacteria by microscopy, culture or PCR. Of the 57 intraocular specimens, conventional microbiological methods could detect a bacterial aetiology in 32 (56.1%), while PCR-DNA probe hybridization could detect 52 (91.2%) specimens. In bacteriologically positive specimens, there was absolute correlation of the Gram reaction between the results of smear and culture methods and PCR-DNA probe hybridization. Of the 25 bacteriologically negative specimens, 20 (80%) were positive by PCR-DNA probe hybridization, of which seven (35%) were gram-positive, 12 (60%) gram-negative and one (5%) positive by both. Glimpses into the  literature suggest that  PCR and DNA probe hybridization to determine the Gram reaction of the bacterium in intraocular fluids is a specific and sensitive method in the diagnosis of bacterial endophthalmitis.
Recently, Bispo et al 36 have developed a Real Time PCR assay to detect, quantitate and  discriminate Gram positive and Gram negative bacterial pathogens in intraocular specimens. The Real Time PCR assay developed detected 1pg/μl of E. coli  and
S. epidermidis DNA. This assay demonstrated good correlation with culture-proven results. With the use of these methods, bacterial detection was improved from 47.6%
to 95.3% demonstrating them to be sensitive , rapid test for quantitation of bacteria causing endophthalmitis.

Multiplex PCR in infectious endophthalmitis

A, novel multiplex PCR (mPCR) was developed by Bagyalakshmi et al 34 and applied on to intraocular specimens to detect eubacterial, P. acnes  genomes targeting 16SrRNA region and panfungal genome targeting 28SrRNA. mPCR was evaluated against individual uniplex PCR and results were on par with them. mPCR has several advantages over individual PCR: It is cost-effective as it reduces the total cost by one-third, offers considerable reduction in time required for generating reports and eliminates the need to use separate thermalcyclers for individual uPCR. The consumption of PCR reagents is minimized by carrying out the amplification in a single tube with no additional Taq polymerase required to amplify three genomes simultaneously. The annealing temperature for mPCR was determined based on the melting temperature, length (18-24 bases) and GC content of the primers. The annealing temperature of 60oC was optimal for amplifying all three infectious genomes. This novel thermal profile was designed to amplify the three infectious genomes by using 25 cycles for the first round and a reduction of five cycles for the second round without affecting the sensitivity and specificity of the procedure. Moreover, the time period needed for reporting the results was reduced by two hours compared to individual PCRs. By application of mPCR rapid detection was available within five hours of specimen collection as against 8 hours required for each uPCR. Furthermore, the results of mPCR correlated well with intraocular specimens which were culture positive for bacteria. These findings are comparable to our earlier findings10  and as well as to those published by Hykin et al  19,  Lohmann et al 20 and Carroll et al, 25 . This novel mPCR was useful in diagnosing the infectious agent in the minimal amount of template DNA available. Moreover, this novel mPCR has several advantages over uPCR such as, detection of two or more targets in a single tube, minimizing the use of PCR reagents, reduction in time and the need for a single PCR machine for amplification.
Application of PCR based denaturing High performance Liquid chromatography (dHPLC) in culture negative bacterial endophthalmitis
To establish the aetiology of infectious bacterial endophthalmitis Aarthi et al 39  have developed a PCR based dHPLC technique for rapid separation of mixed bacterial genomes.  In a study conducted on eubacterial PCR positive intraocular specimens a novel denaturing high performance liquid chromatography (dHPLC) based technique which allows rapid high resolution of PCR products was optimized. The application of this PCR based dHPLC approach  for direct detection  and identification from the eubacterial PCR amplified products of aqueous and vitreous aspirates from patients with endophthalmitis proved to be useful in initiating appropriate therapy. In this study 116 intraocular specimens were subjected to PCR based dHPLC  to separate mixed bacterial genomes and further identified by PCR based DNA sequencing. Sixty nine different bacteria were identified using dHPLC  based DNA sequencing of which predominant ones were Gram positive bacteria and cannot be cultured by conventional methods. Forty nine different bacteria detected in the study which were of soil and environmental origin were reported as the etiological agent of infectious endophthalmitis for the first time in literature .The different bacteria which were identified by dHPLC based DNA sequencing in the intraocular specimens that are of environmental origin and not known to cause any human infection is provided in Table 2 and the list of bacteria detected in intraocular specimens which were known to cause infections other than endophthalmitis is provided in Table 3

Table 2 : List of bacteria identified by dHPLC based DNA sequencing in the intraocular specimens that are of environmental origin not known to cause any human infections

BACTERIA

Vitreous aspirate (nos)

Aqueous aspirate (nos)

GRAM POSITIVE BACTERIA
Nonomuraea sp
Planotetrospora sp
Epulopiscium sp
Serinicoccus
Candidatus Rhodoluna lacicola
Crocinobacterium jejui
Pavlova lutheri
Krasilnikovia cinnamonea
Phycibacter  jejuensis
Leucobacter komagatae
Leifsonia pindariensis
Pimelobacter sp
Methanobrevibacter ruminantium
GRAM NEGATIVE BACTERIA
Bradyrhizobium sp
Methylocystis sp
Tepidiphilus sp
Empedobacter bravis
Ronia tepidophila
Azoarcus sp
Labrenzia sp
Phyllobacterium myrsinacearum
Delftia sp
Hydrogenophaga sp

 

1
1
1
1
1


1

1
1

1
1
1
1

1



 



1


1
1
1
1


1
1

1



1
1
1
1
1
1

List of bacteria identified by dHPLC based DNA sequencing in the intraocular specimens that are of environmental origin not known to cause any human infections

BACTERIA

Vitreous aspirate (nos)

Aqueous aspirate (nos)

GRAM POSITIVE BACTERIA
Nonomuraea sp
Planotetrospora sp
Epulopiscium sp
Serinicoccus
Candidatus Rhodoluna lacicola
Crocinobacterium jejui
Pavlova lutheri
Krasilnikovia cinnamonea
Phycibacter  jejuensis
Leucobacter komagatae
Leifsonia pindariensis
Pimelobacter sp
Methanobrevibacter ruminantium
GRAM NEGATIVE BACTERIA
Bradyrhizobium sp
Methylocystis sp
Tepidiphilus sp
Empedobacter bravis
Ronia tepidophila
Azoarcus sp
Labrenzia sp
Phyllobacterium myrsinacearum
Delftia sp
Hydrogenophaga sp

 

1
1
1
1
1


1

1
1

1
1
1
1

1



 



1


1
1
1
1


1
1

1



1
1
1
1
1
1

 

The results of this study indicated that  polymicrobial infections were found to be those known to cause human infections and also those not reported to have caused any human infections. These were of soil / environmental origin that were not reported to cause any human infections, dHPLC was used to separate the PCR products for further processing to identify these polymicrobial agents. An earlier study by Therese et al 41 conducted in collaboration with Alcon Research & Development, Fort Worth, TX, USA, showed similar results of presence of bacterial agents not known to cause human infections and particularly infectious endophthalmitis. The reason for the presence of these environmental bacteria in intraocular specimens was not known in most of the patients since no history of presurgical prophylactic management of the eyes were not been available prior to the collection of aqueous humor or the vitreous fluid. The applications of the WAVE microbial analysis system capitalizes on the ability of the system to unequivocally identify more than one bacterial species in a given sample.

Reverse Transcriptase PCR to determine bacterial viability

Though there are many published literature on the use of qualitative and quantitative PCRs on the detection and quantitation of bacterial genome, RNA based assays are not developed to aid direct detection of the presence of live infectious bacteria. Such assays would be suggestive of  active bacterial  infection to promote the rationale use of antibiotics. There are various studies where 16SrRNA gene has been used  to correlate the cellular viability of the bacterium42,43,44,45,46,47 Previous study conducted by Aarthi et al 39 in culture negative endophthalmitis revealed novel bacteria causing endophthalmitis. To determine whether these bacteria have the ability to thrive in the intraocular environment a novel Reverse Transcriptase PCR assay was developed and applied on intraocular specimens. Thirty five intraocular specimens (19 vitreous fluid and 16 aqueous humor) collected from patients with typical infectious endophthalmitis were subjected to conventional and molecular microbiological investigations. Culture negative, eubacterial genome PCR positive intraocular specimens were subjected to denaturing high performance liquid chromatography (dHPLC) for separation of mixed genomes and subsequently identified by PCR based DNA sequencing. In parallel, RT-PCR was performed to detect the presence of viable bacteria in intraocular specimens.
Among 35 intraocular specimens, single bacterial genome was detected in 9 (25.7%) and two or more genomes in 26 (74.28%) intraocular specimens. Eubacterial genome was detected by RT-PCR in 29 (82.85%) specimens. PCR based dHPLC followed by PCR based DNA sequencing revealed the presence of 65 bacterial genomes in 35 intraocular specimens. Since the intraocular specimens included in the study revealed the presence of mixed bacterial genomes, PCR based dHPLC was performed to separate the mixed amplicons. PCR based DNA sequencing revealed the presence of bacteria known to cause endophthalmitis as well as bacteria which are of environmental origin not known to cause endophthalmitis. Interestingly five novel  bacterial genera which were not known to cause endophthalmitis comprising of 2 Gram positive –Terrabacter, Facklamia species and 3 Gram negative – Xylella, Duganella, Synechococcus species were identified by PCR based dHPLC. This is suggestive of the organism’s ability to survive in the intraocular environment and produce disease progression through their elaborated virulence mechanisms. These organisms prove to be emerging pathogens causing infectious endophthalmitis. The use of dHPLC based identification of bacterial genome would be extremely useful in a diagnostic set up since the presence of mixed genomes can be separated easily and identified. Further, timely institution of specific antibiotics would prove useful in better management of infectious endophthalmitis.

Further investigations are required to monitor the mode of entry of these  bacteria into the eye, their interaction with the immune cells in the ocular environment and their virulence mechanisms adapted. Developing such studies would aid in formulating newer and effective antibacterial agents which would prove useful in successful clinical management of infectious endophthalmitis.

Future perspectives

The utilization of molecular methods has been explored in ophthalmology field, especially for the diagnosis of endophthalmitis, because they represent a diagnostic approach with a marked increase in positivity in relation to conventional methods. Due to the small quantity of specimen collected, and consequently less quantity of microorganisms detected in the aqueous and vitreous humors, the nested PCR technique is indicated for the diagnosis of endophthalmitis, by increasing substantially the sensitivity of bacterial DNA detection. The amount of bacterial DNA that can be detected by nested PCR can be as low as 1 fg. Therefore, the real-time PCR technology could be a potential technique for use in ophthalmology. A more rapid result in relation to the etiology of the intraocular inflammatory process, permitting the introduction of an early more specific antibiotic therapy, improving the prognosis, reducing the toxicity of treatments, and preventing the inappopriate use of antibiotics, which would thereby minimize the potential emergence of resistant bacterial strains. In addition, due to the
possibility of quantifying bacterial DNA present in the specimen, its application can contribute to the differentiation between true infection and a possible contamination of the anterior chamber by microorganisms present in the conjunctival flora in patients recently submitted to intraocular surgery.

Further studies are required to gather data on susceptibility of bacteria causing intraocular infection. In culture negative cases of endophthalmitis, no information can be obtained with regard to the drug resistance mechanism of  the infecting bacteria. Molecualr assays targeting the drug resistance genes expressed by the spectrum of bacterial agents causing endophthalmitis needs to be investigated further. Such studies would be extremely useful in developing multiplex assays to detect the presence of bacteria along with the  expression of  resistance genes.

Conclusion

An early and accurate diagnosis of endophthalmitis is an essential factor for therapeutic success. Molecular biology techniques applied in the laboratory elucidation of
bacterial endophthalmitis have been shown to be effective, and the confirmation or elimination of the involvement of microorganisms as the causative agents, with greater sensitivity, makes it possible to determine a better clinical management for these
cases. The use of such techniques increases substantially the laboratory confirmation of suspected endophthalmitis cases, with the special advantage to detect microorganisms that are difficult or impossible to culture.

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