Avolio, Manuela; Diamante, Paola; Modolo, Maria Luisa; De Rosa, Rita; Stano, Paola; Camporese, Alessandro
Bone (1) defined sepsis as systemic inflammatory response syndrome (SIRS) plus infection, severe sepsis as sepsis associated with organ dysfunction, hypoperfusion, or hypotension, and septic shock as sepsis with arterial hypotension, despite adequate fluid resuscitation. These general definitions are now widely used in practice and serve as the basis for the inclusion criteria of numerous clinical trials.
The current criterion standard for bloodstream microbial detection and identification is blood culture (BC), followed by Gram stain, subculturing, and antibiotic susceptibility testing. The major limitation of culture is the time required to complete the process, which ranges from 1 to 5 days or more. This timeline is inconsistent with the need to obtain rapid answers to guide therapy. Moreover, an inadequate sample volume, sampling during antibiotic therapy, or the number of repeated blood draws usually affect the sensitivity of BC. Blood cultures are reported to be negative in more than 50% of the cases with true bacterial or fungal sepsis (1–3).
Diagnosis of sepsis is difficult as its clinical signs often overlap with noninfectious condition. These signs are tachycardia, leukocytosis, tachypnea, and pyrexia and define the SIRS clinical picture. Systemic inflammatory response syndrome can be found in various conditions including trauma, burns, pancreatitis, and post–cardiac arrest syndrome. This means that critically ill patients often have a SIRS syndrome (2). The SIRS concept is valid to the extent that a systemic inflammatory response can be triggered by a variety of infectious and noninfectious conditions. It is very important for clinicians and researchers to have the proper tools to recognize and diagnose infective SIRS promptly (1).
Research is ongoing to find new markers to better discriminate SIRS related to infection from SIRS unrelated to infection. Molecular techniques are becoming more and more useful to decrease laboratory turnaround time so that results can be available to the clinicians at an earlier stage. However, these tests remain to be evaluated and validated in clinical settings.
Conventional laboratory signs of sepsis and acute phase protein biomarkers (C-reactive protein, procalcitonin, cytokines) are sensitive and easy to use, but often also very nonspecific (3, 4) Molecular-based technologies are emerging as promising tools, in addition to BCs, for rapid identification of the etiological agents’ DNA in blood during an infective SIRS. However, several limitations exist (i.e., costs and the need for special equipment), which negatively affect the implementation of these techniques for routine laboratory diagnostics. Nevertheless, molecular diagnostic, despite inherent limitations, reflects currently the most promising avenue to decrease time to result and to influence decision making for antibiotic therapy in the septic host.
In our preliminary study (5), we have shown a high correlation between a multi-pathogen probe-based real-time polymerase chain reaction (Light Cycler SeptiFast [SF] assay, Roche Diagnostics GmbH, Mannheim, Germany) assay with respect to conventional BC results; thus, we hypothesized that molecular approach should be adopted in addition to traditional BCs to support early evidence-based treatment decisions in all patients admitted to emergency room (ER) with suspected diagnosis of sepsis.
In the present study, we have evaluated prospectively the diagnostic value of microbial DNA detected in a heterogeneous cohort of patients suspected of having infective SIRS, and its additional value respect to the traditional approach in order to obtain, as quick as possible, an accurate etiological diagnosis of infective SIRS.
MATERIALS AND METHODS
Patient inclusion criteria
Over a period of about 3 years (from September 2008 to December 2011), 830 adult patients (aged >18 years) admitted to the ER and to the intensive care unit (ICU) of the S. Maria Degli Angeli Hospital (Pordenone, Italy) with suspected bloodstream infections and at least two criteria of the systemic inflammatory response syndrome (SIRS) (6) with temperatures greater than 38°C or less than 36°C, heat rate greater than 90 beats/min, PaCO2 less than 32 mmHg, or white blood cell count greater than 12,000 or less than 4,000 cells/µL were included in the study.
Five hundred twenty-five of 830 requests for SF polymerase chain reaction (PCR) were comparable with BC; in the remaining 305 cases, the clinicians did not request BC assay if patients were already under antibiotic treatment at the moment of blood sample collecting, or they had already previous BC-negative results, such as in long-term, critically ill inpatients (nonresponder patients).
LightCycler SF PCR
A total of 830 patients were analyzed with LightCycler SF. The LightCycler SF Test M Grade assay detects a wide range of bacterial and fungal pathogens (7). Blood collected in EDTA tubes was lysed according to the manufacturer’s instructions (LightCycler SF Test M Grade; Roche Diagnostics). DNA extraction was performed in a laminar flow cabinet situated in a dedicated pre-PCR room to prevent contamination. DNA was extracted by use of a SF Lys kit on a MagNA Lyser platform (Roche Diagnostics), followed by silica matrix purification. We performed multiplex PCR using the LightCycler SF MGRADE test, version 2.0 (Roche Diagnostics), an in vitro nucleic acid amplification test for the detection and identification of DNA from bacterial and fungal microorganisms (see the SF master list in Table 1). Internal controls for the amplification step are included with the assay. Gram-positive and gram-negative organisms were targeted via the internal transcribed spacer between the 16S and 23S rRNA genes, whereas fungi were amplified by using the 18S and 5.8S rDNA sequence. SeptiFast identification software (SIS; version 1.1) was used to identify the organisms on the SF master list by melting curve analysis. The processing time was in total around 6.5 h and consisted of 30 min for sample thawing, 90 min for DNA extraction, 45 min for PCR setup, and 3.5 h for the PCR itself, followed by 5 min for automated software analysis.
In this study, SF assays were performed once daily for samples collected in the previous 24 h.
Analytical sensitivity (limits of detection) of the PCR test
The analytical sensitivity of the PCR test is 30 colony-forming units (CFUs)/mL for organisms listed in Table 1, except coagulase-negative staphylococci (CoNSs) (Staphylococcus epidermidis and Staphylococcus haemolyticus), Streptococcus species (Streptococcus pyogenes and Streptococcus agalactiae), and Candida glabrata, for which the minimum sensitivity is 100 CFUs/mL (manufacturer’s instruction).
A total of 525 patients were analyzed by BC (3150 BC bottles). Blood cultures were analyzed using Bact/Alert 3D automated system (bioMérieux, Marcy l’Etoile, France). Briefly, from Monday to Friday from 7:30 am at 5:00 pm and on Saturdays and Sunday from 7:30 am to 12:30 am, when the standard aerobic and/or standard anaerobic BC incubated bottles gave a positive signal, Gram staining was carried out. An aliquot of positive BCs was plated onto solid media and incubated for at least 24 h; identification was carried out with the Vitek 2 system or API (bioMérieux) according to laboratory-defined standard procedures.
Definition of pathogen
Microorganisms detected by SF were considered to be pathogens if results coincided with those of the BC analysis and/or, according to the American College of Clinical Pharmacy/Society of Critical Care Medicine Consensus Conference Committee definition of infection (3), if the species is generally accepted as a common etiologic agent of the patient’s type of infection (8). Coagulase-negative staphylococci detected in only one bottle from two BC sets were not considered pathogens in the absence of clinical and/or laboratory data suggesting their pathogenetic role (9).
Clinically or laboratoristic documented infection
In SF+/BC− and in SF+/BC not requested cases, microorganisms identified by SF were interpreted as true pathogens in the presence of at least one of the following criteria (10): (i) if a microorganism was detected only by SF, culture results from samples taken from the suspected infection site, if available, were evaluated (e.g., central venous catheter, urine, sputum, or bronchoalveolar lavage fluid or from another normally sterile site). If these culture tests revealed the presence of the same organism, it was considered a pathogen; (ii) if a microorganism was detected in only SF without any other culture support, the clinical picture was evaluated. The microorganism was considered a pathogen if a site of infection was documented clinically and/or radiologically in concomitance with the positive SF finding and the microorganism identified by SF was consistent with a potential pathogen according to the site of infection (11).
Skin disinfection was performed with chlorhexidine digluconate alcoholic spray formulation (Citroclorex 2%; Esoform, Rovigo, Italy) (12). A BC bottle consisted of 20 mL of blood obtained either by venipuncture or from an intravenous access device. A maximum of three sets (six bottles) of BCs for patients, obtained during a 24-h period and arrived simultaneously at laboratory, was included, according to Lee et al. (13).
During a febrile episode, a single venipuncture was used to draw samples for 2 × 3 bottles of Bact/Alert (bioMérieux). Immediately after the blood was drawn for the BC (8–10 mL/bottle), a 1.5 mL of whole blood was collected in sterile EDTA-KE tubes (Sarstedt, Nümbrecht, Germany) for the molecular method. SeptiFast result was compared with the results obtained with all culture bottles taken on different time points during a 24-h period and arrived simultaneously in our laboratory. For the definition of contaminants in our laboratory, we follow the workup indicated by Weinstein (14).
Diagnostic performances of different methods
Subdivision and analysis of six different “subgroups” of SF results are shown in Figure 1 (SF+/BC+, SF−/BC−, SF+/BC−, SF−/BC+, SF+/BC not requested, SF−/BC not requested). Out of the 830 requests for SF PCR assay, 170 resulted positives and 660 negatives. As described in Materials and Methods, only 525 of 830 requests for SF PCR were comparable with BC; in the remaining 305 cases, the clinicians did not request BC assay if patients were already under antibiotic treatment at moment of blood sample collecting, or they already have previous BC-negative results, such as in long-term, critically ill inpatients (nonresponder patients).
SeptiFast versus BC
The overall concordance between the SF−/BC−, SF+/BC+ subgroups (a total of 525 patients) resulted to be 86.4%. We excluded from the analysis the 10 cases of CoNS detected in only one bottle from two BC sets, thus considered as contaminants according to laboratory-defined standard procedures (14). We also excluded from the analysis seven microorganisms (Achromobacter species, Bacteroides capillosus, Bacteroides thetaiotaomicron, Corynebacterium urealyticum, Escherichia fergusonii, Fusobacterium species, Salmonella typhi) detected in BC, but not in SF because of the lack of specific DNA targets in SF master list (Table 1).
Among cases with either SF or BC requests (525 cases), 356 (88.6%) of 402 were negative by both methods (SF−/BC−), and 46 (11.4%) were negative discrepant results with respect to BC (SF−/BC+). Table 2 shows SF-negative (SF−) discrepant results compared with BC-positive results for each microorganism.
Overall, 83 (67.5%) of the 123 SF+ cases showed concordant results by both methods (SF+/BC+); 40 (32.5%) cases found no correlation with BC results (SF+/BC−) (Table 3).
Rule-in diagnostic value of SF+ results
Focusing our attention on SF+/BC− and SF+/BC not requested subgroups summarized in Table 4, 87 SF+ results were analyzed and interpreted on the basis of clinically or laboratoristic signs of documented infection (see Materials and Methods). Clinical and laboratory data (clinical diagnosis and microbiological data from samples other than blood or additional blood samples) supported the etiology of sepsis in 72 of 87 cases positive by SF only, whereas in 15 of 87 neither clinically nor laboratory infection was documentable.
In the absence of a laboratory reference standard for the diagnosis of sepsis, we evaluated SF+ results assuming that (i) the BC result is 100% accurate; (ii) the BC/CDI/LDI is 100% accurate; (iii) the findings by BC or SF are 100% accurate excluding contaminant isolates and/or DNA not detectable by the SF method (15). In Table 5, the first column shows only the subgroup SF+/BC+ (n = 83 patients); the second column shows the subgroup SF+/BC+ plus SF+/BC− and SF+/BC− not requested (total n = 155 patients) with clinically or laboratoristic documented infection (in 15 of 170 SF+ patients, neither clinically nor laboratory infection was documentable). On the basis of these definitions, the specificity of SF calculated was 0.960 (95% confidence interval [CI], 0.939–0.975), and the positive predictive value (PPV) was 0.912 (95% CI, 0.867–0.945). The sensitivity of SF calculated was 0.795 (95% CI, 0.756–0.824), and the negative predictive value (NPV) was 0.899 (95% CI, 0.880–0.913). The sensitivity and the NPV of SF with respect to BC/CDI/LDI standard are not available because of lack of clinical/laboratoristic information about the SF−/BC− subgroup of patients.
Time to result (SF versus BC)
Turnaround time for final microbial identification of pathogens of BC isolates was compared with time for microbial DNA detection by SF. Mean and median time to result were calculated for SF, preliminary Gram stain, and definitive culture results from the arrival of the sample in the laboratory (Table 6). In recent years, we reorganized our laboratory in order to cover all working days, 7 days a week: from Monday to Friday, from 7:30 am to 5:00 pm and from 7:30 am to 12:30 am in the weekend.
Moreover, our Clinical Pathology Department offers a stat lab open 24 h/24 h to receive and store specimens for microbiology. With regard to BC, the Gram stain communication and the culture definitive results are guaranteed 7 days a week, at the above times. With regard to SF assay, all the technical staff is trained to perform PCR test, although currently, only one analytic session is guaranteed every 24 h on 7 days a week.
In this study, the longest median time to result was observed for samples arriving on Saturdays because in the first phase of this study the technical staff was not yet all able to perform PCR.
As shown in Table 6, a median of 36 h was necessary to obtain Gram staining. Besides, a final identification of the pathogens was achieved in a median time of at least 80 h, calculated from the arrival of the sample to the laboratory. We obtained a molecular analytical result in a median of 15 h (range, 6–36 h) calculated on the basis that SF had been performed once daily, as described in Materials and Methods. The time advantage was even more important for SF−/BC− samples in which a median time of 15 h (range, 6–36 h) was necessary to give a negative result versus 5 days for BCs. In Figure 2, the Kaplan-Meier curve for time-to-reported-result is represented for SF, Gram stain, and culture (BC negative and SF negative were excluded from this analysis).
Sepsis is a leading cause of morbidity and mortality in hospitalized patients worldwide (6). Microbiological culture provides the main route for infections diagnosis but by its nature cannot provide time-critical results that can affect early management (16). There is growing interest in the potential use of real-time PCR (RT-PCR) technology in diagnosing bloodstream infection by detecting pathogen DNA in blood samples within a few hours. Polymerase chain reaction–based techniques allow more rapid and sensitive detection of pathogens compared with conventional BC. Nevertheless, combined detection rate of both methods was significantly higher compared with PCR or BC alone (17). Culture and pathogen DNA are not equivalent measures of infection telling us different but complementary things about infection. The CFUs measured by culture microbiology represent only the viable organisms that survive the plating process and do not count dead cells, cells that cannot form colonies, or free microbial DNA that may have been liberated from lysed cells in the blood compartment (18, 19). Polymerase chain reaction may give a positive result in the absence of intact pathogens because it does not distinguish between DNA associated with viable bacteria and DNA originally from intact bacteria in the circulation, which have been destroyed as a result of host immune responses and/or recent antibiotic administration (16). There are several possible reasons for frequently reported so-called “false” positives in which PCR shows evidence of pathogen DNA in the absence of culturable organisms. Given the sensitivity of the PCR technique, it is important to rule out the possibility that a “false” positive occurs as a result of environmental contamination, although these events can be minimized by adoption of strict procedures for sample collection and processing (7, 16).
A major problem in studies comparing PCR-based diagnostics with BC is interpretation of pathogens detected by PCR that are not found in corresponding BCs, being BC a poor criterion standard (20, 21). To distinguish between false- and true-positive results, most investigators analyzed additional information available on these patients such as further additional microbiological tests and levels of inflammatory parameters: the majority of pathogens detected by PCR but not by BC could be identified as true positive because they were found by culture of specimen obtained from the infectious focus (17).
Several studies, while considering the numerous molecular approach limits, show that presence of microbial DNA in the bloodstream is a significant and prognostic event even if the accompanying BC remains negative (16, 20–22), thus suggesting that DNA may be a useful biomarker of the presence of extracirculatory site infection in these patients.
In a recent review Chang et al. (23) took into consideration 34 studies (6.012 patients) on LightCycler SF to diagnose suspected sepsis, concluding that the LightCycler SF has higher rule-in than rule-out diagnostic value. According to the evidence of Chang et al. (23), in the presence of a positive SF result, a clinician can confidently begin appropriate antimicrobial therapy while forgoing unnecessary additional diagnostic testing. However, although it is possible to describe potential benefits by including PCR results in the treatment decisions, currently there is no evidence that PCR improves patient important outcomes (23).
In our study, we evaluated 830 adult patients admitted in ER and ICU with suspected infective SIRS. In more than 50% (87/170) of critically ill patients strongly suspected of having sepsis, for which BC was negative or not requested, we improve the microbiological documentation for an etiological diagnosis only by the molecular method in a median time of 15 h, with specificity and PPV of 96% and 91,2% respectively.
The present study has two following main strengths: (a) to our knowledge, it is the biggest cohort of ICU and ED undifferentiated patients with positive result to SF (170 positives/830 total patients); this relatively undifferentiated patient group is in contrast to the patient populations used in other studies with particularly high risk of developing bloodstream infections; (b) our study describes in routine settings a realistic and feasible workflow model, which implies the need for all staff members trained to be trained in advanced skills in molecular biology for a 24-h/7-day service.
Our data of sensitivity and specificity (0.79 of 0.96, respectively) support the results of Chang and colleagues’ meta-analysis (0.75 of 0.92, respectively) (23). Thus, the major limitations of our data are the suboptimal sensitivity of SF, but we strongly underline the high rule-in diagnostic value of SF molecular assay. The high rule-in diagnostic value of SF molecular assay and feasibility of a workflow model in routine settings (described and shown in our study) are, in our opinion, the two main critical requirements to determine, in the future, how exactly then are these results critical on patient outcomes.
Molecular-based technologies have several limitations (i.e., costs and the need for special equipment), which negatively affect the implementation of these techniques for routine laboratory diagnostics. The development of completely automated systems (from extraction to read-out), which are now not available, may overcome this limitation. Meanwhile, the MagNA Pure Compact Instrument (Roche Diagnostics) should be a good solution for fast and easy automation of nucleic acid purification from small sample numbers. It performs one to eight nucleic acid purifications from a broad variety of sample materials in 20 to 45 min suitable for gene expression analysis using PCR and RT-PCR on the LightCycler System. Recently we have adopted this automatic extraction system, and we have observed an advantage of 1 to 2 h in terms of total process time to the time totaled of around 6.5 h by using the manual extraction (see Materials and Methods) (data not shown).
This study does not involve a clinical-therapeutic implication as it is limited by its observational nature. Although it is possible to describe potential benefits of including PCR results in the treatment decisions, we cannot conclude that PCR-driven treatment decisions improve antimicrobial therapy in a way that infection is controlled earlier or outcome is improved. We think that in order to reap the full benefits of PCR-based pathogen detection technologies, preliminary adjustments to laboratory staffing and workflows might be necessary. Therefore, it is unclear how to better integrate molecular techniques into clinical pathways. Moreover, clinical utility may depend on the population prevalence of septicemia, with potentially lower levels being detected in the ED setting than in the ICU setting. High-quality research into the real-clinical-time application of these results is a critical future step in the development of this technology. Although SF does not provide information on the susceptibility of microorganisms, the species identification of pathogen (with a PPV of 91.2%) in a median time of 15 h may be useful in improving microbiological documentation and on this basis in the reassessment of ongoing antimicrobial therapy, especially in nonresponder patients, on the basis of epidemiological data.
We think that we should consider DNAemia as a specific biomarker of infective SIRS non–culture based, which can guide an antibiotic empiric therapy. The DNAemia should be considered in our opinion as a very useful tool to guide the clinicians to distinguish an infective from an uninfective SIRS and, on the basis of epidemiological context, to guide better the more appropriate empiric therapy. Obviously, antibiotic susceptibility is a crucial test, but it needs at least 48 to 72 h from the collection of a sample. We never assume that BC should not be performed. Strengths and limitations of molecular methods had been explored in our previous work, and we further realized that conventional microbiology remains currently indispensable (5).
According to Dark (21), the performance of SF in different patient groups and clinical environments may vary significantly, and careful and specific evaluation is required for each scenario (19, 23–30), also evaluating the economic impact of an RT-PCR technique, in patients with sepsis (31).
Further studies are needed to monitor the kinetics of bacteria DNA appearance and clearance from the blood during infection and antibiotic treatment. It is important to consider that “false” positives may have biological significance and provide information of diagnostic value. For example, freely circulating pathogen DNA may be a biomarker of infection at extracirculatory sites due to shedding of pathogen DNA into the circulation (16). We think that molecular assay may be precious for patients with persistent signs of infections who may benefit from modification of empirical antibiotic therapy.
In conclusion, BC is an indispensable test, as rightly emphasized in literature, but it stands to reason that SF and BC are two different tools; thus, we need both for their different diagnostic potential role. Therefore, we believe that the two tests do not need to be more compared any longer; in order to reap the full benefits of PCR-based pathogen detection technologies, preliminary adjustments to laboratory staffing and workflows are strictly necessary; direct molecular detection of pathogens in blood has impact on clinical utility for the analysis of pathogens in patients with a background of pretreatment with antibiotics before blood collection because of the severity of their condition (4); we highlight the role of pathogen DNAemia as time-critical, high-specificity, etiological, non–culture-based rule-in diagnostic biomarker in patients with presumed.
The authors thank Francesco Marchetti for his precious role in this study.
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