Introduction

In current clinical practice, procalcitonin (PCT) has developed into a promising new biomarker for the early detection of systemic bacterial infections. PCT is a 116-amino acid residue first explained by Le Moullec et al. in 1984; its diagnostic significance was not recognized until 1993.[1] In 1993, Assicot et al. demonstrated a positive correlation between high serum levels of PCT and patients with positive findings for bacterial infection and sepsis (e.g., positive blood cultures). Further, they demonstrated that PCT did not elevate in viral infections and that serum levels of PCT would decrease following the administration of appropriate antibiotic therapies.[2] 

Other inflammatory biomarkers, such as C-reactive protein, lack the specificity to accurately distinguish between bacterial and non-bacterial infections.[3] Therefore, PCT assays, with a specificity of 79%, have been developed and utilized to more accurately determine if a systemic inflammatory reaction is caused by a bacterial species.[4]

The United States Food and Drug Administration has approved using PCT assays for initiating or discontinuing antibiotics in lower respiratory tract infections (LRTIs) and for discontinuing antibiotics in patients with sepsis.[5] Numerous studies have evaluated PCT-based treatment algorithms in these settings and found them safe compared to standard care.[6] In particular, using PCT assays allows cessation of antibiotic therapy without increased morbidity and mortality. This makes PCT a potentially helpful tool for preventing the emergence of antibiotic-resistant organisms while still ensuring appropriate treatment for serious bacterial infections.[4]

PCT should not be used as the sole determinant for antimicrobial therapy.[7] The results of a PCT assay should be placed in the context of the clinical scenario considering the possible site of infection, the likelihood of bacterial infection, the severity of illness, and other pertinent clinical data.[8]

Pathophysiology

Under normal homeostasis, pre-procalcitonin undergoes initial synthesis by thyroid C cells. Later this peptide is transformed into procalcitonin via cleavage of a 25-amino acid signal sequence by endopeptidases. The end product calcitonin, the 32-amino acid hormone responsible for serum calcium regulation, is formed following conversion by the enzyme prohormone convertase.[9] Typically, physiological conditions result in very low serum PCT levels of less than 0.05 ng/mL. However, the synthesis of PCT can be increased up to 100 to 1000 fold due to circulating endotoxins or cytokines such as interleukin (IL)- 6, tumor necrosis factor (TNF)-alpha, and IL-1b, which act on various tissues.[10]

The extra-thyroid synthesis of PCT occurs in the liver, pancreas, kidney, lung, intestine, and leukocytes; notably, the synthesis of PCT is suppressed within these tissues in the absence of bacterial infection.[11] In contrast, cytokines released following viral infection, such as interferon (INF)-gamma, will lead to the down-regulation of PCT, thus highlighting another advantage of PCT assays.[12]

Specimen Requirements and Procedure

Human serum or plasma specimens can be used to determine procalcitonin (PCT). For accurate results, serum and plasma specimens should be free of fibrin, red blood cells, and other particulate matter.[13] Serum specimens from patients receiving anticoagulant or thrombolytic therapy may contain fibrin due to incomplete clot formation.[14] Using heat-inactivated specimens, pooled specimens, grossly hemolyzed specimens, specimens with obvious microbial contamination, and specimens with fungal growth is not recommended. The use of plasma is recommended for rapid turnaround of results.[15]

The PCT specimens should be free of bubbles. Remove bubbles with an applicator stick before analysis. Use a new applicator stick for each specimen to prevent cross-contamination. To ensure consistency in results, recentrifuge specimens before testing if they contain fibrin, red blood cells, or other particulate matter.[16] The EDTA plasma and serum specimens stored frozen at -70^oC or colder have demonstrated stability for up to 18 months.[17]

Diagnostic Tests

Kinetics

Procalcitonin (PCT) serum levels have been shown to increase 6 to 12 hours following initial bacterial infections and increase steadily in the two to four hours following the onset of sepsis.[18][10] The half-life of PCT is between 20 to 24 hours; therefore, when a proper host immune response and antibiotic therapy are in place, PCT levels decrease by 50% over 24 hours.[11]

Chemical Assays

In current clinical practice, several chemical assays have been developed to detect PCT serum levels at varying sensitivities, most displaying functional sensitivity around 0.06 ng/mL.[3] One of the first commercially available assays was a homogenous immunoassay that utilized time-resolved amplified cryptate emission technology.[12] The assay comprises a sheep polyclonal anti-calcitonin antibody and a monoclonal anti-katacalcin antibody which binds to the calcitonin and katacalcin amino acid sequence of PCT via the sandwich method. The assay uses 20 to 50 mL of plasma or serum and takes 19 minutes to complete; results are typically obtained within one hour following the serum draw.[19]

Testing Procedures

All currently available assays for the quantification of PCT are based on immunoassay techniques.[13] The chemiluminescent microparticle immunoassay (CMIA) technology is used to quantitatively determine PCT in human serum and plasma.[20] Samples and anti-PCT-coated paramagnetic microparticles are combined and incubated. The PCT present in the sample binds to the anti-PCT-coated microparticles. The mixture is washed. Anti-PCT acridinium-labeled conjugate is added to create a reaction mixture and incubated. Following a wash cycle, pre-trigger and trigger solutions are added.[21] The resulting chemiluminescent reaction is measured as relative light units (RLUs). There is a direct relationship between the amount of PCT in the sample and the RLUs detected by the system optics.[20]

A point-of-care test for PCT  is available using an immunochromatographic technique. A colored band appears on the test strip 30 minutes after applying 200 microliters of serum or plasma; the intensity of the band is read against a reference card. The results are reported as less than 0.5, 0.5 to 2.0, 2.0 to 10, and greater than 10 μg/L.[22] However, the semi-quantitative nature of these results may limit clinical use when a change in the PCT trend is important to monitor the patient’s clinical status. It may still be valuable when quantitative measurements are unavailable within a reasonable period, such as one to three hours.[23]

Interfering Factors

Although procalcitonin (PCT) assays have shown promising results over the years, several limitations still require consideration before implementing these tests in everyday clinical practice.[13] For instance, it has been shown that PCT serum levels can also become elevated during times of noninfectious conditions, such as trauma, burns, certain carcinomas (medullary C-cell, small cell carcinoma of the lung, and bronchial carcinoid), immunomodulator therapy that increases proinflammatory cytokines, cardiogenic shock, during the first two days of life, during peritoneal dialysis treatment, and in patients with cirrhosis, particularly Child-Pugh Class C.[4] Furthermore, PCT levels may be falsely elevated in patients suffering from various degrees of chronic kidney disease, which can, in turn, alter baseline results, making the determination of an underlying bacterial infection difficult to establish.[24] Thus, the clinician needs to rule out the above scenarios to ensure no confounding issues may be obscuring the PCT measurements.[25]

The PCT assay may exhibit interference when a sample is collected from a person consuming a supplement with a high dose of biotin (also termed as vitamin B7 or B8, vitamin H, or coenzyme R).[26] It is recommended to ask all patients with an indication for PCT testing about biotin supplementation. Patients should be cautioned to stop biotin consumption at least 72 hours before collecting a sample.[27] A hook effect may occur with extremely high PCT concentrations, resulting in a much lower reported value.[28] 

The cost-effectiveness of PCT assays also needs to be considered, as overuse in the emergency setting leads to extraneous costs. The average price of the test is roughly $9.44, which is relatively inexpensive. However, this cost does not consider the amount that insurance charges, nor does it include sample acquisition costs.[29] Salinas et al. discovered that of 142,644 PCT assays performed in a calendar year, 44.1% could have been avoided based on clinical presentation and outcome, saving $594,390 annually.[30]

Within the intensive care setting, Kip et al. performed a randomized controlled trial to determine the cost-effectiveness of PCT assays among septic patients. They determined that PCT assays improved mortality rates and decreased the clinical course of antibiotics. However, they found costs for patients tested with PCT assays averaged $2704 greater than patients who did not undergo PCT testing.[31] Therefore, clinicians should exercise caution when ordering PCT assays to ensure cost-effective medical practice.[29]

Results, Reporting, and Critical Findings

Procalcitonin (PCT) has a set half-life of 20 to 24 hours which provides clinicians and researchers with a rough timeline of when levels should begin to decrease following physiological control of the systemic infection. When physiologic control is reached, PCT should decrease by approximately 50% over 24 hours.[10] The current clinical practice utilizes a variety of PCT cut-off levels to determine the initiation and discontinuation of antibiotic therapy. The clinical scenario and setting play a fundamental role in which the cut-off level should be employed. However, most research has shown that PCT levels display clinical significance in the range of 0.1 to 0.5 ng/mL.[11] Further, research has shown that PCT levels less than 0.1 ng/mL have been shown to have a high negative predictive value (96.3%) for excluding bacterial infections.[3]

The following clinical scenarios have utilized various PCT cut-off levels to determine the source of an infective process as well as when antibiotic therapy could be utilized or discontinued:[11]

Arthritis

• PCT cut-off level: 0.1 to 0.25 ng/mL
• Role of PCT: To discriminate infective (septic) arthritis from non-infective arthritis.  
• Type of Study: Observational

Bacteremic Infections

• PCT cut-off level: 0.25 ng/mL
• Role of PCT: To rule out bacteremic infections. 
• Type of Study: Observational

Blood Stream Infection (primary)

• PCT cut-off level: 0.1 ng/mL
• Role of PCT: To differentiate between true infection and a contaminated sample. 
• Type of Study: Observational

Acute Bronchitis/Chronic Obstructive Pulmonary Disease (COPD) Exacerbations

• PCT cut-off level: 0.1 to 0.5 ng/mL
• Role of PCT: To reduce (unnecessary) antibiotic exposure in the ED and inpatient setting without adverse outcomes. 
• Type of Study: Randomized controlled trial  

Infective Endocarditis 

• PCT cut-off level: 2.3 ng/mL
• Role of PCT: High diagnostic accuracy for predicting acute endocarditis. 
• Type of Study: Observational

Meningitis:

• PCT cut-off level: 0.5 ng/mL
• Role of PCT: To differentiate viral from bacterial meningitis and reduce antibiotic exposure.
• Type of Study: Before-after

Neutropenia 

• PCT cut-off level: 0.1 to 0.5 ng/mL
• Role of PCT: To identify systemic bacterial infections within neutropenic patients. 
• Type of Study: Observational

Pneumonia

• PCT cut-off level: 0.1 to 0.5 ng/mL
• Role of PCT: To reduce antibiotic exposure during hospitalization without adverse outcomes.

Postoperative Fever

• PCT cut-off level: 0.1 to 0.5 ng/mL
• Role of PCT:  To differentiate postoperative infections from non-infectious fevers. 
• Type of Study: Observational

Postoperative Infections

• PCT cut-off level: 0.5 to 1.0 ng/mL
• Role of PCT: To minimize antibiotic treatment in surgical intensive care settings without detrimental outcomes. 
• Type of Study: Randomized controlled trial

Severe Sepsis With or Without Shock

• PCT cut-off level: 0.25 to 0.5 ng/mL
• Role of PCT: To limit antibiotic treatment in intensive care settings without detrimental outcomes.
• Type of Study: Randomized controlled trial

Upper Respiratory Tract Infections

• PCT cut-off level: 0.1 to 0.25 ng/mL
• Role of PCT: To limit antibiotic treatment in intensive care settings without detrimental outcomes.
• Type of Study: Randomized controlled trial

Urinary Tract Infections

• PCT cut-off level: 0.25 ng/mL
• Role of PCT: To determine the extent of renal involvement. 
• Type of Study: Observational

Ventilator-associated Pneumonia

• PCT cut-off level: 0.1 to 0.25 ng/mL
• Role of PCT: To minimize antibiotic treatment without detrimental outcomes. 
• Type of Study: Randomized controlled trial

Once the cut-off level is established, the timing and frequency of PCT measurement to assess adequate infection control should be determined.[32] Current clinical data suggests that PCT serum levels should be remeasured after 6 to 24 hours, absent evidence of spontaneous clinical improvement such as the resolution of hemodynamic instability. Following antibiotic initiation, the recommendation is that PCT values be assessed every one to two days to ensure adequate coverage. Further, antibiotic courses should be discontinued as soon as PCT levels drop below 0.1 ng/mL or 80 to 90% below the initial measurement.[11]

Algorithms have been established for emergency and intensive care settings, providing clinicians with a quick method for determining when to initiate or discontinue antibiotics.[13] For example, an algorithm has been established to determine when to start antibiotic therapy in the emergency department for patients with respiratory tract infections.[33] Recommendations are that antibiotics be utilized when PCT levels are above 0.25 ng/mL; PCT levels should be repeated on days three, five, and seven. Antibiotics should be discontinued when PCT levels fall below 0.25 ng/mL or drop by 80 to 90%. If the PCT remains elevated, then consider new treatment options.[11]

In the intensive care unit, an algorithm has been instituted to determine when antibiotic treatment should be discontinued in patients with sepsis.[34] The algorithm recommends that antibiotic coverage be discontinued when PCT levels drop below 0.5 ng/mL or decrease 80% from the peak value. However, if PCT levels remain elevated (over 0.5 ng/mL), continuing the antibiotic course or changing the treatment entirely is advised.[11] These algorithms have been used successfully in clinical trials and have proven to reduce overall antibiotic use, thus improving antibiotic stewardship. However, further research is needed to ensure these results can be adequately repeated on a larger scale and by utilizing more clinical trials versus observational studies.[35]

Clinical Significance

It is well-documented that early diagnosis of a bacterial infection can decrease mortality and morbidity among all patients.[36] Efficient diagnosis of bacterial infections allows clinicians to initiate antibiotic therapy when deemed appropriate, thus preventing the misuse and overuse of antibiotics. As antibiotic resistance continues to rise, it has become increasingly important for clinicians to determine different algorithms and laboratory tests that help sustain current antibiotic parameters.[37]

Unfortunately, most first-line tests for determining infection, such as blood cultures and C-reactive protein, lack the efficiency and specificity needed to treat patients promptly. Therefore, procalcitonin (PCT) serum assays have been developed to provide healthcare providers with an earlier detection method to determine the origin of a systemic inflammatory response (e.g., bacterial versus non-bacterial). Early detection, in turn, limits the development of antibacterial resistance and patient exposure to antibiotics when they are no longer warranted.[11] 

The prognostic value of PCT has also shown clinical significance by providing clinicians with a positive correlation between disease severity and elevated PCT serum levels, especially within septic patients.[12] Although PCT assays have shown great promise, the cost-effectiveness of these tests continues to be debated. Current research has shown that these tests are already being overused because there are currently no adequate guidelines for when these tests should and should not be obtained.[30] Therefore, the clinical significance of these tests needs to be more thoroughly researched on a large scale and through randomized clinical trials so that guidelines can be implemented to ensure the practice of cost-effective medicine.[31]

Quality Control and Lab Safety

For non-waived tests, laboratory regulations require, at the minimum, analysis of at least two levels of control materials once every 24 hours. If necessary, laboratories can assay QC samples more frequently to ensure accurate results. Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance.[38] To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency (such as once per month), the labs can develop an individualized quality control plan (IQCP) that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[39] Westgard multi-rules are used to evaluate the quality control runs. In case of a rule violation, proper corrective and preventive action should be taken before patient testing.[40] 

The laboratory must participate in the external quality control or proficiency testing (PT) program because it is a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the Clinical Laboratory Improvement Amendments (CLIA) regulations. It is helpful to ensure the accuracy and reliability of the laboratory concerning other laboratories performing the same or comparable assays.[41] Required participation and scored results are monitored by CMS and voluntary accreditation organizations. The PT plan should be included as an aspect of the quality assessment (QA) plan and the overall quality program of the laboratory.[42]

Consider all specimens, control materials, and calibrator materials as potentially infectious. Exercise the normal precautions required for handling all laboratory reagents. Disposal of all waste material should be in accordance with local guidelines. Wear gloves, a lab coat, and safety glasses when handling human blood specimens. Place all plastic tips, sample cups, and gloves that come into contact with blood in a biohazard waste container.[43] Discard all disposable glassware into sharps waste containers. Protect all work surfaces with disposable absorbent bench top paper, discarded into biohazard waste containers weekly or whenever blood contamination occurs. Wipe all work surfaces weekly.[44]

Enhancing Healthcare Team Outcomes

As bacterial drug resistance continues to rise across the globe, it has become of utmost importance to enhance antibiotic stewardship.[37] Procalcitonin (PCT) provides healthcare providers with a more specific marker for determining the presence of bacterial infections when compared to current measures. Therefore, PCT assays can be utilized to determine if antibiotics need to be initiated, discontinued, or changed based on changing serum levels, thus decreasing the overall use or misuse of antibiotics.[4] Moreover, the assays have also been useful as a prognostic indicator for patients in the critical care setting. However, further research needs to be performed to determine if PCT assays are adequate for this purpose.[11]

PCT assays have utility in several clinical scenarios; current research suggests that PCT levels are most useful in acute exacerbations of chronic obstructive pulmonary disease (COPD) to determine when and if antibiotics should be initiated.[11] The current European Respiratory Society and American Thoracic Society guidelines state that using antibiotics in the setting of COPD exacerbations is controversial because research showing improvement in clinical outcomes is inadequate. Therefore, they recommend further effectiveness studies and the use of biomarkers to determine when antibiotics are clinically appropriate.[45] A biomarker, such as PCT, can be used to determine if antibiotics are appropriate in an acute COPD exacerbation; this improves antibiotic stewardship and reduces morbidity associated with unnecessary antibiotic use.[37] 

Overall, PCT levels provide a promising laboratory measurement for identifying bacterial infections. However, the utility of this test is limited by the clinical setting and patient population. Therefore, further research must be conducted before implementing PCT guidelines for everyday clinical practice.