Abstract

Sepsis is the systemic response to infection by microbial organisms. A differential diagnosis of infection caused by either bacteria or other microbial organisms is essential for effective treatment and prognostic assessment. Current clinical laboratory methods in the diagnosis of bacterial infections are either non-specific or require longer turnaround times. Procalcitonin (PCT) is a biomarker that exhibits greater specificity than other proinflammatory markers (eg, cytokines) in identifying patients with sepsis and can be used in the diagnosis of bacterial infections. In this article, we review the current knowledge of PCT and its use in the clinical laboratory setting.

Identifying whether the cause of inflammation in patients is of bacterial origin has been an important area of development in the clinical laboratory. Several clinical laboratory tests have been applied to the diagnosis of sepsis.1 The broth culture method is the gold standard for the diagnosis of bacterial infection, but a definitive result can take 24 hours or more before a conclusive diagnosis. A number of the inflammatory markers, such as leukocyte cell count, C reactive protein (CRP), and cytokines (TNF-α, IL-1β, or IL-6), have been applied in the diagnosis of inflammation and infection, but their lack of specificity has generated a continued interest to develop more specific clinical laboratory tests.2 One promising marker has been procalcitonin (PCT), whose concentration has been found to be elevated in sepsis. Owing its specificity to bacterial infections, PCT has been proposed as a pertinent marker in the rapid diagnosis of bacterial infection, especially for use in hospital emergency departments and intensive care units. Since its identification and association with sepsis in the 1990s, a large number of studies involving PCT and its clinical application have been conducted. A test to determine PCT levels has been available in Europe for several years and recently was approved by the FDA for use in the United States.3

Biochemistry of PCT

Procalcitonin is a 116 amino acid peptide that has an approximate MW of 14.5 kDa and belongs to the calcitonin (CT) superfamily of peptides. It can be divided into 3 sections including the amino terminus of the PCT region, immature calcitonin, and calcitonin carboxyl-terminus peptide-1 (CCP-1, also called katacalcin) (Figure 1).4 Procalcitonin is encoded by CALC-1 gene located on chromosome 11. Cleavage in 1 site of the primary transcript of CALC-1 gene produces pre-PCT, which further undergoes proteolytic cleavage of its signal sequence to produce PCT.5,6 The other members of the CT superfamily of peptides include calcitonin gene-related peptide I, II (CGRP-I, CGRP-II), amylin, and adrenomedullin. Calcitonin gene-related peptide I is also encoded by CALC-1 gene and is generated by alternative splicing of the primary transcript of CALC-1 mRNA. Calcitonin gene-related peptide II, amylin, and adrenomedullin are encoded by other genes (Table 1).

Procalcitonin expression occurs in a tissue-specific manner. In the absence of infection, transcription of the CALC-1 gene for PCT in the non-neuroendocrine tissue is suppressed, except in the C cells of the thyroid gland where its expression produces PCT, the precursor of CT in healthy and non-infected individuals.5 The synthesized PCT then undergoes post-translational processing to produce small peptides and mature CT, which is generated as a result of the removal of the C-terminal glycine from the immature CT by peptidylglycine α-amidating monooxygenase (PAM).7 Mature CT is stored in secretary granules and is secreted into the blood to regulate the calcium concentration. In the presence of microbial infection, non-neuroendocrine tissues also express the CALC-1 gene to produce PCT. A microbial infection induces a substantial increase of CALC-1 gene expression in all parenchymal tissue and differentiated cell types in the body producing PCT.8 Its levels increase significantly in severe systemic infections, as compared to other parameters of microbial infections.9 The function of PCT synthesized in the non-neuroendocrine tissues under microbial infection is presently unclear; however, its detection has helped in the differential diagnosis of inflammatory processes.

Overview of Sepsis

Sepsis refers to the systemic response to infection by microbial agents, such as bacteria, fungi, and yeast, where the patient typically develops fever, tachycardia, tachypnea, and leukocytosis. Microbiologic cultures from the blood or the infection site are frequently, although not invariably, positive. Severe sepsis is associated with the hypoperfusion or dysfunction of at least 1 organ. When severe sepsis is accompanied by hypotension or multiple organ system failure, the condition is known as septic shock. Epidemiological studies indicate an incidence of approximately 750,000 sepsis cases per year in the United States.10 The signs and symptoms of sepsis are highly variable and are influenced by many factors, including the virulence and bioburden of the pathogen, the portal of entry, and the host susceptibility. Primary sites are respiratory tract infections, followed by genitourinary infections, and gastrointestinal infections.11 Recently there has been an increasing number of reports on bacterial infections of hospitalized patients due to increased nosocomial infections from catheterization and immunosuppressive therapies, in addition to increased causes of methicillin-resistant Staphylococcus aureus (MRSA).11 Distinguishing inflammation due to bacterial, other microbial infection, or organ rejection is important in the treatment of the immune reaction in hospitalized patients. A common problem in the clinical practice is that the signs and symptoms of bacterial and viral infections are widely overlapping, especially in respiratory tract infections. Occasionally the diagnostic uncertainty still remains, even after obtaining a patient history, performing a physical examination, chest x-ray test, and laboratory tests. Thus, a laboratory test with more specificity would significantly improve the clinical differential diagnosis in these cases. In addition, the differential diagnosis of infection would help in deciding whether treatment with antibiotics would be beneficial. In this regard, approximately 75% of all antibiotic doses are prescribed for acute respiratory infections that have a predominantly viral etiology.5 The excessive use of antibiotics is the main cause for the spread of antibiotic-resistant bacteria. Thus, decreasing the use of antibiotics is essential in combating the increase of antibiotic-resistant micro-organisms.

Figure 1

Schematic showing the relationship of procalcitonin, calcitonin, and calcitonin carboxypeptide-1.

Figure 1

Schematic showing the relationship of procalcitonin, calcitonin, and calcitonin carboxypeptide-1.

Diagnostic Methods for Sepsis

The traditional method of diagnosis for sepsis includes culturing blood, urine, cerebrospinal fluid (CSF), or bronchial fluid specimens and usually takes 24 to 48 hours. Unfortunately, clinical symptoms frequently manifest themselves in the absence of a positive culture. The traditional clinical signs of infection and the routine laboratory tests for sepsis, such as CRP or leukocyte count, lack diagnostic accuracy and are sometimes misleading. In severe infections, most classical pro-inflammatory cytokines, such as TNF-α, IL-1β, or IL-6, are increased only briefly or intermittently, if at all. In view of the above diagnostic and therapeutic dilemmas, a more unequivocal test for the differential diagnosis of infection and sepsis is of paramount importance.

Table 1

The Relationship Between the Calcitonin Superfamily of Peptides

Chromosome 11 11 11 12 11 
Genes CALC-1 CALC-II CALC-III CALC-IV CALC-V 
Peptides *PCT I CGRP-II Pseudogene (non-translated gene)31 Amylin Adrenomedullin 
 *PCT II     
 CGRP-I     
Chromosome 11 11 11 12 11 
Genes CALC-1 CALC-II CALC-III CALC-IV CALC-V 
Peptides *PCT I CGRP-II Pseudogene (non-translated gene)31 Amylin Adrenomedullin 
 *PCT II     
 CGRP-I     
*

PCT II differs from PCT I by 8 amino acids at the C terminus.

PCT Measurement in the Diagnosis of Bacterial Infection

Since the mid 1990s, there has been an increasing use of PCT measurements in identifying systemic bacterial infections.3 The short half-life (25–30 hours in plasma) of PCT, coupled with its virtual absence in health and specificity for bacterial infections, gives it a clear advantage over the other markers of bacterial infection.3,4 Studies have also shown that an increase in PCT levels is minimal in viral infections while levels increase rapidly after a single injection with endotoxin.12,13 Furthermore, elevations in PCT are not associated with specific bacterial strains, although in a study by Rowther and colleagues, strains from septic patients with serum PCT levels >2 ng/mL were identified and listed.14 Recently, Jacquot and colleagues demonstrated that rapid measurement of PCT could help rule out nosocomial infection in newborns hospitalized in intensive care units.15

In another study, de Jager and colleagues investigated the value of measuring PCT levels in the blood of patients infected with community-acquired pneumonia (CAP) caused by Legionella pneumophila in comparison to other conventional parameters such as CRP and WBC counts.16 They found that initial high levels of PCT were indicative of a more severe disease, and this was reflected in a longer patient stay in the intensive care unit (ICU) and/or in-hospital death. Furthermore, persistently increased levels of PCT were always indicative of an unfavorable outcome. Thus, determination of PCT levels provided valuable information on patient prognosis that could not be determined from conventional inflammatory parameters such as CRP and WBC counts. Furthermore, the use of PCT measurements to efficiently treat patients with antibiotics has been shown to decrease patient hospital stay. Kristoffersen and colleagues reported that in those patients with chronic obstructive pulmonary disease, a single serum PCT determination at the time of admission reduced the mean length of stay from 7.1 days to 4.8 days.17

Measurement of PCT

Procalcitonin can be measured using a quantitative homogenous assay (BRAHMS, Hennigsdorf, Germany). The assay is based on Time Resolved Amplified Cryptate Emission (TRACE) technology (Figure 2). A nitrogen laser at 337 nm is directed at a sample containing PCT and 2 fluorescently labeled antibodies recognizing different epitopes of the PCT peptide. The principal of the assay is based on the transfer of non-radiative energy between “donor” and “acceptor” molecules. The donor molecule, upon excitation, emits a long-lived fluorescent signal in the milli-second range at 620 nm, while the acceptor molecule upon excitation emits a short-lived signal in the nano-second range at 665 nm. When both molecules are brought into close proximity by binding to PCT, the resultant signal is amplified at 665 nm and prolonged to last for a few micro-seconds. This prolongation ensures the signal can be measured after the background fluorescence (common in biological samples) has decayed. In the BRAHMS’ assay, the donor molecule is an Europium Cryptate labeled polyclonal sheep antibody recognizing epitopes in the immature CT region, while the acceptor molecule is an XL665 labeled monoclonal antibody raised against the CCP-1 region of PCT.18,19

Figure 2

Schematic showing the principles of TRACE technology.

Figure 2

Schematic showing the principles of TRACE technology.

Samples suitable for the assay can be serum or plasma using either EDTA or heparin as the anticoagulants but not citrate since this has been shown to underestimate PCT levels.18

PCT Measurements in Other Diseases

Usefulness of PCT as a diagnostic marker in other inflammatory states has also been evaluated and reviewed by Becker and co-workers.20 In malaria, PCT levels are elevated in both severe and uncomplicated Plasmodium falciparum malaria but could not be used to differentiate between the 2 types and was thus of limited use in its diagnosis.21,22 The value of PCT in the diagnosis of pulmonary tuberculosis (PTB) has also been investigated and shown to have little value.19 Baylan and co-workers found that serum PCT levels were slightly high on admission in patients with active PTB in comparison with controls and patients on anti-tuberculous chemotherapy. Although this difference was statistically significant, the PCT levels of most cases with PTB (58.7%) were below the usual cut-off level (0.5 ng/mL).23 Hence, PCT was not a reliable indicator in the diagnosis of active PTB and could not be substituted for microbiological, epidemiological, clinical, and radiological data.23 Nyamande and Lalloo, however, found that PCT levels could be useful in distinguishing community-acquired pneumonia (CAP) due to common bacteria, such as Mycobacterium tuberculosis (TB) and Pneumocystis jirovecii (PJP) in a high HIV prevalence setting where atypical presentations often confounded the empirical clinical diagnosis.24 Their study showed that PCT levels differ significantly in patients with CAP due to TB, PJP, and bacteria.

Procalcitonin determinations have been found considerable value in identifying whether inflammation following an organ transplant is due to bacterial infection or organ rejection. Mendonca and colleagues demonstrated that in liver transplant recipients, plasma PCT levels were significantly increased in infected patients in comparison to those that had acute liver rejection in whom the levels were similar to those of non-complicated patients.25 Madershahian and co-workers showed PCT levels following heart transplantation could serve as a useful marker of prognosis.26 Procalcitonin levels were found to be consistently low (<10 ng/mL) in patients with an uneventful course following transplant but more frequently increased in patients with postoperative complications and even associated with an increased mortality early postoperatively when values exceed 80 ng/mL. Thus, PCT levels in the first few days following cardiac transplantation could help identify patients at risk for complications, when concentrations exceeded the “normal” post-transplant range.26

Pitfalls in PCT Measurement

There are also reports in the literature where elevations in PCT levels were not connected with bacterial infections. Addisonian crisis caused by adrenal failure has been associated with elevated PCT levels.27 Increased PCT levels, comparable to what is observed in severe sepsis, were also seen in transplant patients receiving pan T-cell antibody therapy.28 More recently, Brodska and colleagues reported marked elevations in PCT and CRP levels in patients scheduled for hematopoietic stem cell transplantation and receiving anti-thymocyte globulin during conditioning.29

Summary

Over the past 15 years, the use of PCT in identifying the bacterial or non-bacterial origin of systemic inflammation has been gaining widespread support, and it is likely this trend will continue. Although PCT has proved to be an interesting marker of sepsis, its physiological role still remains uncertain. There is evidence suggesting the use of PCT in monitoring patients with sepsis leads to reduced morbidity and mortality of these patients. Administration of PCT to septic hamsters increased their death rate, while anti-PCT antibody administration increased their survival rate.30 The almost lack of PCT expression in the healthy state and its expression in virtually every organ during sepsis fuels the notion it is intrinsically associated with the characteristic “organ shutdown” of severe sepsis. Further research designed at elucidating the role of PCT in sepsis would help in understanding the pathogenesis of sepsis with the aim to develop better and more efficacious therapeutic regimens.

The authors would like to thank Chris Ciotti, Stephen Barnes, and Jim Bromley (BRAHMS USA) for helpful discussions on technical and clinical aspects of the PCT assay.

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