This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
The bloodstream is normally sterile in healthy individuals, therefore the presence of micro organisms is considered potentially serious Hawkey and Lewis 2004. Bacteria may enter the bloodstream from an extra vascular source of infection within the body or from a surface site colonised with normal flora entering through broken skin or through mucous membranes(Hawkey and Lewis 2004). Blood contains many antimicrobial components including lysozyme, leucocytes, immunoglobulins and complement components. Bacteria are normally removed from the bloodstream by these host defenses. Only when host defenses are overwhelmed or evaded, does a bloodstream infection become apparent. The culture of micro organisms and Yeast from blood is important in the clinical laboratory diagnosis of bloodstream infection (BSI). Depending on the causative agent this process is described as either bacteraemia, fungaemia, viraemia or parasitaemia (Ryan and Ray 2010).
Transient bacteraemias may occur when there is trauma to a body site that has normal flora as occurs after surgery and tooth brushing. The organisms are soon cleared by the immune system and such transient bacteraemias have no clinical significance (Ryan and Ray 2010).
Septicaemia refers to systemic disease associated with the presence and persistence of pathogenic micro organisms or their toxins in the blood (Ryan and Ray 2010). Symptoms are often non specific including fever, chills and hypotension. A BSI is potentially life threatening and is recognized globally as having a high mortality rate and increasing in incidence (Pechorsky, Nitzan et al. 2009). The causative agents of BSI include both Gram positive and Gram negative micro organisms.
Etiology of bloodstream infection
Patients with BSI include representative of all age groups. The etiology of BSI has changed significantly over the last 20 years (Gaibani, Rossini et al. 2009). Traditionally Gram negative bacteria (GNB) were more frequently isolated than Gram positive bacteria (GPB) in a BSI. Recently however the reverse trend is occurring with GPB and Yeast being the most common cause of BSI, probably a result of increased use of intravascular and other medical devices, an increase in invasive procedures and the use of immunosuppressant agents (Kollef 2000). Cases of BSI are usually a result of overflow of the causative agent from an extra vascular infection. In these cases the organisms are drained by the lymphatic system or from the infection site after which they reach the capillaries. The most common source of organisms causing BSI appear concomitantly with other serious infections such as urinary tract infection (UTI), respiratory tract infection (RTI), endocarditis, kidney and bowel infections (Pechorsky, Nitzan et al. 2009).
1.3 Epidemiology of Bloodstream infection
Approximately 1,500 cases of blood stream infection are reported in the Ireland each year. The most prevalent bacteria causing BSI in Ireland have been reported. These include Pseudomonas aeruginosa (P.aeruginosa), Enterococcus faecalis (E.faecalis), Staphylococcus aureus (S.aureus) and Klebsiella pneumonia (K.pneumoniae).
1.4 Diagnosis of bloodstream infection
Blood cultures have an important role in the diagnosis of serious infection. Since bloodstream infections have a significant impact on the morbidity and mortality of patients, accurate and rapid blood culture data have an important role in the diagnosis of serious infection reducing mortality and healthcare costs. The accurate isolation of the etiological agent of a bloodstream infection is one of the most important functions performed by the clinical microbiology laboratory.
Current culture based methods are the gold standard, based on phenotypical identification of the causative agent of a bloodstream infection. While they are of great diagnostic value these tests have a number of inherent limitations that restrict their ability to rapidly identify the species and susceptibility. Culture based identification techniques of pathogens causing a BSI can take between 1-3 days.
Because of this, clinicians often employ empiric treatment approaches, using broad spectrum antimicrobial therapies that can be costly, ineffective and contribute to an increase in drug resistance (Dellinger, Carlet et al. 2004).
1.4.1 Overview of the standard microbiological techniques
The current clinical microbiology standard of identifying the pathogens causing a blood stream infection involves using culture based techniques that have not changed much since their introduction into use. This phenotypic method of identifying organisms has a number of inherent limitations that prevent it from providing rapid results, including time required for organism growth which prevent these methods providing valuable information on bacterial or fungal species of blood culture positive samples within the first few hours.
The normal work flow within the laboratory for the investigation of a bloodstream infection involves a positive blood culture being typically detected by an automated microbial detection system within 5 days from the time of collection. A number of systems are available including BACTEC 9240, ESP II and the BD BACTEC FX.
The BACTEC FX continuous monitoring blood culture system is employed in this laboratory, Department of Medical Microbiology, University Hospital Galway.
1.4.2 Principle of BACTEC FX
The principle of this system is based on the sample being inoculated directly into a blood culture bottle (aerobic, anaerobic and pediatric). These blood cultures are then entered into the BACTEC FX instrument for incubation at 37oC. Each blood culture bottle contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms. The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the increasing amount of CO2. A positive reading indicates the presumptive presence of viable microorganisms in the blood culture bottle.
1.4.3 Processing positive blood cultures
From the point of a blood culture turning positive it can take up to 36 hours to determine the species and susceptibility of the causative agent using conventional culture techniques. The immediate management of any positive blood culture bottle includes prompt Gram staining, which allows classification of the micro organism as either Gram positive or Gram negative. The positive blood culture is also inoculated on to blood and chocolate agar and incubated for 24 hours followed by incubation for a further 24 hours. The positive blood culture is also inoculated onto media (type depends on Gram stain result) for provisional susceptibility. Inoculation on to blood and chocolate agar is performed in order to obtain colonies that would be subjected to identification following the standard laboratory procedure; that growth is identified phenotypically and full susceptibility testing is performed.
1.5 Empiric treatment and antimicrobial resistance
While awaiting the identification of the causative agent of a BSI, clinicians use broad spectrum antibiotics to treat BSI, including Flucoxacillin; a broad spectrum antibiotic used in the treatment of Staphylococcal infection. In recent years the increase in multi drug resistant pathogens has highlighted the importance for rapid organism identification. Both antibiotic-resistant gram-negative bacilli and gram-positive bacteria are reported as important causes of BSI. Gram-negative bacilli such as E.coli and P.aeruginosa are the leading causes of GNB BSI, while MRSA and VRE are the major causes of Gram positive infection. Figures released by the HPSC show that the number of cases of E.coli BSI infection -resistant to fluoroquinolone antibiotics increased in Ireland from 5% in 2002 to 17% in October 2005, while 30-40% of S.aureus BSIs are caused by MRSA.
The marked increase in the incidence of infections due to antibiotic-resistant organisms in recent years is of significant concern caused by excessive and inappropriate use of antibiotics which accelerates the emergence and spread of antibiotic-resistant bacteria.
Studies have shown that BSI caused by antibiotic-resistant bacteria result in greater mortality, longer hospitalization, and higher costs than infections caused by antibiotic-susceptible bacteria (Kollef 2000). Bloodstream infection caused by S.aureus was found to have a median hospital day of 4 days while MRSA BSI had a median hospital stay of 12 days. Previous studies have also shown that treatment of blood stream infections with early, appropriate antimicrobial therapy has been shown to reduce mortality among patients with gram-negative bacteremia and, when initiated early, to have a favorable effect on outcome in critically ill patients with BSI (Kollef 2000).
The opportunity to move to a targeted antimicrobial therapy sooner using rapid microbiological and molecular techniques would therefore decrease the time patients are exposed to unnecessary treatment, potentially decreasing their length of stay and preventing antimicrobial resistance.
This can be achieved by focusing on alternative techniques to improve the detection of pathogens in the blood stream. Of these new techniques some are designed to reduce the time to identification after detection of pathogens by conventional blood culture. These rapid methods for earlier identification of the causative agent of a blood stream infection not only help the clinician to adjust antimicrobial therapy reducing antibiotic resistance, they also benefit the healthcare institution resulting in cost savings.
Figure 1.1: Workflow of culture based techniques and new molecular techniques for the identification of pathogens causing blood stream infection. (Courtesy of AdvanDx)
1.6 Available molecular techniques for the identification of pathogens causing bloodstream infections
The introduction of a more rapid, accurate testing method for the identification of the causative agent causing a blood stream infection would have significant implications for infection control practices, clinical therapies and healthcare costs.
Recently, new molecular assays have become available commercially that rapidly identify bloodstream pathogens, giving clinical laboratories an excellent opportunity to improve care of patients with BSI.
Several molecular diagnostic tools such as universal amplification and sequencing, nucleic acid based fluorescence hybridization probes and DNA micro arrays are available. In general all these techniques identify and differentiate pathogens on a genotypic, rather than phenotypic basis by detecting species specific products directly from positive blood cultures. This therefore would eliminate the time needed for bacterial growth that is required by current culture based identification techniques.
Among the commercial assays available for identification of pathogens from positive blood cultures is Hyplex BloodScreen, a multiplex PCR assay with the subsequent identification of several bacterial species such as S. aureus, MRSA, S.pneumoniae and E.coli by hybridization in an ELISA-like format. Another commercially available assay is Prove-it Sepsis; a DNA microarray assay for the identification of pathogens causing BSI. The turnaround time for this assay is 3 h. Other assays available include Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF), Peptide Nucleic Acid fluorescent insitu hybridization (PNA FISH) a novel method using fluorescent labeled Peptide nucleic acid probes and the Xpert MRSA/SA blood culture assay for the rapid detection of S.aureus and MRSA from positive blood cultures.
1.6.1 Xpert MRSA/SA blood culture assay
The GeneXpert system is a qualitative diagnostic assay for the rapid identification of Staph aureus/MRSA in patients with positive blood cultures. This system is provided by Cepheid and was first described in 2007. It is an automated RT-PCR assay for unique gene specific amplification of S.aureus and MRSA DNA from positive blood cultures, and fluorogenic target specific hybridization for the detection of amplified DNA (Enns 2008). The principle of this assay is based on Polymerase chain reaction (PCR), which is used to amplify target DNA. In Real Time PCR (RT PCR) the inclusion of fluorescent labeled DNA probes allows quantification of target DNA in real time i.e. allows visualization of the increase in DNA as it is amplified.
The Xpert system integrates sample purification, nucleic acid hybridization and detection of target sequence in a single disposable cartridge. Primers and probes in the assay detect proprietary sequences of the gene for Methicillin resistance (mecA), Staph protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) inserted into the Staph. aureus chromosomal attB site (Rossney, Herra et al. 2008).
The assay is performed in chambers of a single use disposable cartridge, sample preparation time is minimal and the RT PCR assay time is less than 1 hour. This system provides identification of S. aureus and MRSA within 1 hour. This assay is the first commercially available assay for the simultaneous detection of S.aureus and MRSA.
The assay has shown sensitivity and specificity of 100% and 98.6% respectively in a study performed on wound samples and blood cultures (Wolk, Struelens et al. 2009). The rapid detection of MRSA along with early implementation of an appropriate intervention has been reported to reduce the incidence of MRSA (Rossney, Herra et al. 2008).
1.6.2 Peptide nucleic acid insitu hybridization (PNA FISH)
PNA FISH is a modification of the well known traditional staining procedure fluorescence insitu hybridization (FISH) technique; for the rapid and specific identification of micro organisms directly from positive blood cultures(Oliveira, Procop et al. 2002). Peptide nucleic acid (PNA) molecules are DNA mimics in which the negatively charged sugar-phosphate backbone of DNA is replaced with a non-charged polyamide or peptide backbone (Rigby, Procop et al. 2002). PNA probes contain the same nucleotide bases as DNA, adenine (A), cytosine (C), guanine (G), and thymine (T) and follow standard Watson-Crick base-pairing rules while hybridizing to complementary nucleic acid sequences (Oliveira, Procop et al. 2002; Rigby, Procop et al. 2002).
This modification has added a number of improvements over the traditional DNA probe including improved hybridization characteristics. PNA FISH assay uses fluorescent labeled PNA probes targeting ribosomal RNA (rRNA) in growing bacteria and Yeast containing regions of highly conserved, species-specific sequences. PNA FISH assays are based on complementary hybridization between a commercially available probe (AdvanDx and bioMérieux, Inc.) and rRNA of common microbes found in positive blood cultures, such as S.aureus, E.faecalis and E.coli. PNA FISH is performed by preparing a smear of a positive blood culture and provides results within 2 hours. The species of the causative organism of a BSI is determined by examining the colour of the fluorescent probes, along with the morphological characteristics of the organism. Based on this information clinicians can select appropriate antimicrobial therapy for patients within hours of a positive blood culture result, rather than days with current routine culture identification techniques. Studies from various laboratories demonstrate that PNA fish is highly sensitive and specific. Recent work carried out by Oliveira et al., 2003 showed PNA FISH identified S. aureus directly from blood cultures with a sensitivity and specificity of 98%. Use of PNA FISH for differentiation between S. aureus and CoNS has also been shown to reduce antimicrobial usage and hospital length-of-stay by two days (Forrest et al., 2008) (Oliveira, Brecher et al. 2003).
A study of the Yeast Traffic light PNA FISH assay demonstrated that a rapid identification of Candida albicans in blood cultures allowed substantial cost savings, due principally to caspofungin usage (Forrest, Roghmann et al. 2008).
SA-CNS PNA FISH Overview.png
Figure : Principle of PNA FISH assay
The therapeutic implications of PNA FISH include appropriate and effective therapy being administered earlier to improve clinical outcomes for patients with infections due to critical pathogens (S. aureus, E. faecium and P. aeruginosa) and to reduce unnecessary antibiotic use and lower hospital costs.
New advanced molecular methods are offering rapid detection and identification of pathogens causing BSI, with turnaround times not possible by conventional laboratory identification techniques. The purpose of this study was to compare the performance of the Xpert SA/MRSA blood culture assay, which is run on the GeneXpert Real Time PCR platform (Cepheid) and a novel method of peptide nucleic acid fluorescent insitu hybridization (PNA FISH) to conventional laboratory identification techniques for positive blood cultures.
Chapter 4: Discussion
Every year in Ireland over 1500 patients acquire bloodstream infections (BSIs) while in hospital care. Blood stream infections have a significant impact on morbidity and mortality of patients (Scola and Raoult 2009). Therefore methods for rapid and accurate identification of the causative pathogen of a bloodstream infection are critical for patient care. A blood culture is the gold standard for the diagnosis of a BSI, a method which may take several days for identification. This time delay in organism identification by conventional methods results in clinicians treating suspected BSIs with broad spectrum antibiotics while awaiting the patient's blood culture result from the clinical Microbiology laboratory. This practice while necessary for patient care has undesirable effects such as increasing antibiotic resistance.
Recently new molecular assays have become available that rapidly identify the causative micro organisms of a BSI. These new molecular assays give the clinical Microbiology laboratory a previously unavailable opportunity to improve patient care and reduce healthcare costs. Using this information, clinicians can potentially administer appropriate antimicrobial therapy to patients with a BSI within hours of blood culture positivity rather than days with routine conventional culture identification techniques. Studies such as those carried out by (Forrest, Roghmann et al. 2008) have shown a 1.8 day earlier administration of appropriate antibiotic therapy using the molecular assay PNA FISH resulting in reduced mortality and healthcare costs for patients with BSI .
This study characterized the performance of 2 molecular assays; the Xpert MRSA/SAâ„¢ blood culture assay and PNA FISH for use within the clinical Microbiology laboratory compared to conventional identification techniques for the identification of pathogens causing BSI. A total of 230 positive blood cultures that signaled positive by the BD BACTECâ„¢ FX between March and May 2011 were tested. A total of (n=80) patient positive blood cultures with Gram positive cocci in clusters were tested using the Xpert MRSA/SAâ„¢ blood culture assay.
48 positive blood cultures of the 80 tested using the Xpert MRSA/SAâ„¢ blood culture assay were also tested using the AdvanDx S.aureus/CNS PNA FISH Â® assay.
Evaluation of the AdvanDx E.faecalis/OE PNA FISHÂ® assay was performed on (n=50) positive blood cultures with Gram stain results indicating gram positive cocci in chains. Evaluation of the AdvanDx GNR traffic light PNA FISHÂ® was performed on (n= 50) positive blood cultures with gram stain results revealing Gram negative bacilli. Finally (n= 50) positive blood cultures with gram stain results revealing Gram positive yeast cells were tested using the AdvanDx Yeast traffic light PNA FISHÂ® assay. These results were then compared to conventional culture identification results.
4.1 Xpert MRSA/SA â„¢ blood culture assay
The first part of this study evaluated the Xpert MRSA/SA blood culture assay for the identification of S.aureus and MRSA from positive blood cultures. The Xpert MRSA/SA blood culture assay is a real time PCR assay which is being increasingly utilized in hospital laboratories because it is very simple method to perform with fast results. During testing internal control results including the sample processing controls, was positive for each assay which confirmed all results obtained were valid. S.aureus was present in 17 (21.25%) positive blood cultures tested (12 MSSA and 5 MRSA) (See figure 3.2). Laboratory methods for detecting MRSA and S. aureus from blood cultures require overnight incubation time and do not support rapid decisions for selection of the most appropriate therapeutic interventions. Rapid identification of MRSA infection is critical to infection control measures, with delays in detection resulting in either late institution of infection control measures and resultant transmission of MRSA between patients increasing healthcare costs (Warren, Liao et al. 2004). In Ireland 40-50 % of S.aureus bacteraemias are caused by MRSA; this is significantly higher than our European counterparts. Recent information published by the HPSC has shown BSIs caused by S.aureus have a median hospital stay of 4 days while MRSA BSI has a median hospital stay of 12 days. Therefore methods for rapid identification of MRSA would result in infection control procedures being implemented sooner and a reduction in healthcare costs.
The remaining samples were identified as MRSA negative/SA negative by the Xpert and subsequently identified as CoNS (77.50%) on culture.
CoNS are frequently isolated from blood cultures, where they may be only a contaminant. Of the positive blood cultures identified as CoNS, 47.5% were positive for the presence of the mecA gene. Information which may be clinically important if the CoNS isolate was determined to be clinically significant. CoNS isolates positive for mecA gene cannot be treated with flucoxacillin; therefore a more expensive therapeutic agent is required.
The Xpert MRSA/SA blood culture assay in this study demonstrated 100% specificity for the detection of S.aureus/MRSA which is similar to other studies testing the Xpert MRSA/SA blood culture assay (Wolk, Struelens et al. 2009). The Xpert MRSA/SA blood culture assay has a high specificity due to the presence of three assay targets that limit the potential for false-positive results due to SCCmec variants with missing or incomplete mecA genes, which have been described with other commercial assays that do not target the mecA gene" (Donnio, Oliveira et al. 2005; Francois, Bento et al. 2007). One Invalid result was obtained on the Xpert for a positive blood culture tested. Organism was identified by routine identification as CoNS (S.auricularis). Inhibition of the Xpert MRSA/SA blood culture assay resulting in Invalid results has been observed in the presence of anticoagulants such as Heparin and EDTA (Ethylene diamine tetra acetic acid). The assay can be performed by a single Medical scientist and allow same day results with batching of specimens. The Xpert MRSA/SA assay provided positive blood culture results in approximately 50 minutes. Limitations of the assay include only the identification of S.aureus and or MRSA from positive blood cultures. Future developments for Xpert assays include the availability of identifying fungal BSI using an Xpert fungal assay which is due to be commercially available in 2013-2014. Currently available assays which can also be run on the GeneXpert Real Time PCR platform include the Xpert C.difficile assay for the detection and identification of toxin producing C.difficile from stool samples and the identification of vanA for VRE from rectal swabs.
4.2 Peptide nucleic acid insitu hybridization (PNA FISH)
Peptide nucleic acid fluorescent insitu hybridization (PNA FISH) provides accurate identification of organisms using fluorescent probes that bind to complementary sequences of rRNA in microbes such as S.aureus, E.coli, C.albicans and E.faecium. The assay is performed using smears made from positive blood culture bottles and the interpretation of results is carried out by microscopy to provide identification of organisms in a time not possible by conventional methods. For this PNA FISH assay positive samples were determined as multiple bright fluorescent cells in multiple fields of view. For each batch run a control slide was included, for all batches the commercially available control slide when viewed on microscopy had multiple bright fluorescent cells in multiple fields of view, therefore the patient results were accepted as valid.
The S.aureus/CNS PNA FISH assay was performed on positive blood cultures that were simultaneously tested using the Xpert MRSA/SA assay. S.aureus was identified in 12 samples, CoNS in 32, while 4 positive blood cultures were an S.aureus/CoNS mix. A 100% correlation was found between PNA FISH results and conventional techniques, while those identified as S.aureus positive on FISH were also positive for the presence of S.aureus by the Xpert assay. A drawback of S.aureus/CNS PNA FISH in comparison to Xpert MRSA/SA assay does not provide Methicillin resistance information therefore conventional susceptibility testing would need to be performed to identify if an S.aureus positive sample by PNA FISH carried the mecA gene and was therefore MRSA.
E.faecalis/OE PNA FISH assay identified 9 samples as positive for E.faecalis, 19 blood cultures positive for other Enterococci, while 14 positive blood cultures with Gram stain results of GPC Chains had a negative result. These blood cultures were identified by conventional techniques as positive for S.pneumoniae, Strep Group A and Strep group B. Enterococci are a common cause of BSI with E. faecalis being the predominant species followed by E. faecium (Gharibi, Tajbakhsh et al. 2010).
Because resistance to Vancomycin in E. faecalis is less common compared to resistance found in E. faecium, the implementation of PNA FISH allowing differentiation of E.faecalis from other Enterococci would be important as it may prevent costly infection control measures associated with Vancomycin resistance.
Blood stream infections caused by Gram negative micro organisms are associated with significant mortality and represent approximately 58% of all BSIs reported in Ireland in 2010. For the GNR Traffic light PNA FISH assay a total of (from 17 patient and 33 simulated positive blood cultures) 18 E.coli, 7 P.aeruginosa and 13 K.pneumoniae isolates were identified. A total of 8 GNB mixed cultures produced mixed fluorescence results on microscopy. Blood culture samples which produced no fluorescence upon testing with the GNR Traffic light were simulated positive samples containing wild strain Rauoltella ornitholytica (2) and Klebsiella oxytoca (2). These organisms were assayed to investigate the specificity of the K.pneumoniae probe as a study carried out by (Peters, Savelkoul et al. 2006) found fluorescence was observed with the K. pneumoniae probe for an isolate of Klebsiella oxytoca. During this study the specificity of the K.pneumoniae probe was found to be 100%. A difficulty identified during this study was the fluorescence of the K.pneumoniae probe. On viewing patient and simulated positive blood cultures with K.pneumoniae definite identification was difficult as the fluorescence was weak however it was sufficiently evident compared to the background fluorescence thereby signifying a positive result. There was 100% agreement between the GNR Traffic light PNA FISH assay and routine identification techniques. One sample which contained a mixture of K.pneumoniae and stenotrophomonas species was negative for stenotrophomonas by the PNA FISH method. GNR Traffic light PNA FISH was not designed to distinguish this organism. This sample highlights a limitation of the current assay which without routine culture identification this organism would not have been identified. This could potentially impact severely on the patient whereby treatment of a BSI caused by K. pneumonia would not have provided antimicrobial cover against the Stenotrophomonas species which was also cultured from this sample.
GNR Traffic light PNA FISH results informs the medical scientist that a member of the Enterobacteriacae (E.coli or K.pneumoniae) versus Pseudomonas species is causing a BSI, providing potentially important information for guiding more appropriate therapy earlier as Pseudomonas treatment differs to that of an Enterobacteriacae BSI. PNA FISH provides an improvement over Gram stain morphology results alone.
Given the increasing resistance to antimicrobial agents especially among the GNBs, the advent of PNA FISH assays that can identify pathogens within hours represents a potential advantage over traditional methods which may take 1-3 days (Morgan, Marlowe et al. 2010).
Immunocompromised patients such as: transplantation and oncology patients are especially at risk for contracting Candidaemia. Identification of the infecting Candida species causing BSI is used to guide appropriate antifungal therapy, conventional laboratory identification methods can take up to 5 days or longer. Yeast Traffic Light PNA FISH enabled rapid detection Candida species directly from positive blood cultures including C.albicans /C. parapsilosis (32) and C.glabrata (14) in less than 2 hours instead of several days. A recent study by Della-Latta et al., demonstrated that rapid identification of Candida species using PNA FISH can significantly impact antifungal selection and care for patients with candidemia. In the study, rapid identification of C.albicans led to a switch to the antifungal drug fluconazole for 70% of the patients that had been on caspofungin, a newer, more broad-spectrum but more expensive antifungal drug. At the same time, rapid identification of C.glabrata, a Candida species with high levels of resistance to fluconazole, led to an 81% switch to caspofungin for those patients that had otherwise been given fluconazole (Della-Latta, al et al. 2008). Based on the study results, the authors concluded that the Yeast Traffic light PNA FISH assay can impact the appropriate selection of the most effective antifungal therapy, thereby making it a clinically relevant diagnostic assay for BSI.
Our results corroborate with previous studies of PNA FISH assays targeting BSI microbial pathogens as performed by Oliveira et al., 2002. This study found PNA FISH easy to perform and highly specific. The potential clinical utility of PNA FISH assays for specific identification of micro organisms such as K. pneumoniae, S.aureus and E.faecalis which are some of the major pathogens causing BSI should enable clinicians to provide early, effective and appropriate antifungal therapy for patients with BSI. A limitation of the current PNA FISH assays is that they do not allow the identification of organisms such as Streptococcus pneumonia; as such identification is possible by conventional techniques.
With a further extension in the number of PNA probes available allowing pathogen identification, this would rectify this shortcoming of PNA FISH. PNA FISH provided pathogen identification results within 2 hours.
While there was a noted majority of Bactec Aerobic medium and Anaerobic medium blood culture bottles tested in the study in comparison to Bactec PAED medium (figure 3.14), no performance difference for specificity was observed between bottle types. The benefits of implementing these new molecular assays for rapid identification of pathogens causing BSI as shown not only has the potential to improve patient care and reduce the use of empirical antibiotic treatment in the healthcare setting. These assays for the clinical laboratory provide an opportunity for critical testing to be performed in the laboratory with long term cost savings. The PNA FISH assay and the Xpert MRSA/SA blood culture assay are performed on positive blood cultures and provide results within 1 and 2 hours respectively. This substantial time gain in identification would allow laboratory personnel to be available to perform other laboratory work. A limitation of both assays is conventional culture and susceptibility testing would still have to be performed on all positive blood cultures. Limitations are found in both methods. The Xpert MRSA/SA blood culture assay costs approximately Ð„31 per assay while PNA FISH assays cost between Ð„21 and Ð„26 per assay. While PNA FISH is cheaper than Xpert assay; for an organism such as S.aureus it does not distinguish MSSA from MRSA as can be identified by the Xpert MRSA/SA blood culture assay. As previously highlighted the Xpert assay is limited to S.aureus i.e. it cannot identify other pathogen causing BSI, similarly the limited number of PNA probes does not allow the identification of other pathogens causing BSI such as S.pneumoniae (8% of all cases of BSI reported in Ireland, 2008). The short time to identification result represents a significant advantage in comparison to conventional identification techniques and the ease of use of both methods would allow easy incorporation into the routine working day of the laboratory. Therefore both methods are worth considering for implementation into use in the Medical Microbiology laboratory.
Chapter 5: Conclusion
This study demonstrates the effectiveness of new molecular assays for the detection and identification of micro organisms and yeast in positive blood cultures. The Xpert MRSA/SA blood culture assay allows for rapid identification of S.aureus and or MRSA from positive blood cultures. In Ireland 40-50 % of S.aureus bacteraemias are caused by MRSA; therefore rapid identification of MRSA would result in infection control procedures being implemented sooner and a reduction in healthcare costs. Similarly PNA FISH assays based on complementary hybridization of PNA probes and species specific rRNA sequences allows for the identification of a number of pathogens causing BSI. Both assays provide results in hours of blood culture positivity which is a significant time reduction in comparison to routine conventional identification techniques. This decrease in turnaround time through molecular testing would result in effective antimicrobial therapy being administered sooner and a reduction in antimicrobial resistance caused by empiric antimicrobial usage. The ease of use of both assays found in this study would allow easy integration into routine laboratory use.
Della-Latta, e. al, et al. (2008). Impact of Rapid Identification of C. albicans and C. glabrata Directly from Blood Cultures using PNA FISH Technology on Selection of Antifungal Therapy. Poster 1382. ECCMID. Barcelona, Spain.
Dellinger, R. P., J. M. Carlet, et al. (2004). "Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock." crit care med 32(3): 858-873.
Donnio, P.-Y., D. C. Oliveira, et al. (2005). "Partial Excision of the Chromosomal Cassette Containing the Methicillin Resistance Determinant Results in Methicillin-Susceptible Staphylococcus aureus." J. Clin. Microbiol. 43(8): 4191-4193.
Enns, R. K. (2008). "Xpert MRSA/SA blood culture assay kit insert." Retrieved 7/5/2011, from http://www.cepheid.com/media/files/eu/brochures/Xpert%20MRSASA_BC_EU_Brochure.pdf.
Forrest, G. N., M.-C. Roghmann, et al. (2008). "Peptide Nucleic Acid Fluorescent In Situ Hybridization for Hospital-Acquired Enterococcal Bacteremia: Delivering Earlier Effective Antimicrobial Therapy." Antimicrob. Agents Chemother. 52(10): 3558-3563.
Francois, P., M. Bento, et al. (2007). "Evaluation of Three Molecular Assays for Rapid Identification of Methicillin-Resistant Staphylococcus aureus." J. Clin. Microbiol. 45(6): 2011-2013.
Gaibani, P., G. Rossini, et al. (2009). "Blood culture systems: rapid detection--how and why?" Int J Antimicrob Agents. 34(suppl.4): S13-15.
Gharibi, S., S. Tajbakhsh, et al. (2010). "Evaluation of flourescent insitu hybridization for rapid diagnosis of enterococcal wound infection " African Journal of Microbiology Research 4(23): 2498-2502.
Hawkey, P. and D. A. Lewis (2004). Medical bacteriology: a practical approach, Oxford University Press.
Kollef, M. H. (2000). "Inadequate Antimicrobial Treatment: An Important Determinant of Outcome for Hospitalized Patients." Clinical Infectious Diseases 31(Supplement 4): S131-S138.
Morgan, M., E. Marlowe, et al. (2010). "Multicenter Evaluation of a New Shortened Peptide Nucleic Acid Fluorescence In Situ Hybridization Procedure for Species Identification of Select Gram-Negative Bacilli from Blood Cultures." J. Clin. Microbiol. 48(6): 2268-2270.
Oliveira, K., S. M. Brecher, et al. (2003). "Direct Identification of Staphylococcus aureus from Positive Blood Culture Bottles." J. Clin. Microbiol. 41(2): 889-891.
Oliveira, K., G. W. Procop, et al. (2002). "Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes." J. Clin. Microbiol. 40(1): 247-251.
Pechorsky, A., Y. Nitzan, et al. (2009). "Identification of pathogenic bacteria in blood cultures: Comparison between conventional and PCR methods." Journal of Microbiological Methods 78(3): 325-330.
Peters, R. P. H., P. H. M. Savelkoul, et al. (2006). "Faster Identification of Pathogens in Positive Blood Cultures by Fluorescence In Situ Hybridization in Routine Practice." J. Clin. Microbiol. 44(1): 119-123.
Rigby, S., G. W. Procop, et al. (2002). "Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles." J. Clin. Microbiol. 40(6): 2182-2186.
Rossney, A. S., C. M. Herra, et al. (2008). "Evaluation of the Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Assay Using the GeneXpert Real-Time PCR Platform for Rapid Detection of MRSA from Screening Specimens." J. Clin. Microbiol. 46(10): 3285-3290.
Ryan, K. J. and C. G. Ray (2010). Sherris medical microbiology, McGraw Hill Medical.
Scola, B. L. and D. Raoult (2009). "Direct Identification of Bacteria in Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry." PLoS ONE 4(11).
Warren, D. K., R. S. Liao, et al. (2004). "Detection of Methicillin-Resistant Staphylococcus aureus Directly from Nasal Swab Specimens by a Real-Time PCR Assay." J. Clin. Microbiol. 42(12): 5578-5581.
Wolk, D. M., M. J. Struelens, et al. (2009). "Rapid Detection of Staphylococcus aureus and Methicillin-Resistant S. aureus (MRSA) in Wound Specimens and Blood Cultures: Multicenter Preclinical Evaluation of the Cepheid Xpert MRSA/SA Skin and Soft Tissue and Blood Culture Assays." J. Clin. Microbiol. 47(3): 823-826.