Rapid identification of bacterial pathogens using a PCR- and microarray-based assay

Järvinen, Anna-Kaarina; Laakso, Sanna; Piiparinen, Pasi; Aittakorpi, Anne; Lindfors, Merja; Huopaniemi, Laura; Piiparinen, Heli; Mäki, Minna

doi:10.1186/1471-2180-9-161

Cited by 103 publications

(77 citation statements)

References 28 publications

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“…The assay is easy to process, reducing the risk of human error and providing high throughput and reliability for the routine laboratory diagnosis. 74,76,77 The main strength of the test is that it offers essential information for the treatment of septic patients 18 hours faster than traditional methods. Several papers have been presented indicating a high sensitivity and specificity (95% and 99%, respectively), although a lower diagnostic sensitivity (62%) has also been observed, nevertheless in a previous study a significantly lower diagnostic sensitivity (62%) was observed.…”

Section: Prove-it™ Sepsismentioning

confidence: 99%

In vitro diagnosis of sepsis: a review

Marcello

Tumolo²,

Donno³

et al. 2016

PLMI

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Sepsis, severe sepsis and septic shock, systemic inflammatory response, and other related manifestations represent a relevant medical problem with high morbidity and mortality, despite the improvements in diagnosis, treatment, and preventive measures over the last few decades. The limited knowledge of the pathophysiology in association with the lack of in vitro diagnostic methods for the certain and quick determination of the causative microbiological agents and their antibiotic resistance means the condition is still critical and of high impact in health care. The current gold standard method to detect the sepsis-causing pathogens, which is based on blood culture, is still insufficiently sensitive and slow. The new culture-independent molecular biology-based techniques can lead to the identification of a broad range of microorganisms and resistance markers within a few hours and with high sensitivity and specificity; nevertheless, limitations of, for example, the polymerase chain reaction-based methods still hamper their application in the clinical routine. This review summarizes the in vitro diagnostic methods and their approach in the clinical diagnosis of the bloodstream infections, and explores their advantages and disadvantages at the current state of the art. A quick analysis of the future prospective in multiplex technologies for microbiological diagnosis of sepsis is also provided.

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Section: Prove-it™ Sepsismentioning

confidence: 99%

In vitro diagnosis of sepsis: a review

Marcello

Tumolo²,

Donno³

et al. 2016

PLMI

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“…Also, it indicates that there were bands in lane number 3 (cDNA amplified with primer S4F, S4R), which referred to the presence of a single band with a molecular weight of (324bp); this reveals that the microorganism which caused the infection was belonged to S. aureus keratitis. Finally, lane numbers 4,5 showed no bands when the cDNA was amplified with (PA-SS-F, PA-SS-R) (9Y, 9Z) primers for P. aeruginosa and S. pneumonie respectively. The specimens gave a positive result in the second set of the PCR method (using the universal fungal primer) when has been amplified by the C. albicans species specific primer.…”

Section: Isolation and Identification Of Microbial Keratitis Causativmentioning

confidence: 99%

“…In such methods, the pathogen is simultaneously detected and identified, which results in more rapid diagnoses than those obtained by conventional culturing methods and obviates the need for additional culture tests. Rapid diagnostics can also reduce the use of antimicrobial agents in addition to allowing a faster switch to the most optimum treatment, thus reducing both sideeffects and costs [4].…”

Section: Introductionmentioning

confidence: 99%

Diagnosis of Microbial Keratitis Causatives by Polymerase Chain Reaction

Ameen¹,

Al-Shakarchi

Al-Shaibani

2013

JNUS

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In order to evaluate the efficiency of using Polymerase Chain Reaction (PCR) in the identifications of microorganisms causing microbial keratitis, 20 corneal scraping samples were collected from patients who attended the Eye Casualty Unit at the Southampton General Hospital in the United Kingdom. Samples cultured on blood agar and chocolate agar incubated at 37 ͦ C for 24hrs and on sabrouad agar at 28 ͦ C for one week. PCR procedure was performed with the primer paired that targeted to the 16S rRNA for bacterial species and 18S rRNA gene for fungal species, in addition to the species specific primer for the most common microbial keratitis causatives microorganisms. Results in the regards showed that out of the 20 presumed cases of keratitis, PCR showed positivity in 75% of them, from these 55% were due to the fungal infection and 20% of the cases indicated that the keratitis belonged to bacterial infections: In comparison, only 25% of positivity was obtained by the cultural method. The species specific primer showed that half of the 20% bacterial infection cases were caused by S. aureus and the other 10% referred to S.epidermidis infection. While the candida albicans primer gave a positive result only in 72% of the original percentage (55%), the rest 28% may belong to the other fungal infection. Depending on the above results, it can be concluded that PCR not only proved to be an effective rapid method for the diagnosis of bacterial and fungal keratitis, but was also more accurate and sensitive method than the culture methods.

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“…123,124 The modified version of the assay used 10μL PCR product instead of using the standard volume of 3μL PCR product for hybridization, though all other steps were followed as stated in the protocol.…”

Section: Assessment Of Illnessmentioning

confidence: 99%

“…123 Analysis by Proveit™ Advisor software includes simultaneous identification of bacterial targets and evaluation of the control probes included in the array. Results were detected by signals from the hybridization of PCR products with target specific oligonucleotides on the array.…”

Section: Microarray Scanning and Analysismentioning

confidence: 99%

Chronic Otitis Media: Microbial Environment During Viral Infection and Genetics of Host Susceptibility

Allen

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Otitis media (OM), inflammation of the middle ear, is an upper respiratory tract infection (URTI) caused by viral and bacterial infections. OM is the most common pediatric disease and the leading cause of pediatric health care visits, antibiotic usage and surgery. A subset of OM affected children develops chronic otitis media with effusion (COME) and/or recurrent otitis media (ROM), with significant consequences on hearing and development. The aim of this research was to find novel risk factors that increase OM susceptibility by looking at both bacterial communities during URTI and host genetics. To discover novel OM host risk factors, a multi-stage approach was used. First, a genome-wide association study (GWAS) of COME/ROM was conducted using a familyii based population. The most significantly associated SNP, rs1110060, is located on chromosome 15 in an intron of kinesin family member 7 (KIF7). Association at SNP rs10497394, an intergenic SNP on chromosome 2, was replicated in an independent family-based population of OM. Fine-mapping of associated regions of chromosomes 2 and 15 was carried out using targeted resequencing and subsequent genotyping. Six SNPs were significantly associated (P<2.6x10 -4 ), two in the chromosome 2 region and four in the chromosome 15 region. Explorations of the functional potential of these two loci were carried out. Investigation of chromatin marks revealed an enhancer region on chromosome 2 in the human middle ear epithelial cell line, HMEEC-1. The -G‖ risk allele of the most significantly-associated chromosome 15 SNP, rs1110060, was found to impact expression of IQGAP1 in adenoids (P=0.026). Also, both lipopolysaccharide (LPS) and TGFβ treatment were found to regulate expression of KIF7 in HMEEC-1.The data presented in this dissertation show the complex nature of the microbiome of the nasopharynx during URTI, and we have identified novel regions and genes involved in susceptibility to OM. Moreover, functional assays support the participation of previously unsuspected mechanisms in OM pathogenesis. In summary, this research has provided insights into OM susceptibility from both host and bacterial perspectives.iii Acknowledgements

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Rapid identification of bacterial pathogens using a PCR- and microarray-based assay

Cited by 103 publications

References 28 publications

In vitro diagnosis of sepsis: a review

In vitro diagnosis of sepsis: a review

Diagnosis of Microbial Keratitis Causatives by Polymerase Chain Reaction

Chronic Otitis Media: Microbial Environment During Viral Infection and Genetics of Host Susceptibility

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