The presence of circulating micro- organisms in the bloodstream is either part of the natural history of a disease or a reflection of serious uncontrolled infection. Depending on the causative agent this process is described as bacteraemia, fungaemia, viraemia or parasitaemia. (Ryan & ray, 2010)
The bloodstream is normally sterile in healthy individuals, the presence of micro organisms is considered is considered potentially serious. Transient bacteraemias may occur when there is trauma to a body site that has normal flora as occurs after tooth brushing. The organisms are soon cleared by the immune system and such transient bacteraemias have no clinical significance.
Septicaemia refers to systemic disease associated with the presence and persistence of pathogenic micro organisms or their toxins in the blood. Septicaemia is potentially life threatening and is recognized globally as having a high mortality rate and increasing in incidence. The causative agents of septicaemia include both gram positive and gram negative micro organisms. Cases of septicaemia 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 and reaches the capillaries. The most common source of organisms causing bacteraemia appear concomitantly with other serious infections such as urinary tract infection (UTI), respiratory tract infection (RTI), endocarditis, kidney and bowel infections.
Approximately 250,000 cases of blood stream infection are reported in the United States each year. The most prevalent bacteria that might cause bacteraemia have been reported. In Ireland these include Coagulase negative Staphylococci (CoNS) mainly Staphylococcus epidermidis (S.epidermidis). Bacteraemia caused by Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S.aureus) and Klebsiella pneumonia (K.pneumo) are long recognized in Ireland as a source of nosocomial bacteraemia.
Organism
% of BC+1
S. aureus
30%
CNS
70%
E. faecalis
45%
E. faecium
20%
Streptococcus sp.
35%
E. coli
35%
K. pneumoniae
20%
P. aeruginosa
15%
Other GNRs
30%
Candida sp.
5%
Other
5%
Figure 1: Prevalence of bacteria isolated from blood cultures of hospitalized patients in the United States in 2002.
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.
Because of this, treating clinicians are forced to use empiric antimicrobial therapy to cover all suspected pathogens while awaiting definitive reports from the laboratory. Therefore the introduction of rapid microbial testing techniques for blood stream infections in the clinical setting is necessary for both patient and healthcare payer perspectives.
1.2 Overview of the standard microbiological techniques and their drawbacks
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. While a number of systems are available the Becton Dickinson BD FX continuous monitoring blood culture system is employed in this laboratory.
The principle of this system is based on the sample to be tested inoculated into a blood culture bottle (aerobic, anaerobic and paed) which is entered into the BACTEC FX instrument for incubation and periodic reading. Each blood culture bottle contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms or the consumption of oxygen needed for the growth 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 or the decreasing amount of O2 present in the vial. A positive reading indicates the presumptive presence of viable microorganisms in the blood culture bottle.
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. 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 blood culture medium is also inoculated on to blood and chocolate agar and incubated for 24 hours. Inoculation on to blood and chocolate agar is performed in order to obtain colonies that would be subjected to identification. The positive blood culture is further processed following the standard laboratory procedure.
Previous studies have shown that treatment of blood stream infections with early, appropriate antimicrobial therapy reduces morbidity, mortality and subsequent healthcare costs. Prescribing broad spectrum antimicrobials before final culture and susceptibility is also reported to have reduced mortality and healthcare costs, the opportunity to move to a targeted antimicrobial therapy sooner using rapid microbiological and molecular techniques could 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 sooner they also benefit the healthcare institution resulting in cost savings.
In recent years the increase in multi drug resistant pathogens has highlighted the importance for rapid organism identification.
For many pathogens differentiation to species level is crucial an example is Enterococcus faecium (E.faecium). Isolates of E.faecium are resistant to ampicillin, which is however efficient for the treatment of Enterococcus faecalis (E.faecalis).
Correspondingly within the genus staphylococcus which is responsible for up to 50% of all cases of sepsis, CoNS require different therapy to Staphylococcus aureus (S.aureus). CoNS are commonly isolated from the blood but only approximately 20% of such isolates represent true infection, the remaining being contaminants. True CoNS infections are nearly always associated with the presence of foreign bodies such as vascular catheters.
Blood cultures with S.aureus as the isolate are associated with significant mortality.
Blood stream infections caused by candida species is presumptively diagnosed by the presence of gram positive yeast cells on gram stain of the contents of the positive blood culture. Empirical antimicrobial therapy which include common antifungals are effective against most candida species including Candida albicans (C.albicans). However there is an increasing prevalence of other candida species causing infection such as Candida glabrata and candida krusei with different resistance patterns to common antifungals such as fluconazole. This has necessitated more rapid identification of yeast species in blood cultures.
1.3 Currently available rapid molecular techniques
