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A SYBR Green based multiplex Real-Time PCR assay for rapid detection and differentiation of ocular bacterial pathogens
Journal of Microbiological Methods 171 (2020) 105875
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A SYBR Green based multiplex Real-Time PCR assay for rapid detection and differentiation of ocular bacterial pathogens
T
Deepthi KrishnanNair Geethaa, Balaji Sivaramana, Ram Rammohanb, Narendran Venkatapathyb, ⁎ Prabagaran Solai Ramatchandiranea, a b
Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, Tamil Nadu, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Ocular infections Staphylococcus aureus Streptococcus pneumonia Pseudomonas aeruginosa Multiplex real-time PCR Melting curve analysis
Purpose: Ocular bacterial pathogenesis is a serious sight threatening infection due to several bacterial species like Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa which are predominant. It is necessary to expedite diagnosis of pathogens for early treatment. Hence, a SYBR Green based multiplex RealTime PCR assay coupled with melting curve analysis has been developed for rapid detection and differentiation of Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa in a single reaction. Methods: The assay was designed for simultaneous detection and differentiation of pathogens based on their distinct melting curve. The analytical specificity, sensitivity and reproducibility of the assay were examined using various reference strains. Clinical validation was carried out with 100 ocular samples collected from patients suffering from ocular infections. Result: Each reaction tested for the targets individually generated three non overlapping melting curves with well alienated peaks corresponding to each gene. Among 100 ocular samples tested, 40 samples diagnosed with positive results in RT-PCR. Thus assay showed 100% specificity with high sensitivity and reproducibility. Conclusion: The developed assay consistently established as a rapid and accurate diagnosis of ocular bacterial pathogens compared to the conventional laboratory techniques. Such precise method would aid greatly in clinical management of devastating ocular infections.
1. Introduction Ocular bacterial infections together with distressing visual consequences are considered as more prevalent eye complication worldwide. Among them, foremost ocular diseases namely of conjunctivitis, keratitis and endophthalmitis are generally associated with either monobacterial or polybacterial infections. These infections may transpire through pathogens that are found in ocular surface due to incessant contact with outer environment and their further intra ocular access during trauma or surgery. In some cases, hematogenous dissemination of pathogens from other sites of infection may also occur. Recent developments in molecular techniques based on DNA sequence facilitate better understanding of bacterial profile in various ocular infections at elevated resolution. Even though diverse bacteria are causing ocular infections, in particular Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa are predominant in majority of the patients leading to visual threat (Teweldemedhin et al., 2017). Therefore, the need for prompt diagnosis of pathogens is
⁎
mandatory to administer appropriate treatment immediate after the onset of symptoms. Despite the limitations, traditional culture and stain based methods are regarded as gold standards that are widely used in routine diagnostic protocols (Sharma, 2012). However, most often time delayed diagnosis or substantial misidentification of pathogens lead to surrogate treatment of suspected infections based on their concomitant evidences (Hong et al., 2015). In the recent past, several PCR based techniques have been developed as diagnostic tool aspiring for rapid and precise detection when compared to conventional methods. Furthermore, to ensure precision and cost reduction, multiplex PCR approaches targeting more than one pathogen simultaneously are prevalently attempted (Aslam et al., 2003; Kawasaki et al., 2005; Omiccioli et al., 2009; Prabagaran et al., 2017). Most such approaches necessitate time consuming post PCR analysis and further risks of false positive detection due to amplicon contamination. To address these concerns, LAMP and real-time PCR (RT-PCR) techniques were developed for detection and discrimination of
Corresponding author at: Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu 641 046, India. E-mail address: [email protected] (P. Solai Ramatchandirane).
https://doi.org/10.1016/j.mimet.2020.105875 Received 20 October 2019; Received in revised form 16 February 2020; Accepted 17 February 2020 Available online 19 February 2020 0167-7012/ © 2020 Published by Elsevier B.V.
Journal of Microbiological Methods 171 (2020) 105875
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2.4. Validation of primer specificity and PCR optimization
pathogens. Among them, though economic, LAMP technique can detect only one pathogen at a time which limits its multiplex assay. Conversely, RT-PCR based approach is regarded appropriate for multiplex assay that measures the fluorescent signals generated either with TaqMan probe or SYBR Green dyes. While considering as a routine advanced and reliable diagnostic tool, fluorescent labeled TaqMan probe based RT-PCR was deemed impracticable due to its expensive assay cost. To overcome this aspect, the present study was focused on SYBR Green based multiplex RT-PCR assay (Whitcombe et al., 1998) coupled with melting curve analysis for rapid detection and differentiation of S. aureus, S. pneumoniae and P. aeruginosa in a single reaction (Tong and Giffard, 2012).
All primer pairs were screened for their specificity and checked for possibility of cross amplification against all the available type strains. Initially the primers were validated by performing SYBR Green based simplex RT-PCR reaction followed by melting curve analysis. The amplified products were confirmed by agarose gel electrophoresis along with 100 bp DNA ladder (Thermo Scientific, Waltham, Massachusetts, United States) as a molecular marker and their corresponding Tm values. Those primers with non specific amplification and nearly closer melting temperatures were eliminated. Thus primers with 100% specificity and non overlapping melting temperature (Tm) values alone were selected for further experiments. Subsequently, the multiplex reactions were optimized by testing at different concentrations with pooled set of primers in single reaction.
2. Material and methods 2.1. Bacterial strains
2.5. Multiplex real-time PCR The following strains were incorporated in the study as standards including Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (NRRL B-4268), Staphylococcus hominis (MTCC 4435), Streptococcus pneumoniae (ATCC 49619), Streptococcus viridians (clinical isolate), Streptococcus pyogenes (clinical isolate), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (MTCC 3231), Escherichia coli (MTCC 443), Acinetobacter baumannii (ATCC 17978), Bacillus subtilis (MTCC 2391). All strains were maintained as glycerol stocks in nutrient broth which served as positive control for the assays.
The SYBR Green based multiplex RT-PCR assay was carried out in single tube containing 10 μl of SYBR Green qPCR Master Mix (Takara), 1 μl of each forward and reverse primers (5pM), 2 μl of DNA template (ranging from 1 to 50 ng/reaction) and finally the reaction was made up to 20 μl using nuclease free water. PCRs were performed in RT-PCR cycler (Applied Biosystems' 7500 RT-PCR system, Foster City, CA, USA) with an initial denaturation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s. Immediately after the PCR amplification, the melting curve analysis was performed at temperature ranges from 60 °C to 95 °C by raising 0.5 °C at each sec. To avoid contamination from external sources, PCR reaction without genomic DNA was included as negative control in each run. Also the human β-globin gene was amplified separately as an endogenous positive control for all 100 clinical samples to confirm that they possessed DNA of sufficient quality for amplification (Bispo et al., 2011).
2.2. Bacterial DNA extraction Genomic DNA was extracted from the type strains using QIAamp DNA mini kit (Qiagen, Germany). All the strains were subjected to 16S rRNA gene sequencing towards confirmation of the respective species. The extracted DNA was stored at −20 °C for performing further PCR experiments.
2.3. Primer design
2.6. Analytical sensitivity of the assay
The conserved regions of S. aureus, S. pneumoniae and P. aeruginosa genomes were retrieved from GenBank database and the target regions were selected in silico based on melting temperature predicted by uMelt software (Dwight et al., 2011). Optimal regions were theoretically identified for PCR amplification based on melting temperature of the product and GC content. A set of species specific primers that uniquely amplify S. aureus, S. pneumoniae and P. aeruginosa were designed using primer 3plus software (http://www.bioinformatics.nl/cgi-bin/ primer3plus/primer3plus.cgi). Subsequently, three additional primer pairs that were proven from earlier studies were also incorporated for in silico melting curve prediction (Table 1).
The reaction efficiency and sensitivity of each primer pair in the developed multiplex RT-PCR assay were determined by amplifying plasmids carrying known concentration of DNA. The amplified products of S. aureus, S. pneumoniae and P. aeruginosa were separately ligated into pJET1.2/blunt cloning vector (Thermo Scientific, Waltham, Massachusetts, United States) for propagation in Escherichia coli DH5α competent cells. The isolated plasmid carrying target DNA were diluted 10 fold from concentration ranging 10 ng to 1 pg using nuclease free water. Singleplex reaction was carried out in triplicates using 1 μl of serially diluted plasmid. The PCR efficiency for each primer pair was calculated from the slope of standard curve.
Table 1 List of oligonucleotide primers. Target Species
Target gene
Primers
Sequence
Amplification size
Source/ Reference
S. aureus
nuc
This study
lytA
141
This study
P. aeruginosa
lasR
195
This study
S. aureus
nuc
154
(Kilic et al., 2010)
S. pneumoniae
lytA
275
(Messmer et al., 1997)
P. aeruginosa
oprL
5′-AACAGTATATAGTGCAACTTCAA-3′ 5′-CTTTGTCAAACTCGACTTCAA-3′ 5′-CACTCAACTGGGAATCCGC-3′ 5′-CCAGGCACCATTATCAACAGG-3′ 5′ GTTCGGCCTGTTGCCTAAG 3′ 5′ AACTCGTGCTGCTTTCGC 3′ 5′-GTTGCTTAGTGTTAACTTTAGTTGTA-3′ 5′-AATGTCGCAGGTTCTTTATGTAATTT-3 5′-ATCCAAAAGACAAGTTTGAGA-3′ 5′-CTGGATAAAGGCATTTGATAC-3′ 5′-CGAGTACAACATGGCTCTGG-3′ 5′-ACCGGACGCTCTTTACCATA-3′
248
S. pneumoniae
Sa-nucl-F Sa-nucl-B Sp-Alyt-F Sp-Alyt-R Pa-lasR-F Pa-lasR-R Sa-nuc-F Sa-nuc-R Sp-lyt-F Sp-lyt-R Pa-oprL-F Pa-oprL-R
117
(Feizabadi et al., 2010)
2
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for each triplicate was assayed (Bispo et al., 2018). The developed assays showed competence over DNA concentration ranging from 10 ng to 1 pg per reaction. The reaction efficiency of primer pairs was determined as 105% for nuc gene (Fig. 2), 110% for lytA gene (Fig. 3) and 107% for oprL genes (Fig. 4).
2.7. Intra and inter test reproducibility assay Reproducibility of the SYBR Green based RT-PCR assay was measured by checking Ct values and Tm variations of genomic DNA in two different concentrations (10 ng and 10 pg) based on the triplicate reaction performed in singleplex format for all samples on the same run and independently in three consecutive runs. The data obtained from intra and inter tests were articulated as mean and standard deviation. In addition, the percentage of coefficient variation was also calculated for triplicates of each template concentration.
3.3. Intra and inter assay variability To determine the reproducibility of the developed assay, both intra and inter assay percentage coefficient of variation (CV) of Ct and Tm values for all three samples were analyzed. The intra assay variation was determined from each sample in two different concentrations simultaneously using triplicates. Inter assay variability was obtained from the same test on three different days. Generally, intra assay % CVs extent expected to be < 10 are acceptable with inter assay % CVs of < 15. The developed assay showed intra assay variability of Ct measurement ranged from 0.5 to 5% and inter-assay % CV varied from 2 to 5% for all three targets. However, both intra and inter assay variability for Tm measurement was found to be between 0 and 0.1% (Table 2).
2.8. Assay validation using clinical samples To validate the realistic efficacy and accuracy of the SYBR Green based multiplex RT-PCR assay, a total of 100 ocular samples were tested for the diagnosis of these three pathogens. These ocular samples were collected from patients with keratitis (36 corneal scrape), postoperative endophthalmitis (24 Aqueous humor (AH) & 9 Vitreous fluid (VF), posttraumatic endophthalmitis (2AH & 1 VF), endogenous endophthalmitis (1AH) and preoperative cases (24 conjunctival swab) presented at Aravind Eye Hospital, Coimbatore, India. The research was carried out in accordance with the tenets of the Declaration of Helsinki. All the 100 samples were primarily subjected to routine microbiological identification of infectious organisms by culturing them on various agar plates and broths (blood agar, chocolate agar, potato dextrose agar, brain-heart infusion broth and thioglycollate broth). Blood agar and chocolate agar plates were incubated aerobically with 5% carbon dioxide at 37 °C for 24 to 48 h. Thioglycollate and brain-heart infusion broth were incubated aerobically at 37 °C for 24 to 48 h. Potato dextrose agar plates were incubated at 27 °C for a maximum of 3 weeks. Plates showed visible colonies were recognized by Analytical Profile Index strip Method (Moore et al., 2017). Subsequently, the plates without any growth were considered as culture negative and discarded. Besides intraocular samples collected from five non infected individuals served as control which were processed similar to that of other samples. DNA was extracted from all the samples using QIAamp DNA mini kit (Qiagen, Germany). Each DNA was subjected to PCR in triplicate with all three primer pairs specific for S. aureus, S. pneumoniae and P. aeruginosa.
3.4. Clinical validation During microbiological identification of 100 samples collected from ocular patients, 36 showed presence of one or more among S. aureus, S. pneumoniae or P. aeruginosa infection. Other bacterial or fungal infections were established in 32 samples. Remaining 32 samples were microbiologically diagnosed as culture negative. In order to clinically validate the developed multiplex RT-PCR assay for detection of S. aureus, S. pneumoniae and P. aeruginosa, 100 ocular samples were analyzed (Table 3). The developed assay diagnosed positive results in 40 samples tested. The assay effectively detected pathogens in corneal scraping (n = 23), aqueous fluid (n = 10), vitreous humor (n = 4) and conjunctival swab (n = 3). Among the 40 positive cases, S. aureus, S. pneumoniae and P. aeruginosa were detected in 13, 14 and 12 samples respectively as single pathogen and remaining one sample with both S. aureus and S. pneumoniae detection. The mean Ct value for S. aureus detection assorted from 7.21 to 19.21. S. pneumoniae infection was diagnosed from a mean Ct value of 8.11 to 22.66. Likewise, P. aeruginosa mean Ct values were diverged from 6.17 to 24.41. All those positively diagnosed samples generated Tm values corresponding to respective gene amplification. Those positive samples were further confirmed by resolving the amplified products through agarose gel electrophoresis (Supplementary file). Remaining 60 samples yielded negative results in SYBR Green based multiplex RT-PCR assay in accordance with the result of culture method. We also analyzed analytical sensitivity, analytical specificity, positive predictive value (PPV) and negative predictive value (NPV) of our real-time PCR assay as compared to the culture based identification in hospital. While, the real-time PCR based identification showed 100% for all four diagnostic parameters, culture based diagnosis showed 90%, 100%, 100%, and 93.8%, respectively. Hence, the developed multiplex real-time PCR based technology proved to be exceptionally functional diagnostic routine for ocular infections when compared with conventional methods.
3. Results 3.1. Primer specificity and assay optimization Preliminary experiments evaluated the specificity of selected primer pairs with all candidate organisms and checked for cross reactivity of Sa-nucl and Pa-lasR primer pairs which produced non specific amplification. Even though rest of the primer pairs Sa-nuc, Sp-lyt, Sp-Alyt and Pa-oprL showed 100% specificity, Sp-Alyt was eliminated since melting curve was closer to the Sa-nuc gene. All the three targets tested individually with Sa-nuc, Sp-lyt, and Pa-oprL primer pairs generated three different melting curves with well alienated peaks equivalent to each gene (Fig. 1). The average melting temperature of amplicons generated from the targeted genes corresponding to S. aureus, S. pneumoniae and P. aeruginosa were 75, 80.80 and 85.70 respectively. These values were approximately in accordance with the Tm value predicted using uMelt software. In compliance with the singleplex amplification and melting curve analysis, multiplex reaction with all the targets and primers were optimized to diagnose target bacteria, if any, in a single assay with their corresponding Tm values.
4. Discussion Diagnosis of ocular infections usually relies on conventional microbiological methods, which is more time consuming and laborious procedure. Hence, development of rapid, sensitive and reliable tools for diagnosis of pathogens causing ocular infection is deemed to be of major concern associated with timely management of visual impediments. PCR based diagnosis makes it possible to detect pathogens directly from samples with more specificity and sensitivity than conventional methods. However, most of the PCR based diagnosis follows
3.2. Analytical sensitivity of the assay Standard curves were obtained for each gene individually with a regression coefficient of approximately 0.99. The lowest titre with identical melting curve for expected Tm value with reliable Ct values 3
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Fig. 1. Melting curve profiles and corresponding amplification analysis of nuc, lytA and oprL genes from type strains Staphylococcus aureus (SA), Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) respectively following SYBR Green based real-time singleplex PCR. Lane M: 100 bp DNA Ladder. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Primer efficiency and validation of nuc gene targeting Staphylococcus aureus. Standard curve (a), melting profile (b) and agarose gel (c) for various plasmid dilutions. Lane M: 100 bp DNA Ladder; Lane 1–5: varying concentrations of plasmid from 10 ng to 1 pg.
the
conventional singleplex or even multiplex reactions that necessitate post PCR procedures for identification which is also time consuming and may lead to carryover contaminations. Although TaqMan based multiplex RT-PCR available as a diagnostic tool for pathogen detection,
multiplexing capability of such method is constrained by the requirement of different filters to detect each fluorescent agent besides its elevated expenditure (Bispo et al., 2018). Hence, in this study 4
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Fig. 3. Primer efficiency and validation of lytA gene targeting Streptococcus pneumoniae. Standard curve (a), melting profile (b) and agarose gel (c) for various plasmid dilutions. Lane M: 100 bp DNA Ladder; Lane 1–5: varying concentrations of plasmid from 10 ng to 1 pg.
pathogen causing visual threats (Pinna et al., 2009; Fleiszig and Evans, 2002; Weed et al., 2013). An investigation on bacterial keratitis over five years identified S. pneumoniae contributing 38% of infections followed by P. aeruginosa (29%) and S. aureus (4%) (Mascarenhas et al., 2014). Based on these backgrounds, the candidate organisms were chosen to unravel ocular infections. Among the battery of primer pairs tested, three individual primer sets that yielded Tm with a difference of > 5 °C with 100% specificity and high reproducibility were chosen. Specificity of the assay was analyzed by monitoring melting curve of amplified PCR products. The developed assay identified pathogens with their appropriate melting curve using DNA extracted from keratitis, endophthalmitis and conjunctival swab samples. Precise melting temperatures corresponding to S. aureus, S. pneumoniae, and P. aeruginosa at 75 ± 0.5 °C, 88.80 ± 0.5 °C and 85.7 ± 0.5 °C respectively were repeated consistently in all triplicates in each runs. Another merit of this assay is that it can be detected within three hours including DNA isolation, while culture techniques necessitate minimum of 24 h for conclusive decision. Validation of assay with 36 corneal scraping samples collected from keratitis patients showed melting peak in 23 cases, which is in perfect agreement with conventional culture results. Besides, the importance of multiplexing PCR was demonstrated by sample COR34, which was identified as Gram Positive cocci in conventional diagnostic laboratory detection. However, SYBR Green based multiplex RT-PCR assay showed melting peak corresponding to both S. aureus and S. pneumoniae confirming the presence of polybacterial infection leading to keratitis. A total of 40 samples collected from endophthalmitis patients showed positive amplification in 10 samples with SYBR Green based multiplex RT-PCR assay. Conversely, while a postoperative endophthalmitis sample (POE14) showed the presence of Viridans Streptococci through culture based identification, RT-PCR assay diagnosed it as S. pneumoniae with a mean Ct of 17.64 (Table 3) which upon
we developed SYBR Green based multiplex RT-PCR which offers timely detection of pathogens in a consistent and economic means. Several studies reported occurrence of Gram Positive cocci, in particular S. aureus followed by S. pneumoniae and Coagulase negative staphylococci causing ocular infections in South India. Along with Staphylococci and Streptococci, other organisms such as Corynebacterium, Haemophilus, Moraxella and Neisseria were also involved in various ocular infections. Among gram-negative bacilli, Pseudomonas aeruginosa prevalence was reported in many cases followed by Klebsiella spp., Enterobacter spp., and Citrobacter spp. (Reddy et al., 2010; Ramesh et al., 2010). However, the bacterial etiology of ocular infections may diverge with geographical site based on the confined population. Streptococcus pneumoniae was reported as predominant pathogen causing corneal infection in Tiruchirapalli and Madurai, whereas Pseudomonas aeruginosa was leading in Coimbatore (Bharathi et al., 2010). Hence, it is necessary to understand the bacterial etiology of confined population to create a rational preference for instigation of suitable therapy. As far as, S. aureus, S. pneumoniae and P. aeruginosa are most extensive isolates from ocular infections according to various reports, which necessitate their rapid detection. S. aureus is the most virulent bacterium in Staphylococcus genus possessing multifactorial pathogenecity against host system (Zecconi and Scali, 2013). S. aureus often causes keratitis and endophthalmitis associated with visual acuity or even blindness (Major Jr et al., 2010; O'Neill et al., 2014; Chang et al., 2015; Sadaka et al., 2017; O'Callaghan, 2018). While S. pneumoniae is significantly pathogenic and fastidious, Streptococcus spp. contribute ocular infections with aggressive clinical course and poor visual prognosis owing to their potential pneumococcal virulence factors (Miller et al., 2004; Benton and Marquart, 2018; Clere-Jehl et al., 2019). Similarly, P. aeruginosa also poses a large repertoire of cell associated and extracellular pathogenic mechanisms that make it as most common 5
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Fig. 4. Primer efficiency and validation of oprL gene targeting Pseudomonas aeruginosa. Standard curve (a), melting profile (b) and agarose gel (c) for various plasmid dilutions. Lane M: 100 bp DNA Ladder; Lane 1–5: varying concentrations of plasmid from 10 ng to 1 pg. Table 2 Validation of real-time PCR primers through intra and inter assay coefficient. Concentration per reaction
Intra-assay
a
Inter-assay
Ct
nuc 10 ng 0.01 ng lytA 10 ng 0.01 ng oprL 10 ng 0.01 ng
Tm
b
Ct
Tm
Mean
SD
CV (%)
Mean (°C)
SD
CV (%)
Mean
SD
CV (%)
Mean (°C)
SD
CV (%)
7.42 13.44
0.36 0.17
5.00 1.00
75.23 75.18
0.09 0.04
0.10 0.07
7.75 14.80
0.49 0.45
5.00 3.70
75.03 75.00
0.04 0.00
0.07 0.00
7.79 13.48
0.03 0.11
0.50 5.00
81.16 81.06
0.04 0.09
0.07 0.10
7.17 12.00
0.27 0.25
4.00 2.60
81.20 81.00
0.04 0.00
0.07 0.00
7.87 13.95
0.08 0.45
1.00 5.00
85.80 86.06
0.00 0.04
0.00 0.07
8.18 14.71
0.36 0.24
5.00 2.00
85.90 86.10
0.09 0.09
0.10 0.10
a- Calculated from 3 technical replicates. b- Calculated from reaction tested over 3 consecutive days. Ct- cycles threshold; SD - standard deviation; CV- coefficient of variation.
Thus the developed SYBR Green based multiplex RT-PCR assay could be an alternative tool to examine the infectious pathogens accessible in this panel with a benefit of rapid and accurate detection than the conventional laboratory techniques. On the other hand, it can also be simplified as a conventional multiplex PCR in case of resource limited laboratories where detection of pathogens can be through amplicon analysis using agarose gel electrophoresis. Overall, the developed SYBR Green based multiplex RT-PCR assay established rapid and accurate diagnosis of ocular bacterial pathogens in an economic manner than the expensive hybridization probe based
sequencing also confirmed as S. pneumoniae (99.91%) proving the specificity of our developed assay. Conjunctiva remains as primary cause for further infection to cornea and other internal ocular regions due to its massive contact with surrounding environment. With respect to 24 conjunctival swab samples, two showed melting peak corresponding to S. aureus (CON12, CON21) and one (CON6) with P. aeruginosa. Thus, 12.5% of the samples showed positive results for both culture and PCR signifying such diagnosis as a preoperative procedure on conjunctival swab samples before any ocular surgery. 6
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Table 3 Culture identification and SYBR Green based multiplex real-time PCR assay results from 100 ocular patients. Case
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 P41 P42 P43 P44 P45 P46 P47 P48 P49 P50 P51 P52 P53 P54 P55 P56 P57 P58 P59 P60 P61 P62 P63 P64 P65 P66 P67 P68 P69 P70 P71 P72
Sample Name
COR1 COR2 COR3 COR4 COR5 COR6 COR7 COR8 COR9 COR10 COR11 COR12 COR13 COR14 COR15 COR16 COR17 COR18 COR19 COR20 COR21 COR22 COR23 COR24 COR25 COR26 COR27 COR28 COR29 COR30 COR31 COR32 COR33 COR34 COR35 COR36 POE1 POE2 POE3 POE4 POE5 POE6 POE7 POE8 POE9 POE10 POE11 POE12 POE13 POE14 POE15 POE16 POE17 POE18 POE19 POE20 POE21 POE22 POE23 POE24 PTE1 PTE2 EE1 EE2 EE3 EE4 POE25 POE26 POE27 POE28 POE29 POE30
Culture identification
Culture Negative Streptococcus pneumoniae Coagulase negative staphylococci Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Streptococcus pneumoniae Gram negative bacilli Culture Negative Streptococcus pneumoniae Pseudomonas aeruginosa Culture Negative Streptococcus pneumoniae Streptococcus pneumoniae Pseudomonas aeruginosa Viridans streptococci Culture Negative Pseudomonas aeruginosa Streptococcus pneumoniae MRSA Enterobacter aerogenes Staphylococcus aureus Pseudomonas aeruginosa Staphylococcus aureus Coagulase negative staphylococci Streptococcus pneumoniae Culture Negative Gram Positive Cocci Culture Negative Bacillus sp. Staphylococcus aureus Gram Positive Cocci Gram Positive Cocci Coagulase negative Culture Negative Klebsiella sp. Culture Negative Coagulase negative staphylococci Coagulase negative staphylococci Culture Negative Pseudomonas sp. Viridans streptococci Streptococcus pneumoniae Streptococcus pneumoniae Culture Negative Coagulase negative staphylococci Coagulase negative staphylococci Viridans streptococci Coagulase negative staphylococci Pseudomonas aeruginosa Streptococcus pneumoniae Bacillus sp. Viridans streptococci Viridans streptococci Streptococcus pneumoniae Staphylococcus aureus Culture Negative Culture Negative Streptococcus pneumoniae Coagulase negative staphylococci Aspergillus fumigatus Haemophilus sp. Pseudomonas aeruginosa Staphylococcus aureus Pseudomonas aeruginosa Culture Negative Bacillus sp. Culture Negative Coagulase negative staphylococci Streptococcus pneumoniae
CT Mean
− 22.66 − 22.28 15.39 24.41 20.88 14.69 8.87 − − 11.77 8.88 − 11.48 11.26 9.19 − − 9.74 12.20 9.25 − 10.30 15.91 10.76 − 18.74 − 11.30 − − 10.11 18.78 19.21 − − − − − − − − − 15.15 13.98 − − − 17.64 − 7.76 15.20 − − − 10.02 7.90 − − 8.56 − − − 6.17 10.45 6.49 − − − − 8.11
Tm Mean (°C)
RT−PCR Result
SA
SP
PA
SA
SP
PA
− − − − − − − − − − − − − − − − − − − − − 75.40 − 75.20 − 75.00 − − − 75.00 − − 75.00 74.80 74.90 − − − − − − − − − − − − − − − − − − − − − − 74.70 − − − − − − − 74.90 − − − − − −
− 81.00 − − − − − − 81.00 − − 81.10 − − 81.00 81.00 − − − − 80.80 − − − − − − 80.50 − − − − − 80.00 − − − − − − − − − − 80.70 80.80 − − − 80.56 − − 80.80 − − − 81.10 − − − 81.20 − − − − − − − − − − 81.00
− − − 85.80 85.90 85.80 85.70 86.10 − − − − 85.80 − − − 85.50 − − 85.80 − − − − 85.50 − − − − − − − − − − − − − − − − − − − − − − − − − − 85.50 − − − − − − − − − − − − 85.70 − 86.10 − − − − −
− − − − − − − − − − − − − − − − − − − − − + − + − + − − − + − − + + + − − − − − − − − − − − − − − − − − − − − − − + − − − − − − − + − − − − − −
− + − − − − − − + − − + − − + + − − − − + − − − − − − + − − − − − + − − − − − − − − − − + + − − − + − − + − − − + − − − + − − − − − − − − − − +
− − − + + + + + − − − − + − − − + − − + − − − − + − − − − − − − − − − − − − − − − − − − − − − − − − − + − − − − − − − − − − − − + − + − − − − −
(continued on next page) 7
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Table 3 (continued) Case
P73 P74 P75 P76 P77 P78 P79 P80 P81 P82 P83 P84 P85 P86 P87 P88 P89 P90 P91 P92 P93 P94 P95 P96 P97 P98 P99 P100
Sample Name
POE31 POE32 POE33 PTE3 CON1 CON2 CON3 CON4 CON5 CON6 CON7 CON8 CON9 CON10 CON11 CON12 CON13 CON14 CON15 CON16 CON17 CON18 CON19 CON20 CON21 CON22 CON23 CON24
Culture identification
Coagulase negative staphylococci Culture Negative Pseudomonas aeruginosa Staphylococcus aureus Coagulase negative staphylococci Culture Negative Culture Negative Culture Negative Culture Negative Pseudomonas aeruginosa Culture Negative Gram negative bacilli Culture Negative Gram negative bacilli Culture Negative Staphylococcus aureus Culture Negative Culture Negative Coagulase negative staphylococci Culture Negative Culture Negative Culture Negative Culture Negative Culture Negative Staphylococcus aureus Culture Negative Culture Negative Culture Negative
CT Mean
− − 7.99 9.27 − − − − − 12.50 − − − − − 13.00 − − − − − − − − 7.21 − − −
Tm Mean (°C)
RT−PCR Result
SA
SP
PA
SA
SP
PA
− − − 75.10 − − − − − − − − − − − 74.90 − − − − − − − − 75.20 − − −
− − − − − − − − − − − − − − − − − − − − − − − − − − − −
− − 85.90 − − − − − − 85.20 − − − − − − − − − − − − − − − − − −
− − − + − − − − − − − − − − − + − − − − − − − − + − − −
− − − − − − − − − − − − − − − − − − − − − − − − − − − −
− − + − − − − − − + − − − − − − − − − − − − − − − − − −
Case P1 to P36 is Corneal Scraping Samples (COR), Case P37 to P66 include Vitreous Fluid from Postoperative Endophthalmitis (POE), Posttraumatic Endophthalmitis (PTE), and Endogenous Endophthalmitis (EE) patients. Case P67 to P76 comprises Aqueous humor from POE and PTE patients. Case P77 to P100 contains Conjunctival Swab Samples (CON).
qPCR. Hence, it can be recommended as a routine diagnostic tool in eye hospitals for early diagnosis towards appropriate treatment against devastating ocular infections. Additionally, the rapidity, sensitivity and inexpensiveness of this technique would emphasize its practicability in other infections as well.
infectious diseases. Interdisc. Perspect. Infect. Dis. 2018. Bharathi, M.J., Ramakrishnan, R., Shivakumar, C., Meenakshi, R., Lionalraj, D., 2010 Oct 16. Etiology and antibacterial susceptibility pattern of community-acquired bacterial ocular infections in a tertiary eye care hospital in South India. Indian J. Ophthalmol. 58, 497–507. Bispo, P.J., de Melo, G.B., Hofling-Lima, A.L., Pignatari, A.C., 2011 Feb 1. Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays. Invest. Ophthalmol. Vis. Sci. 52 (2), 873–881. Bispo, P.J., Davoudi, S., Sahm, M.L., Ren, A., Miller, J., Romano, J., Sobrin, L., Gilmore, M.S., 2018 Jan 1. Rapid detection and identification of uveitis pathogens by qualitative multiplex real-time PCR. Invest. Ophthalmol. Vis. Sci. 59 (1), 582–589. Chang, V.S., Dhaliwal, D.K., Raju, L., Kowalski, R.P., 2015 Jun. Antibiotic resistance in the treatment of Staphylococcus aureus keratitis: a 20-year review. Cornea. 34 (6), 698. Clere-Jehl, R., Merdji, H., Derhy, D., Helms, J., 2019 Feb 27. Blindness as an uncommon complication of Streptococcus pneumoniae systemic infection. Intensive Care Med. 45 (2), 263–265. Dwight, Z., Palais, R., Wittwer, C.T., 2011 Feb 7. uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics. 27 (7), 1019–1020. Feizabadi, M.M., Majnooni, A., Nomanpour, B., Fatolahzadeh, B., Raji, N., Delfani, S., Habibi, M., Asadi, S., Parvin, M., 2010 Dec 1. Direct detection of Pseudomonas aeruginosa from patients with healthcare associated pneumonia by real-time PCR. Infect. Genet. Evol. 10 (8), 1247–1251. Fleiszig, S.M., Evans, D.J., 2002 Sep. The pathogenesis of bacterial keratitis: studies with Pseudomonas aeruginosa. Clin. Exp. Optom. 85 (5), 271–278. Hong, B.K., Lee, C.S., Van Gelder, R.N., Garg, S.J., 2015 May. Emerging techniques for pathogen discovery in endophthalmitis. Curr. Opin. Ophthalmol. 26 (3), 221. Kawasaki, S., Horikoshi, N., Okada, Y., Takeshita, K., Sameshima, T., Kawamoto, S., 2005 Mar. Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157: H7 in meat samples. J. Food Prot. 68 (3), 551–556. Kilic, A., Muldrew, K.L., Tang, Y.W., Basustaoglu, A.C., 2010 Apr 1. Triplex real-time polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci and determination of methicillin resistance directly from positive blood culture bottles. Diagn. Microbiol. Infect. Dis. 66 (4), 349–355. Major Jr., J.C., Engelbert, M., Flynn Jr., H.W., Miller, D., Smiddy, W.E., Davis, J.L., 2010. Staphylococcus aureus endophthalmitis: antibiotic susceptibilities, methicillin resistance, and clinical outcomes. Am J. Ophthalmol. 149, 278–283 e271. Mascarenhas, J., Lalitha, P., Prajna, N.V., Srinivasan, M., Das, M., D’Silva, S.S., Oldenburg, C.E., Borkar, D.S., Esterberg, E.J., Lietman, T.M., Keenan, J.D., 2014 Jan 1. Acanthamoeba, fungal, and bacterial keratitis: a comparison of risk factors and
Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest No conflicting relationship exists for any author. Acknowledgments The author K. G. Deepthi acknowledges University Grant Commission, New Delhi for her BSR fellowship (UGC/BSR/No.F.25.1/ 2014-15/7-25/2007). The authors are thankful to UGC (Govt. of India) for the financial support to establish infrastructure in the Department of Biotechnology grant vide UGC/SAP/No.F.3-20/2013. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mimet.2020.105875. References Aslam, M., Hogan, J., Smith, K.L., 2003 Jun 1. Development of a PCR-based assay to detect Shiga toxin-producing Escherichia coli, listeria monocytogenes, and Salmonella in milk. Food Microbiol. 20 (3), 345–350. Benton, A.H., Marquart, M.E., 2018. The role of pneumococcal virulence factors in ocular
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Brahmadathan, K.N., Mani, M., 2017 Aug 1. Molecular diagnosis of Salmonella typhi and its virulence in suspected typhoid blood samples through nested multiplex PCR. J. Microbiol. Methods 139, 150–154. Ramesh, S., Ramakrishnan, R., Bharathi, M.J., Amuthan, M., Viswanathan, S., 2010 Jun 12. Prevalence of bacterial pathogens causing ocular infections in South India. Indian J. Pathol. Microbiol. 53, 281–286. Reddy, A., Garg, P., Alam, M., et al., 2010. Comparison of in vitro susceptibilities of grampositive cocci isolated from ocular infections against the second and fourth generation quinolones at a tertiary eye care Centre in South India. Eye. 24, 170–174. Sadaka, A., Durand, M.L., Sisk, R., Gilmore, M.S., 2017 Jan 2. Staphylococcus aureus and its bearing on ophthalmic disease. Ocul. Immunol. Inflamm. 25 (1), 111–121. Sharma, S., 2012 Feb. Diagnosis of infectious diseases of the eye. Eye. 26 (2), 177. Teweldemedhin, M., Gebreyesus, H., Atsbaha, A.H., Asgedom, S.W., Saravanan, M., 2017 Dec. Bacterial profile of ocular infections: a systematic review. BMC Ophthalmol. 17 (1), 212. Tong, S.Y., Giffard, P.M., 2012 Nov 1. Microbiological applications of high-resolution melting analysis. J. Clin. Microbiol. 50 (11), 3418–3421. Weed, M.C., Rogers, G.M., Kitzmann, A.S., Goins, K.M., Wagoner, M.D., 2013 Jun 24. Vision Loss After Contact Lens-Related Pseudomonas Keratitis. Eye Rounds org. Whitcombe, D., Newton, C.R., Little, S., 1998 Dec 1. Advances in approaches to DNAbased diagnostics. Curr. Opin. Biotechnol. 9 (6), 602–608. Zecconi, A., Scali, F., 2013 Feb 1. Staphylococcus aureus virulence factors in evasion from innate immune defenses in human and animal diseases. Immunol. Lett. 150 (1–2), 12–22.
clinical features. Am J. Ophthalmol. 157 (1), 56–62. Messmer, T.A., Whitney, C.G., Fields, B.S., 1997 Jun 1. Use of polymerase chain reaction to identify pneumococcal infection associated with hemorrhage and shock in two previously healthy young children. Clin. Chem. 43 (6), 930–935. Miller, J.J., Scott, I.U., Flynn Jr., H.W., Smiddy, W.E., Corey, R.P., Miller, D., 2004 Aug 1. Endophthalmitis caused by Streptococcus pneumoniae. Am J. Ophthalmol. 138 (2), 231–236. Moore, S.G., Ericsson, A.C., Poock, S.E., Melendez, P., Lucy, M.C., 2017 Jun 1. 1. Hot topic: 16S rRNA gene sequencing reveals the microbiome of the virgin and pregnant bovine uterus. J. Dairy sci. 100 (6), 4953–4960. O’Callaghan, R., 2018 Mar. The pathogenesis of Staphylococcus aureus eye infections. Pathogens. 7 (1), 9. Omiccioli, E., Amagliani, G., Brandi, G., Bruce, I.J., Magnani, M., 2009 Jun. Simultaneous direct detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk samples by magnetic extraction and multiplex PCR. J. Rapid Methods Autom. Microbiol. 17 (2), 195–213. O’Neill, E.C., Yeoh, J., Fabinyi, D.C., Cassidy, D., Vajpayee, R.B., Allen, P., Connell, P.P., 2014 Sep 1. Risk factors, microbial profiles and prognosis of microbial keratitis-associated endophthalmitis in high-risk eyes. Graefes Arch. Clin. Exp. Ophthalmol. 252 (9), 1457–1462. Pinna, A., Usai, D., Sechi, L.A., et al., 2009. An outbreak of post-cataract surgery endophthalmitis caused by Pseudomonas aeruginosa. Ophthalmol 116, 2321–2326 e2324. Prabagaran, S.R., Kalaiselvi, V., Chandramouleeswaran, N., Deepthi, K.N.,
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A SYBR Green based multiplex Real-Time PCR assay for rapid detection and differentiation of ocular bacterial pathogens
T
Deepthi KrishnanNair Geethaa, Balaji Sivaramana, Ram Rammohanb, Narendran Venkatapathyb, ⁎ Prabagaran Solai Ramatchandiranea, a b
Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, Tamil Nadu, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Ocular infections Staphylococcus aureus Streptococcus pneumonia Pseudomonas aeruginosa Multiplex real-time PCR Melting curve analysis
Purpose: Ocular bacterial pathogenesis is a serious sight threatening infection due to several bacterial species like Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa which are predominant. It is necessary to expedite diagnosis of pathogens for early treatment. Hence, a SYBR Green based multiplex RealTime PCR assay coupled with melting curve analysis has been developed for rapid detection and differentiation of Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa in a single reaction. Methods: The assay was designed for simultaneous detection and differentiation of pathogens based on their distinct melting curve. The analytical specificity, sensitivity and reproducibility of the assay were examined using various reference strains. Clinical validation was carried out with 100 ocular samples collected from patients suffering from ocular infections. Result: Each reaction tested for the targets individually generated three non overlapping melting curves with well alienated peaks corresponding to each gene. Among 100 ocular samples tested, 40 samples diagnosed with positive results in RT-PCR. Thus assay showed 100% specificity with high sensitivity and reproducibility. Conclusion: The developed assay consistently established as a rapid and accurate diagnosis of ocular bacterial pathogens compared to the conventional laboratory techniques. Such precise method would aid greatly in clinical management of devastating ocular infections.
1. Introduction Ocular bacterial infections together with distressing visual consequences are considered as more prevalent eye complication worldwide. Among them, foremost ocular diseases namely of conjunctivitis, keratitis and endophthalmitis are generally associated with either monobacterial or polybacterial infections. These infections may transpire through pathogens that are found in ocular surface due to incessant contact with outer environment and their further intra ocular access during trauma or surgery. In some cases, hematogenous dissemination of pathogens from other sites of infection may also occur. Recent developments in molecular techniques based on DNA sequence facilitate better understanding of bacterial profile in various ocular infections at elevated resolution. Even though diverse bacteria are causing ocular infections, in particular Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa are predominant in majority of the patients leading to visual threat (Teweldemedhin et al., 2017). Therefore, the need for prompt diagnosis of pathogens is
⁎
mandatory to administer appropriate treatment immediate after the onset of symptoms. Despite the limitations, traditional culture and stain based methods are regarded as gold standards that are widely used in routine diagnostic protocols (Sharma, 2012). However, most often time delayed diagnosis or substantial misidentification of pathogens lead to surrogate treatment of suspected infections based on their concomitant evidences (Hong et al., 2015). In the recent past, several PCR based techniques have been developed as diagnostic tool aspiring for rapid and precise detection when compared to conventional methods. Furthermore, to ensure precision and cost reduction, multiplex PCR approaches targeting more than one pathogen simultaneously are prevalently attempted (Aslam et al., 2003; Kawasaki et al., 2005; Omiccioli et al., 2009; Prabagaran et al., 2017). Most such approaches necessitate time consuming post PCR analysis and further risks of false positive detection due to amplicon contamination. To address these concerns, LAMP and real-time PCR (RT-PCR) techniques were developed for detection and discrimination of
Corresponding author at: Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu 641 046, India. E-mail address: [email protected] (P. Solai Ramatchandirane).
https://doi.org/10.1016/j.mimet.2020.105875 Received 20 October 2019; Received in revised form 16 February 2020; Accepted 17 February 2020 Available online 19 February 2020 0167-7012/ © 2020 Published by Elsevier B.V.
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2.4. Validation of primer specificity and PCR optimization
pathogens. Among them, though economic, LAMP technique can detect only one pathogen at a time which limits its multiplex assay. Conversely, RT-PCR based approach is regarded appropriate for multiplex assay that measures the fluorescent signals generated either with TaqMan probe or SYBR Green dyes. While considering as a routine advanced and reliable diagnostic tool, fluorescent labeled TaqMan probe based RT-PCR was deemed impracticable due to its expensive assay cost. To overcome this aspect, the present study was focused on SYBR Green based multiplex RT-PCR assay (Whitcombe et al., 1998) coupled with melting curve analysis for rapid detection and differentiation of S. aureus, S. pneumoniae and P. aeruginosa in a single reaction (Tong and Giffard, 2012).
All primer pairs were screened for their specificity and checked for possibility of cross amplification against all the available type strains. Initially the primers were validated by performing SYBR Green based simplex RT-PCR reaction followed by melting curve analysis. The amplified products were confirmed by agarose gel electrophoresis along with 100 bp DNA ladder (Thermo Scientific, Waltham, Massachusetts, United States) as a molecular marker and their corresponding Tm values. Those primers with non specific amplification and nearly closer melting temperatures were eliminated. Thus primers with 100% specificity and non overlapping melting temperature (Tm) values alone were selected for further experiments. Subsequently, the multiplex reactions were optimized by testing at different concentrations with pooled set of primers in single reaction.
2. Material and methods 2.1. Bacterial strains
2.5. Multiplex real-time PCR The following strains were incorporated in the study as standards including Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (NRRL B-4268), Staphylococcus hominis (MTCC 4435), Streptococcus pneumoniae (ATCC 49619), Streptococcus viridians (clinical isolate), Streptococcus pyogenes (clinical isolate), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (MTCC 3231), Escherichia coli (MTCC 443), Acinetobacter baumannii (ATCC 17978), Bacillus subtilis (MTCC 2391). All strains were maintained as glycerol stocks in nutrient broth which served as positive control for the assays.
The SYBR Green based multiplex RT-PCR assay was carried out in single tube containing 10 μl of SYBR Green qPCR Master Mix (Takara), 1 μl of each forward and reverse primers (5pM), 2 μl of DNA template (ranging from 1 to 50 ng/reaction) and finally the reaction was made up to 20 μl using nuclease free water. PCRs were performed in RT-PCR cycler (Applied Biosystems' 7500 RT-PCR system, Foster City, CA, USA) with an initial denaturation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s. Immediately after the PCR amplification, the melting curve analysis was performed at temperature ranges from 60 °C to 95 °C by raising 0.5 °C at each sec. To avoid contamination from external sources, PCR reaction without genomic DNA was included as negative control in each run. Also the human β-globin gene was amplified separately as an endogenous positive control for all 100 clinical samples to confirm that they possessed DNA of sufficient quality for amplification (Bispo et al., 2011).
2.2. Bacterial DNA extraction Genomic DNA was extracted from the type strains using QIAamp DNA mini kit (Qiagen, Germany). All the strains were subjected to 16S rRNA gene sequencing towards confirmation of the respective species. The extracted DNA was stored at −20 °C for performing further PCR experiments.
2.3. Primer design
2.6. Analytical sensitivity of the assay
The conserved regions of S. aureus, S. pneumoniae and P. aeruginosa genomes were retrieved from GenBank database and the target regions were selected in silico based on melting temperature predicted by uMelt software (Dwight et al., 2011). Optimal regions were theoretically identified for PCR amplification based on melting temperature of the product and GC content. A set of species specific primers that uniquely amplify S. aureus, S. pneumoniae and P. aeruginosa were designed using primer 3plus software (http://www.bioinformatics.nl/cgi-bin/ primer3plus/primer3plus.cgi). Subsequently, three additional primer pairs that were proven from earlier studies were also incorporated for in silico melting curve prediction (Table 1).
The reaction efficiency and sensitivity of each primer pair in the developed multiplex RT-PCR assay were determined by amplifying plasmids carrying known concentration of DNA. The amplified products of S. aureus, S. pneumoniae and P. aeruginosa were separately ligated into pJET1.2/blunt cloning vector (Thermo Scientific, Waltham, Massachusetts, United States) for propagation in Escherichia coli DH5α competent cells. The isolated plasmid carrying target DNA were diluted 10 fold from concentration ranging 10 ng to 1 pg using nuclease free water. Singleplex reaction was carried out in triplicates using 1 μl of serially diluted plasmid. The PCR efficiency for each primer pair was calculated from the slope of standard curve.
Table 1 List of oligonucleotide primers. Target Species
Target gene
Primers
Sequence
Amplification size
Source/ Reference
S. aureus
nuc
This study
lytA
141
This study
P. aeruginosa
lasR
195
This study
S. aureus
nuc
154
(Kilic et al., 2010)
S. pneumoniae
lytA
275
(Messmer et al., 1997)
P. aeruginosa
oprL
5′-AACAGTATATAGTGCAACTTCAA-3′ 5′-CTTTGTCAAACTCGACTTCAA-3′ 5′-CACTCAACTGGGAATCCGC-3′ 5′-CCAGGCACCATTATCAACAGG-3′ 5′ GTTCGGCCTGTTGCCTAAG 3′ 5′ AACTCGTGCTGCTTTCGC 3′ 5′-GTTGCTTAGTGTTAACTTTAGTTGTA-3′ 5′-AATGTCGCAGGTTCTTTATGTAATTT-3 5′-ATCCAAAAGACAAGTTTGAGA-3′ 5′-CTGGATAAAGGCATTTGATAC-3′ 5′-CGAGTACAACATGGCTCTGG-3′ 5′-ACCGGACGCTCTTTACCATA-3′
248
S. pneumoniae
Sa-nucl-F Sa-nucl-B Sp-Alyt-F Sp-Alyt-R Pa-lasR-F Pa-lasR-R Sa-nuc-F Sa-nuc-R Sp-lyt-F Sp-lyt-R Pa-oprL-F Pa-oprL-R
117
(Feizabadi et al., 2010)
2
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for each triplicate was assayed (Bispo et al., 2018). The developed assays showed competence over DNA concentration ranging from 10 ng to 1 pg per reaction. The reaction efficiency of primer pairs was determined as 105% for nuc gene (Fig. 2), 110% for lytA gene (Fig. 3) and 107% for oprL genes (Fig. 4).
2.7. Intra and inter test reproducibility assay Reproducibility of the SYBR Green based RT-PCR assay was measured by checking Ct values and Tm variations of genomic DNA in two different concentrations (10 ng and 10 pg) based on the triplicate reaction performed in singleplex format for all samples on the same run and independently in three consecutive runs. The data obtained from intra and inter tests were articulated as mean and standard deviation. In addition, the percentage of coefficient variation was also calculated for triplicates of each template concentration.
3.3. Intra and inter assay variability To determine the reproducibility of the developed assay, both intra and inter assay percentage coefficient of variation (CV) of Ct and Tm values for all three samples were analyzed. The intra assay variation was determined from each sample in two different concentrations simultaneously using triplicates. Inter assay variability was obtained from the same test on three different days. Generally, intra assay % CVs extent expected to be < 10 are acceptable with inter assay % CVs of < 15. The developed assay showed intra assay variability of Ct measurement ranged from 0.5 to 5% and inter-assay % CV varied from 2 to 5% for all three targets. However, both intra and inter assay variability for Tm measurement was found to be between 0 and 0.1% (Table 2).
2.8. Assay validation using clinical samples To validate the realistic efficacy and accuracy of the SYBR Green based multiplex RT-PCR assay, a total of 100 ocular samples were tested for the diagnosis of these three pathogens. These ocular samples were collected from patients with keratitis (36 corneal scrape), postoperative endophthalmitis (24 Aqueous humor (AH) & 9 Vitreous fluid (VF), posttraumatic endophthalmitis (2AH & 1 VF), endogenous endophthalmitis (1AH) and preoperative cases (24 conjunctival swab) presented at Aravind Eye Hospital, Coimbatore, India. The research was carried out in accordance with the tenets of the Declaration of Helsinki. All the 100 samples were primarily subjected to routine microbiological identification of infectious organisms by culturing them on various agar plates and broths (blood agar, chocolate agar, potato dextrose agar, brain-heart infusion broth and thioglycollate broth). Blood agar and chocolate agar plates were incubated aerobically with 5% carbon dioxide at 37 °C for 24 to 48 h. Thioglycollate and brain-heart infusion broth were incubated aerobically at 37 °C for 24 to 48 h. Potato dextrose agar plates were incubated at 27 °C for a maximum of 3 weeks. Plates showed visible colonies were recognized by Analytical Profile Index strip Method (Moore et al., 2017). Subsequently, the plates without any growth were considered as culture negative and discarded. Besides intraocular samples collected from five non infected individuals served as control which were processed similar to that of other samples. DNA was extracted from all the samples using QIAamp DNA mini kit (Qiagen, Germany). Each DNA was subjected to PCR in triplicate with all three primer pairs specific for S. aureus, S. pneumoniae and P. aeruginosa.
3.4. Clinical validation During microbiological identification of 100 samples collected from ocular patients, 36 showed presence of one or more among S. aureus, S. pneumoniae or P. aeruginosa infection. Other bacterial or fungal infections were established in 32 samples. Remaining 32 samples were microbiologically diagnosed as culture negative. In order to clinically validate the developed multiplex RT-PCR assay for detection of S. aureus, S. pneumoniae and P. aeruginosa, 100 ocular samples were analyzed (Table 3). The developed assay diagnosed positive results in 40 samples tested. The assay effectively detected pathogens in corneal scraping (n = 23), aqueous fluid (n = 10), vitreous humor (n = 4) and conjunctival swab (n = 3). Among the 40 positive cases, S. aureus, S. pneumoniae and P. aeruginosa were detected in 13, 14 and 12 samples respectively as single pathogen and remaining one sample with both S. aureus and S. pneumoniae detection. The mean Ct value for S. aureus detection assorted from 7.21 to 19.21. S. pneumoniae infection was diagnosed from a mean Ct value of 8.11 to 22.66. Likewise, P. aeruginosa mean Ct values were diverged from 6.17 to 24.41. All those positively diagnosed samples generated Tm values corresponding to respective gene amplification. Those positive samples were further confirmed by resolving the amplified products through agarose gel electrophoresis (Supplementary file). Remaining 60 samples yielded negative results in SYBR Green based multiplex RT-PCR assay in accordance with the result of culture method. We also analyzed analytical sensitivity, analytical specificity, positive predictive value (PPV) and negative predictive value (NPV) of our real-time PCR assay as compared to the culture based identification in hospital. While, the real-time PCR based identification showed 100% for all four diagnostic parameters, culture based diagnosis showed 90%, 100%, 100%, and 93.8%, respectively. Hence, the developed multiplex real-time PCR based technology proved to be exceptionally functional diagnostic routine for ocular infections when compared with conventional methods.
3. Results 3.1. Primer specificity and assay optimization Preliminary experiments evaluated the specificity of selected primer pairs with all candidate organisms and checked for cross reactivity of Sa-nucl and Pa-lasR primer pairs which produced non specific amplification. Even though rest of the primer pairs Sa-nuc, Sp-lyt, Sp-Alyt and Pa-oprL showed 100% specificity, Sp-Alyt was eliminated since melting curve was closer to the Sa-nuc gene. All the three targets tested individually with Sa-nuc, Sp-lyt, and Pa-oprL primer pairs generated three different melting curves with well alienated peaks equivalent to each gene (Fig. 1). The average melting temperature of amplicons generated from the targeted genes corresponding to S. aureus, S. pneumoniae and P. aeruginosa were 75, 80.80 and 85.70 respectively. These values were approximately in accordance with the Tm value predicted using uMelt software. In compliance with the singleplex amplification and melting curve analysis, multiplex reaction with all the targets and primers were optimized to diagnose target bacteria, if any, in a single assay with their corresponding Tm values.
4. Discussion Diagnosis of ocular infections usually relies on conventional microbiological methods, which is more time consuming and laborious procedure. Hence, development of rapid, sensitive and reliable tools for diagnosis of pathogens causing ocular infection is deemed to be of major concern associated with timely management of visual impediments. PCR based diagnosis makes it possible to detect pathogens directly from samples with more specificity and sensitivity than conventional methods. However, most of the PCR based diagnosis follows
3.2. Analytical sensitivity of the assay Standard curves were obtained for each gene individually with a regression coefficient of approximately 0.99. The lowest titre with identical melting curve for expected Tm value with reliable Ct values 3
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Fig. 1. Melting curve profiles and corresponding amplification analysis of nuc, lytA and oprL genes from type strains Staphylococcus aureus (SA), Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) respectively following SYBR Green based real-time singleplex PCR. Lane M: 100 bp DNA Ladder. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Primer efficiency and validation of nuc gene targeting Staphylococcus aureus. Standard curve (a), melting profile (b) and agarose gel (c) for various plasmid dilutions. Lane M: 100 bp DNA Ladder; Lane 1–5: varying concentrations of plasmid from 10 ng to 1 pg.
the
conventional singleplex or even multiplex reactions that necessitate post PCR procedures for identification which is also time consuming and may lead to carryover contaminations. Although TaqMan based multiplex RT-PCR available as a diagnostic tool for pathogen detection,
multiplexing capability of such method is constrained by the requirement of different filters to detect each fluorescent agent besides its elevated expenditure (Bispo et al., 2018). Hence, in this study 4
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Fig. 3. Primer efficiency and validation of lytA gene targeting Streptococcus pneumoniae. Standard curve (a), melting profile (b) and agarose gel (c) for various plasmid dilutions. Lane M: 100 bp DNA Ladder; Lane 1–5: varying concentrations of plasmid from 10 ng to 1 pg.
pathogen causing visual threats (Pinna et al., 2009; Fleiszig and Evans, 2002; Weed et al., 2013). An investigation on bacterial keratitis over five years identified S. pneumoniae contributing 38% of infections followed by P. aeruginosa (29%) and S. aureus (4%) (Mascarenhas et al., 2014). Based on these backgrounds, the candidate organisms were chosen to unravel ocular infections. Among the battery of primer pairs tested, three individual primer sets that yielded Tm with a difference of > 5 °C with 100% specificity and high reproducibility were chosen. Specificity of the assay was analyzed by monitoring melting curve of amplified PCR products. The developed assay identified pathogens with their appropriate melting curve using DNA extracted from keratitis, endophthalmitis and conjunctival swab samples. Precise melting temperatures corresponding to S. aureus, S. pneumoniae, and P. aeruginosa at 75 ± 0.5 °C, 88.80 ± 0.5 °C and 85.7 ± 0.5 °C respectively were repeated consistently in all triplicates in each runs. Another merit of this assay is that it can be detected within three hours including DNA isolation, while culture techniques necessitate minimum of 24 h for conclusive decision. Validation of assay with 36 corneal scraping samples collected from keratitis patients showed melting peak in 23 cases, which is in perfect agreement with conventional culture results. Besides, the importance of multiplexing PCR was demonstrated by sample COR34, which was identified as Gram Positive cocci in conventional diagnostic laboratory detection. However, SYBR Green based multiplex RT-PCR assay showed melting peak corresponding to both S. aureus and S. pneumoniae confirming the presence of polybacterial infection leading to keratitis. A total of 40 samples collected from endophthalmitis patients showed positive amplification in 10 samples with SYBR Green based multiplex RT-PCR assay. Conversely, while a postoperative endophthalmitis sample (POE14) showed the presence of Viridans Streptococci through culture based identification, RT-PCR assay diagnosed it as S. pneumoniae with a mean Ct of 17.64 (Table 3) which upon
we developed SYBR Green based multiplex RT-PCR which offers timely detection of pathogens in a consistent and economic means. Several studies reported occurrence of Gram Positive cocci, in particular S. aureus followed by S. pneumoniae and Coagulase negative staphylococci causing ocular infections in South India. Along with Staphylococci and Streptococci, other organisms such as Corynebacterium, Haemophilus, Moraxella and Neisseria were also involved in various ocular infections. Among gram-negative bacilli, Pseudomonas aeruginosa prevalence was reported in many cases followed by Klebsiella spp., Enterobacter spp., and Citrobacter spp. (Reddy et al., 2010; Ramesh et al., 2010). However, the bacterial etiology of ocular infections may diverge with geographical site based on the confined population. Streptococcus pneumoniae was reported as predominant pathogen causing corneal infection in Tiruchirapalli and Madurai, whereas Pseudomonas aeruginosa was leading in Coimbatore (Bharathi et al., 2010). Hence, it is necessary to understand the bacterial etiology of confined population to create a rational preference for instigation of suitable therapy. As far as, S. aureus, S. pneumoniae and P. aeruginosa are most extensive isolates from ocular infections according to various reports, which necessitate their rapid detection. S. aureus is the most virulent bacterium in Staphylococcus genus possessing multifactorial pathogenecity against host system (Zecconi and Scali, 2013). S. aureus often causes keratitis and endophthalmitis associated with visual acuity or even blindness (Major Jr et al., 2010; O'Neill et al., 2014; Chang et al., 2015; Sadaka et al., 2017; O'Callaghan, 2018). While S. pneumoniae is significantly pathogenic and fastidious, Streptococcus spp. contribute ocular infections with aggressive clinical course and poor visual prognosis owing to their potential pneumococcal virulence factors (Miller et al., 2004; Benton and Marquart, 2018; Clere-Jehl et al., 2019). Similarly, P. aeruginosa also poses a large repertoire of cell associated and extracellular pathogenic mechanisms that make it as most common 5
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Fig. 4. Primer efficiency and validation of oprL gene targeting Pseudomonas aeruginosa. Standard curve (a), melting profile (b) and agarose gel (c) for various plasmid dilutions. Lane M: 100 bp DNA Ladder; Lane 1–5: varying concentrations of plasmid from 10 ng to 1 pg. Table 2 Validation of real-time PCR primers through intra and inter assay coefficient. Concentration per reaction
Intra-assay
a
Inter-assay
Ct
nuc 10 ng 0.01 ng lytA 10 ng 0.01 ng oprL 10 ng 0.01 ng
Tm
b
Ct
Tm
Mean
SD
CV (%)
Mean (°C)
SD
CV (%)
Mean
SD
CV (%)
Mean (°C)
SD
CV (%)
7.42 13.44
0.36 0.17
5.00 1.00
75.23 75.18
0.09 0.04
0.10 0.07
7.75 14.80
0.49 0.45
5.00 3.70
75.03 75.00
0.04 0.00
0.07 0.00
7.79 13.48
0.03 0.11
0.50 5.00
81.16 81.06
0.04 0.09
0.07 0.10
7.17 12.00
0.27 0.25
4.00 2.60
81.20 81.00
0.04 0.00
0.07 0.00
7.87 13.95
0.08 0.45
1.00 5.00
85.80 86.06
0.00 0.04
0.00 0.07
8.18 14.71
0.36 0.24
5.00 2.00
85.90 86.10
0.09 0.09
0.10 0.10
a- Calculated from 3 technical replicates. b- Calculated from reaction tested over 3 consecutive days. Ct- cycles threshold; SD - standard deviation; CV- coefficient of variation.
Thus the developed SYBR Green based multiplex RT-PCR assay could be an alternative tool to examine the infectious pathogens accessible in this panel with a benefit of rapid and accurate detection than the conventional laboratory techniques. On the other hand, it can also be simplified as a conventional multiplex PCR in case of resource limited laboratories where detection of pathogens can be through amplicon analysis using agarose gel electrophoresis. Overall, the developed SYBR Green based multiplex RT-PCR assay established rapid and accurate diagnosis of ocular bacterial pathogens in an economic manner than the expensive hybridization probe based
sequencing also confirmed as S. pneumoniae (99.91%) proving the specificity of our developed assay. Conjunctiva remains as primary cause for further infection to cornea and other internal ocular regions due to its massive contact with surrounding environment. With respect to 24 conjunctival swab samples, two showed melting peak corresponding to S. aureus (CON12, CON21) and one (CON6) with P. aeruginosa. Thus, 12.5% of the samples showed positive results for both culture and PCR signifying such diagnosis as a preoperative procedure on conjunctival swab samples before any ocular surgery. 6
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Table 3 Culture identification and SYBR Green based multiplex real-time PCR assay results from 100 ocular patients. Case
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 P41 P42 P43 P44 P45 P46 P47 P48 P49 P50 P51 P52 P53 P54 P55 P56 P57 P58 P59 P60 P61 P62 P63 P64 P65 P66 P67 P68 P69 P70 P71 P72
Sample Name
COR1 COR2 COR3 COR4 COR5 COR6 COR7 COR8 COR9 COR10 COR11 COR12 COR13 COR14 COR15 COR16 COR17 COR18 COR19 COR20 COR21 COR22 COR23 COR24 COR25 COR26 COR27 COR28 COR29 COR30 COR31 COR32 COR33 COR34 COR35 COR36 POE1 POE2 POE3 POE4 POE5 POE6 POE7 POE8 POE9 POE10 POE11 POE12 POE13 POE14 POE15 POE16 POE17 POE18 POE19 POE20 POE21 POE22 POE23 POE24 PTE1 PTE2 EE1 EE2 EE3 EE4 POE25 POE26 POE27 POE28 POE29 POE30
Culture identification
Culture Negative Streptococcus pneumoniae Coagulase negative staphylococci Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Streptococcus pneumoniae Gram negative bacilli Culture Negative Streptococcus pneumoniae Pseudomonas aeruginosa Culture Negative Streptococcus pneumoniae Streptococcus pneumoniae Pseudomonas aeruginosa Viridans streptococci Culture Negative Pseudomonas aeruginosa Streptococcus pneumoniae MRSA Enterobacter aerogenes Staphylococcus aureus Pseudomonas aeruginosa Staphylococcus aureus Coagulase negative staphylococci Streptococcus pneumoniae Culture Negative Gram Positive Cocci Culture Negative Bacillus sp. Staphylococcus aureus Gram Positive Cocci Gram Positive Cocci Coagulase negative Culture Negative Klebsiella sp. Culture Negative Coagulase negative staphylococci Coagulase negative staphylococci Culture Negative Pseudomonas sp. Viridans streptococci Streptococcus pneumoniae Streptococcus pneumoniae Culture Negative Coagulase negative staphylococci Coagulase negative staphylococci Viridans streptococci Coagulase negative staphylococci Pseudomonas aeruginosa Streptococcus pneumoniae Bacillus sp. Viridans streptococci Viridans streptococci Streptococcus pneumoniae Staphylococcus aureus Culture Negative Culture Negative Streptococcus pneumoniae Coagulase negative staphylococci Aspergillus fumigatus Haemophilus sp. Pseudomonas aeruginosa Staphylococcus aureus Pseudomonas aeruginosa Culture Negative Bacillus sp. Culture Negative Coagulase negative staphylococci Streptococcus pneumoniae
CT Mean
− 22.66 − 22.28 15.39 24.41 20.88 14.69 8.87 − − 11.77 8.88 − 11.48 11.26 9.19 − − 9.74 12.20 9.25 − 10.30 15.91 10.76 − 18.74 − 11.30 − − 10.11 18.78 19.21 − − − − − − − − − 15.15 13.98 − − − 17.64 − 7.76 15.20 − − − 10.02 7.90 − − 8.56 − − − 6.17 10.45 6.49 − − − − 8.11
Tm Mean (°C)
RT−PCR Result
SA
SP
PA
SA
SP
PA
− − − − − − − − − − − − − − − − − − − − − 75.40 − 75.20 − 75.00 − − − 75.00 − − 75.00 74.80 74.90 − − − − − − − − − − − − − − − − − − − − − − 74.70 − − − − − − − 74.90 − − − − − −
− 81.00 − − − − − − 81.00 − − 81.10 − − 81.00 81.00 − − − − 80.80 − − − − − − 80.50 − − − − − 80.00 − − − − − − − − − − 80.70 80.80 − − − 80.56 − − 80.80 − − − 81.10 − − − 81.20 − − − − − − − − − − 81.00
− − − 85.80 85.90 85.80 85.70 86.10 − − − − 85.80 − − − 85.50 − − 85.80 − − − − 85.50 − − − − − − − − − − − − − − − − − − − − − − − − − − 85.50 − − − − − − − − − − − − 85.70 − 86.10 − − − − −
− − − − − − − − − − − − − − − − − − − − − + − + − + − − − + − − + + + − − − − − − − − − − − − − − − − − − − − − − + − − − − − − − + − − − − − −
− + − − − − − − + − − + − − + + − − − − + − − − − − − + − − − − − + − − − − − − − − − − + + − − − + − − + − − − + − − − + − − − − − − − − − − +
− − − + + + + + − − − − + − − − + − − + − − − − + − − − − − − − − − − − − − − − − − − − − − − − − − − + − − − − − − − − − − − − + − + − − − − −
(continued on next page) 7
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Table 3 (continued) Case
P73 P74 P75 P76 P77 P78 P79 P80 P81 P82 P83 P84 P85 P86 P87 P88 P89 P90 P91 P92 P93 P94 P95 P96 P97 P98 P99 P100
Sample Name
POE31 POE32 POE33 PTE3 CON1 CON2 CON3 CON4 CON5 CON6 CON7 CON8 CON9 CON10 CON11 CON12 CON13 CON14 CON15 CON16 CON17 CON18 CON19 CON20 CON21 CON22 CON23 CON24
Culture identification
Coagulase negative staphylococci Culture Negative Pseudomonas aeruginosa Staphylococcus aureus Coagulase negative staphylococci Culture Negative Culture Negative Culture Negative Culture Negative Pseudomonas aeruginosa Culture Negative Gram negative bacilli Culture Negative Gram negative bacilli Culture Negative Staphylococcus aureus Culture Negative Culture Negative Coagulase negative staphylococci Culture Negative Culture Negative Culture Negative Culture Negative Culture Negative Staphylococcus aureus Culture Negative Culture Negative Culture Negative
CT Mean
− − 7.99 9.27 − − − − − 12.50 − − − − − 13.00 − − − − − − − − 7.21 − − −
Tm Mean (°C)
RT−PCR Result
SA
SP
PA
SA
SP
PA
− − − 75.10 − − − − − − − − − − − 74.90 − − − − − − − − 75.20 − − −
− − − − − − − − − − − − − − − − − − − − − − − − − − − −
− − 85.90 − − − − − − 85.20 − − − − − − − − − − − − − − − − − −
− − − + − − − − − − − − − − − + − − − − − − − − + − − −
− − − − − − − − − − − − − − − − − − − − − − − − − − − −
− − + − − − − − − + − − − − − − − − − − − − − − − − − −
Case P1 to P36 is Corneal Scraping Samples (COR), Case P37 to P66 include Vitreous Fluid from Postoperative Endophthalmitis (POE), Posttraumatic Endophthalmitis (PTE), and Endogenous Endophthalmitis (EE) patients. Case P67 to P76 comprises Aqueous humor from POE and PTE patients. Case P77 to P100 contains Conjunctival Swab Samples (CON).
qPCR. Hence, it can be recommended as a routine diagnostic tool in eye hospitals for early diagnosis towards appropriate treatment against devastating ocular infections. Additionally, the rapidity, sensitivity and inexpensiveness of this technique would emphasize its practicability in other infections as well.
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