The prognostic value of endotoxemia detection has been studied in more than forty studies 1234567891011121314151617181920212223242526272829303132333435363738394041. Conflicting conclusions became apparent from the earliest studies undertaken 242541.

The prognostic value remains unresolved despite 17 large studies including more than 2,000 patients 112124252627293031323335363738394041. On the one hand, in six studies endotoxemia was predictive of septicaemia onset or severe illness 1121243840 and hospital mortality 112426 among studies of hospitalized patients unrestricted to an ICU setting. On the other hand, 13 studies including eight among patients restricted to ICU settings found the detection of endotoxemia either did not predict organ dysfunction or mortality 25273031333941, predicted mortality but not organ dysfunction 35, predicted organ dysfunction but not mortality 29323738, or predicted mortality only when the level of endotoxemia was combined within a lipo-polysaccharide cytokine score 36. In only three 112124 of these 17 studies did the mortality difference between the groups positive versus negative for endotoxemia exceed 20 percentage points.

Several additional clinical observations indicate that the inter-relation between endotoxemia, gram negative (GN) bacteremia and outcome is not simple 424344. Less than two thirds of patients with GN bacteremia have endotoxemia detected and vice versa 42. The concordance with GN bacteremia varies with GN bacteremia species type 43. The structure function activity of lipid-A, the biologically active component of endotoxin differs for different GN bacteria 45. Furthermore, the impact of underlying risk of death is a key factor in the clinical setting 4647 but is difficult to investigate in laboratory studies 4849505152. These factors illustrate the 'disconnect' between attempts to study sepsis in animal models versus the clinical setting 50. The objective here is to evaluate the GN bacteremia species type and underlying patient risk as possible confounding factors of the prognostic value of endotoxemia as detected using the limulus assay in published clinical studies of patients across a broad spectrum of risk.

Thirty-five studies 1234567891011121314151617181920212223242526272829303132333435 were found of which 14 studies were supplemented with data provided by personal communication (Table 1). The survival outcome was reported for a total of 3,235 patients among these 35 studies of which 432 (13%), 272 (8%), 1,091 (34%) and 1,440 (44%) were in groups one to four, respectively. Patient inclusion for nine of the studies was based on various criteria for sepsis in adult patients in an ICU setting. A total of 26 of the 35 studies were published within the 1980s and 1990s. The largest study 35 provided mortality data stratified in relation to endotoxemia detection at two breakpoints.

The species types of GN bacteremia isolates were identified for 31 of the 35 studies [See Additional file 1]. Among the mono-microbial GN bacteremias, there were 174 (26%), 134 (22%), 74 (12%), and 94 (15%) bacteremias with Escherichia coli, Enterobacteriaceae other than E. coli (for example, Klebsiella species, Enterobacter species), Pseudomonas aeruginosa, and Neisseria meningitides, respectively. After excluding studies restricted to specified infections, there were 497 GN bacteremias with species type known among which there was an uneven distribution of E. coli versus P. aeruginosa identified among the GN bacteremias of group one versus group two; E. coli was less common in group one than in group two (92 of 303 (30%) versus 82 of 194 (42%), P = 0.007; chi-square test). By contrast, P. aeruginosa was more common in group one than in group two (53 of 303 (17%) versus 23 of 194 (12%), P = 0.09; chi-square test). This reciprocal mal-distribution was also apparent among the nine studies of adults with sepsis in an ICU setting (data not shown).

This reappraisal of this somewhat dated and disparate literature was undertaken to clarify the following as possible confounding variables toward the prognostic value of endotoxemia: ICU versus non-ICU setting, underlying patient risk, and the species types and distributions of GN bacteremia isolates within the studies. It uses L'Abbe plots to address these questions and adds 20 studies 12346789101213141516171819223335 with 9 received as personal communications, not included in a previous meta-analyses 57.

There are four findings from this analysis. The mortality risk in each of groups one (co-detection of endotoxemia with GN bacteremia), groups two (GN bacteremia alone) and groups three (endotoxemia alone) versus group four (neither) are generally either non-significant, or borderline (OR <2) when derived from only the nine studies in an ICU setting. However, when the 26 non-ICU studies are considered, the risks versus group four are similar to those derived from studies in an ICU setting with the exception of groups one (co-detection of endotoxemia with GN bacteremia), which is markedly increased. Note that these ORs for mortality risk as estimated here are all less than those found in the previous analysis that contained fewer studies 57.

Second, the I² associated with each summary OR are all 0% with the exception of that associated with the OR for group one versus group four derived from all 35 studies. With this latter exception, this absence of heterogeneity is surprising given the diversity of patient groups, underlying risk, and numbers and settings of these studies that have been conducted and published over a period exceeding three decades.

Third, the underlying patient risk, as reflected in the group four mortality proportion, was higher for studies of patients with sepsis in ICU settings versus studies in other settings (Figure 5a, b, and Figure 5c), as might be expected. However, with the notable exception of the co-detection of group one (endotoxemia with GN bacteremia), the additional risk for each of group two (GN bacteremia alone) and group three (endotoxemia alone) versus group four (neither) is generally similar in the ICU versus the non-ICU setting.

Finally, there are important differences in the prevalence and species types of GN bacteremias among these studies. Of the patients included within the studies of this meta-analysis (not counting GN bacteremias from studies of specified infections or studies in which the species types of GN bacteremia was not stated), 21% had GN bacteremia detected (group one and group two) of which 33% were E. coli whereas only 16% were P. aeruginosa. However, there are reciprocal differences in the frequency of P. aeruginosa versus E. coli among the bacteremias of groups one and two. The reciprocal distribution is a consequence of differences in endotoxemia detection rates, being more common for GN bacteremias with P. aeruginosa versus E. coli. This reciprocal distribution for different GN bacteremia species types is apparent among a broader collection of studies that used the limulus assay 43. Likewise, among 57 GN bacteremias found in a therapeutic trial in patients with septic shock selected on the basis of a positive detection of endotoxemia using the limulus assay, 15 versus only 12 of the GN bacteremias were P. aeruginosa versus E. coli, respectively 58.

The relative frequencies of bacteremias with P. aeruginosa versus bacteremias with E. coli among the studies are of interest for two reasons. The mortality risk for these two common bacteremias differs 59. For example, in the literature experience over thirty years to 2004, the mortality associated with P. aeruginosa bacteremia was typically 32% versus the mortality associated with E. coli bacteremia which was typically 19% 60. The basis for this higher mortality risk is multi-factorial with patient 4761 and treatment 6263 factors contributing. Also of interest are the structural differences in lipid-A, the biologically active component of endotoxin (lipopolysaccharide, LPS) 45, specific for P. aeruginosa versus E. coli. The different lipid-A structures of P. aeruginosa versus E. coli confer potency differences which are apparent in vitro 6465, and in vivo 66. While the clinical significance of these potency differences is unclear, the resulting unequal distributions of bacteremias among groups one and two uncovered here may confound the apparent relationship between endotoxemia and mortality risk.

This overview is based on a summation of disparate observational studies in an attempt to identify confounders underlying the disparate observations regarding endotoxemia and mortality risk in the literature. This analysis is unable to identify the mechanism for any increased risk. Many relevant patient specific details such as age and co-morbidities for the patients of the four groups in each study were not available. In this analysis, a variable proportion of blood cultures that were classified as negative for GN bacteria would have yielded gram positive bacteremias or fungemias. The detection of endotoxemia in association with blood culture isolates other than GN bacteremias is a commonly reported finding 2541. In this respect, the prognostic impact of blood stream infections other than GN bacteremias in relation to the co-detection with endotoxemia has not been addressed here. Moreover, the origin of endotoxemia for patients in group three is uncertain and the possibility of endotoxin originating from other sources, such as gut barrier breakdown, as is presumed to occur for non-septic forms of shock, needs to be considered 67. Also, it needs to be noted that endotoxemia and GN bacteremia are each either episodic or dynamic 68 phenomena and the criteria for a positive detection of each will have differed among the studies.

The additional mortality associated the co-detection of endotoxemia and GN bacteremia (group one) was less apparent in the studies of patients at high versus low underlying risk (Table 1). This finding here at a group level of analysis resembles other recent findings at an individual patient level of analysis among bacteremias of all species types occurring in an ICU setting 46. However, this inference requires caution for three reasons.

Firstly, it needs to be clarified as to whether the increased risk is absolute 47 or relative 46.

Second, the proportion of GN bacteremia types that are other than E. coli is more variable in studies outside of the ICU setting. In particular, there were five non-ICU studies 511122124 with an unusually high proportion of isolates other than E. coli among the GN bacteremias.

Third, it should be cautioned that the L'Abbé plots are useful merely as simple graphical methods to facilitate visual comparisons of the group mortality proportion over the range of underlying risk as found in individual studies within a meta-analysis 56. The issues underlying the statistical testing for variation in either additional risk or treatment effect in relation to underlying risk are not simple 6970. In particular, where linear regression has been used within L'Abbe plots to explore heterogeneity over a range of underlying risk in other contexts, regression to the mean is an important consideration as noted among treatment studies of mild hypertension 71.

In this analysis, group four, which is usually the largest group in each study, serves not only as the reference group for each study in the derivation of the ORs (Table 2) but also as a surrogate marker for the underlying mortality risk within each study (Figures 5a, b, and Figure 5c). However, this proportion will be an imprecise marker in small studies and studies with less restrictive patient selection. As a consequence of this imprecision, proportion estimates from small studies will have reference and comparator groups that by chance will be either more or less extreme than the average and hence, the accompanying comparator or reference group, respectively, will tend to be closer to the mean 72. This regression to the mean will cause these outlier studies to have a disproportionate impact on the overall relationship with underlying risk and will tend to inflate any deviation of a derived regression relationship with underlying risk away from the null. In the analysis here however, linear regression has not been used and the studies that trended away from the null in the L'Abbe plots were generally neither the smaller studies nor the studies with high event rates among group four.

There were insufficient studies that had used assays other than the limulus assay, or patient groups that had received anti-endotoxin antibodies to include these studies so as to enable a study of these variables by meta-regression. Only seven studies 11121314151617 were limited to specific GN bacteremia species types. Also, many relevant study level details, such as method of blood culture used and antibiotic therapy protocols were not available. Anti-endotoxin antibodies are detectable in patients with severe sepsis and septic shock and the kinetics of these antibodies over time differs between survivors and non-survivors 73. However, the impact of these anti-endotoxin antibodies on both the detection of endotoxemia and possibly also on patient outcome within the studies examined here is uncertain. A more detailed examination adjusting for relevant prognostic variables contributing to underlying patient level risk and also the inter-relation between various GN bacteria known to have differing lipid-A structures would require an individual patient data meta-analysis.

With these findings, it is now possible to re-appraise the relationship between endotoxemia detection and mortality risk as observed in the broader literature. With the patient populations dichotomized into endotoxemia positive versus negative, only five 511122124 of the 35 studies included in this analysis found differences in mortality proportions exceeding 20 percentage points (data not shown) and in all five of these studies there was an unusually high proportion of isolates other than E. coli (for example, P. aeruginosa or N. meningitidis) among the GN bacteremias and none of these five studies were restricted to ICU populations. There were a further four large studies in an ICU setting 36373839 that had not been included in this analysis as the mortality proportions were not extractable into a 2 × 2 × 2 contingency table format or used endotoxemia assays other than the limulus assay. None of these four additional studies found differences in mortality proportions exceeding 15 percentage points between endotoxemia positive versus negative patients. While one study had E. coli accounting for two of the four GN bacteremias 38, the GN bacteremia species types were not stated in the other three studies. One large non-ICU study did not report mortality as an outcome 40.

While some of the studies were conducted more than thirty years ago, they remain of interest due to the unresolved conflicting conclusions that continue to emerge. These large studies are difficult and expensive to undertake. An understanding of the previous uncertainties would assist in the planning of any further studies to be undertaken.

The extent to which the species type and prevalence of GN bacteremia confounds the relationship between the detection of endotoxemia and outcome and in the evaluation of anti-endotoxin and other adjuvant therapies for sepsis warrants further consideration 747576.

The underlying patient risk and the GN bacteremia species types within a study may be unrecognized confounders in the interpretation of the predictive value of endotoxemia. This may not only help to resolve conflicting observations in the clinical literature but may also help to bridge the 'disconnect' with animal models of sepsis. Clarification will help toward defining the exact role of endotoxemia within the pathogenesis of GN sepsis and the evaluation of anti-endotoxin and other adjuvant therapies for sepsis.

• There are conflicting conclusions regarding the prognostic value of endotoxemia detection among more than forty studies in various ICU and non-ICU settings.

• Using the limulus assay for endotoxemia detection, less than two thirds of patients with GN bacteremia have endotoxemia detected and vice versa.

• The mortality risk in association with the detection of endotoxemia or GN bacteremia either alone or together versus the detection of neither is generally either non-significant or borderline (OR <2) when derived from only the nine studies in an ICU setting.

• The co-detection of GN bacteremia and endotoxemia is most predictive of increased mortality risk versus the detection of neither but only in studies undertaken outside of an ICU setting.

• Variation in GN bacteremia species types and underlying risk are likely unrecognized confounders in the individual studies.

CI: confidence interval; GN: gram negative; LPS: lipopolysaccharide; ND: no data; OR: odds ratio.

The authors declare that they have no competing interests. Financial support was provided by The University of Melbourne. The funder had no role in the design, analysis or writing of this study or in its submission for publication.

JCH contributed to the study design, literature search, data analysis, writing and final approval of the manuscript for submission. BG, GO and EM contributed to the literature search, provision of original data and final approval of the submission. All authors read and approved the final manuscript.