MINISTRY OF EDUCATION University of Medicine and Pharmacy of Craiova

DOCTORAL SCHOOL

DOCTORAL THESIS

ABSTRACT

THE ROLE OF BIOMARKERS IN DIAGNOSIS OF

SEPSIS

PhD SUPERVISOR:

PROF. UNIV. DR. PURCARU FLOREA

PhD Student:

DOGARU SEBASTIAN

CRAIOVA

2021

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TABLE OF CONTENTS

1. INTRODUCTION …………………………. …………. ……………… ...1

2. CURRENT STATE OF KNOWLEDGE ……………. ………. …… ……2

2.1. General notions…. …………………………………………………… ... 2

2.2. Scores used to evaluate sepsis …………………………… ……………..2

2.3. Biomarkers used in sepsis …………………………………………….. ..3

2.4. Integration of concepts ……………………………………………… ....4

3. OWN CONTRIBUTIONS ……… .. ……………………………………..4

3.1. WORKING HYPOTHESIS AND GENERAL OBJECTIVES ……….. 4

The purpose of the paper …………………………………………………....4

3.2. RESEARCH METHODOLOGY ………………………………………4

3.3. RESULTS …………………………………………………………….. 5

4. DISCUSSIONS …………………………………………………………..7

5. CONCLUSIONS …………………………………………………………8

6. SOURCES OF FUNDING ………………………………………… ….10

7. SELECTIVE BIBLIOGRAPHY ………………………. …………….. 10

Keywords: sepsis, Emergency Department, early diagnosis for sepsis, POCT,

biomarkers, presepsin, early diagnosis scores, combined score

1. Introduction Sepsis is a major public health problem globally and one of the

most complex pathologies that are encountered in emergency departments and

intensive care; it remains a major cause of mortality despite the impressive

resources used and the progress made in understanding it.

From diagnosis (the team of authors who participated in the development of

the last Sepsis Guidelines 2016 chose to call Sepsis-3 this definition to suggest that

the field is very dynamic) to attempts to find a relevant biomarker (in 2010 were

under study about 178 biomarkers), shows that it is an area where much remains to

be done.

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The definition of Sepsis-3 (2016) is the expression of the new approach -

the severity of the infection, more specific and less sensitive, different from the

previous approach based on SIRS criteria which, less specific but more sensitive,

focused on the infection process. After the presentation at the emergency

department, the first hour is paramount. The study reflected this approach:

evaluation and investigations performed (NEWS2 and qSOFA scores were

assessed and for patients who met the SIRS criteria, supplementation with a POCT

biomarker, presepsin) - aimed at obtaining an early diagnosis (about 20 minutes),

which is in line with current trends in rapid diagnosis (Sepsis Update 2018).

By combining this sensitive, but less specific, early warning score(NEWS2)

with a more specific biomarker (presepsin), we obtained a new score with which I

was able to diagnose a statistically significant number of sepsis cases earlier.

2. The current state of knowledge

2.1. General notions The first mention of the notion of sepsis belongs to

Hippocrates (Corpus Hippocraticum), but the notion of sepsis has evolved and been

refined as new scientific discoveries have become more widespread. The new

definition is the result of the third consensus conference in 2016 which re-

evaluated the definitions for sepsis and septic shock in force until then; In 2018,

Sepsis Update was released, which emphasized the importance of diagnosis and

initiation of treatment as early as possible, with the diagnosis and treatment being

performed in the first hour after presentation. Changes in the treatment of patients

with sepsis were highlighted in 2020 through treatment guidelines for the critical

patient with COVID-19.

2.2. Scores used for the assessment of sepsis The diagnosis of sepsis only when

life-threatening organ dysfunctions are found, according to the current definition,

leaves little time for intervention and should be anticipated whenever possible.

Under the conditions of the emergency department an optimal time should be about

20 minutes, the time required for patient assessment, monitoring, anamnesis, taking

blood samples and possibly obtaining point-of-care results. Accurate assessment at

first contact with the patient can optimize management and improve prognosis.

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We noticed that depending on the time available, the location of the patient

and the purpose in mind, influenced the way in which the information was used,

the complexity of the score or the followed parameters. That is why they were

elaborated scores for different environments such as: scores designed for rapid

diagnosis, in suboptimal conditions (qSOFA, SIRS); early warning scores (MEWS,

NEWS, NEWS2); scores developed for sections dedicated to a specific pathology

(PSI, CURB-65); scores used for rapid reaction in environments with increased

response and intervention capacity - emergency department (qSOFA, MEDS,

REMS, RAPS); scores for complex evaluation, more elaborate used in the intensive

care unit (SOFA, APACHE II, PIRO)

Another observation can be made: depending on the environment in which

they are used, there are scores designed to have a high sensitivity, but with low

specificity that leave more time for intervention (SIRS, NEWS, MEWS, RAPS,

REMS) or severity scores, involving organ dysfunctions, which leaves little time

available for the therapeutic act, which must be performed quickly, in sections with

rapid response capacity (qSOFA, MEDS).

2.3. Biomarkers used in sepsis that are cost-effective, time-tested, clinically

applicable, and evidence-based and routinely available are C-reactive protein,

procalcitonin, and presepsin. Presepsin has been studied mainly because it is

readily available on point-of-care equipment that can deliver the result in about 20

minutes. This is in line with the latest guidelines for the treatment and diagnosis of

sepsis, which considers time to be a very important resource and can significantly

optimize patient management in the emergency department and can be used

successfully in the early detection of acute myocardial infarction. of acute heart

failure or in risk stratification in patients who have undergone heart surgery. The

kinetics of presepsin is another advantage that cannot be overlooked. Presepsin

appears earlier in the circulation after the aggression of the pathogen compared to

procalcitonin, which makes it more suitable for optimizing the management of the

patient with sepsis in the emergency department.

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2.4. Integrating concepts By using an early warning score, which has been

verified over time that provides sensitivity and by using a specific biomarker with

fast dynamics that provides specificity, time would be managed more efficient.

These qualities in my opinion would be met by the NEWS2 score, which non-

invasively evaluates respiratory, cardiovascular, neurological and metabolic

parameters that can offer an early warning, in correlation with presepsin, which can

be obtained quickly in 17 minutes; together with blood count and acid-base balance

obtained at presentation can very accurately assess the patient's clinical condition.

3. Own contributions

3.1. Working hypothesis and general objectives In the thesis "The role of

biomarkers in the early diagnosis of sepsis" we aimed to demonstrate how

sepsis can be diagnosed faster, in a cost-effective manner. For this purpose we

used a National Early Warning Score 2 (NEWS2) early warning score. By

combining this score that monitors the patient's condition for access to adequate

medical resources with a biomarker, presepsin, which grows rapidly in infections,

obtained quickly at the patient's bed, we were able to accurately diagnose early a

significant number of cases of sepsis. To stratify the risk and assess the adverse

prognosis for patients presenting to the emergency room, we used the score MEDS

(Mortality in the Emergency Department in Sepsis). The secondary objectives of

the doctoral research were: evaluation of the role of presepsin having in mind the

new definition of sepsis, determination of presepsin values in correlation with the

assessed severity with different severity scores, reassessment of new threshold

values of monitoring / prognosis scores, evaluation of risk factors for adverse

evolution, the impact of comorbidities on the prognosis.

3.2. Research methodology The type of study was retrospective, observational,

which based on the literature studied was optimal for the studied population,

conducted at a tertiary center of diagnosis and treatment, which had inclusion and

exclusion criteria well specified from the beginning; the study was conducted over

2 years and included 125 patients diagnosed with systemic inflammatory response

syndrome. From the initial group, a population of 30 patients who were diagnosed

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with sepsis was selected, according to the Sepsis-3 definition. In order to diagnose

sepsis early, we applied various scores to which we stopped after the critical

analysis of the literature: qSOFA- for the diagnosis of sepsis, an early warning

score (NEWS2) and prognosis score (MEDS) were assessed. To increase

specificity, we used a specific biomarker, presepsin. The result was the

development of a composite score, obtained by aggregating the results provided by

the NEWS2 score and the presepsin values adjusted to the values provided by the

ROC curve, for life-threatening organ dysfunctions. Although this was not the

original purpose, it was practically a comparative assessment of patients diagnosed

with sepsis, according to the definitions of Sepsis-2 and Sepsis-3, observed using

scores to which a biomarker was added to diagnose an infection early, before it

becomes life threatening. The secondary outcome was the determination of

presepsin threshold values, consistent with the new definition of sepsis. The

research was carried out based on the opinion of the ethics commission at the

Central Military Emergency University Hospital "Dr. Carol Davila" Bucharest,

after signing the informed consent to prepare the presentation, in accordance with

the ethical principles set out in the Helsinki Declaration. Patient evaluation was

performed retrospectively, with centralization of data in a Microsoft Excel version

version 2019. Statistical analysis of the data used was performed using the SPSS

(Statistical Package for the Social Sciences) software package, version 20.0 (IBM

Corporation, Armonk, NY, USA). The concentration of the soluble fragment of

CD14 (presepsin) was measured using an immunological test based on a

chemoluminescent enzyme (PATHFAST ™ Presepsin) performed on a Point-of-

Care analyzer (Mitsubishi Chemical Medience Corporation, Tokyo, Japan).

3.3. Results The group included 125 patients who were diagnosed with systemic

inflammatory response syndrome, diagnosed with sepsis, according to the

definition of Sepsis-2. According to the Sepsis-3 definition, a group of 30 patients

was selected who met 2 of the 3 qSOFA criteria, corresponding to the new

definition. In conclusion, the follow-up group included 30 patients with sepsis,

analyzed compared to 95 patients who constituted the control group. The

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distribution of patients was analyzed according to demographic characteristics (age

and sex) and we found that males and advanced age have a slight prevalence;

comorbidities that have influenced the subsequent evolution of patients as sepsis,

septic shock or death have been found predominantly in patients with pre-existing

organ dysfunction; the gateway of the pathogen that was predominantly the

urogenital tract; of the scores used for evaluation and laboratory analyzes, as well

as in terms of complex monitoring on devices and systems that ultimately led to the

analysis of mortality in the studied cohort. Respiratory failure had the greatest

impact on the poor prognosis, as all these patients died. In the studied literature,

respiratory failure is recognized as an important risk factor, but it is not always

fatal in the context of sepsis.

The main goal was the early diagnosis of sepsis, which is why we

developed a compound score based on an early warning score, the NEWS2 score

and the presepsin for which we obtained the best values for the AUROC curve for

sepsis detection 0.9721 (IC95% 5.69-7.65), which translates into early detection of

sepsis according to the definition of Sepsis-3

AUROC curves for NEWS2, MEDS, PSP scores and for derived scores (NEWS2PSP,

NEWS2PSP2, NEWS2PSP3, NEWS2PSP4) in sepsis (AUROC 0.971% 95 CI 5.69-7.65 for

NEWS2PSP4

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By using this algorithm for 16 patients, the NEWS2 score was modified

(12.8%). For 10 out of 95 patients (10.52%) undiagnosed with sepsis and for 6

patients out of 30 (20%) of patients with sepsis, more points were added to the

NEWS2 score, which means that more patients could be diagnosed with sepsis,

before the infection becomes life-threatening. The comparative values for the

AUROC curves in sepsis for the established NEWS2, MEDS scores and the studied

compound scores are shown below.

Sepsis

AUROC %95 CI

NEWS2 0,962 5,54- 7,51

MEDS 0,875 4,39- 7,36

PSP 0,603 41,32- 255,17

NEWS2PSP 0,924 6,36- 9,45

NEWS2PSP2 0,953 5,69- 7,84

NEWS2PSP3 0,956 5,95- 7,65

NEWS2PSP4 0,971 5,69- 7,65

AUROC curve values for NEWS2, MEDS, presepsin derived scores and NEWS2PSP,

NEWS2PSP2, NEWS2PSP3 and NEWS2PSP4 derived scores in sepsis (AUROC 0.971% 95 CI

5.69-7.65 for NEWS2PSP4)

Based on the data obtained from the studied group, we tried to find

correlations according to the patient's sex and we did not find any statistically

significant influence on the incidence of sepsis, while old age showed a positive

correlation with sepsis, with values adjusted according to the definition of sepsis-3.

For septic shock the values of the AUROC curves were better for the derived

scores NEWS2PSP2 and NEWS2PSP3 0.975 with slightly different 95CI% with

values of 6.31-10.19, respectively 6.22-9.96 while for mortality the score MEDS (a

score developed specifically to predict mortality in the emergency department)

confirmed the reliability of AUROC 0.762 (95CI% 2.55-6.01).

Through the detailed statistical analysis by linear regression, some of the

data obtained during the study of the group of patients included in the study were

nuanced.

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4. Discussions Sepsis is a complex pathology, intensively researched, with a major

impact on patients, with a still very high mortality, although declining; even if

cured, patients are burdened with distant comorbidities and an increased risk of

subsequent death. The different scores available are appropriate to the purpose for

which they were designed; application without discernment, in situations other than

those for which they were designed lead to errors. For Romania at present, the

resources available in the emergency units are more evolved and in order to make a

faster diagnosis, they must be used as efficiently as possible. Biomarkers that are

used for diagnosis must have rapid kinetics to capture the pathophysiological

changes that occur. For emergency units it would be useful to diagnose as early as

possible.

The data in the literature to which we had access did not study sepsis from

this point of view: early detection with the help of a specific biomarker with rapid

kinetics, before organ dysfunctions become life-threatening, to which a monitoring

score should be added for the necessary sensitivity to testing. From the data I have

is the first study of this type.

The research also aimed to establish new threshold values for presepsin

according to the current definition of sepsis for which the predictive value is

maintained in combination with the composite score obtained.

A secondary objective that was achieved was the possibility of applying this

composite score for the early determination of septic shock. The fact that this is

possible at lower presepsin values may be another new direction of research, as

normally the worsening of the patient's condition should be reflected in a higher

score value.

Also, during the research we confirmed the value of the MEDS score for

the ominous prognosis at 28 days. It was best for predicting sepsis mortality at the

emergency department.

5. Conclusions The main purpose of the study was early detection of sepsis. The

dynamics of definitions and discoveries in the field suggested the use of a

monitoring score for sensitivity, to which I added a specific biomarker with fast

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dynamics. For approximately 10% of patients with systemic inflammatory response

syndrome, early detection of sepsis. Put in perspective, the results obtained mean a

step in the right direction for a disease with still very high mortality, given the

chronic lack of beds in any hospital and intensive care.

Biomarkers in sepsis according to the new definition no longer have a

place. However, the emphasis on time makes early diagnosis all the more

necessary. Presepsin, which has rapid kinetics, fulfills this purpose. In this context,

I had to determine the new threshold values for which presepsin could provide

more time for therapeutic intervention. Reinterpreting these values to detect sepsis

early was a useful element with immediate applicability in the clinic and is at the

same time a contribution of its own.

The same biomarker and rapid algorithm can be used to predict septic

shock. However, it is still necessary to investigate why the results obtained are at

lower values of presepsin, compared to the values at which sepsis is diagnosed.

To accurately predict 28-day mortality, we used a score designed for this purpose,

which we validated from the perspective of the new definition of sepsis, the MEDS

score, its validation being a new contribution of its own.

The evaluation of the risk factors for the unfortunate evolution led to an

unexpected conclusion: institutionalized patients presented an increased mortality,

an own contribution, which may be a feature of our country in terms of reduced

intra-family surveillance due to increased migration of the active population. We

found, somewhat expected, that respiratory failure has a strong impact on the

prognosis.

The influence of the age and sex on the evolution of sepsis remains to be

investigated, the data obtained not being very well substantiated.

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In conclusion, I would like to systematize the main conclusions resulting

from the doctoral activity:

1. We designed a new score for the early detection of sepsis, by analysis and

evaluated the previously known scores

2. We investigated the effectiveness of this score in the cohort described in this

study, observing encouraging results

3. This study allowed a complete evaluation of the scores used for diagnosing,

monitoring the patient with acute pathology and the prognosis of sepsis in a

Romanian population

4. Following this study we identified multiple benefits both from a medical

perspective and cost effective in optimizing the management of patients with sepsis

5. Despite the limitations of the study, I consider that these results presented above

are a starting point for future further scientific research.

6. Sources of funding We had no sources of funding

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