Quantitative data from the SeptiFast real-time PCR is associated with disease severity in patients with sepsis

Bloodstream infections are associated with high morbidity and mortality rates worldwide [1, 2]. Prompt administration of adequate antibiotics is crucial for successfully treating severe sepsis and septic shock [3, 4]. Blood culture (BC) is the current ‘gold standard’ for diagnosing bloodstream infections, but the method has several limitations. Firstly, the method has a low sensitivity both to slow-growing and fastidious organisms [5] and when antibiotics have been given prior to culture [6, 7]. Previous studies have shown that a positive BC is only found in about 30% of patients with severe sepsis and septic shock [8]. Secondly, it takes between 24 to 72 hours before the pathogen is identified. By implementing new techniques, such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), the time to pathogen identification can be significantly shortened, and it has been shown that this reduction has a significant effect on the time to adequate antibiotic treatment [9, 10].

For better, more appropriate initial antibiotic treatment, techniques that can confirm the infectious aetiology as soon as possible are needed. Using polymerase chain reaction (PCR) analysis to detect bacterial DNA in blood samples provides a result within hours, and is virtually unaffected by the prior use of antibiotics [5]. Additionally, PCR enables the quantification of bacterial DNA.

For broad, routine use of PCR techniques in sepsis diagnostics, there is a need for validated commercial assays. There are already a few commercial tests available for the molecular identification of pathogens directly on blood samples [11–13]. Among such tests, the Light Cycler SeptiFast Test (Roche Diagnostics) is, to date, the most used and investigated in clinical studies [14], and it has been approved for clinical use in Europe. The method has been evaluated in a systematic review [15], and was found to be specific, with a high rule-in value for the early detection of septic patients. The SeptiFast test is used for pathogen identification, but the result does not include any quantitative information.

The diagnosis of sepsis is sometimes difficult, and for the clinician it is a major challenge to, as early as possible, identify patients at risk from septic shock and death. In addition to the clinical evaluation, laboratory tests are currently the main diagnostic tools used in the initial assessment. Thus, a rapid molecular test that can contribute to predicting the risk of developing severe sepsis or septic shock would be of significant clinical value. Previous studies have shown that a high bacterial DNA load in the blood is related to a more severe disease, an increased risk of developing septic shock, and consequently a poor prognosis [16–20]. Given this, our aim was to study whether quantitative data from the commercial SeptiFast test could inform disease severity.

For this purpose, we used a study cohort of patients with positive Septifast test results from a recent prospective study [21]. The manufacturer provided access to Crossing point (Cp) values for all positive PCR results. A Cp value represents the PCR cycle at which DNA is significantly amplified, and it is generally accepted as an indirect and inverse marker of the DNA load.

In the present study, our aim was to investigate whether the level of the Cp values could differentiate cases with severe sepsis/septic shock from those with non-severe sepsis.

To our knowledge, this is the first study which evaluates quantitative data from a commercial PCR assay for bacterial species in blood samples.

The results of the study indicate that SeptiFast Cp values do correlate to disease severity in community onset bloodstream infections. When the SeptiFast test is used for pathogen identification and PCR positivity is reported, Cp values can probably be useful in identifying patients with severe sepsis/septic shock at an early stage of the disease. A low Cp value, with a cut-off level at 17.5 cycles as proposed in this study, might be used effectively as a predictive indicator in the clinical assessment. In addition to providing rapid information about the aetiology, a positive Septifast test with a Cp value <17.5 could be an early signal of the risk for a more severe disease and indicate that the BC will probably also be positive later on.

Concordance between BC and SeptiFast PCR has been shown to be moderate [21, 23–27], with a high frequency of PCR positive results not being confirmed by a positive BC, and vice versa. A positive PCR but a negative BC can occur due to fastidious microbes, sub-optimal culture conditions or antibiotic treatment before sampling. False-positive PCR results can occur due to contamination from sampling tubes or reagents. To minimise such risk, the EDTA tubes used in the study were endotoxin and DNA-free, and high-quality PCR reagents, free of bacterial or fungal DNA, were used in the SeptiFast test, as recommended by the manufacturer.

PCR positivity, even when BC remains negative, was recently found to correlate to disease severity [27]. This suggests that such a result probably often represents a relevant bloodstream infection that BC may have failed to detect.

In the present study there were several positive PCR tests with high Cp values and without microbiological verification, and it is difficult to determine their clinical significance. Many of the BC negative cases had other microbiological or clinical support, but not all of them [21].

A study cohort where all PCR positive samples were supported by a positive BC, or other microbiological or clinical verification, would have been optimal.

One possible explanation for a negative PCR despite a positive BC might be that the sample volume of approximately one mL, compared with 20 mL in BC, is not always sufficient to detect bacterial DNA, when the bacterial load is low. This is a limitation with SeptiFast and other PCR assays for bacterial identification directly on blood samples, leading to suboptimal sensitivities. Other reasons for a false-negative SeptiFast test might be competition for the PCR by human DNA in the blood, problems with the selection of species-specific targets in the test, or, for Gram-positive bacteria, especially Streptococcus species, a detection level set too high.

The most apparent advantage of molecular methods compared with BC is the possibility of a rapid diagnosis of aetiology with results available within hours. The time required to conduct a SeptiFast analysis is about 6 hours, but this assumes optimal laboratory conditions, which is difficult to obtain in everyday clinical practice.

A disadvantage with PCR techniques compared with BC is the lack of drug susceptibility testing. However, genetic determinants of drug resistance can be found by PCR. SeptiFast has an optional step where S. aureus positive samples are tested for the presence of the mecA gene in a subsequent run. We did not carry out this step in our study as the frequency of methicillin resistant S. aureus (MRSA) is low in Sweden, and all BC positive cases with S. aureus were methicillin sensitive in the present study.

Today, little is known about the bacterial DNA load in S. aureus bacteraemia [19]. In terms of the focus of S. aureus bloodstream infection, a correlation has been shown between time to positivity in BC, used as a surrogate marker for bacterial load, and endovascular infection sources, such as endocarditis [28]. It was recently shown that a persistently positive SeptiFast test can predict endovascular complications in patients with S. aureus bloodstream infections [29]. The endocarditis group in our study was very small, only four patients. Despite this, a correlation was found showing that Cp values seem to be lower in endocarditis than in other S. aureus bloodstream infections. Such correlation might be of significant clinical value as there is always a considerable risk of endocarditis in S. aureus blood stream infections. The Cp value could indicate when endocarditis should be suspected, and help distinguish which patients should be subjected to further diagnostics.

Our study has several limitations, the most important of which is the limited size of the study cohort. Further studies are needed to confirm the correlation between Cp values and disease severity, preferably with larger study cohorts and a higher proportion of patients with severe sepsis/septic shock. For certain pathogens the sample size was too small to enable any statistical analyses for the specific species. We have chosen to present results for the largest groups of specific pathogens, for Gram-positive and Gram-negative pathogens, and for the entire study cohort. The manufacturer of the SeptiFast test uses different Cp cut-offs for different pathogens, and pathogenicity and growth manner differ between species. Consequently, the associations we found for groups of pathogens put together are less reliable than the pathogen-specific associations.

The positive predictive values are calculated based on this study cohort, where the prevalence of severe sepsis was about 30%, and cannot be generalised. The suggested cut-off level at 17.5 cycles is calculated on this specific study population and needs further validation in other studies before it can be applicable in a clinical context. This value is also lower compared to other real-time PCR tests since the fluorescence in the SeptiFast test is not measured during the first six cycles.

The Cp values of the SeptiFast test can only serve as a positive predictive tool, as high Cp values cannot rule out severe sepsis/septic shock. In a real scenario, there would certainly be a number of cases with severe sepsis/septic shock and negative PCR, due to the limited sensitivity of the SeptiFast test [21]. As we lack a control group with such patients, we have not been able to calculate negative predictive values, which is a limitation.

Concerning the 12 samples that were positive for Gram-negative bacteria with Cp values >25, we unfortunately have no access to continuous variables. We have given them a Cp value of 25.0, which is a statistical concern. However, this should not affect the associations found, as data is presented in medians, and all calculations compare median values and not means.

Studies have shown that PCR identification using the SeptiFast test has the potential to become a cost-effective component in managing sepsis [30–32]. The possible benefits of a more rapid diagnosis of sepsis aetiology would be earlier adequate antimicrobial treatment and reduced mortality. However, there remains a need to optimise the commercial assays for this purpose, where the most important issue would be to obtain better sensitivities. In addition, we emphasise the need for further evaluation of quantitative data and its clinical implications.

Today the results of commercial PCR tests for sepsis diagnostics do not give information about the amount of DNA of the identified pathogen. Our results indicate that such quantification data would provide valuable information to the user concerning the clinical significance of a positive test. As an improvement of such tests, we suggest introducing automatic presentation of Cp values (or Ct values when other PCR platforms than the LightCycler are used) as a part of the test result. The manufacturer of The SeptiFast test can easily derive Cp values, and it should be possible to routinely share this information with the user. It would be even better in terms of easier interpretation, if a calculation of the bacterial DNA load, measured in copies/mL, was included in the test result.