Sepsis is defined as the host response to infection and it is an important cause of morbidity and mortality around the world 1 2 . The surviving sepsis campaign issued a call for global action against sepsis and pointed out diagnosis as a fundamental challenge 3 4 . In early stages of the process, the source of infection may be unclear and the related systemic response indistinguishable of no-infectious diseases. Consequently, clinicians often miss or delay this diagnosis. This is especially worrying; since there is strong evidence supporting that early treatment is associated with greater clinical success 5 . An ideal “Gold Standard” is not available for sepsis diagnosis, as microbiology is not enough sensitive and laboratory tests are not specific for using as reference standards. The lack of any reference standard has been overcome by using techniques that avoid the need for comparison with a single accurate test. These techniques can be broadly divided into latent class analysis (LCA) and Bayesian analysis 6 . LCA has been used widely in psychiatry as well as other disciplines 7 8 9 10 11 but, it has not been yet applied to the evaluation of sepsis.

On the other hand, sepsis is associated with the simultaneous activation of the inflammatory and coagulation cascades, and most of their components are markers or mediators in the host response 12 13 . From this close interplay between inflammation and coagulation, which is a recognized way toward organ dysfunction and mortality 14 , emerges the rationale to characterize the host response to infection. Three potential biomarkers have shown regular presence in systemic infections: C-reactive protein (CRP), procalcitonin (PCT), and D-dimer (DD); the latter as an unspecific signal of coagulation activation 15 16 17 18 19 . So far, however, no large prospective studies support any of them as a single independent criterion for sepsis. We aimed to estimate the diagnostic accuracy of these three biomarkers as diagnostic tests for sepsis, with the application of the latent class analysis, in patients at the ER admittance with a presumptive infection as main diagnosis.

Prospective single center study on the diagnostic accuracy of a test. The study protocol and a pre-specified nested analysis were previously published 20 21 .

Enrollment began on August 2007 and concluded on February 2009. A total of 1,795 patients were eligible and 1,030 were excluded, most of them because of more than 24 hours of hospitalization before recruitment and refusal to participate (Figure 1). Among 765 patients included, 683 (89%) had a suspected infection as admission diagnosis, 56 (7%) fever of unknown cause, 20 (3%) delirium or encephalopathy of unknown origin, and 6 (1%) unexplained hypotension. There were 377 males (49%), the mean age was 51.4 years (SD = 20), and the median time of symptoms before consultation was 72 hours (IQR = 24 to192 hours). There was no comorbidity in 307 (40%) of the participants, and the most frequent previous diseases were diabetes mellitus (n = 146, 19%), chronic obstructive pulmonary disease (n = 94, 12%), chronic renal failure (n = 88, 11%), use of corticosteroids or chemotherapy during the past 3 months (n = 70, 9%) and trauma or surgery in the previous month (n = 53, 7%). As suspected sources of infection, the most frequent were respiratory (n = 179, 23%) and skin and soft tissues (n = 174, 23%), followed by urinary tract (n = 127, 17%), intrabdominal (n = 93, 12%), undetermined (n = 94, 12%) and others (n = 96, 13%). The median SOFA and APACHE II score were 2 (IQR = 1-4) and 9 (IQR = 5-14), respectively, hospital length of stay was 9 days (IQR = 5 to 17 days), ICU admission was required in 66 patients (9%) and the overall 28-day mortality rate was 12% (n = 91). Due to logistic or technical reasons CRP, DD and PCT at admission were measured in748 (98%), 744 (97%) and 747 (98%) patients, respectively. The median and IQR values for these test were CRP = 9.4 mg/dl (3.5-20), DD = 1673 ng/ml (982–1841) and PCT = 0.4 ng/ml (0.1-3.65).

According to the expert committee 505 patients (66%) were classified with sepsis, 112 (15%) with infection but without sepsis and 148 (19%) without infection. The kappa-statistic measure for multi-rater agreement between experts was 0.65 for sepsis-no sepsis and 0.73 for infection with and without sepsis. Figure 1 shows this classification and their respective biomarker values, and Table 1 shows the main characteristics by group. Among infected patients (with and without sepsis, n = 617) a microbiologic diagnosis was confirmed in 104 patients (17%) by blood culture, in 135 (22%) by urinary culture and in 145 (26%) by other samples. Microorganisms isolated in blood were E. coli (n = 29, 28%), S. aureus (n = 17, 16%), coagulase-negative staphylococci (n = 15, 14%) and others (n = 43, 42%); and in urine were E. coli (n = 79, 59%), K. pneumoniae (n = 22, 16%) and others (n = 34, 25%). The main admission diagnosis in the sepsis group (n = 505) was community-acquired pneumonia (n = 115, 23%), followed by urinary tract infection (n = 87, 17%) and soft tissue infection (n = 79, 16%).The main alternative diagnosis in 148 patients without infection were cancer (n = 22,15%), chronic obstructive pulmonary disease (n = 15, 10%), acute pulmonary edema (n = 13, 9%), metabolic diseases (n = 13, 9%), biliary diseases (n = 10, 7%), and others (n = 75, 50%).

The values are expressed as median (IQR, Observations available) or number (percentage). MAP, Mean Arterial Pressure, WBC, White Blood Cells, COPD, Chronic obstructive pulmonary disease, CRF, Chronic renal failure.

*Kruskal-Wallis for continuous variables and Chi-square for proportions.

According to the ROC curve analysis, the cut-offs points with the best sensitivity and specificity for CRP, DD, and PCT to discriminate at admission between sepsis and not sepsis (infection without sepsis or not infection) were 7.8 mg/dl, 1616 ng/ml, and 0.3 ng/ml, respectively. Their operating characteristics, at both measurement times, are shown in Table 2. Analyses combining pairs of tests or using changes in the first 24 hours (Δ24) did not show any improvement in diagnostic accuracy. Similar results were seen using the alternative reference standards (data no shown).

Values between parentheses are cut-off points and 95% Confidence Interval, respectively. LR, Likelihood ratio.

The LCA analysis with the same three tests identified a “cluster” of 187 patients, among those defined as sepsis by the expert committee, with several characteristics suggesting a more severe condition as well as better microbiological confirmation compared to the rest of the study population. According to standard definitions, 70% (n = 131) of these patients had severe sepsis without circulatory failure and 5% (n = 9) had septic shock (Table 3) (Additional file 1). It was not possible to classify 46 patients because of missing values in any of CRP, DD or PCT. Assuming this cluster of 187 patients as the new prevalence of sepsis based on the LCA gold standard, the ROC curve analysis identified new cut-off points for the tests and suggesting a better discriminatory ability for PCT with a value of 2 ng/ml (Table 4).

The values are expressed as median (IQR, observations available) or number (percentage).

Missing = patients without information in any of PCT, CRP or DD; Cluster 1 = patients identified by LCA as a sepsis-like syndrome; Cluster 2 = the remaining of the study population; MAP, Mean Arterial Pressure; WBC, White Blood Cells; PaFi, PaO₂/FiO₂; COPD, Chronic obstructive pulmonary disease; CRF, Chronic renal failure.

*Kruskal-Wallis for continuous variables and Chi-square for proportions.

Values between parentheses are cut-off points and 95% Confidence Interval, respectively. LR, likelihood ratio.

Our results suggest that, under a “classical” definition of sepsis, three typical biomarkers (CRP, PCT and DD) are not capable enough to differentiate septic from non-septic patients in the ER. Indeed, the kappa-statistic measure for multi-rater agreement between experts for this definition was 0.65 for sepsis-no sepsis and 0.73 for infection with and without sepsis, which underlines the limitations for clinical diagnosis in this condition. Using another analytic approach, however, a higher cut-off point for PCT (2 ng/ml) is able to identify and to exclude a specific population more severely ill and with better microbiological confirmation. To the best of our knowledge, this is the first research that incorporates the novel concept of a latent class to the process of diagnosis in sepsis.

The performance of a diagnostic test is judged by how accurately the test result can identify a diseased or no diseased person. The true disease status is the “gold standard” against which a test should be compared. However, there are many conditions for which the definitive diagnosis is very difficult or expensive to establish. This is especially true for the diagnosis of a complex clinical condition as sepsis, in which even within the construct of “systemic response to infection” there is not a real “gold standard” against which the diagnostic criteria can be calibrated 22 28 29 . Psychological and social sciences have a long tradition in coping with primary study objects that are not directly observable. Constructs such as intelligence, fear or trust can only be measured indirectly. Inference proceeds by modeling the relationship between observable and latent variables in such a way that the parameters of interest are estimable from the implied relations between observable variables. When the unobservable variable is categorical, the term latent class analysis (LCA) applies 6 7 . In other words, LCA postulates the existence of an unobserved categorical variable that divides the population of interest in to classes. Members of the population with a set of observed variables will respond differently depending on the latent class to which they belong. This technique can be applied to the problems related to diagnostic testing, with the unobserved categorical variable being “disease present” or “disease absent” 20 .

Given the established interplay between inflammation and coagulation in sepsis 14 30 31 32 , it is reasonable to characterize the host response to infection as a potential diagnosis tool on the basis of three recognized markers of these two cascades. The sensible mathematical model of the latent diagnostic classification, using individuals’ values of CRP, DD and PCT, was able to identify a subset of patients attended in the ER with suspicion of infection and with clear differences in clinical status, microbiological profile and 28-day mortality. Although this subset was identified among those patients classified as sepsis by the expert committee, there is not a unique clear cut-off in any variable or test that may define the cluster specifically as severe or bacteremic sepsis (Table 3). Furthermore, among these three potential biomarkers, PCT proved to be the most contributor to the “new” standard of more severe disease but with a higher cut-off point than that usually suggested. Our main result, consequently, is that PCT is useful to identify a subgroup of more severely ill septic patients attending the ER. Such a finding was previously reported by Hausfater P et al. 33 , whom studied 243 patients with body temperature of 38.5°C or greater attended in the adult emergency department of an academic tertiary care hospital. They found, using standard statistical methods, that PCT is an independent variable that can predict whether a febrile episode has a bacterial origin, and that at a threshold of 2 μg/l it is independently associated with critical illness. The coincidence with our findings is remarkable, despite the fact that their study population was extremely different: all the patients consulted by a febrile episode, 29% of them were immunocompromised, and only 81% were hospitalized in that consultation.

Needless to say, sepsis is not an illness but a syndrome suspected mainly on clinical criteria, and the misdiagnosis of sepsis is associated with an extremely adverse outcome. Consequently, we are not proposing a new methodological approach for sepsis diagnosis. Instead, we are identifying a new cut-off point for procalcitonin to be able to detect more severely ill patients. This goal was not achieved by the conventional clinical gold standard with expert consensus and, in this way, LCA is just an instrument to show that we can improve the process of sepsis diagnosis in the emergency room. The recent literature is full of studies evaluating PCT for sepsis diagnosis 16 17 34 35 , but in the setting of the ER there are less investigations testing its potential usefulness. In a secondary care hospital of Finland, a population of 539 patients admitted to the ER with suspicion of infection and with clinician’s order for blood cultures was studied 16 . In assessing how the parameters differentiated all sepsis patients (n = 358) from patients with no sepsis (n = 181), AUC-ROC for PCT was 0.73 (95% CI 0.69 – 0.78), and PCT emerged as the best marker for severe sepsis with an AUC-ROC = 0.77 (95% CI 0.71 – 0.84). Riedel et al. evaluated the usefulness of PCT as a diagnostic predictive marker of bacteremia and sepsis in 259 patients who had blood cultures obtained in the ER of a tertiary medical center in Baltimore 35 . In16 patients there was evidence of bacteremia and 12(75%) patients had a PCT level of more than 0.1 ng/ml. The PCT cut-off value that maximizes the AUC-ROC (0.79) was 0.1475 ng/ml., but with sensitivity just in 75% and specificity of 79.8%. In a recent meta-analysis 36 , Wacker C. et al. analyzed 30 reports, although only two from ER, accounting for 3244 patients. Bivariate analysis yielded a mean sensitivity of 0.77 (95% CI = 0.72–0.81) and specificity of 0.79 (95% CI = 0.74–0.84) and the area under the receiver operating characteristic curve was 0.85 (95% CI = 0.81–0.88). The median cut-off for PCT of the studies included was 1.1 ng/ml (IQR = 0.5–2.0) and the absence of a threshold effect suggests that a cut-off between 1.0 and 2.0 ng/ml, close to our findings, is helpful for discrimination of patients with sepsis from other inflammatory conditions. However, the studies had substantial heterogeneity (I ² = 96%, 95% CI = 94–99) and none of the subgroups investigated like population, admission category, assay used, severity of disease, and description and masking of the reference standard, could account for that heterogeneity. They concluded that the test may be helpful for diagnosis of sepsis in critically ill patients, but it must be interpreted in context with information from careful medical history, physical examination and microbiological assessment.

Our study has several limitations. First, this is a single center study in a specific geographic location with some particularities from an epidemiological point of view 2 37 , which are obstacles for external validity. Moreover, the pre-test probability of sepsis should be significantly different in patients admitted to the ER and in patients admitted to ICU, even with the same clinical suspicion of bacterial infection, and this is an acknowledged consideration in the use and interpretation of any diagnostic test. As we mentioned before, the clinical diagnostic “gold standard” utilized here performed poorly, as the concordance between experts was 0.65 for sepsis-no sepsis and 0.73 for infection with and without sepsis. This weakness, indeed, underlines the limitations for clinical diagnosis in this condition. On the other hand, LCA also has its limitations as “gold” standard. Under this approach, sepsis is not formally defined but rather is a mathematically defined entity that does not necessarily correspond with a clinically relevant status. Additionally, LCA modeling requires sophisticated analytic techniques and software, and the full model or the hypothetical “true” state of disease cannot be fully tested with the observed data. Finally, although blood sampling was performed immediately after the patient was admitted to the study, he/she could be in the ER at any time within the last 24 hours before recruitment. This is important because biomarker’s kinetic, notably PCT, and their levels may varying considerably during 24 hours.

In summary, the “holy grail” of sepsis diagnosis is an evolving process and the fine exercise of clinical suspicion should be complemented by appropriate laboratory test. In this scenario, PCT emerges as an acceptable choice underscoring both microbiology and prognosis in selected patients. A higher level of PCT seems related more strongly with these two components of the infectious phenomena.

Funded by COLCIENCIAS (Grant 1115-343-19153) and Estrategia de Sostenibilidad 2013–2014 Universidad de Antioquia. The authors declare that they have no competing interests and the investigation was conducted with ethical adherence to investigations in humans.

FJ and GD conceived and designed the study and obtained research funding. FJ, GD, MV, CA, CG, AG, SO, SO and AH supervised the conduction of the study and data collection. FJ, GD, MV undertook recruitment of participating patients and managed the data, including quality control. FJ provided statistical advice on study design and analyzed the data. FJ chaired the data oversight committee. FJ drafted the manuscript and all authors contributed substantially to its revision and final approval of the version to be published. FJ and GD take responsibility for the paper as a whole. All authors read and approved the final manuscript.