Prognostic value of plasma pentraxin 3 levels in patients with septic shock admitted to intensive care.

OBJECTIVE: To evaluate the usefulness of a new marker, pentraxin, as a prognostic marker in septic shock patients.
MATERIALS AND METHODS: Single-centre prospective observational study that included all consecutive patients 18 years or older who were admitted to the intensive care unit (ICU) with septic shock. Serum levels of procalcitonin (PCT), C-reactive protein (CRP) and pentraxin (PTX3) were measured on ICU admission.
RESULTS: Seventy-five septic shock patients were included in the study. The best predictors of in-hospital mortality were the severity scores: SAPS II (AUC = 0.81), SOFA (AUC = 0.79) and APACHE II (AUC = 0.73). The ROC curve for PTX3 (ng/mL) yielded an AUC of 0.70, higher than the AUC for PCT (0.43) and CRP (0.48), but lower than lactate (0.79). Adding PTX3 to the logistic model increased the predictive capacity in relation to SAPS II, SOFA and APACHE II for in-hospital mortality (AUC 0.814, 0.795, and 0.741, respectively). In crude regression models, significant associations were found between in-hospital mortality and PTX3. This positive association increased after adjusting for age, sex and immunosuppression: adjusted OR T3 for PTX3 = 7.83, 95% CI 1.35-45.49, linear P trend = 0.024.
CONCLUSION: Our results support the prognostic value of a single determination of plasma PTX3 as a predictor of hospital mortality in septic shock patients.

Introduction

In 2017, the World Health Organization established sepsis as a global priority [1]. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) defines sepsis as life-threatening organ dysfunction caused by a dysregulated host response to infection [2]. It is a major cause of admission to the intensive care unit (ICU) and a leading cause of mortality and critical illness worldwide; it is responsible for more than 5 million deaths annually, with an estimated global mortality of 30% [3].

Sepsis and septic shock are medical emergencies, so prompt identification and appropriate management in the initial hours after onset improve outcomes [4], especially among high-risk patients. Thus, biomarkers allowing early stratification and recognition of patients at higher risk of mortality are needed. The two biomarkers that have been most widely studied and used in patients with sepsis are the short pentraxin C-reactive protein (CRP), and procalcitonin (PCT), the prehormone of calcitonin. Nevertheless, even they have limited abilities to distinguish sepsis from other inflammatory conditions or to predict outcome [5, 6]. PCT can be also used to support shortening of the duration of antimicrobial therapy in sepsis patients (weak recommendation, low quality of evidence), according to the latest Surviving Sepsis Campaign (SCC) [4].

In recent years, pentraxin 3 (PTX3), an acute phase protein, has emerged as a promising biomarker of sepsis. It is a prototypical member of the long pentraxin subfamily and a key component of humoral innate immunity. PTX3 is expressed in a number of tissues, particularly dendritic cells and macrophages, in response proinflammatory stimuli [7, 8]. Additionally, it is stored in neutrophil granules and localises in neutrophil extracellular traps [9]. Once released, PTX3 acts by recognising microbes, activating complement and facilitating pathogen recognition by phagocytes, thus promoting pathogen clearance, tuning inflammatory responses and promoting tissue remodelling [7, 8].

In healthy subjects, plasma PTX3 levels are barely detectable (<2 ng/mL) [10], but can quickly rise in inflammatory and infectious conditions [8]. Levels are elevated in critically ill patients, with a gradient from systemic inflammatory response syndrome (SIRS) to sepsis and septic shock [11]. Thus, it has been proposed as prognostic marker for sepsis [11-17]. In a systematic review and meta-analysis, PTX3 significantly predicted disease severity and mortality in sepsis [18].

The aim of this prospective study was to assess the prognostic value of a single determination of PTX3 in septic shock patients in relation to hospital mortality, comparing it with the prognostic value of a single determination of the classical biomarkers CRP and PCT, on ICU admission. We also aimed to evaluate whether its addition to severity scores could improve their prognostic accuracy.

Materials and methods

Study design and setting

We performed a single-centre prospective observational study of 75 patients admitted to the medical ICU of Marqués de Valdecilla University Hospital in Spain between April 2015 and April 2016. All consecutive patients 18 years or older who were admitted to ICU with septic shock, according to the Sepsis-2 definition, were eligible to participate. The latest Sepsis-3 definitions were applied and all patients were re-classified according to these new definitions. The criteria for septic shock included the requirement for a vasopressor to maintain a mean blood pressure of 65 mmHg and serum lactate level >2 mmol/L. Exclusion criteria included patients under 18 years of age, as well as individuals with recent cardiac arrest or decision to withdraw life-sustaining treatment; patients who did not have septic shock at the time of ICU admission but who subsequently developed it were also excluded.

Clinical and demographic characteristics were recorded for all patients, and included age, sex and immunosuppression (AIDS, neutropenia [neutrophil count <1 × 109/L], glucocorticoid exposure [>0.5 mg/kg for >30 d] and/or immunosuppressive or cytotoxic medications, solid organ transplantation, allogeneic or autologous stem cell transplantation, haematological malignancy, or solid tumour). Acute Physiology and Chronic Health Evaluation II (APACHE II) score at 24 h, Simplified Acute Physiology Score (SAPS II) and Sequential Organ Failure Assessment (SOFA) score at admission were also recorded.

The study was approved by the Clinical Research Ethics Committee of Cantabria (CEIC: 2014.159), and written informed consent was obtained from each participating patient or their legal representatives, according to the Declaration of Helsinki.

Assay methods

An additional blood sample was drawn coinciding with the first extraction for clinical purposes upon admission of the patient to the ICU. Serum and plasma samples were collected as specified for the different assays, and aliquots for this specific study were stored at −80°C until analysis.

Plasma PTX3 levels were measured by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Quantikine Human Pentraxin 3/TSG-14; R&D Systems Europe, Abingdon, UK). The sensitivity of the assay is 0.026 ng/mL. Specificity is <0.5% cross-reactivity observed with available related molecules; <50% cross-reactivity observed with species tested. Intra-assay reproducibility of the method is <2.6% and interassay reproducibility is <6.9%.

Serum PCT levels were measured by electrochemiluminescence immunoassay (ECLIA) on a Cobas e411 autoanalyzer (Roche Diagnostics, Mannheim, Germany); serum CRP was determined by immunoturbidimetric assay; and plasma lactate by an enzymatic assay using a Dimension EXL autoanalyzer (Siemens Health Care Diagnostics, Gwynedd, UK).

Statistical analysis

Categorical and discrete variables were expressed as counts (percentage). Continuous variables were expressed as median and interquartile ranges (IQR). Statistical differences between groups were assessed with the Chi-square test using Yates´ correction or Fisher´s exact test, when appropriate, for categorical variables. Continuous variables were compared with the Mann-Whitney U test. The correlation between continuous variables was calculated using Spearman´s rank correlation coefficient (rho).

To determine and compare the predictive value of the biomarkers and severity scores, receiver-operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was calculated. The outcome variable was in-hospital mortality.

To estimate the strength of associations, the biomarkers and severity scores were divided into dichotomous variables (low versus high values) according to the median and adjusted odds ratios (OR) with their 95% confidence intervals (CI); 95% CIs for in-hospital mortality were calculated using unconditional logistic regression. The following potential confounders were pre-established for inclusion in the models: age (as a continuous variable), sex and immunosuppression status (yes/no).

In addition, exposure-response trends (biological gradient) were estimated, using a logistic regression model with all potential confounders, categorizing the prognostic factors as ordinal variables according to tertiles.

The level of statistical significance was set at 0.05 and all tests were two-tailed. Data were analysed using SPSS statistical software package 24.0 (SPSS, Inc., Chicago, IL, USA).

Results

Study population

Seventy-five septic shock patients were included in the study (53 men and 22 women) with a median age at admission of 64 years (Table 1). At ICU admission, median APACHE II, SOFA and SAPS II scores were 22, 10 and 42, respectively. The most common sites of infection were the lungs (41.3%) and abdomen (32%). The ICU mortality rate was 24% and the in-hospital mortality rate was 28%. Non-survivors were more severely ill, as reflected by significantly higher severity scores and higher number of organ dysfunctions on ICU admission; mechanical ventilation and haemodiafiltration were required in a higher percentage of non-survivors compared to survivors.

Table 1

Baseline characteristics of the study population, in relation to in-hospital mortality.

Characteristics	Overall population	Survivors	Non-survivors	p value
	n = 75	n = 54	n = 21
Age (years), MD (IQR)	64 (49–74)	63 (47.2–70)	71 (59.5–76.5)	0.08
Male sex, n (%)	53 (70.7)	37 (68.5)	16 (76.2)	0.58
Immunosuppression, n (%)	25 (33.3)	14 (25.9)	11 (52.4)	0.05
Charlson score, MD (IQR)	4 (2–6.6)	3.9 (1.1–6.4)	5.6 (3.4–7.2)	0.28
Site of infection, n (%):				0.05
Lung	31 (41.3)	22 (40.7)	9 (42.9)
Abdomen	24 (32)	15 (27.8)	9 (42.9)
Urinary tract	9 (12)	9 (16.7)	0 (0)
Skin-soft tissues	5 (6.7)	5 (9.3)	1 (4.8)
Catheter	3 (4)	2 (3.7)	1 (4.8)
Others	3 (4)	1 (1.9)	1 (4.8)
Nosocomial infection, n (%)	46 (61.3)	33 (61.1)	12 (57.1)	0,79
APACHE II, MD (IQR)	22 (15–28)	19.5 (14–25.2)	25 (22–30)	0.006
SAPS II, MD (IQR)	42 (32–63)	36 (29.5–50.2)	61 (53.5–69)	<0.001
SOFA, MD (IQR)	10 (7–13)	9 (7–12)	14 (10–17.5)	<0.001
Organ dysfunction (number), MD (IQR)	3 (2–4)	2 (1–3.2)	4 (3–5)	<0.001
Mechanical ventilation, n (%)	39 (52)	19 (35.2)	20 (95.2)	<0.001
ARDS, n (%)	11 (14.7)	3 (5.6)	8 (38.1)	<0.001
CVVHDF, n (%)	11 (14.7)	4 (7.4)	7 (33.3)	0.009
Bacteraemia, n (%)	23 (30.7)	15 (27.8)	8 (38.1)	0.41
SvcO2 (%)	72 (61.4–78.6)	72.7 (62.1–78.7)	74.2 (60–78.6)	0.99
Lactate (mmol/L), MD (IQR)	2.7 (1.3–4.6)	2.1 (1–3.5)	4.6 (2.7–8.5)	<0.001
PCT (ng/ml), MD (IQR)	11.8 (1.5–40.6)	9.6 (1.3–51.3)	12.9 (3.7–24.7)	0.47
CRP (mg/dl), MD (IQR)	22.8 (11.1–25)	23 (11.1–25)	20.8 (8.4–25)	0.56
Pentraxin-3 (ng/ml), MD (IQR)	63.8 (24.2–187)	41.8 (14.3–124.3)	114.4 (56.1–250)	0.01
ICU mortality, n (%)	18 (24)	----	----	----
Hospital mortality, n (%)	21 (28)	----	----	----
ICU stay (days), MD (IQR)	4.9 (2.9–9.9)	5.9 (2.9–9.9)	4.9 (1.9–9.9)	0.10

Apache II: Acute Physiology and Chronic Health disease Classification System II; SAPS: Simplified Acute Physiology Score; SOFA: Sequential Organ Failure Assessment; ARDS: Acute respiratory distress syndrome; CVVHDF: Continuous venovenous haemodiafiltration; SvcO2: Central venous oxygen saturation; PCT: Procalcitonin; CRP: C-reactive protein; VDBP: Vitamin D binding protein. MD (IQR); median (interquartile range). P results based on Mann–Whitney U test.

PTX3 levels were higher than normal in all patients, with a median PTX3 level of 63.8 (24.2–187) ng/mL. Median PCT and CRP levels were 11.8 ng/mL (1.5–40.6) and 22.8 (11.1–25) ng/mL, respectively. Plasma PTX3 concentrations were higher in non-survivors compared to survivors (114.4 vs. 41.8 ng/mL, P = 0.01). However, serum PCT and CRP concentrations showed no statistically significant differences between these groups.

At ICU admission, plasma PTX3 levels showed a statistically significant positive correlation with the APACHE II score (rho = 0.402, P<0.001), SOFA score (rho = 0.378, P = 0.001), SAPS-II score (rho = 0.449, P<0.001) and PCT (rho = 0.329, P = 0.005), but not with CRP (rho = 0.157, P = 0.187), ICU length of stay (rho = 0.104, P = 0.82) or hospital length of stay (rho = 0.041, P = 0.73).

Prognostic value of PTX3: ROC and regression analysis

Among the prognostic factors studied, the best rates of prediction of in-hospital mortality were the severity scores: SAPS II (AUC = 0.81), SOFA (AUC = 0.79) and APACHE II (AUC = 0.73). The ROC curve for PTX3 (ng/mL) yielded an AUC of 0.70, higher than the AUC for PCT (0.43) and CRP (0.48) but lower than the AUC for lactate (0.79) (Table 2) (S1 Fig). Adding PTX3 to the logistic model slightly increased the predictive capacity in relation to SAPS II, SOFA and APACHE II for in-hospital mortality (AUC 0.814, 0.795, and 0.741, respectively) (Table 3).

Table 2

Area under the curve (AUCs) with 95% confidence intervals (CIs) for severity scores and biomarkers in relation to in-hospital mortality.

	AUCa	(95%	CI)
APACHE II	0.728	0.612	0.843
SAPS II	0.814	0.713	0.915
SOFA	0.785	0.672	0.899
PCT (ng/ml)	0.534	0.398	0.671
CRP (mg/dl)	0.481	0.339	0.623
Lactate (mmol/L)	0.794	0.692	0.895
Pentraxin-3 (ng/ml)	0.698	0.575	0.822

a AUC denotes Area Under the Curve ROC; 95% CI Confidence interval at 95%.

Table 3

Area under the curve (AUCs) with 95% confidence intervals (CIs) estimated through regression models for different combinations of APACHE II, SAPS II, SOFA and pentraxin-3 in relation to in-hospital mortality.

	AUCa	(95%	CI)
APACHE II + Pentraxin-3 (ng/ml)	0.741	0.624	0.858
SAPS II + Pentraxin-3 (ng/ml)	0.817	0.719	0.916
SOFA + Pentraxin-3 (ng/ml)	0.795	0.689	0.901

a AUC denotes Area Under the Curve ROC; 95% CI Confidence interval at 95%.

Tables 4 and 5 show the strength of associations (OR) and exposure-trends (biological gradient) between severity scores and biomarkers, including PTX3, in relation to in-hospital mortality. When categorising the prognostic factors according to the median and tertiles, the results were consistent with the predictive capacity determined using the AUCs. With respect to biomarkers, lactate was statistically significantly associated with in-hospital mortality, but the associations were higher for PTX3 compared to PCT and CRP.

Table 4

Associations between severity scores and in-hospital mortality.

		Survivors	Non-survivors
Severity scores	Cut-off points	N = 114	N = 25	OR	(95%	CI)	ORa	(95%	CI)
APACHE II (Median)
Low (reference)	≤ 22	38	6	1.00	--		1.00	--
High	23+	16	15	5.94	1.95	18.06	6.59	1.82	23.93
APACHE II (Tertiles)
Low (reference)	≤ 17	23	3	1.00	--		1.00	--
Medium	18–25	18	8	3.41	0.79	14.72	2.92	0.57	14.94
High	26+	13	10	5.90	1.37	25.36	6.22	1.18	32.67
p linear trend				p = 0.016			p = 0.029
SAPS II
High (reference)	≤ 42	36	2	1	--		1	--
Low	43+	18	19	19	3.98	90.69	23.362	3.972	137.411
SAPS II (Tertiles)
High (reference)	≤ 35	26	1	1	--		1	--
Medium	36–56	17	7	10.71	1.21	94.96	11.01	1.13	107.37
Low	57+	11	13	30.73	3.57	264.49	48.67	4.46	530.95
p linear trend				p<0.001			p = 0.001
SOFA (Median)
High (reference)	≤ 10	36	6	1	--		1	--
Low	11+	18	15	5.00	1.66	15.07	13.10	2.58	66.47
SOFA (Tertiles)
High (reference)	≤ 8	24	2	1	--		1	--
Medium	9–12	20	6	3.60	0.65	19.84	2.75	0.41	18.40
Low	13+	10	13	15.60	2.96	82.17	59.80	5.13	696.73
p linear trend				p = 0.001			p = 0.001

ORa: Odds ratio adjusted for age, sex and immunosuppression.

Table 5

Associations between biomarkers and in-hospital mortality.

		Survivors	Non-survivors
Biomarkers	Cut-off points	N = 114	N = 25	OR	(95%	CI)	ORa	(95%	CI)
PCT (ng/ml) (Median)
Low (reference)	≤ 11.80	30	8	1.00	--		1.00	--
High	11.81+	24	13	2.03	0.72	5.7	2.87	0.86	9.58
PCT (ng/ml) (Tertiles)
Low (reference)	≤ 4.00	20	5	1.00	--		1.00	--
Medium	4.01–22.60	15	10	2.67	0.75	9.45	2.31	0.6	8.9
High	22.61+	19	6	1.26	0.33	4.84	1.73	0.39	7.75
p linear trend				p = 0.753			p = 0.422
CRP (mg/dl) (Median)
Low (reference)	≤ 22.70	26	12	1.00	--		1.00	--
High	22.71+	28	9	0.7	0.25	1.92	0.82	0.27	2.46
CRP (mg/dl) (Tertiles)
Low (reference)	≤ 13.00	17	8	1.00	--		1.00	--
Medium	13.01–25.00	32	12	0.797	0.273	2.325	0.998	0.311	3.202
High	25.01+	5	1	0.425	0.042	4.263	0.39	0.033	4.65
p linear trend				p = 0.466			p = 0.615
Lactate (mmol/L) (Median)
Low (reference)	≤ 24.00	33	6	1.00	--		1.00	--
High	24.01+	21	15	3.93	1.32	11.73	4.48	1.36	14.75
Lactate (mmol/L) (Tertiles)
Low (reference)	≤ 18	26	1	1.00	--		1.00	--
Medium	18.01–32.00	16	7	11.38	1.28	101.22	9.53	1.00	90.40
High	32.01+	12	13	28.17	3.29	240.81	36.47	3.85	345.38
p linear trend				p<0.001			p<0.001
Pentraxin-3 (ng/ml) (Median)
Low (reference)	≤ 63.80	30	8	1.00	--		1.00	--
High	63.81+	24	13	2.03	0.72	5.7	2	0.64	6.24
Pentraxin-3 (ng/ml) (Tertiles)
Low (reference)	≤ 31.90	23	2	1.00	--		1.00	--
Medium	31.91–116.60	16	9	6.47	1.23	34.01	6.05	1.05	34.93
High	116.61+	15	10	7.67	1.47	39.99	7.83	1.35	45.49
p linear trend				p = 0.015			p = 0.024

ORa: Odds ratio adjusted for age, sex and immunosuppression.

Among the severity scores (SOFA, SAPS II and APACHE II), significant crude positive associations were obtained for in-hospital mortality, crude OR at the highest tertile (OR T3): OR T3 for SAPS II = 30.73, 95% CI 3.57–264,49 P trend <0.001; OR T3 for SOFA = 59.80, 95% CI 0.65–19.84, P trend = 0.001; and OR T3 for APACHE II = 5.90, 95% CI 1.37–25.36, P trend = 0.016). After adjusting for age, sex and immunosuppression, these positive associations increased for all severity scores.

Regarding biomarkers, non-significant associations with no dose-response P trends were found for PCT and CRP. In crude regression models, significant associations with significant dose-response trends were found between in-hospital mortality and PTX3. This positive association increased after adjusting for age, sex and immunosuppression: adjusted OR at the highest tertile (ORa T3) for PTX3 = 7.83, 95% CI 1.35–45.49, P trend = 0.024. Lactate was the biomarker with highest associations: adjusted OR at the highest tertile (ORa T3) for lactate = 36.47, 95% CI 3.85–345.38, P trend <0.001 (Table 5).

Discussion

In our study, we evaluated the prognostic value of PTX3, PCT and CRP in patients with septic shock defined according to Sepsis-3 criteria. Our findings suggest that plasma PTX3 could be a potential predictor of mortality, having, according to our results, a superior prognostic value compared to PCT and CRP. Our results also support severity scores—classical markers used to predict mortality—as predictors of mortality, particularly SAPS II. This reproducible accuracy of the severity scores supports the validity of our results for PTX3. We also found that PTX3 was elevated and showed a stronger correlation with disease severity, organ dysfunction and other clinical parameters than CRP or PCT in septic shock.

In accordance with a previous study, CRP remained equally high in both survivors and non-survivors [12]. Although PTX3 and CRP belong to the same pentraxin family, PTX3 differs from CRP in terms of gene organization and localisation, ligand recognition, cellular source and inducing signal [7, 8]. PTX3 is an acute phase protein secreted by various cells in response to proinflammatory signals, unlike the short pentraxin CRP, which is produced in the liver and induced by IL-6. Under normal physiological conditions, plasma PTX3 levels are low (<2 ng/mL) but increase rapidly in sepsis as a result of neutrophil degranulation—up to 100 ng/mL depending on the severity of disease [19]—with levels maintained through de novo production by endothelial cells and some monocytic cells [20, 21]. PTX3 increases within 6–8 h of response to infection, compared to 24–30 h for CRP.

The association between PTX3 and the increase in in-hospital mortality remained after adjusting for the main confounding factors, namely age, sex, and immunosuppression, supporting the independence of this association. Moreover, an exposure-response pattern (a dose-response trend) was found, where higher levels were associated with a higher risk of mortality. Patients with high levels (third tertile) had an approximately eight-fold risk of in-hospital mortality compared to patients with lower PTX3 levels. However, the predictive accuracy of a single PTX3 determination, in relation to the AUC determined by the ROC curves, was lower than the severity scores (SAPS II, SOFA, APACHE II), but superior to the classically used biomarkers (CRP, PCT) in predicting the risk of mortality in septic shock patients. The predictive accuracy in relation to severity scores was slightly increased by adding PTX3 to the logistic models. The clinical relevance of this slight increase merits further attention in future studies, to elucidate whether systemic PTX3 levels would have prognostic value and could help to determine the prognosis of patients with septic shock, complementing disease severity classification systems and other biological markers.

Sepsis occurs when the release of proinflammatory mediators in response to an infection extends beyond the boundaries of the local environment, leading to a more generalised response, so it is difficult to describe it with a single measure. The predictive scoring systems used to predict mortality in ICU patients have major limitations, including poor generalisability, deterioration over time and possibly lead-time bias [22-24], and they also tend to be used more in research than in routine clinical practice. For this reason, although PTX3 did not perform as well as severity scores, especially SAPS II, it should not be excluded for use as a predictor of outcome. CRP is sensitive but not very specific, since it is increased in all inflammatory disorders, while PCT differentiates between infectious and non-infectious causes of critical illness better than CRP [25]. Nevertheless, they provide limited information in relation to patient prognosis in critically ill patients [26, 27]. A meta-analysis of 21 studies with a total of 6007 patients concluded that the initial PCT level was of limited prognostic value in patients with sepsis [28]. In another meta-analysis of 25 studies with 2353 patients, Arora et al. found that, in a subgroup of patients with severe sepsis and septic shock, there was no difference in PCT values between survivors and non-survivors on day 1 (P = 0.062) [29]. In addition, serum levels may be altered by clinical and demographical conditions [30-32]. Our data showed a significant correlation between PCT and PTX3 at ICU admission, but PCT was not an independent marker for in-hospital mortality, nor was CRP. At ICU admission, the AUC value of a single PTX3 determination was significantly much better than CRP and PCT in predicting in-hospital mortality. Moreover, PTX3 levels correlated better with disease severity and organ dysfunction than PCT or CRP.

Our study findings are consistent with previous studies that examined PTX3 as a prognostic marker for sepsis [11-17]. Levels of PTX3 have been correlated with disease severity, organ dysfunction and markers of coagulation activation and, when compared with other biomarkers (e.g. IL-6, TNFα and CRP), have shown a stronger correlation with clinical parameters [33]. In a systematic review and meta-analysis, PTX3 was identified as a marker of sepsis severity and predictor of mortality, but with limited specificity [18]. In a prospective study with 112 patients with septic shock, baseline PTX3 levels were an independent predictor of 28-day mortality, unlike CRP and PCT [17]. In contrast to our study, Mauri et al. reported that, although high PTX3 levels over the first 5 days from onset of sepsis were correlated with poorer outcomes, the initial PTX3 levels at ICU admission did not differ between survivors and non-survivors [12]. Additionally, Caironi et al., in a large multicentre randomised controlled trial, found that plasma PTX3 concentrations were higher in non-survivors compared to survivors on day 1 and were correlated with severity. While PTX3 levels on day 7 showed a significant predictive value for 90-day mortality, PTX3 levels on day 1, after adjustment for all confounders, were not associated with 90-day mortality in patients with severe sepsis or septic shock [15]. These conflicting outcomes may due to the heterogeneity of different study subjects. Both studies were designed with patients with severe sepsis and septic shock according to the first criterion of sepsis based on the concept of SIRS. In addition, patients were randomised at enrolment. In contrast, our clinical study was conducted on a homogeneous group of patients who met the updated definition of septic shock. Additionally, the timing of mortality analysis was different (90-day mortality vs. in-hospital mortality), and in both studies, a sandwich ELISA developed in-house was used to determine PTX3 levels.

It should be pointed out that our results are also consistent with recent prospective studies, according to the Sepsis-3 definitions [34-37]. In contrast to our study, Hu et al. reported that PCT is a moderate predictor of 28-day mortality in patients with sepsis and septic shock.

Our results also showed that elevated lactate levels were highly associated with in-hospital mortality in septic shock, in line with other studies [4, 38–41]. Lactate has been widely used as a marker of altered tissue perfusion. Adult patients with septic shock can be identified using the clinical criteria of hypotension requiring the use of vasopressors to maintain mean blood pressure of 65 mmHg or greater and having a serum lactate level greater than 2 mmol/L persisting after adequate fluid resuscitation [2]. However, an elevated lactate level is a sensitive marker for cellular dysfunction in sepsis [42, 43], but is non-specific, since it can be elevated in other types of shock, such as cardiogenic, obstructive or hypovolaemic shock, and can be affected by several factors, such as liver disease [41, 44].

The present study has several advantages. We performed a prospective study with a very homogeneous group of patients who met septic shock criteria in accordance with the latest Sepsis-3 definitions at the time of admission to the ICU. Furthermore, the prognosis was established very promptly because it was based on the fact that blood samples were obtained on ICU admission.

This study also has some limitations, namely that the generalisability of our findings is constrained by the fact that this was a single centre observational study with a small sample size.

Conclusions

In summary, our results support the available published studies suggesting the prognostic value of a single determination of plasma PTX3 as a predictor of hospital mortality in septic shock patients, defined according to the latest Sepsis-3 criteria.

Further multicentre studies with larger sample size are needed in order to generalise these results, elucidating the existence of subgroups of patients with specific characteristics in whom this biomarker could demonstrate higher accuracy in assessing the risk of mortality or increased severity. This would allow us to better understand the predictive potential of PTX3 in comparison to other existing biomarkers such as lactate, PCT or CRP or severity scores.

ROC curves for severity scores and biomarkers with respect to ‘in-hospital mortality’.

(DOC)

Click here for additional data file.

9 Oct 2020

PONE-D-20-28810

PROGNOSTIC VALUE OF PLASMA PENTRAXIN 3 LEVELS IN PATIENTS WITH SEPTIC SHOCK ADMITTED TO INTENSIVE CARE

PLOS ONE

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

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**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: - Describing Pentraxin 3 as a new marker for sepsis is not correct , many studies has discussed its role in sepsis . may be the Gap between the study period ( 2015-2016) and the submission for publishing is a factor . this time gap make most of the results of this study NOT Novel which is an important issue for publication.

- A major drawback in this study is lack of control group

-In Table one how can you make a statistical comparison between survival with UTI and non-Survival ( count = zero)

- what were the results of cultures( blood, urine, etc)

- Ptx3 levels increase in other NON-SEPSIS condition as hypoxia, renal insult . DO you exclude those patients??

- what is the initial diagnosis of your patients

- it is recommended to include figures of ROC

Reviewer #2: In the present study, Perez-San Martin et al. present data showing the prognostic potential of PTX3 in a cohort of 75 septic shock patients. They state that adding PTX3 to severity scores increased the predictive capacity of the latter for mortality, and that a single determination of plasma PTX3 on ICU admission was a better prognostic marker compared with PCT and CRP.

The study is relatively limited in terms of number of patients involved, but it confirms the general messages of previous studies on the prognostic potential of PTX3 in sepsis.

Major points:

- The authors state that adding PTX3 to severity scores increased the predictive capacity of the latter for mortality. However, this increase is really limited and AUC reported in Tables 2 and 3 are almost identical. If there is any advantage, is this statistically significant?

-The authors should better underline the novelties of this study compared to papers published in the last years on this marker in sepsis, which are collected in the review by Porte et al (Frontiers Immunol, 2019), in addition to ref 18. Among them, there are studies with larger sample size.

-The authors discuss the difference with results reported in ref 12 and 15, pointing to the value of PTX3 levels at ICU admission found in this study, but not in the studies of ref 12 and 15, where the authors state that levels on day 5 and 7, respectively, correlated with severity, but PTX3 levels on day 1, did not. In this regard, it would be important to discuss criteria for ICU admission, and the time between sepsis diagnosis and PTX3 test, since these factors could explain the difference with previous studies.

-It would be important to add figures, e.g. for AUC and correlation analysis.

-It would be interesting to perform further analysis based on the characteristics of patients. For instance, is there any difference in PTX3 levels/predictive capacity depending on the infection site?

Reviewer #3: The authors discuss the prognostic role of a single determination at the time of ICU admission of 3 biomarkers (PCT, CRP, PTX-3) in septic shock patients. They conclude that, after adjustment for confounding, PTX-3 has a prognostic value superior to the other two: the higher this biomarker, the higher the mortality.

I have several concerns.

1) The authors state that biomarkers are needed to stratify the patients according to the risk of death. Indeed, severity scores gather several clinical characteristic to convert them into a risk of mortality. PCT and CRP, being inflammatory markers, are certainly related to the prognosis, however their clinical use is usually different: early recognition of infection, follow-up of a certain treatment, antibiotic stewardship. Do you really think that such biomarkers could perform better than a severity score in prognostication? In Table 2 and 3 the authors present these findings, but they do not discuss them appropriatedly.

2) The classical biomarker for sepsis prognostication is lactate, which is not discussed at all.

3) Premise: I am not a statistician. The ROC/AUC approach is ok, but I think that a survival analysis should be used rather than a logistic regression. The exposure-response trend with three groups does not add much to the analysis performed with two groups. Table 4 and 5 are redundant.

Additional remarks

- The biological and clinical role of the biomarkers should be clearly presented either in the Introduction or in the Discussion section without repetition

- Try to discuss the discrepancies with Caironi's and Mauri's studies

- Lack of a non-septic control group is not a limitation of the study

- Conclusions, first sentence: PTX-3 is an actual, not potential prognostic factor. The sentence does not mean anything in this way.

**********

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Reviewer #2: No

Reviewer #3: No

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11 Nov 2020

Point-by-point responses

Reviewer #1: - Describing Pentraxin 3 as a new marker for sepsis is not correct , many studies has discussed its role in sepsis . may be the Gap between the study period ( 2015-2016) and the submission for publishing is a factor . this time gap make most of the results of this study NOT Novel which is an important issue for publication.

- A major drawback in this study is lack of control group

Response: The design is a cohort study: the patients with the lower values (below the median) act as the non-exposed (reference group) in this design, and the outcome (the event) is in-hospital mortality, comparing the risk of this event between exposed and non-exposed.

-In Table one how can you make a statistical comparison between survival with UTI and non-Survival ( count = zero)

Response: The variable is “Source of infection” with the following qualitative categories: Lung, Abdomen, Urinary tract, Skin-soft tissues, Catheter, Others. The p value comes from a Chi2 test for the overall variable covering all categories, and comparing them between survival and non-survival groups.

- what were the results of cultures( blood, urine, etc):

Response: We have information about the source of infection but we do not have information about the specific microorganism responsible for the infection. We believe that given the small sample size the conclusions that can be drawn from this information are limited. Even so, if the reviewer considers this information indispensable, we could try to complete it.

- Ptx3 levels increase in other NON-SEPSIS condition as hypoxia, renal insult . DO you exclude those patients??

Response: Indeed, as explained in the section corresponding to methods, only patients who met criteria for septic shock were included in the study. No patient was included who did not have an infection as the origin of his symptoms. Therefore, patients with other symptoms were excluded.

- what is the initial diagnosis of your patients

Response: In all cases the initial diagnosis and the reason for ICU admission was septic shock.

- it is recommended to include figures of ROC

Response: We agree, we have performed a figure of ROC for all biomarkers including Lactate, as S Fig 1. It is showed below.

S Fig 1. ROC Curves for Severity Scores and biomarkers with respect to ‘in hospital mortality’.

Reviewer #2: In the present study, Perez-San Martin et al. present data showing the prognostic potential of PTX3 in a cohort of 75 septic shock patients. They state that adding PTX3 to severity scores increased the predictive capacity of the latter for mortality, and that a single determination of plasma PTX3 on ICU admission was a better prognostic marker compared with PCT and CRP.

The study is relatively limited in terms of number of patients involved, but it confirms the general messages of previous studies on the prognostic potential of PTX3 in sepsis.

Major points:

- The authors state that adding PTX3 to severity scores increased the predictive capacity of the latter for mortality. However, this increase is really limited and AUC reported in Tables 2 and 3 are almost identical. If there is any advantage, is this statistically significant?

Response: We agree that there is not a substantial increase between Tables 2 and 3 (adding PTX3), however we think that the fact that PTX3 increase the AUC of each severity score, deserve future attention, because for the other studied biomarkers (PCT and CRP), we did not find these findings. Further studies must deep in the clinical relevance of these findings, to elucidate whether an apparent low increase in AUC may be clinically relevant. We have added in the discussion a sentence about it.

Now it reads: “The predictive accuracy in relation to severity scores was slightly increased by adding PTX3 to the logistic models. The clinical relevance of this slight increase deserve further attention in future studies, to elucidate whether systemic PTX3 levels would have prognostic value and may help to determine the prognosis of patients with septic shock, complementing disease severity classification systems and other biological markers”.

-The authors should better underline the novelties of this study compared to papers published in the last years on this marker in sepsis, which are collected in the review by Porte et al (Frontiers Immunol, 2019), in addition to ref 18. Among them, there are studies with larger sample size.

Response: We agree with the observation made by the reviewer. We have included a paragraph in this section that includes both the bibliographic citation and the articles to which the reviewer refers and whose results have been compared with our own.

-The authors discuss the difference with results reported in ref 12 and 15, pointing to the value of PTX3 levels at ICU admission found in this study, but not in the studies of ref 12 and 15, where the authors state that levels on day 5 and 7, respectively, correlated with severity, but PTX3 levels on day 1, did not. In this regard, it would be important to discuss criteria for ICU admission, and the time between sepsis diagnosis and PTX3 test, since these factors could explain the difference with previous studies.

Response: we agree with the comments made by the reviewer. It is possible that the factors cited influence the different results observed. For this reason we have included changes to the text in this regard and now it reads “These conflicting outcomes may due to the heterogeneity of different study subjects. Both studies were designed with patients with severe sepsis and septic shock according to the first criterion of sepsis based on the concept of systemic inflammatory response syndrome (SIRS). In addition, patients were randomized at enrollment. However, our clinical study was conducted on a homogeneous group of patients who met the updated definition of septic shock. On the other hand, the timing of mortality analysis was different (90-day mortality vs. in-hospital mortality). Furthermore, in both studies a sandwich ELISA developed in-house was used to determine PTX3 levels.”

-It would be important to add figures, e.g. for AUC and correlation analysis.

Response: We agree. We have performed a figure of ROC Curves for Severity Scores and biomarkers (including Lactate) with respect to ‘in hospital mortality’ (see please S Fig 1 and response to review 1).

-It would be interesting to perform further analysis based on the characteristics of patients. For instance, is there any difference in PTX3 levels/predictive capacity depending on the infection site?

Response: PTX3 mean and median levels, seems to be higher in patients with abdominal or lung infection than those with urinary tract infection.

Pentraxin-3 (ng/ml)

Source of infection

Mean

Median

N

SD

P25

P75

Lung

94,47

70,40

31

81,45

22,00

151,80

Abdomen

126,71

103,95

24

96,15

35,20

250,00

Urinary tract

41,32

14,30

9

78,91

4,60

29,70

Others

105,08

67,10

11

101,29

11,50

250,00

Total

99,97

63,80

75

91,05

24,20

187,00

Regarding predictive capacity depending on the infection site, as there is not events (zero in-hospital mortality) in urinary tract infection patients (see please table 1 and response to reviewer 1), it is not possible to perform a ROC curve for the specific category of urinary tract infection patients. On the other hand, a subgroup analysis would make the N even lower, which is another limitation. We agree with the reviewer that a subgroup analysis would enrich the analysis strategy. However, because of the aforementioned, we think it is preferable not to present these results in the manuscript.

Reviewer #3: The authors discuss the prognostic role of a single determination at the time of ICU admission of 3 biomarkers (PCT, CRP, PTX-3) in septic shock patients. They conclude that, after adjustment for confounding, PTX-3 has a prognostic value superior to the other two: the higher this biomarker, the higher the mortality.

I have several concerns.

1) The authors state that biomarkers are needed to stratify the patients according to the risk of death. Indeed, severity scores gather several clinical characteristic to convert them into a risk of mortality. PCT and CRP, being inflammatory markers, are certainly related to the prognosis, however their clinical use is usually different: early recognition of infection, follow-up of a certain treatment, antibiotic stewardship. Do you really think that such biomarkers could perform better than a severity score in prognostication? In Table 2 and 3 the authors present these findings, but they do not discuss them appropriatedly.

Response: We agree, due to the small sample size, the unicentric character, the existing bibliography, and the constructive arguments given by the reviewer, we have changed the conclusions and the discussion

Now, the new text of conclusions in the abstract and discussión is:

“Abstract Conclusion: Our results support the prognostic value of a single determination of plasma PTX3 as a predictor of hospital mortality in septic shock patients”.

“Discussion conclusions: In summary, our results, support the available published studies suggesting the prognostic value of a single determination of plasma PTX3 as a predictor of hospital mortality in septic shock patients, defined according to the latest Sepsis-3 criteria.

Further studies with larger sample size and multicentre character are needed in order to generalise these results, to elucidate the existence of subgroups of patients with specific characteristics in whom this biomarker could demonstrate higher accuracy in assessing the risk of mortality or increased severity, deepening finally in the knowledge the PTX3 predictive potential in comparison to other existing biomarkers such as Lactate, PCT or CRP or severity scores”.

In the discussion section, the new sentences referring to Table 2 and 3 results are:

“The predictive accuracy in relation to severity scores was slightly increased by adding PTX3 to the logistic models. The clinical relevance of this slight increase deserve further attention in future studies, to elucidate whether systemic PTX3 levels would have prognostic value and may help to determine the prognosis of patients with septic shock, complementing disease severity classification systems and other biological markers”.

2) The classical biomarker for sepsis prognostication is lactate, which is not discussed at all.

Response: We agree, we studied lactate with good results. As our main objective was to compare PTX3 with PCT and CRP we were undecided about including information on this classic biomarker. Now we have included this information.

3) Premise: I am not a statistician. The ROC/AUC approach is ok, but I think that a survival analysis should be used rather than a logistic regression. The exposure-response trend with three groups does not add much to the analysis performed with two groups. Table 4 and 5 are redundant.

Response: Survival analysis is also a valid approach through a Cox regresión model. However, it estimates Hazard Ratios (HR) instead of Odds Ratios. We decided to use a logistic regression model because it estimates OR allowing for better comparisons with other studies from biomarkers.

We agree that Table 4 is redundant with Table 5 in the sense that both tables show crude and adjusted ORs. However, as we have added Lactate results in Table 5 we think that combining both tables would produce a too large table, more difficult to read.

Additional remarks

- The biological and clinical role of the biomarkers should be clearly presented either in the Introduction or in the Discussion section without repetition

Response: we agree with the suggestion made by the reviewer. we have included the requested information in both sections.

- Try to discuss the discrepancies with Caironi's and Mauri's studies

Response: we agree with the suggestion made by the reviewer. We have modified this paragraph and now it reads “In contrast to our study, Mauri et al. reported that, although high PTX3 levels over the first 5 days from onset of sepsis were correlated with poorer outcomes, the initial PTX3 levels at ICU admission did not differ between survivors and non-survivors (12). Additionally, Caironi et al., in a large multicentre randomised controlled trial, found that plasma PTX3 concentrations were higher in non-survivors compared to survivors on day 1 and were correlated with severity. While PTX3 levels on day 7 showed a significant predictive value for 90-day mortality, PTX3 levels on day 1, after adjustment for all confounders, were not associated with 90-day mortality in patients with severe sepsis or septic shock”

- Lack of a non-septic control group is not a limitation of the study

Response: our idea was that in an ICU control group without sepsis, PTX-3 higher levels would be less associated with mortality, supporting the hypothesis of being a selective predictor in sepsis. Nevertheless, we agree that is not directly related with our main objective (focused on sepsis) and we have deleted this limitation.

- Conclusions, first sentence: PTX-3 is an actual, not potential prognostic factor. The sentence does not mean anything in this way.

Response: We agree we have changed conclusions in abstract and discussion section.

Submitted filename: Response to reviewers.doc

Click here for additional data file.

16 Nov 2020

PONE-D-20-28810R1

PROGNOSTIC VALUE OF PLASMA PENTRAXIN 3 LEVELS IN PATIENTS WITH SEPTIC SHOCK ADMITTED TO INTENSIVE CARE

PLOS ONE

Dear Dr. Suberviola,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the following points:

- the language used in the revised sections of the manuscript needs to be improved. In particular:

1. I suggest using an alternative phrase for describing/comparing the magnitude of AUC. Consider using "lower" instead of "smaller".

2. Please consider rephrasing the Conclusion sentence of the Abstract: instead of "Our results support the prognostic value", consider the following: "Our results suggest that the prognostic value of... may support..."

3.  Please rephrase the following: "But even they have limited abilities..."

4. Please, try rephrasing the following sentence: "Lactate was the biomarker with higher associations..."

5. "Our results also support the severity scores..." Please consider using "Our results also suggest that..."

6. Please consider rephrasing: "slight increase"

7. Please revise the following sentence: "These conflicting outcomes may due to the heterogeneity..."

I suggest proofreading/editing the manuscript from a proofreader with full professional proficiency in scientific English.

Please submit your revised manuscript by Dec 31 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Aleksandar R. Zivkovic

Academic Editor

PLOS ONE

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27 Nov 2020

Attached to this email I am sending you a new manuscript that includes all the changes suggested by the reviewers. In addition, English has been reviewed by a professional translator specialized in scientific articles.

- The language used in the revised sections of the manuscript needs to be improved. In particular:

1. I suggest using an alternative phrase for describing/comparing the magnitude of AUC. Consider using "lower" instead of "smaller".

We have made the suggested change.

2. Please consider rephrasing the Conclusion sentence of the Abstract: instead of "Our results support the prognostic value", consider the following: "Our results suggest that the prognostic value of... may support..."

We have made the suggested change

3. Please rephrase the following: "But even they have limited abilities..."

We have made the suggested change

4. Please, try rephrasing the following sentence: "Lactate was the biomarker with higher associations..."

We have made the suggested change

5. "Our results also support the severity scores..." Please consider using "Our results also suggest that..."

We have made the suggested change

6. Please consider rephrasing: "slight increase"

We have made the suggested change

7. Please revise the following sentence: "These conflicting outcomes may due to the heterogeneity..."

We have made the suggested change

We trust that the new manuscript meets the requested specifications and we apologize for any inconvenience that the inappropriateness of its format may have created.

Best regards.

Borja Suberviola

Submitted filename: RESPOND TO REVIEWERS.docx

Click here for additional data file.

30 Nov 2020

PROGNOSTIC VALUE OF PLASMA PENTRAXIN 3 LEVELS IN PATIENTS WITH SEPTIC SHOCK ADMITTED TO INTENSIVE CARE

PONE-D-20-28810R2

Dear Dr. Suberviola,

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Kind regards,

Aleksandar R. Zivkovic

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

2 Dec 2020

PONE-D-20-28810R2

Prognostic value of plasma pentraxin 3 levels in patients with septic shock admitted to intensive care

Dear Dr. Suberviola:

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on behalf of

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PLOS ONE