Sepsis is a major cause of high mortality in the intensive care unit. Therefore, an early diagnosis is required to reduce the mortality due to sepsis-related multiple organ dysfunction (1-3). Markers of inflammation such as C-reactive protein (CRP) and white blood cell count have proven to be far from ideal in identifying critically ill patients who need antimicrobial therapy, and therefore, new markers are required for the early diagnosis of sepsis (4, 5).
Procalcitonin (PCT), a 12.6-kd and 114-amino-acid polypeptide, is a precursor of calcitonin, a hormone produced in medullary C-cells of the thyroid gland allegedly associated with calcium metabolism (6). Upon systemic infection, PCT is produced and secreted by virtually all parenchymal cells (7, 8). The circulating levels of several calcitonin precursors, including PCT, but not mature calcitonin, increase several-thousand-fold in microbial infections and in various forms of inflammation (9). This increase, specifically the time course, correlates with the severity of the condition and mortality (10-12). Procalcitonin levels on admission have been reported to be useful for predicting the severity and outcome of patients with pneumonia (13).
Fungal infections are opportunistic infections that occur during aggressive medical treatment in patients who have serious underlying diseases and who are in an immunologic deficient state. The high mortality rate associated with fungal infection after organ transplantation is due to the difficulty in making an early diagnosis. Because clinical management of fungal infection depends on the rapid and unambiguous identification of pathogens, early detection of fungal pathogens using polymerase chain reaction (PCR) has recently been investigated (14-16). In several bacterial infections, early diagnosis of sepsis by PCR is also required (17, 18). Recently, an analysis of PCT was made to determine the cutoff value for bacterial infections in the treatment with antibiotics (19).
In the present study, PCT levels will be measured in patients suspected of having either bacteremia or fungemia, and their correlation with the results of blood cultures, PCR, and the CRP level will be examined to establish the cutoff value of PCT for the diagnosis of bacteremia.
MATERIALS AND METHODS
The study population consisted of 116 patients suspected of having bacteremia or fungemia because of continuous high fever during more than 3 days: 50 patients who had undergone liver transplantation, 13 with pneumonia, 8 with hematologic malignancies, 5 with heart failure, 5 with other solid tumors, 4 with renal failure, 4 with burns, 3 who had undergone bone marrow transplantation, 3 with hepatic cell carcinoma, 3 with esophageal cancer, 3 with autoimmune disease, 3 with deep vein thrombosis, 2 with aortitis, 2 with liver cirrhosis, 2 with pleurisy, 2 with gastric ulcer, 1 with acute myocardial infarction, 1 with diabetes, 1 with liver abscess, and 1 with hemophagocytic syndrome. All of these patients were managed at Mie University School of Medicine, between June 1, 2003, and December 31, 2006 (median age, 59 years; 25%-75% percentile, 48-68 years old; female-male sex = 41:75). The patients who had a tumor-induced fever, drug-induced fever, or fever due to autoimmune diseases were excluded. The study protocol was approved by the Human Ethics Review Committee of Mie University School of Medicine, and a signed consent form was obtained from each subject. This study was carried out in accordance with the Helsinki Declaration.
Procalcitonin levels were measured by VIDAS BRAHMS PCT (BioMérieux, Marcy L'Etoile, France) using a mini-VIDAS (BioMérieux). C-reactive protein was measured using the N-assay TIA CRP-S kit (Nitto Boseki, Fukushima, Japan), and blood culture for fungi and bacteria was carried out using the BacT/Alert system (BioMérieux, Durham, NC).
Amplification of fungus and bacteria DNA by PCR
Blood samples were obtained from each subject, and DNA was extracted from 100 μL of whole-blood cells. Polymerase chain reaction was performed in a 25-μL reaction mixture containing 15 mM Tris (pH 8.0); 50 mM KCl; 1.5 mM MgCl2; 100 μM each of dATP, dCTP, dGTP, and dTTP; and 0.25 U of Taq Gold DNA polymerase (Applied Biosystems, Foster City, Calif) using primer sets for fungi (Fung-F: 5′-TTCGATGGTAGGATAGTGGCC-3, forward; B4R: 5′-TGATCGTCTTCGATCCCCTA-3′, reverse) and for bacteria (UN-F: 5′CAGCAGCCGCGCTAATAC-3′, forward; UN-R: 57-CCGTCAATTCCTTTGAGTTT-3′, reverse).
The fungi-specific PCR protocol was as follows: 10 min at 95°C, 40 cycles of 30 sec at 95°C, 30 sec at 55°C, 60 sec at 72°C, and finally, 10 min at 72°C. The bacteria-specific PCR protocol was as follows: 10 min at 95°C, 40 cycles of 30 sec at 95°C, 30 sec at 59°C, 60 sec at 72°C, and finally, 10 min at 72°C. Polymerase chain reaction products were separated by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining and fluorescence transillumination. The fungi-specific PCR was designed to detect various pathogenic fungi, and the primer pair Fung-F and B4R, which amplify the 18S rRNA region, were used (20). The bacteria-specific PCR was designed to detect various pathogenic bacteria, and the primer pair UN-F and UN-R, which amplify the 16S rRNA region, were used (21).
The identification of fungi or bacteria was carried out by direct sequencing of pathogenic DNA using ABI PRISM 310 (Applied Biosystems).
The median values (25% - 75% percentile) of data are given. Differences between two groups were examined for statistical significance using the Mann-Whitney U test. The correlation between two variables was tested by Pearson correlation analysis. P < 0.05 denoted a statistically significant difference. The usefulness of PCT and CRP for the diagnosis of bacteremia was examined using a receiver operating characteristic (ROC) analysis (22). In the analysis of the outcome, χ2 test was used to assess independence of two variables.
Blood culture for bacteria was positive in 65 patients and negative in 51 patients: 19 patients tested positive for coagulase-negative staphylococci, 7 for methicillin-resistant Staphylococcus aureus (MRSA), 3 for Enterococcus, 3 for streptococcus, 14 for Enterobacteriaceae, 1 for Salmonella, 1 for Pseudomonas aeruginosa, 3 for anaerobic bacteria, and 14 for other bacteria (Table 1). Procalcitonin levels were significantly higher in the patients with a positive blood culture (median, 6.2 μg/L; 25%-75% percentile, 1.0-18.5 μg/L) than in those with a negative culture (median, 0.3 μg/L; 25%-75% percentile, 0.1-0.7 μg/L; P < 0.001) (Fig. 1). The PCR assay for bacteria DNA was positive in 63 patients and negative in 53 patients: 21 patients tested positive for coagulase-negative staphylococci, 9 for MRSA, 3 for Enterococcus, 3 for Streptococcus, 14 for Enterobacteriaceae, 1 for Salmonella, 1 for P. aeruginosa, 3 for anaerobic bacteria, and 8 for other bacteria. Procalcitonin levels were significantly higher in the patients positive for bacteria DNA (median, 4.5 μg/L;25%-75% percentile, 1.0-16.9 μg/L) than in those who tested negative (median, 0.3 μg/L; 25%-75% percentile, 0.1-0.7 μg/L; P < 0.001; Fig. 2). Both the blood culture and PCR were positive in 50 patients, either blood culture or PCR was positive in 27, and neither of them was positive in 39. Procalcitonin levels were the highest in the patients who tested positive for both blood culture and PCR (median, 7.8 μg/L; 25%-75% percentile, 1.7-22.4 μg/L; P < 0.001), whereas there was no significant difference between the patients who tested positive for one of the two tests (median, 0.4 μg/L; 25%-75% percentile, 0.3-3.6 μg/L) and those who tested negative for both tests (median, 0.3 μg/L; 25%-75% percentile, 0.1-0.7 μg/L, not significant [NS]; Fig. 3). In the 39 patients who tested negative for both bacterial tests, the PCR for fungi was positive in 13 and negative in 26. Procalcitonin levels were significantly higher in those with a positive PCR for fungi (median, 0.7 μg/L; 25%-75% percentile, 0.3-8.3 μg/L) than in those with a negative one (median, 0.1 μg/L; 25%-75% percentile, 0.1-0.4 μg/L; P < 0.01; Fig. 4).
Of the 65 patients whose blood culture tested positive for bacteria, PCT levels were markedly high in 63 and low in 2: one with MRSA infection and the other with methicillin-resistant coagulase-negative staphylococci infection. Of the 51 patients whose blood culture tested negative for bacteria, PCT levels were low in 43 and high in 8 (Table 2). Six of these 8patients were positive for fungus DNA.
Procalcitonin levels correlated with the level of CRP (y = 1.96 + 1.05x, r = 0.498; P < 0.05). Bacterial infection was classified and subsequently compared according to the following four patterns: (a) positive blood culture, (b) positive PCR, (c) positive blood culture and more than 3.5 mg/mL of CRP, and (d) positive blood culture and PCR (Table 3). In case of positive blood culture or PCR, the odds ratio was not markedly high, but it was markedly high when both tests were positive or when either of the two tests was positively associated with more than 3.5 mg/dL of CRP. In the ROC analysis for the diagnosis of bacteremia, the area under the curve (AUC) was significantly higher for PCT (0.895) than for CRP (0.705; P < 0.01; Fig. 5). Receiver operating characteristic analysis showed that 3.5 mg/dL of CRP was the appropriate cutoff value for bacteremia. The appropriate cutoff values of PCT were 0.38 μg/L for the high negative predictive value (NPV) and 0.83 μg/L for the high positive predictive value (PPV) (Table 3). In the above findings, the symptoms, and the response to antibiotics, 64 patients were diagnosed as having clinical bacteremia. The combination of PCT with a blood culture clinically increases the sensitivity and odds ratio for the diagnosis of bacteremia (Table 4).
In this study, 34 patients died, and of them, 11 patients died within 21 days. The cause of death was an infection in 10 of the 11 patients. The final mortality rates were as follows: 23.3% for patients with a negative blood culture versus 33.8% for those with a positive blood culture (NS), 26.4% for patients with negative PCR versus 31.7% for those with positive PCR (NS), 22.2% for patients with low PCT levels versus 35.5% for those with high PCT levels (NS). Regarding the 21-day mortality rate, the sensitivity, specificity, and odds ratio were the highest in PCT (Table 5). The combination of PCT with a blood culture or PCR increases the sensitivity for mortality.
Procalcitonin levels were significantly higher in patients who tested positive for either blood culture or PCR for bacteria, thus confirming the usefulness of the PCT assay for the diagnosis of bacterial infection (11, 12). In addition, the PCT levels were high in patients who were positive for fungus DNA, thus suggesting that the PCT levels might increase in not only bacterial infection, but also in fungal infection. A markedly high frequency of fungal infections was reported in recipients of a liver transplant (23-25) and in those with leukemia (26). Fungal infection is also a serious condition in critically ill patients.
In this study, the PCT levels were low in two cases, thus suggesting a risk of bacterial contamination from the skin. As a result, when both a blood culture and PCR were positive for bacteria, the possibility of bacteremia might therefore be high. It is currently recommended that, for the diagnosis of bacteremia, a low-virulence pathogen must be isolated in at least two blood cultures performed on separate occasions using samples from different sites. In Japan, patients suspected of having a severe infection are usually treated with antibiotics before the identification of the pathogen. Therefore, many infections without identification exist in Japan, and these conditions make the clinical examination such as laboratory test or antibiotics unclear.
The ROC analysis for the diagnosis of bacteremia shows that PCT is better than CRP and that the appropriate cutoff value of PCT was 0.38 μg/L for the high NPV and 0.83 μg/L for the high PPV. According to antibiotic therapy under PCT guidance, antibiotic therapy is encouraged when the PCT level is more than 0.25 μg/L and is greatly encouraged when it is more than 0.5 μg/L (19).
The patient with a high CRP level who test positive either in the blood culture or bacteria DNA is usually considered to have bacteremia. These findings suggest that a CRP level of more than 3.5 mg/dL is associated with either a positive blood culture or positive PCR for bacteria or positive results of both tests as indicative of bacteremia. In addition, the direct amplification of rRNA genes has been recommended for the diagnosis of bacterial infections in addition to blood culture (27). Because none of the markers of infection is by itself of absolute diagnostic value, several markers such as PCT and CRP should be measured together with blood culture or PCR for micropathogens.
Blood culture is clinically the main index of sepsis, and final diagnosis is obtained by a positive blood culture; however, blood culture usually requires a long time to complete. In contrast, PCT can be performed quickly, and the fine-span adjustment of PCT determination is longer than that of blood culture. As mortality due to sepsis is significantly high, use of antibiotics is the most important method for treating sepsis. Indeed, PCT levels are related to the outcome, and the combination of PCT with blood culture or PCR increases the sensitivity for predicting a poor outcome. In clinical settings, PCT, blood culture, and PCR might be useful for severe sepsis. As a result, early treatment with antibiotics is therefore recommended in patients with high PCT levels.
Procalcitonin is useful for the diagnosis of bacteremia, and in combination with blood culture and PCR, the diagnostic value of either PCT or CRP for bacteremia was found to increase.
1. Jensen JU, Heslet L, Jensen TH, Espersen K, Steffensen P, Tvede M: Procalcitonin
increase in early identification of critically ill patients at high risk of mortality. Crit Care Med
2. Garnacho-Montero J, Garcia-Garmendia JL, Barrero-Almodovar A, Jimenez-Jimenez FJ, Perez-Paredes C, Ortiz-Leyba C: Impact of adequate empirical antibiotic therapy on the outcome of patients admitted to the intensive care unit with sepsis. Crit Care Med
3. Alberti C, Brun-Buisson C, Chevret S, Antonelli M, Goodman SV, Martin C, Moreno R, Ochagavia AR, Palazzo M, Werdan K, et al.: Systemic inflammatory response and progression to severe sepsis in critically ill infected patients. Am J Respir Crit Care Med
4. Herzum I, Renz H: Inflammatory markers in SIRS, sepsis and septic shock. Curr Med Chem
5. Rau BM, Kemppainen EA, Gumbs AA, Büchler MW, Wegscheider K, Bassi C, Puolakkainen PA, Beger HG: Early assessment of pancreatic infections and overall prognosis in severe acute pancreatitis by procalcitonin
(PCT): a prospective international multicenter study. Ann Surg
6. Becker KL, Nylen ES, White JC, Muller BC, Snider RH Jr: Clinical review 167: procalcitonin
and the calcitonin gene family of peptides in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors. J Clin Endocrinol Metab
7. Meisner M, Müller V, Khakpour Z, Toegel E, Redl H: Induction of procalcitonin
and proinflammatory cytokines in an anhepatic baboon endotoxin shock model. Shock
8. Müller B, Christ-Crain M: Biomarkers in respiratory tract infections: diagnostic guides to antibiotic prescription, prognostic markers and mediators. Eur Respir J
9. Christ-Crain M, Müller B: Procalcitonin
in bacterial infections-hype, hope, more or less? Swiss Med Wkly
10. Nylen ES, O'Neill W, Jordan MH, Snider RH, Moore CF, Lewis M, Silva OL, Becker KL: Serum procalcitonin
as an index of inhalation injury in burns. Horm Metab Res
11. Whang KT, Steinwald PM, White JC, Nylen ES, Snider RH, Simon GL, Goldberg RL, Becker KL: Serum calcitonin precursors in sepsis and systemic inflammation. J Clin Endocrinol Metab
12. Assicot M, Gendrel D, Carsin H, Raymond J, Guilbaud J, Bohuon C: High serum procalcitonin
concentrations in patients with sepsis and infection. Lancet
13. Krüger S, Ewig S, Marre R, Papassotiriou J, Richter K, von Baum H, Suttorp N, Welte T; CAPNETZ Study Group: Procalcitonin
predicts patients at low risk of death from community-acquired pneumonia across all CRB-65 classes. Eur Respir J
14. Buchman TG, Rossier M, Merz WG, Charache P: Detection of surgical pathogens by in vitro
DNA amplification. Part I. Rapid identification of Candida albicans
by in vitro
amplification of a fungus-specific gene. Surgery
15. Shin JH, Nolte FS, Morrison CJ: Rapid identification of Candida
species in blood cultures by a clinically useful PCR method. J Clin Microbiol
16. de Aguirre L, Hurst SF, Choi JS, Shin JH, Hinrikson HP, Morrison CJ: Rapid differentiation of Aspergillus
species from other medically important opportunistic molds and yeasts by PCR-enzyme immunoassay. J Clin Microbiol
17. Fenollar F, Raoult D: Molecular diagnosis of bloodstream infections caused by non-cultivable bacteria. Int J Antimicrob Agents
30(Suppl 1):S7-S15, 2007.
18. Ammann RA, Zucol F, Aebi C, Niggli FK, Kühne T, Nadal D: Real-time broad-range PCR versus blood culture
. A prospective pilot study in pediatric cancer patients with fever and neutropenia. Support Care Cancer
19. Christ-Crain M, Jaccard-Stolz D, Bingisser R, Gencay MM, Huber PR, Tamm M, Müller B: Effect of procalcitonin
-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, single-blinded intervention trial. Lancet
20. Makimura K, Murayama S, Yamaguchi H: Detection of wide range of medically important fungi by the polymerase chain reaction
. J Clin Microbiol
21. Sprengart ML, Fatscher HP, Fuchs E: The initiation of translation in E. coli
: apparent base pairing between the 16srRNA and downstream sequences of the mRNA. Nucleic Acids Res
22. Goldstein BJ, Mushlin AI: Use of a single thyroxine test to evaluate ambulatory medical patients for suspected hypothyroidism. J Gen Intern Med
23. Becker KL, Snider R, Nylen ES: Procalcitonin
assay in systemic inflammation, infection, and sepsis: clinical utility and limitations. Crit Care Med
24. Echániz-Quintana A, Pita-Fernández S, Otero-Ferreiro A, Suárez-López F, Gómez-Gutiérrez M, Guerrero-Espejo A: Risk factors associated with invasive fungal infection
in orthotopic liver transplantation. Med Clin Med Clin (Barc)
25. Said A, Safdar N, Lucey MR, Knechtle SJ, D'Alessandro A, Musat A, Pirsch J, Kalayoglu M, Maki DG: Infected bilomas in liver transplant recipients, incidence, risk factors and implications for prevention. Am J Transplant
26. Karp JE, Merz WG, Charache P: Response to empiric amphotericin B during antileukemic therapy-induced granulocytopenia. Rev Infect Dis
27. Rantakokko-Jalava K, Nikkari S, Jalava J, Eerola E, Skurnik M, Meurman O, Ruuskanen O, Alanen A, Kotilainen E, Toivanen P, et al.: Direct amplification of rRNA genes in diagnosis of bacterial infections. J Clin Microbiol
Bacterial infection; procalcitonin; fungal infection; blood culture; polymerase chain reaction