Bacterial bloodstream infection (bBSI) represents any form of invasiveness of the blood circulatory system caused by bacteria and can lead to death among critically ill patients. Thus, there is a need for rapid and accurate diagnosis and treatment of patients with septicemia. So far, different molecular diagnostic tools have been developed. The majority of these tools focus on amplification based techniques such as polymerase chain reaction (PCR) which allows the detection of nucleic acids (both DNA and small RNAs) that are specific to bacterial species and sequencing or nucleic acid hybridization that allows the detection of bacteria in order to reduce delay of appropriate antibiotic therapy. However, there is still a need to improve sensitivity of most molecular techniques to enhance their accuracy and allow exact and on time antibiotic therapy treatment. In this regard, we conducted a systematic review of the existing studies conducted in molecular diagnosis of bBSIs, with the main aim of reporting on clinical significance and benefits of molecular diagnosis to patients. We searched both Google Scholar and PubMed. In total, eighteen reviewed papers indicate that shift from conventional diagnostic methods to molecular tools is needed and would lead to accurate diagnosis and treatment of bBSI.
Bloodstream infection (BSI) is a life-threatening condition caused by the presence of microorganisms, generally bacteria or fungi, in the blood [
The presence of bacteria and bacterial products in circulating blood has been known for decades. Thus, detection and identification of bacteria based on detection of circulating nucleic acids has been a constant and ongoing challenge [
The current gold standard method of bloodstream microbial detection and identification is the blood culture (BC). The latter is currently based on an automatic and continuous manipulation of liquid culture, followed by gram staining, subculture, and the use of phenotypic methods to identify the bacteria and their associated antibiotic susceptibility. A major disadvantage to culture is the time required to complete the entire described process, which normally ranges from 1 to 5 days or more [
Published papers related to our review topic were carefully searched through Google Scholar and PubMed searching tools. Google Scholar has been of our interest because it covers a broad range of scientific papers in different research areas; the same is for PubMed which covers around 26 million of biomedical papers from Medline and life science journals. Both databases are regularly updated with newly published papers.
The online paper search was conducted on two different dates (February 12 and 27, 2016). Our research was developed based on different searching keywords related to our review topic. We used the following keywords: “bacterial bloodstream infections”, “bBSIs”, “molecular diagnosis of bBSI”, and “clinical significance bBSI”. These three searching keywords or terms were entered in Google Scholar and PubMed. Thus, we considered and reviewed all papers published on clinical significance of molecular diagnostic tools for bBSI.
The papers were examined, extracted, and considered based on different inclusion and exclusion criteria we set. All these criteria were applied to paper titles we got for our first time of searching; therefore all chosen papers for our review have met and satisfied the following criteria: being “published in English,” “providing information on development of molecular diagnostic tools for bBSI and their clinical significance,” or being “published in 2000 to 2016.” However, other retrieved papers were rejected based on the following exclusion criteria: being “published before 2000,” “published in a language other than English,” or “published as a book.” After examining all these aforementioned criteria, papers were considered for review if they were available in full text through PubMed.
A complete description of our review strategy is found on the flowchart presented in Figure
Summary of papers included in this review.
First author | Year of publication | Study period | Study country | Age range | Sample size | Patient’s setting | Type of diagnostic tool | Design | Cases with bBSIs | Performance | Findings |
---|---|---|---|---|---|---|---|---|---|---|---|
Faria | 2015 | Not stated | Canada | Adult | Not stated | Hospital based | Illumina sequencing following PCR amplification of 16S rDNA | Lysis of cells followed by DNA extraction | Not stated | High but not specified | Molecular profiling is more accurate than blood culture |
Jordana-Lluch | 2014 | 2012-2013 | Spain | Not stated | Not stated | Review type of study | PCR/ESI-MS | Use of amplicons | Not stated | Sensitivity 75%, specificity 92% | This molecular technique provides fast diagnosis of clinical samples |
Venkatesh [ | 2010 | Not stated | USA | Neonatal | Not stated | Hospital, ICU | Microarray and PCR | Detection of organism by hybridization and amplification | Not stated | Sensitivity of 98.7%, specificity of 99% for microarray, sensitivity and specificity of 86.4 and 99.0%, respectively, for PCR | These methods are more feasible compared to BC |
Warhurst | 2015 | Not stated | UK | Not stated | Not stated | Review study | SeptiFast real-time PCR | Not stated | Not stated | 60.8% sensitivity, 86.3% specificity | PCR-based assays are more specific but suffer from low sensitivity values |
Carrara | 2013 | 4/2011–9/2011 | Spain | Not stated | 267 | Hospital, ICU | Multiplex PCR assay, the Magicplex Sepsis Test | Not stated | 98 | Sensitivity 65% (52–76%), specificity 92% (87–95%) | Can detect even fastidious bacteria but still needs some improvement to increase sensitivity |
Ecker | 2010 | Not stated | USA | Both adult and children | Not stated | Not stated | PCR-ESI/MALDI-TOF/PCR-EIA | Sample extraction, lysis and enrichment with reagent | Not stated | Sensitivity, specificity, and positive and negative predictive values were 95.0, 98.8, 95.0, and 98.8%, respectively | They are more accurate, sensitive, and fast compared to blood culture |
Boyd | 2014 | 2011–2013 | Canada | Not stated | 245 | Not stated | Multiplex PCR | Use of 16RNA for bacterial identification | Not stated | High specificity but low sensitivity | The use of molecular techniques will improve life in septic patients |
Lecuit | 2014 | Not stated | France | Not stated | Not stated | Review study | Multiplex PCR | Bacterial typing is done by detecting conserved 16S rRNA regions | Not stated | 95% sensitivity, 92% specificity | Further optimization of multiplex PCR is recommended |
Reddy [ | 2010 | Not stated | Tanzania, Malawi, and Kenya | Adults and infants | 221 | Hospital based | PCR | Not stated | 136 | Moderate | Ease of identifying bacterial etiologies |
Lehmann | 2008 | Not stated | Germany | Not stated | 574 | Healthy volunteers | Multiplex real-time PCR | Compare PCR amplicons to the conserved regions | Not stated | Higher performance than blood culture | PCR-based techniques chosen for their sensitivity and specificity |
Wallet | 2010 | Not stated | France | Not stated | Not stated | Hospital, ICU | LightCycler-SeptiFast (LC-SF) | Not stated | Not stated | Sensitive at 78%, specific at 99% | LC-SF is of valuable interest for patients with sepsis |
Bacconi | 2014 | Not stated | Maryland | Not stated | 331 | Hospital samples | PCR/EI-MS | Cells are lysed and followed by DNA extraction by an automated instrument | 35 | 83% sensitivity, 94% specificity | Rapid detection and identification of microbes |
Tennant | 2015 | Not stated | Pakistan/Karachi | Adult | Not stated | Hospital samples | q-PCR | Not stated | Not stated | 40% sensitivity, >90% specificity | Use of multiple methodologies to increase accuracy |
Wang | 2014 | Not stated | Republic of Korea | Both adult and children | Not stated | Hospital, ICU | The real-time PCR TaqMan assay | DNA extraction from a colony of blood culture | Not sated | Sensitivity of 100% and specificity of 89.5% | Molecular techniques are more specific than BC |
Chang | 2013 | 2011–2013 | USA | Adult | 34 | Review type of study | LC-SF, multiplex real-time PCR | Not stated | 18 | High specificity and moderate sensitivity | LC-SF multiplex real-time PCR gives more promising results than BC |
Liesenfeld | 2014 | Not stated | USA | Not stated | Not sated | Review type of study | Emphasis on commercially available molecular techniques | Not stated | Not stated | The most reported one is the PCR of 84% sensitivity and 94% specificity | Molecular methods have advantages in microbial identification, but they must be refined in a good algorithm |
Dark | 2011 | Not stated | UK | Adult | 600 | Hospital, ICU | Multiplex real-time PCR | BC followed by DNA extraction and PCR | Not stated | 95% specific and 87% sensitive | PCR-based techniques are better than blood culture when it comes to assay time, sensitivity, and specificity values |
Jordana-Lluch | 2015 | 2012–2014 | Spain | Not stated | 410 | Hospital based | Multiplex PCR | Automated DNA extraction followed by PCR | Not stated | Sensitivity of 76.9% and specificity of 87.2% | A promising technology to detect a wide range of bacterial microbes |
Flow diagram of paper selection.
The first category of the reviewed papers underlined the development and benefits of PCR-based assays. PCR-based methods have been discussed by 8 of the 18 reviewed papers. Dark et al., 2011, reported the usefulness of PCR techniques mostly by using universal probes, followed by sequencing, and highlighted their high sensitivity and specificity [
The second category of the reviewed papers compared the diagnostic significance of BC to PCR. Some reviewed publications reported on BC as a good technique in diagnosis of bBSI but also presented its various disadvantages such as long turnaround time, easy contamination, and false negative and positive results [
The same comparison of BC to PCR was illustrated through different field trials. Most of the works by Faria et al., 2015, evaluated the Illumina sequencing of PCR amplified 16S rDNA samples collected from intensive care unit (ICU). As part of their findings, they suggested that a molecular approach may enable improved detection of polymicrobial infections. The application of sensitive molecular methods to clinical samples can identify more organisms in samples when compared to BC clinical diagnostics, which is selective for specific organisms. By working on patients’ samples from ICU using real-time PCR, Dark et al., 2011, revealed a high diagnostic specificity and a 3- to 10-fold higher sensitivity for real-time PCR compared to conventional BC [
All the 18 reviewed papers emphasize the need of using molecular techniques for the diagnosis of bBSIs. Warhurst et al., 2015, reported that SeptiFast real-time PCR is more rapid in the detection of BSIs though it has some limitations that must be handled over time [
Faria et al., 2015, indicated that each delay in antibiotic administration decreases the survival chance of the patient; then rapid diagnostic tools are needed and nucleic acid-based technologies and proteomic approaches are taking part in a more accurate diagnosis of bBSI [
Globally, bBSIs are the most common cause of sepsis and characterized by high mortality rates [
In most settings, diagnosis of bBSIs is still based on conventional blood culture followed by the identification and antibiotic susceptibility testing of the grown bacteria. However, blood culture shows a sensitivity rate of only 60% and is not only time-consuming, but also laborious. In addition, it possesses serious biosafety risks since the bacteria are grown in vitro for subsequent microbiological analysis. In the last decades, there have been improvements in enriched growth media towards automated blood culture systems such as Bactec and BacT/Alert. This automated system uses software allowing a quicker detection of grown bacteria in culture; and this has significantly decreased contamination rates [
In this review, it is clearly noticed that PCR-based techniques increase the sensitivity and specificity in the detection of bBSI. In addition, the use of such molecular techniques in diagnosing bBSI has reduced associated risks such as long turnaround time and false negative and positive results and has contributed to easy identification of fastidious bacteria and prevention of empirical therapy. All reviewed papers emphasized more the effectiveness and rapidity of molecular techniques. Reviewed techniques are mainly based on automated DNA extraction, PCR set-up, PCR amplification, amplicons purification, and PCR/ESI-MS. They overall lead to microbial identification from whole blood in not more than 6 hours [
Perfect diagnostic technology is able to identify the infecting organism and also the determinants of antibiotic resistance in a timely fashion so that the administration of appropriate therapy could start soon after diagnostic results. The ideal molecular method would analyze a patient’s blood sample and provide all the information needed to immediately direct the optimal antimicrobial therapy for bBSI [
From a theoretical point of view, PCR-based diagnostic techniques hold promise for sensitive and specific detection of target pathogen within a short time. In contrast, for a good and accurate identification of a pathogen in bBSI, several parallel or serial specific PCR analyses or a more universal PCR assay followed by specific probe hybridization or sequencing of the targeted bacteria would bring more promise [
Broad-range assays, with primers targeting variable regions in the16S rRNA or 18S/23S rRNA gene, present clinical applicability for diagnosis of bBSI due to their short turnaround time and ability to directly detect any noncultivable or cultivable pathogens in patients’ blood sample [
Current microarray-based techniques include Prove-It and Verigene tests. Prove-It consists in multiplex PCR in combination with microarray and can detect 60 bacterial pathogens in a positive culture sample [
Next to microarray-based tools, there is an emergence of various assays targeting biomarkers. Most of these techniques target endotoxins; acute-phase protein biomarkers such as C-reactive protein (CRP), lipopolysaccharide-binding protein (LBP), procalcitonin (PCT), pentraxin, serum amyloid A, ceruloplasmin, and alpha 1 acid glycoprotein; cytokines and chemokines; coagulation biomarkers; soluble receptor and cell surfaces [
Following the results of this systematic review, we believe that next generation molecular tools constitute a new and powerful approach that could identify main species causing bBSIs and detect their respective genetic markers responsible for antibiotic resistance. Molecular diagnostic tools would provide unique possibilities in the surveillance of bBSI. Surveillance studies at all health system levels are important to know the causative agents of bBSI and devise appropriate interventions to control the spread of antibiotic resistance and guide physicians in deciding which adequate antibiotics to prescribe.
In conclusion, it is noteworthy that molecular techniques are now emerging as another promising option for diagnosis of bBSI. In this review, PCR-based assays were highly reported to have significantly changed diagnostics of bBSI by increasing a bit sensitivity, specificity, and test accuracy overall. These techniques are generally good as they yield much better and reliable results in much shorter time than BC. In countries where trials have been conducted, reports have emphasized the accuracy of test results leading to a timely and right antibiotic administration. Although the cost of some of the newly developed techniques is still comparably high to be used in some poor endemic settings, we hope to get cheap, accurate, and fast methods requiring low training soon. This will be achieved through the advances in genomics, metagenomics, transcriptomics, metatranscriptomics, and proteomics together with much collaboration in international health services. Thus, the use of these sophisticated tools will soon shift from research settings and developed world to clinical settings and developing world. This will obviously tackle the challenge of usual delay in test results deliverance when using conventional BC. We are all convinced that elaboration of a quick and affordable tool for detecting bacterial pathogens in patients’ blood sample is of great interest in global public health.
The authors declare that there is no conflict of interests regarding the publication of this paper.