Which Biomarkers Can Be Used as Diagnostic Tools for Infection in Suspected Sepsis?

Abstract

The diagnosis of infection in patients with suspected sepsis is frequently difficult to achieve with a reasonable degree of certainty. Currently, the diagnosis of infection still relies on a combination of systemic manifestations, manifestations of organ dysfunction, and microbiological documentation. In addition, the microbiologic confirmation of infection is obtained only after 2 to 3 days of empiric antibiotic therapy. These criteria are far from perfect being at least in part responsible for the overuse and misuse of antibiotics, in the community and in hospital, and probably the main drive for antibiotic resistance. Biomarkers have been studied and used in several clinical settings as surrogate markers of infection to improve their diagnostic accuracy as well as in the assessment of response to antibiotics and in antibiotic stewardship programs. The aim of this review is to provide a clear overview of the current evidence of usefulness of biomarkers in several clinical scenarios, namely, to diagnose infection to prescribe antibiotics, to exclude infection to withhold antibiotics, and to identify the causative pathogen to target antimicrobial treatment. In recent years, new evidence with “old” biomarkers, like C-reactive protein and procalcitonin, as well as new biomarkers and molecular tests, as breathomics or bacterial DNA identification by polymerase chain reaction, increased markedly in different areas adding useful information for clinical decision making at the bedside when adequately used. The recent evidence shows that the information given by biomarkers can support the suspicion of infection and pathogen identification but also, and not less important, can exclude its diagnosis. Although the ideal biomarker has not yet been found, there are various promising biomarkers that represent true evolutions in the diagnosis of infection in patients with suspected sepsis.

Keywords

infection - sepsis - diagnosis - biomarkers - C-reactive protein - procalcitonin

The modern definition of sepsis begins with the 1991 consensus conference where sepsis resulted from the host's systemic inflammatory response syndrome (SIRS) to a suspected or documented infection.[1] A 2001 consensus conference revisited the sepsis criteria and considered SIRS useful, although overly sensitive and nonspecific, not allowing a precise staging or prognostication of the host response to infection.[2] As a result, the authors expanded the list of signs and symptoms of sepsis to better reflect the clinical response to infection. In this list, for the first time, it was included two serum biomarkers, C-reactive protein (CRP) and procalcitonin (PCT), under the section inflammatory variables, as tools to help clinicians in the diagnosis of sepsis. Besides, the authors acknowledge that the concomitant changes in biochemical/biologic markers could be more consistent than the clinical manifestations of sepsis. And they speculate that in the future the diagnosis of suspected sepsis could be purely supported by biomarkers.[2] In both consensus conferences, the panel of experts defined infection as a pathologic process caused by the invasion of normally sterile tissue or fluid or body cavity by pathogenic or potentially pathogenic microorganisms, although it was recognized that this definition was not perfect.

After the Third International Consensus, sepsis became defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.[3] For clinical operationalization, organ dysfunction can be represented by an increase in the Sequential Organ Failure Assessment score of 2 points or more, which was associated with an in-hospital mortality greater than 10%.[3] In this new definition, there is no reference to the potential usefulness of biomarkers of infection and sepsis. It was only considered the potential use of biomarkers that could help in the early identification of renal and hepatic dysfunction or coagulopathy. The definition of infection was not revisited by the Sepsis 3.0 task force.

Although sepsis incidence and mortality seem to be decreasing worldwide, in 2017 the estimated incidence of sepsis in the world was 48.9 million (95% uncertainty interval: 38.9–62.9) with a sepsis-related death of 11.0 million (10.1–12.0), which represented a total of 19.7% (18.2–21.4) of all global deaths.[4] But the distribution of sepsis incidence and mortality varies markedly across regions with the highest burden in low- and middle-income countries. Although infection is a frequent disease, its definite diagnosis remains difficult. We should always keep in mind that there is no pathophysiological aspect that is pathognomonic of infection and sepsis and the diagnosis of infection results from the intersection of three vectors (systemic manifestations, manifestations of organ dysfunction, and microbiological documentation). Though, the microbiologic confirmation of infection is obtained only after 2 to 3 days of empiric antibiotic therapy and in around half of the cases documentation is never achieved. This uncertainty is at least in part responsible for the overuse and misuse of antibiotics in the community and in hospital, and this practice is probably the main drive for antibiotic resistance.[5] [6] As a result, antibiotics are commonly prescribed in patients without a definitive diagnosis of infection, in particular in the presence of severe infections, since a delay in treatment is associated with worse outcomes, a strategy that, without proper guidelines and monitoring, can contribute to increase antibiotic resistance.[7]

The diagnostic uncertainty of infection and sepsis also results in the treatment with antibiotics of patients that look septic but have a sepsis-like syndrome. With our current tools the concordance between the sepsis diagnosis at intensive care unit (ICU) admission and a posthoc assessment by experts, using strict criteria, is very poor, with >40% of the patients treated as sepsis with an unlikely infection.[8] In this original study, a biomarker, CRP, if elevated increased significantly the likelihood of infection diagnosis at ICU admission. But it was worrisome that patients treated with antibiotics but without infection presented a poorer prognosis compared with those with documented infections, probably because the underlying disease causing the sepsis-like syndrome was not being adequately treated.

All the above-described limitations in the diagnosis of infection, as well as the lack of a gold standard test to diagnose infection, led researchers and clinicians to use several molecular tests, in particular biomarkers from the inflammatory cascade, as surrogate markers of infection.[9] As a result, biomarkers became very popular in clinical practice since they may improve the diagnostic accuracy of infection as well as the assessment of response to antibiotics and in the antibiotic stewardship programs.

A biomarker is defined as a biological characteristic, objectively measured (i.e., with acceptable accuracy and reproducibility), that can be used as an indicator for a physiological or pathological process, or of the activity of a medicine. Before being used clinically, biomarkers need to be evaluated in a three-stage process: analytical validation, qualification, and utilization.[9]

Biomarkers can be divided in two categories: prognostic and predictive biomarkers. In sepsis and infection, >250 biomarkers have been evaluated, and the great majority to assess prognosis. But why should we use a biomarker merely to determine whether a patient has a higher risk of death when the currently available interventions are not able to modify the prognosis? Only a small number of biomarkers have been assessed as predictive biomarkers to give additional clinical information that can be categorized as triage, diagnosis, risk stratification, monitoring clinical course, and antibiotic stewardship.[10] A good predictive biomarker of infection should be absent if the patient is not infected, should appear concomitantly with and ideally preceding the clinical manifestations of infection, and disappear with successful therapy or remain elevated if infection is refractory to treatment.[11] [12]

The “ideal” biomarker has not yet been found. Contrary to other time-dependent medical emergencies like acute myocardial infarction or stroke, there is no test that can reliably rule in or rule out infection and sepsis. As a result, biomarkers should never be used as a stand-alone tool, but integrated in a complete clinical, radiologic, and laboratory evaluation of patients with suspicion of infection. And there is convincing evidence that correctly used biomarkers are useful adjuncts to the clinician at the bedside.

The likelihood of antibiotic prescription by the attending clinician depends on the balance of his/her degree of confidence that an infection is present (pretest probability) against the fear of not treating the potential infection and the negative consequences of that decision. In a context of a high pretest probability, the role of biomarkers in the clinical decision of antibiotic prescription is frequently only confirmatory of the clinical suspicion. The fundamental decision has already been made. But besides the need of a prompt diagnosis of infection, its exclusion is equally important to decrease antibiotic pressure and to target the therapy for the correct underlying disease. In this very important and difficult decision, biomarkers could play a crucial role, ruling in or ruling out an infection, which should be further detailed.

The aim of this review is to provide a clear overview of the current evidence for biomarkers in several clinical scenarios, namely, to diagnose infection to prescribe antibiotics, to exclude infection to withhold antibiotics, and to identify the causative pathogen to target antimicrobial treatment ([Fig. 1]).

Fig. 1 Potential usefulness of biomarkers and molecular tests as diagnostic tools in suspected sepsis. CRP, C-reactive protein; FAIM3:PLAC8, Fas apoptotic inhibitory molecule 3 to placenta-specific 8 ratio; PCT, procalcitonin; PSP, pancreatic stone protein; VOCs, volatile organic compounds. Green = a (+), Yellow = a (+/−), Red = a (−).

Biomarkers to Confirm Infection

The early diagnosis of infection and sepsis is challenging. However, evidence supports that rapid and precise diagnosis of septic patients is crucial, since early and adequate antibiotic therapy is critical to improve prognosis.[13] The diagnosis of infection, most of the times, still relies on the clinical evaluation of the patient and the most typical signs and symptoms, such as fever, cough, tachycardia, polypnea, or basic analytical parameters like leukocytosis, which are also commonly present in many other noninfectious diseases.[14] For these reasons, biomarkers are becoming important to the care of patients with suspicion of infection, since they are noninvasive, rapidly available, and may be followed over patient́s clinical course. Various biological mediators have been proposed, among which PCT and CRP are the most frequently studied.

It has been shown that the information obtained from the assessment of biomarker kinetics can be more informative than the assessment of a single determination. In a previous study, our group found that CRP kinetics before ICU-acquired infection was useful in the prediction of the diagnosis showing that patients presenting a maximum daily CRP increase above 4.1 mg/dL plus a concentration above 8.7 mg/dL had an 88% risk of ICU-acquired infection diagnosis. In addition we found that a patient presenting a steady increase reaching a value >8.7mg/dL (pattern A) or a persistently elevated CRP level always >8.7mg/dL (pattern B) had a very high risk of ICU-acquired infection.[15] Also, in the BioVAP study, we showed again that the kinetics of CRP in the days before ventilator-associated pneumonia (VAP) diagnosis, namely the slope of CRP, could be useful in VAP prediction, that is just a type of ICU-acquired infection. On the opposite, both PCT and Mid-regional proadrenomedullin (MR-proADM) kinetics in the days before VAP diagnosis showed a poor predictive performance.[16] More recently, Garvik et al showed that in patients with blood stream infections (BSIs), CRP presented a significant rise (slope increase to 3.63 mg/dL/d) 3.1 days before BSI diagnosis (defined as the day of positive blood cultures).[17] On the contrary, PCT kinetics before infection diagnosis showed poor diagnostic performance in several studies. Luyt et al in a previous study assessing the course of PCT before VAP diagnosis, both the crude PCT values and its kinetics, showed poor diagnostic accuracy.[18] We have reproduced these findings in the BioVAP study.[16]

Another interesting biomarker is pancreatic stone protein (PSP), a C-type lectin protein that triggers polymorphonuclear cell activation, and PSP has shown proinflammatory activity in vitro.[19] In an unselected cohort of critically ill adults, PSP was found to be superior to PCT and other sepsis biomarkers (soluble CD25 and heparin-binding protein) for the accurate identification of infection and sepsis,[20] and an increase in PSP level precedes the development of sepsis in a cohort of severely burnt patients.[21] Recently, a multicentric international prospective observational study performed in patients with nosocomial sepsis showed that increasing serial PSP preceded the onset of clinical signs of sepsis. Comparing with other commonly use biomarkers, PSP started to increase 5 days before the clinical diagnosis of sepsis, PCT 3 days, and CRP 2 days, respectively.[22]

Single measurements of biomarkers have been extensively studied as a way of obtaining a rapid and precise diagnosis of infection. Several biomarkers have been assessed, especially in the diagnosis of respiratory infections, frequently with conflicting results. Some studies evaluating a single CRP measurement suggest that it could be helpful in the diagnosis of patients with community-acquired pneumonia (CAP)[12] [23] while others suggest that a single CRP value was not helpful, except if we use a higher cut-off (>10 mg/dL).[24] [25] Also, the ability of CRP and PCT to differentiate between ventilator-associated tracheobronchitis (VAT) and VAP was studied.[26] Although VAT presented lower levels of both biomarkers in comparison with VAP, there was a large overlap with both biomarkers at the day of the diagnosis of ventilator-associated lower respiratory tract infections (VA-LRTI). In addition, the level of both biomarkers was not significantly different according to the bacterial etiology either being a gram-positive or gram-negative pathogen, and the presence or absence of multidrug-resistant bacteria.

Concerning PCT, several randomized trials showed that PCT could be used to safely withhold antibiotics in patients with suspected community-acquired respiratory tract infections.[27] However, the role of PCT in CAP diagnosis was challenged in a study to assess whether early chest computed tomographic scan affects diagnosis and management of patients visiting the emergency department with suspected CAP. In this study, it was demonstrated that a lower CRP level was the only helpful marker of a potential false-positive chest X-ray diagnosis. In contrast, with both false-positive and false-negative chest X-rays, PCT was a poor discriminator.[28]

Beyond classical biomarkers, the analysis of the whole-blood leukocyte transcriptome enables the assessment of thousands of molecular signals,[29] suggesting that genome-wide transcriptional profiling of blood leukocytes can be useful to differentiate between infectious and noninfectious causes of severe disease. Scicluna et al performed a study to characterize the blood genomic response in patients with suspected CAP and identify a candidate biomarker for the rapid diagnosis of CAP on ICU admission.[30] They proposed the Fas apoptotic inhibitory molecule 3 to placenta-specific 8 (FAIM3:PLAC8) ratio as a candidate biomarker to assist in the rapid diagnosis of CAP on ICU admission. This biomarker outperformed plasma PCT and interleukin (IL)-8 and IL-6 in discriminating between CAP and no-CAP patients.

Biomarkers to Exclude Infection

The diagnosis of infection and sepsis relies mainly on the clinical judgment. Sometimes the diagnosis is easy and straightforward, but frequently, namely in the early phases of infection, the distinction between the presence or absence of infection is doubtful, namely for young less trained junior doctors.[31] An experienced clinician is more able to achieving an instant recognition by the identification of a single clue or of pattern recognition and in doing so to balance the likelihood that an infection is present against other possible diagnoses, and also assess clinical severity.

Based on main complains and physical examination, clinicians divide the patients according to the clinical severity and the perceived probability of the presence of an infection. Based on this delicate balance, clinicians must decide on antibiotic administration still without a definite diagnosis of infection, in particular in patients with shock.

There are two frequent situations where a biomarker could be useful in clinical decision making: clinically stable patients who present systemic inflammatory manifestations but with signs and symptoms that are not that specific of infection; clinically unstable patients with a low likelihood for the presence of infection. In these clinical situations, the addition of a biomarker that can identify noninfectious sepsis-like conditions with a high sensitivity and a low posttest probability could be useful. As a consequence, this could lead to a decrease in antibiotic prescription and above all to the treatment of a disease that is mimicking infection (e.g., malignancy, autoimmune diseases, etc.).[8]

C-Reactive Protein

When assessing biomarkers there two possible scenarios, assessment of a single value versus assessment of serial measurements. Both scenarios have been evaluated in diagnosis, in a variety of infections as well as in clinical settings, namely emergency departments, medical and surgical wards, and ICUs.

The results of these studies are sometimes contradictory. It is important to stress that the studies are comparable if using similar designs, namely methodologies, inclusion and exclusion criteria, as well as endpoints.[32] [33] This is closely linked to the diagnosis of sepsis or infection.[2] Using only the 1991 Consensus Conference criteria (sepsis, severe sepsis, and septic shock) as the endpoint, it could result in being only assessing clinical severity rather than the evaluation of the diagnostic accuracy of the biomarker for infection itself. The “gold standard,” which should be the presence or absence of documented infection, that is patients with a definite diagnosis of infection with positive cultures as opposed to patients with no infection and no antibiotic therapy, is frequently ignored.[34]

Several studies evaluated the performance of a single measurement of a biomarker in different clinical settings and different infections. In clinical practice, a low level of a CRP, e.g., CRP levels <2–5 mg/dL, may be useful to exclude the diagnosis of infection.[16] [35] [36] A recent study performed in critically ill patients assessing the diagnostic performance of >50 biomarkers for infection clearly show that CRP performed better than any other studied biomarker or panels of biomarkers.[37]

There is a situation that, although uncommon, could be responsible for false-negative CRP values. Since serum CRP is produced exclusively in the liver, patients with severe acute liver failure present very low levels of CRP in spite of being infected.[38] However, patients with chronic liver disease the usefulness of CRP is similar to a patient without cirrhosis.[39]

In the outpatient management of patients with nonspecific symptoms of lower respiratory tract infections, CRP could be used to exclude pneumonia. A systematic review showed that a low CRP, ≤2 mg/dL, has a positive likelihood ratio of 2.1 and a negative likelihood ratio of 0.33.[40] In patients who can be managed in the outpatient clinic and who are not severely ill, with a pretest probability of pneumonia >10%, CRP may be of value to rule out the diagnosis of infection avoiding unnecessary antibiotic therapy.[40]

A cluster randomized trial with four arms was done (standard of care, communication, point-of-care CRP test, both interventions) to further evaluate this approach.[41] The baseline characteristics of the patients were similar between the groups. The rate of prescription of antibiotics was >50% in the standard-of-care group and decrease in CRP and communication groups to 31 and 25% respectively (p = 0.02 and p < 0.01) and to 23% in the combined intervention group, without any negative impact on outcomes nor of the satisfaction with care. These results lead to changes in the guidelines for Dutch general practitioners by the implementation of point-of-care CRP devices in the outpatient clinic as well as specific training in communication skills.

Since biomarkers are not static but on the opposite dynamic, with marked changes in serum concentrations over time, serial measurements could be more informative. Our group demonstrated that daily CRP determinations are useful as a marker of infection prediction in ICU patients admitted for longer than 72 hours. We compared the daily CRP values of patients prior to the development of an ICU-acquired infection with those of patients without infection who were also not receiving antibiotics. In patients who developed an ICU-acquired infection, CRP of the previous days showed a steady and significant increase in the 5 days preceding the diagnosis, whereas in noninfected patients CRP remained almost unchanged.[15] The daily CRP measurements were used to classify patients in different patterns. We identified two patterns (pattern C occurred when the CRP was ≤8.7 mg/dL at the end of the follow-up period and, in the previous days, was at least once above the cut-off value; pattern D occurred when CRP was always ≤8.7 mg/dL) with a very low risk of infection.

In another study assessing CRP kinetics in patients under mechanical ventilation for noninfectious reasons (and not receiving antibiotics). we demonstrated that a patient with an average increase of CRP of 1 mg/dL/d had a 62% greater chance of having a VAP when compared with a patient with no CRP increase.[16] More recently, from a population-based BSI database, we showed that CRP and plasma albumin (PA) concentrations began to change inversely some days before CA-BSI diagnosis, CRP increasing by day −3.1, and PA decreasing by day −1.3.[17] In addition, in a population-based study of acute myeloid leukemia, we also found that serial CRP measurements remain steadily low in the absence of infectious complications.[42]

Other studies in different clinical settings showed similar findings.[43] [44] In other words, these studies showed that serial measurements of biomarkers, namely CRP, could be helpful when infection is not present as well as excluding infection, whenever the levels remain steadily low.

Procalcitonin

Concerning PCT, the major limitation is the frequent finding of patients with documented infections with very low or even undetectable levels, in other words frequent false negatives. This is particularly true in infections considered to be “localized,” like empyema or abscesses.[27] This is a common finding in several studies and trials. Just as an example, in the PRORATA trial, N = 89 patients had a PCT <0.5 μg/L at inclusion and according to the algorithm antibiotics were “discouraged” or “strongly discouraged.” However, the attending physician overruled this recommendation, because they considered patient “infected” despite a low PCT level in 73% of patients.[45] [46]

In patients under mechanical ventilation absolute values as well as kinetics before diagnosis of VAP have repeatedly showed poor performance.[16] [18] [47] And the same was true concerning the diagnosis of early-onset pneumonia in post-cardiac arrest patients under therapeutic hypothermia in whom PCT presented also poor diagnostic performance.[43] [48] [49]

The performance of PCT for the exclusion of bacterial pneumonia was assessed in a systematic review in patients who present with a moderate to high suspicion of pneumonia based on clinical symptoms.[50] At a cut-off of 0.5 μg/L PCT, which according to the recommended algorithm antibiotics are “discouraged” or “strongly discouraged” since infection is considered “unlikely” or “very unlikely,” had a pooled sensitivity of 55% and a specificity of 76%. As a result, these characteristics of PCT are insufficient to withhold antibiotics in a patient with a negative test. For example, a patient with a 50% pretest probability of bacterial pneumonia will yield a 37% posttest probability with a negative test.

Inflammatory Mediators in the Bronchoalveolar Lavage Fluid

Pneumonia is one of the most common infections, either in community or in hospital. Although bronchoscopy with bronchoalveolar lavage (BAL) is the best technique to obtain a good distal respiratory sample to document a lung parenchymal infection, it is seldom done, being reserved for patients with severe pneumonia, in particular those under invasive mechanical ventilation.[51] [52] In addition, BAL cultures take 2 to 3 days before having a definite result, and infection is confirmed in <50% of patients with suspicion of pneumonia.[53]

Lung parenchymal bacterial infection is sought to be associated with a local inflammatory response.[54] [55] The rapid assessment of inflammatory mediators, namely cytokines, could provide clinicians with relevant information for decision making concerning starting or not antibiotics. To exclude pneumonia, clinicians need a diagnostic test with 100% sensitivity and a negative predictive value of 100%, since patients under mechanical ventilation have a high risk of morbidity and mortality when not promptly treated.[56] In a discovery study, tumor necrosis factor-α (TNF-α), IL-1b, IL-6, IL-8, IL-10, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein-1a (MIP-1a), type 1 soluble triggering receptor expressed on myeloid cells (sTREM-1), and monocyte chemoattractant peptide 1 (MCP-1) in serum and BAL fluid (BALF) were measured in patients with suspected VAP.[57] The concentrations of all inflammatory mediators in serum were similar in VAP and non-VAP groups. In contrast, BALF IL-1b, IL-8, G-CSF, and MIP-1a were significantly higher in the VAP group. A patient with suspected VAP and a BALF IL-1b concentration <10 pg/mL presents a negative likelihood ratio of VAP of 0.09, and a posttest probability for VAP of 2.8%. In contrast, a BALF IL-8 concentration >2 ng/mL presents a positive likelihood ratio of 5.03 corresponding to a 61% probability of VAP being present.[57]

Later, in a validation study from the same group, IL-1β, IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9, and human neutrophil elastase were measured in BALF of patients with suspected VAP. All inflammatory markers were significantly elevated in VAP patients in comparison to the non-VAP group.[58] And the best performance for the exclusion of VAP was obtained with the combination of IL-1β plus IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3%, and a remarkable posttest probability of 0% (95% confidence interval: 0–9.2%).

This approach was tested in a randomized trial[59]; however, contrary to the expectations, antibiotics use remained high in patients with suspected VAP, and antibiotics stewardship did not improve with the use of this highly sensitive rule-out test.[60] This means that the prescribing culture, rather than poor test performance, might explain this absence of effect as well as differences in antibiotics prescription between institutions and countries.[61]

Detection of Microbial DNA/RNA

Molecular amplification techniques by polymerase chain reaction (PCR) have largely changed the approach of the detection and diagnosis of viral infections replacing cell culture techniques as a mean of diagnosis. Now PCR is the reference standard diagnostic test for viral identification and sometimes quantification of viral load.[62]

The current coronavirus disease 2019 (COVID19) pandemic increased markedly the availability and capacity of PCR testing. Concerning the detection of severe acute respiratory syndrome coronavirus 2 (SARS CoV2), the rate of false-positive results for molecular tests are uncommon because of intrinsic designs and rigorous quality-control guidelines.[63] In addition, the concordance between different SARS-CoV-2 PCR assays is between 96 and 100%.[64] In contrast, the rate of false negatives is low but depends on viral load and dynamics.[65] However, the interpretation of a negative SARS CoV2 test result should be taken with caution in the exclusion of COVID19 if the posttest probability remains elevated.[66]

The detection of bacterial DNA could potentially be used to exclude diagnosis of infection. In theory a low concentration of bacterial DNA would exclude the possibility of bacterial growth in culture. This is the rationale of all molecular detection, namely DNA amplification by real-time PCR (RT-PCR) in blood cultures.[67] [68] [69] One recognized limitation of these tests is the number of pathogen probes present in the PCR test panel. The false negatives could be explained by a pathogen identified in the blood cultures that is not present in the PCR panel.[68] Consequently, these techniques present a poor negative likelihood ratio, between 0.2 and 0.6, and as a result a low or very low diagnostic accuracy to safely rule out a BSI.[70]

The same rationale was applied in patients with suspected VAP with real-time 16S rRNA gene PCR performed in BALF for rapid exclusion of infection, using cycles to cross threshold (Ct) values as the result of the 16S rRNA PCR test.[71] This test showed a 100% sensitivity at an impressive specificity of 67% in the exclusion of VAP. A test with this performance could be used to withhold antibiotics in two-thirds of the patients who would otherwise unnecessarily receive them. These results require external validation. However, two different studies showed that the bacterial composition and number of 16S rRNA copies did not provide the same diagnostic accuracy.[72] [73] It is important to stress that these two later studies were performed in tracheal aspirate samples and not BALF, and the control group did not present suspicion of VAP.

Omics

Volatile organic compounds (VOCs) can be produced either by invading respiratory pathogens (that could be different if in a state of a colonizer or infectious bug) or the host response and its assessment could be used in the diagnosis of VAP.[74] The advantage is that these metabolites can be easily and noninvasively analyzed in the exhaled breath.[75] This has been also referred to as breathomics. Using gas chromatography-mass spectrometry, it is possible to identify >100 different VOCs. In preliminary studies the results were somewhat contradictory.[74] [76] A European multicenter study on the diagnostic accuracy of breath analysis of VA-LRTI was designed to assess the diagnostic accuracy of exhaled breath analysis.[77] The exhaled breath test had a sensitivity of 98% at a specificity of 49% with a negative predictive value of 96%, allowing to exclude pneumonia in half of the patients with negative cultures.[78]

Another promising strategy is to characterize peripheral blood gene expression to identify subjects with bacterial infection. In ICU patients with CAP suspicion, the ratio of FAIM3 and the PLAC8 was found to be predictive of the presence of infection with positive and negative predictive values of 83 and 81%.[30] Both molecules are negative regulators of apoptosis and can bring light to the pathophysiologic process of CAP. However, at ICU admission, the FAIM3:PLAC8 biomarker ratio does not have a good enough negative predictive value to safely withhold antibiotics in the presence of CAP suspicion.

Similar results were obtained in another study assessing a different set of genes, with a sensitivity of 90% and a specificity of 83%.[79] With the pretest probability of both studies (44 and 75%), it is very difficult to develop a test to withhold antibiotics since it requires a very high accuracy, which is probably not reachable.[80]

Biomarkers to Identify Potential Pathogens

Once we established a presumptive diagnosis of infection, the next decision is the choice of the most adequate antimicrobials.[81] [82] And this choice is made according to the most probable causative pathogens. The fear of missing a potential pathogen or a mechanism of resistance led clinicians to prescribe empiric broad-spectrum antimicrobials that is a double-sword strategy since it carries an increased risk of emergence of resistance as well as a marked impact on microbiota.[83] The availability of tests that could identify the presence of specific pathogens as well as resistance genes could be useful for decision making at the bedside.

Identification of Potential Pathogens

Currently we could divide the tests to identify pathogens in antigen tests and RT-PCR tests. There are antigen tests to detect Streptococcus pneumoniae and Legionella pneumophila.

The pneumococcal antigen test detects the C-polysaccharide antigen from S. pneumoniae in the urine of patients with a sensitivity of 50 to 80% and a specificity of >90%,[84] with a turnaround time around 30 minutes. The test has been validated for urine and cerebrospinal fluid. This is a very important test since S. pneumoniae is the most frequently encountered bacterial agent of CAP and is also a frequent cause of community-acquired BSI (frequently secondary to CAP) and meningitis. A positive test is indicative of a pneumococcal infection in particular pneumonia.[85]

A negative result suggests no current or recent pneumococcal infection, but it does not exclude pneumococcal infection since the antigen present in the urine may be below the detection limit of the test.

Urinary pneumococcal antigen test can remain positive in around 50% of patients with pneumococcal pneumonia in the first month after pneumonia diagnosis or longer.[86] Streptococcus pneumoniae vaccine may also cause false-positive results, especially in patients who have received the vaccination within 5 days of having the test performed.

Although it is recommended to perform the urinary antigen test in severe CAP patients,[85] it seems to have no impact on outcomes and there are some concerns related to narrowing of spectrum of antibiotic therapy in patients with a positive test that could be associated with an increase rate of relapses.[85] [87]

The urinary antigen test for Legionella detects the L. pneumophila serogroup 1-soluble antigen with a sensitivity of 70 to 100% and a specificity of 95 to 100%.[88] Legionella is not a common pathogen of CAP being associated with outbreaks and recent travel. Legionella pneumophila is responsible for the great majority of reported cases of legionellosis, and almost all being caused by L. pneumophila serogroup 1. Since this is a difficult pathogen to identify, the presence of Legionella antigen in the urine make it the ideal specimen for diagnosis. The Legionella antigen is usually detectable in the urine as early as 3 days after onset of symptoms.[89] Therefore, a positive test would justify narrowing antibiotic therapy to, for example, a respiratory fluoroquinolone.

A positive test for L. pneumophila serogroup 1 antigen in urine suggests a current or past infection. But, whenever available, culture is recommended to confirm infection. A negative test suggests no recent or current infection. However, in the early stages of infection, the antigen may not be present in the urine and other L. pneumophila serogroups cannot be ruled out. After a Legionella pneumonia, the duration of excretion of the antigen in the urine varies between patients but can reach up to 1 year after acute infection.

Molecular assays for detection of bacterial pathogens with PCR in blood cultures, sputum, BAL, among other samples can provide more sensitive results as compared with classic cultures.[69] [90] The diagnostic accuracy of these tests in blood samples prior to incubation has not met a broad success because of their medium sensitivity and specificity. However, the application of these tests on positive blood cultures showed excellent performances.[91] In CAP patients, the detection of a great number of copies of DNA from a pathogenic bacterium is also probably associated with infection.[92]

In hospital-acquired pneumonia, namely in patients under invasive mechanical ventilation, the dominance by a single potential pathogen can occur independently of the presence or absence of pneumonia.[72] With this highly sensitive technique, it is possible to detect a much larger number of pathogens compared with conventional cultures.[93] In other words, a widespread use of these sensitive techniques would potentially result in overtreatment.

Finally, these molecular assays can detect intracellular pathogens like Mycoplasma, Legionella, or Chlamydophila that are all complex and difficult to recognize by conventional culture methods.[94] Their identification can be useful since these pathogens are frequently not covered by empiric antibiotic therapy in nonsevere patients. In addition, there are data showing that PCR testing allows antibiotic de-escalation in almost three-fourths of CAP patients, with only 6% escalation.[95]

Viral identification via molecular testing has become the standard methodology largely replacing viral cultures.[90] In viral pneumonia the rate of bacterial co-infection is variable being much more frequent in influenza than in SARS CoV2 infection.[96] As a result, the recommendation in patients with influenza is to not withhold antibiotics.[85] However, in patients with SARS CoV2, as a consequence of the very low rate of bacterial co-infection, a more rational use of early empirical antimicrobial treatment should be sought, limited to the most severe patients, with acute respiratory distress syndrome or shock.[97]

Detection of Antibiotic Resistance

Molecular assays can detect by PCR the presence of genetic mutations on bacterial DNA. The presence of resistance genes could be quickly identified supporting the clinical decision concerning the choice of the spectrum of empirical antibiotic therapy. This is currently done in several laboratories to detect the mecA gene of Staphylococcus aureus or the kpc gene in Enterobacteriaceae.[98] However, it is important to stress that the presence of the resistance gene is not equivalent to being phenotypically expressed. The expression of antibiotic resistance is dependent on several factors, in particular environmental, like the presence of antibiotics. The expression of a resistance gene can only be reliably evaluated by bacterial culture.[99]

Conclusions

The initial diagnosis of infection and sepsis is associated with variable degrees of uncertainty. To help with this frequent problem, researchers and clinicians have evaluated several biomarkers and molecular tests that, when adequately used, could add important information for clinical decision making at the bedside. The information given by the biomarkers can be supporting the suspicion of infection but also, and not less important, excluding the diagnosis. Further research is needed to find the right indication for all present and coming tests that could help us in treating infection and sepsis in a more personalized way, increasing efficacy, safety, and improving outcomes.

Conflict of Interest

P.P. and L.C. received unrestricted research grant from Abionic.

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Publication History

Article published online:
20 September 2021