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A Pilot Evaluation of a Rapid Screening Test for Nosocomial Infection at a Neurosurgical Intensive Care Unit in Sweden

Ramezani, Amir MD∗,†; Darbani, Roya PhD; Eng, Lars H. PhD§; Lönn, Johanna PhD; Yin, Lan PhD§; Nayeri, Fariba MD, PhD§,∥; Theodorsson, Annette MD, PhD∗,†

Author Information
Point of Care: The Journal of Near-Patient Testing & Technology: September 2020 - Volume 19 - Issue 3 - p 63-71
doi: 10.1097/POC.0000000000000208

Abstract

An infection occurs when invading pathogenic microorganisms gain a foothold, succeed in growing, and thereby disturb the normal physiological properties of the tissue.1 To minimize injuries, the microbe responsible for the infection must be identified, its susceptibility to antibiotics determined, and effective therapy initiated immediately.2 The discovery and implementation of antibiotics is tremendously valuable in targeting specific pathogens, effectively eliminating them, and saving lives and resources.3 However, sufficient guidelines and proper diagnostic instruments are not always available to communities to support proper antibiotics administration. Insufficient compliance regarding intake and unregulated administration has, in part, resulted in development of resistant bacterial strains.4 Decreasing the consumption of antibiotics has a direct impact on reducing multidrug-resistant bacteria.5 It is, therefore, urgent to develop screening tests for the early diagnosis of infections before the ultimate identification of the microorganism.

The diagnosis of nosocomial meningitis in patients who have undergone neurosurgical procedures is a substantial medical challenge,6 including its differential diagnosis from ventilator-associated pneumonia (VAP).7 The presence of an elevated number of leukocytes or red blood cells or a high level of lactate in the cerebrospinal fluid (CSF) of an injured brain is a suboptimal indicator of bacterial infections.8 Furthermore, most postoperative neurosurgical patients receiving ventilator support have some degree of suspected infiltration/atelectasis on chest X-ray.9 Bacteriuria is evidently common in catheterized patients.10

Systemic inflammatory response markers such as C-reactive protein (CRP) and procalcitonin may or may not be elevated and do not indicate the site of infection.11 Conventional bacterial cultures have low sensitivity to detect an infection due to antibiotic prophylaxis in severe cases or to the fact that nosocomial infections are commonly caused by slow-growing opportunistic bacteria.12 Furthermore, such infections do not induce CRP or procalcitonin elevation in all of the cases.13 Therefore, irrespective of the routine analysis and culture results, broad-spectrum antibiotics to cover severe nosocomial meningitis are administered to patients that develop fever postneurosurgery.6

The local innate immunity system, which can be assessed based on activated neutrophils, can be used to develop rapid point-of-care tests.14 Neutrophils are the immune system's first line of defense against infection and have conventionally been thought to kill invading pathogens through 2 strategies: engulfment of microbes and secretion of antimicrobials. In 2004, a novel third function was identified: formation of neutrophil extracellular traps (NETs). Neutrophil extracellular traps provide for a high local concentration of antimicrobial components and bind, disarm, and kill microbes extracellularly independent of phagocytic uptake. NETosis is a form of “aggressive” neutrophil cell death that constrains and kills pathogens; in the end, it is the physiological background of what has long been known as pus.15

NETosis is a dynamic process that can come in 2 forms, suicidal and vital NETosis.16 Vital NETosis involves a rapid formation and release of NETs, but does not result in neutrophil death. However, it has been found that neutrophils can continue to phagocytose and kill microbes after vital NETosis, highlighting the neutrophil's antimicrobial versatility.17

We recently assessed the ability of a rapid NETs test designed to detect locally produced substances including extracellular DNA extruded from activated neutrophils18 (please see the Supplementary Video, Supplemental Digital Content 1, http://links.lww.com/POC/A26) to diagnose infection in body fluids. Metachromasia is a characteristic color change exhibited by certain aniline dyes upon binding to chromotropic substances,19 and this phenomenon has been widely used in histology. O-toluidine blue is an excellent metachromatic dye that changes from blue to pink upon binding to high molecular-weight polysaccharides, such as sulfated glycan.20 O-toluidine blue binds to DNA with high affinity. Therefore, the pink dye will then quickly turn back to blue after addition of a proportional amount of polynucleotide (inverted metachromasia). This present pilot study aimed to evaluate the sensitivity and specificity of the rapid NETs test in identifying the site of infection. Leftover CSF, endotracheal secretion, and urine samples were consecutively collected from patients at a neurosurgical intensive care unit (ICU) and analyzed using a rapid strip test within a few hours after sampling.

METHODS

Design: A pilot evaluation of a rapid screening test using left-over samples.

Setting: Neurosurgical ICU at Linköping University Hospital, Sweden.

Cohort subjects and case-concealment: Patients who received ventricular or lumbar drainage during neurosurgery and were admitted postsurgery to the neurosurgical ICU.

Samples: As part of a routine procedure, CSF from all patients that undergo neurosurgery, receive lumbar or ventricular drainage and are admitted to the neurosurgical ICU, is collected and cultured twice a week. We took advantage of this routine procedure to access to CSF material from patients direct after surgery and as long as they stayed at the neurosurgical ICU. In the patients from this cohort who developed fever postsurgery, further cultures from respiratory tract and/or urine were taken. Thus, between November 2015 and November 2017, a total of 199 CSF samples were collected by nurses every Monday and Thursday. Endotracheal aspiration samples (ventilators, n = 73) and urine samples (indwelling catheters, n = 71) were collected from the patients from this cohort who developed fever (Table 1).

TABLE 1 - The Demographic and Laboratory Information About the Identified Groups
Diagnosis
N = Number of Samples
n = Number of Cases
Age Range (Median) Sex (Female/Male) Cause of Brain Operation CRP (Median) CSF WBC × 109 (Median) CSF Lactate, mmol/L
CNS infection
N = 30, n = 19
1–87 (62) 8/11 Shunt-related infection (n = 10)
Complication of meningitis (n = 4)
Brain toxoplasmosis (n = 1)
Brain hemorrhage (n = 4)
5–423 (108) 2.5–8640 (269) 2.1–9.8 (3.3)
Suspected meningitis (not verified)
N = 32, n = 18
41–85 (61.5) 6/12 Pneumocephalus (n = 1)
Brain hemorrhage (n = 11)
Major trauma (n = 4)
Shunt-related infection (n = 1)
Subdural EEG electrodes (n = 1)
5–367 (52) 4.6–3890 (35.1) 1.6–6.1 (3.4)
Other postsurgical infections
N = 66, n = 42
2–84 (63) 16/26 Brain hemorrhage (n = 22)
Major trauma (n = 8)
Shunt-related (n = 3)
Brain tumor (n = 4)
Cerebral vascular disease (n = 2)
Others (n = 3)
3–343 (26) 0–2905 (65) 1.5–7.5 (2.8)
Nosocomial pneumonia
N = 71, n = 38
20–80 (62) 17/21 Brain hemorrhage (n = 28)
Major trauma (n = 6)
Central vascular disease (n = 2)
Brain aneurysm (n = 1)
Shunt-related (n = 1)
10–425 (106) 0.6–660 (19) 1.4–5.2 (3.1)

Case concealment: We used the FDA Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable https://www.fda.gov/media/122648/download. Due to agreement with the personnel, all collected left-over samples in each patient (CSF in all cases and other samples if infection was suspected) were placed immediately in refrigerator (4–8°C). The study nurse transferred the samples into coded tubes consecutively. The study leader (one and the same person) analyzed the samples using a strip test on the same day of sample collection and registered the results in an Excel database. The updated document was submitted regularly to the supervisors. The samples were subsequently kept frozen at −135°C.

The patients were treated according to the guidelines of the clinic. The senior specialist at the Department of Infectious Diseases was consulted for each patient. Because such study was not performed previously, a pilot study was needed to evaluate the results and calculate the sample size for a prospective study. Therefore, the sample collection was paused in November 2017. During April to July 2018, patient journals were studied by project leader and information about age, sex, CSF cells and lactate, CRP, cultures from CSF, blood and endotracheal secretion, the chest X-ray, the antibiotic therapy, and the final diagnosis at discharge was registered for each case using the primary codes. The ethical committee of Linköping University Hospital approved this study (2010/284–32, 2015/429–32) and granted permission to collect the samples and document the unidentified cases by study leader. The study has been registered on clinicaltrials.gov (NCT03252028).

“Rapid NETs Test”

The filter paper is cut into pieces and the strip tests (PEAS Institut, Linköping, Sweden article number 07350063630229, batch PKSR20151102, stability 2 years), used in this study were manufactured for R&D purposes. They consisted of 0.5 × 0.5 cm surfaces containing different levels of dextran sulfate and o-toluidine blue. Three or 5 such surfaces were attached to a plastic support (0.5 × 13 cm), forming a strip. The testing procedure was as follows: the strip test was dipped in the sample for 5 seconds, and then removed and placed on its edge for 5 seconds on a tissue paper to remove excess liquid. The result was observed within 1 minute after the reaction, and the color change was compared with a color chart and the results were documented. The strips were kept and the color change was observed after further 10 and 20 minutes with no significant color change. However, these results were unfortunately not documented in the excel-file.

Statistics

The sensitivity, specificity, negative predictive value, and positive predictive value of the rapid NETs test were calculated by Medcalc (https://www.medcalc.org/calc/diagnostic_test.php). Data from patients were used to evaluate differences between the results before and after therapy. Pearson correlation coefficient analysis was used to analyze the effect of antibiotic therapy (the test performed before compared with after antibiotic therapy) on rapid NETs test result. In 19 patients with VAP, the results from the first and second test occasions were compared using the Wilcoxon matched-pairs signed-rank test. For post hoc power analysis of data, a dichotomous endpoint, one-sample study design was used.21

RESULTS

Empirical Antibiotic Therapy

In total, 75 (64%) of 117 patients included in the study received empirical antibiotic therapy for suspected meningitis when signs of an infection with an unclear focus were observed. The therapy consisted of cefotaxime 3 g 3 to 4 times daily or meropenem 3 g 3 times daily + vancomycin 1 g 2 to 3 times daily. In the rest of the cases, empirical antibiotic therapy for other nosocomial infections, such as cefotaxime 1 g 3 times daily or piperacillin/tazobactam 4 g 3 times daily was initiated. In 8 cases (0.07%), no antibiotics were administered. In 47 cases, the samples were collected before intravenous antibiotic was administered, and in 62 cases, the samples were collected after antibiotic administration. Pearson Correlation Coefficient analysis revealed that there were no significant correlations (R = −0.063, R2 = 0.004, P = 0.49) between antibiotic consumption and the rapid test's screening ability (Supplementary Material 1, Supplemental Digital Content 2, http://links.lww.com/POC/A27).

Identified Groups Therapy

Verified Central Nervous System Infection: Nineteen patients (total, 30 samples; 8 women and 11 men; 1–87 years old; median age, 62 years) had a verified central nervous system (CNS) infection (Table 2). Fifteen samples were collected from CSF in which the cultures yielded growth of bacteria (Table 2, Fig. 1). Ten patients underwent neurosurgery due to shunt-related infection; in these patients, the preoperative cultures yielded growth of Staphylococcus aureus (n = 2), Propionibacterium acnes (n = 3), alpha-hemolytic streptococci (n = 2), Staphylococcus epidermidis (n = 2), and Enterobacter cloacae (n = 1). In patients who underwent neurosurgery due to complications of meningitis, the preoperative cultures yielded growth of Streptococcus pneumoniae (n = 3). Moreover, Escherichia coli (n = 1) was cultured from subdural empyema. One patient suffered from brain toxoplasmosis (n = 1). Four cases developed CNS infection postneurosurgery. Cultures were negative in 3 cases (visible pus/turbid CSF), and sepsis and meningitis caused by Achromobacter spp. were found in 1 case. In 7 cases, 2 to 4 samples on different occasions were collected.

TABLE 2 - The Diagnosis of CNS Infection Was Verified in 19 Cases
CSF CSF CSF
RNT RNT RNT CRP PCT WBC Poly Lactate Microbiologic Assessments of CNS
Sex Age CSF Urine ES mg/L μg/L × 109 × 109 mmol/L
M 87 pos pos neg 133 0.1 3840 3680 9.8 neg
M 76 pos nd nd 99 3.5 346 188 4.4 Streptococcus pneumoniae
F 69 pos pos neg 20 0.1 8.8 0 3.4 Staphylococcus. aureus
F 71 neg nd nd 117 0.1 385 215 7.9 Staphylococcus. aureus
M 70 pos pos neg 260 8.0 12.2 0.6 6.5 Escherichia coli
M 68 pos pos neg 38 0.1 21.4 0.6 2.1 Propionibacterium acnes
F 69 neg neg neg 423 3.3 281 67 3.2 neg (visible pus)
M 69 pos nd nd 5 0.1 25.8 3 2.8 neg (visible pus)
F 65 pos nd nd 198 0.4 2.5 0.5 3.3 Propionibacterium acnes
M 62 pos pos neg 5 0.1 2.8 0 2.1 Toxoplasmosis
F 61 pos pos pos 40 0.1 8640 4080 3.1 Alpha hemolytic streptococci
F 60 pos neg nd 14 0.1 9.7 2.5 2.3 Staphylococcus epidermidis
F 50 pos nd nd 136 0.2 974 916 6.8 Enterobacter cloacae
M 48 pos pos pos 273 13 1295 1095 5.4 Streptococcus pneumoniae
M 48 pos neg neg 192 0.2 257.5 130 4.4 Staphylococcus epidermidis
F 40 pos pos neg 34 0.1 6.5 2.6 2.1 Propionibacterium acnes
M 36 pos pos pos 131 0.2 1950 1716 3.3 Achromobacter
M 15 pos neg neg 11 0.1 295 5 6.5 Alpha hemolytic streptococci
M 1 pos neg nd 161 0.5 na na na Streptococcus pneumoniae
F indicates female; M, male; neg, negative; pos, positive; nd, nondefined; na, non available; RNT, rapid NETs test; ES, endotracheal secretion.

FIGURE 1
FIGURE 1:
Flowchart of the study samples.

CNS infection was not verified: Eighteen patients (total 32 samples, 6 females and 12 males, 41–85 years old, median age 61.5 years) had been treated as CNS infection. However, cultures from CSF did not yield growth of bacteria (Fig. 1) (Supplementary Material 2, Supplemental Digital Content 3, http://links.lww.com/POC/A28).

Nosocomial Pneumonia: In total, 38 patients (17 women; 20–80 years; median age, 62 years) developed pneumonia postneurosurgery. Cerebrospinal fluid samples were collected (n = 71) from these patients. The patients were on ventilators and developed fever, and the chest X-ray revealed infiltrates (n = 37). The cultures from endotracheal secretion revealed growth of bacteria (n = 24) (Table 3), and the CRP (range, 5–491 mg/mL; median, 61 mg/mL) and procalcitonin (range, 0.1–14.0 μg/L; median, 0.2 μg/L) levels were elevated. Endotracheal secretion samples for rapid NETs test analysis were available in 32 patients (total 57 samples) (Supplementary Material 3, Supplemental Digital Content 4, http://links.lww.com/POC/A29). In 19 patients, 2 to 6 samples were collected on different occasions. This group showed significant differences between the rapid NETs test results before and after antibiotic therapy (Fig. 2).

TABLE 3 - The Patients That Had a Verified Diagnosis of Pneumonia
RNT RNT RNT CSF CSF
Sex Age CSF Urine ES CRP PCT LPK Lactate Culture From ES
M 80 neg pos pos 61 0.8 231 5.2 neg
F 78 neg pos pos 122 0.1 168 3.8 neg
F 73 neg pos pos 87 0.2 11.3 3.3 Streptococcus
pyogenes +
Corynebacterium
M 70 neg pos pos 41 nd 270 2.8 neg
F 68 pos pos pos 270 0.3 565 3.4 Hemophylus
influenzae neg
M 68 neg pos pos 32 0.1 12.2 2.1 Hemophylus influenzae
F 68 neg neg pos 132 0.3 6 3.2 Proteus mirabilis +
Serratia marcenses
M 68 neg nd nd 44 0.2 8.9 3.1 Klebsiella
F 66 pos pos pos 27 0.1 67.5 2.5 Neisseria flavenscens
F 65 neg neg pos 169 0.6 20 2.6 neg
M 65 pos pos pos 114 0.1 660 3.2 Enterobakter +
Hemophylus
influenza
M 65 neg neg nd 173 0.1 16.3 2.5 Pseudomonas
aeroginosa
M 64 pos pos pos 243 0.6 6.4 2.9 neg
M 64 neg pos neg 173 0.5 117 5.2 Staphylococcus
aureus
M 63 neg pos pos 347 3.9 37.5 3.3 Alpha hemolytic streptococci
+ citerobacter
F 62 neg pos pos 49 0.1 5.4 2.2 Enterococcus fecalis
F 61 neg neg pos 102 1.3 19 4.0 neg
M 63 neg pos pos 80 0.2 305 4.7 neg
F 62 neg nd pos 28 0.1 1.9 2.9 Pasturella canis +
Escherichia coli
F 59 neg nd pos 28 0.1 37 4.0 Moraxella
catarrhalis
F 60 neg neg pos 10 0.1 129.4 4.3 neg
F 57 neg pos pos 180 2.1 na 3.8 neg
F 54 neg nd nd 101 0.1 40.8 3.1 Staphylococcus
aureus
M 54 neg nd pos 72 0.8 na na Staphylococcus
aureus
M 55 neg nd pos 28 ej 61 3.8 Streptococcus
pneumonae +
Hemophylus
influenzae
M 53 neg pos pos 154 0.4 1.4 4.0 neg
F 52 neg neg pos 127 0.3 1.0 1.7 Hemophylus
influenzae
M 43 neg pos pos 74 0.2 160 4.4 Alpha hemolytic streptococci
M 42 neg pos pos 136 0.2 77 3.5 Hemophylus
influenza
M 40 neg neg pos 127 0.1 3.9 1.9 Hemophylus
Influenza
M 30 neg neg pos 81 0.1 0.6 1.6 Candida albicans +
Candida dublinesis
F 39 neg neg neg 52 0.3 na 3.1 neg
F 38 pos pos pos 138 0.4 155 2.1 Klebsiella
M 33 neg nd nd 110 0.1 2.2 1.4 Enterobacter +
Staphylococcus
aureus
M 35 neg pos pos 272 0.6 6.9 4.0 Serratia marcenses
M 25 neg nd pos 425 14.0 5.0 3.2 neg
M 21 neg pos pos 115 13 5.6 2.1 neg
F 20 neg nd nd 54 0.1 325 3.1 Pseudomonas
aeroginosa +
Hemophylus
influenza
F indicates female; M, male; neg, negative; pos, positive; nd, nondefined; na, nonavailable; RNT, rapid NETs test; ES, endotracheal secretion.

FIGURE 2
FIGURE 2:
In 19 cases, 2 to 6 samples were collected on different occasions during the patients' stay in the ward. There were significant differences between the rapid NETs test results before (occasion 1) and after (occasion 2) antibiotic therapy in this group.

Other Postsurgical Infections: A total of 42 patients (total, 66 samples; 16 women, 2–84 years; median age, 63 years) had postsurgical infections other than CNS infection or VAP. In this group, the CSF cultures yielded no growth, and no radiological evidence of pneumonia was seen (Fig. 1). Five patients experienced septicemia, and blood cultures yielded growth of Serratia marcescens (n = 1), coagulase-negative staphylococci (n = 1), Enterococcus faecalis (n = 1), Staphylococcus aureus (n = 1), and Escherichia coli (n = 1). In 23 patients, urine cultures yielded growth of the following bacteria: Pseudomonas aeruginosa (n = 3), Escherichia coli (n = 8), Enterococcus faecalis (n = 5), Klebsiella pneumonia (n = 2), Enterobacter cloacae (n = 2), and extended-spectrum beta-lactamase producing bacteria (n = 1). Candida albicans was also found (n = 2).

Validity of the Rapid NETs Test to Identify the Focus of Infection

CNS Infection: As the golden standard for diagnosing CNS infection, positive CSF cultures were used. In 3 cases, visible pus or turbid CSF confirmed a diagnosis of CNS infection (n = 19). Patients with nosocomial pneumonia plus other infections (neither CNS infection nor VAP, n = 80) were used as the “Negative” control group. The rapid NETs test identified a CNS infection with 89.5% sensitivity (95% confidence interval [CI], 66.9–98.7%), 92.5% specificity (95% CI, 84.4–97.2%), 97.4% negative predictive value (95% CI, 90.9–99.2%), and 73.9% positive predictive value (95% CI, 56.4–86.2%). Accuracy was 91.9% (Table 4).

TABLE 4 - CNS Infection
RNT Present n Absent n Total
Positive
Verified diagnosis
True Positive a = 17 False Positive c = 6 a + c = 23
Negative False b = 2 True Negative d = 74 b + d = 76
 (VAP, sepsis, other infections) Negative
Total 19 80 99
Analysis of the RNT ability to distinguish CNS infection (verified) shows 89.5% sensitivity (95% CI, 66.9–98.7%), 92.5% specificity (95% CI, 84.4–97.2%), 97.4% negative predictive value (95% CI, 90.9–99.3%), and 73.9% positive predictive value (95% CI, 56.4–86.2%). Accuracy was 91.9%.
RNT indicates rapid NETs test.

In the patients that fulfilled therapy as bacterial meningitis without an objective evidence of meningitis (n = 18), the rapid NETs test showed positive result with 66.6% sensitivity (95% CI, 40.9–86.6%), 92.5% specificity (95% CI, 84.4–97.2%), 92.5% negative predictive value (95% CI, 86.4–95.6%), and 66.6% positive predictive value (95% CI, 46.4–82.1%).

Nosocomial Pneumonia: Patients who fulfilled the criteria for nosocomial pneumonia and extraendotracheal secretion samples were available (n = 32) were used as positive controls. The control group comprised patients with CNS infection plus other infections (neither CNS infection nor VAP, n = 38) in which the rapid NETs test was performed on endotracheal secretion. Analysis revealed that the rapid NETs test could distinguish pneumonia from other postsurgical infections with 93.8% sensitivity (95% CI, 79.2–99.2%), 86.8% specificity (95% CI, 71.9–.95.6%), 94.3% negative predictive value (95% CI, 81.1–98. 5%), and 90% positive predictive value (95% CI, 80.5–95.9%). Accuracy was 90.0% (Table 5).

TABLE 5 - Ventilator-Associated Pneumonia
RNT Present n Absent n Total
Positive
Verified pneumonia
True Positive a = 30 False Positive c = 5 a + c = 35
Negative (Meningitis, sepsis, other infections) False Negative b = 2 True Negative d = 33 b + d = 35
Total 32 38 70
Analysis of the RNT ability to distinguish respiratory infection shows 93.75% sensitivity (95% CI, 79.2–99.2%), 86.8% specificity (95% CI, 71.9–95.6%), 94.3% negative predictive value (95% CI, 81.1–98.5%), and 90% positive predictive value (95% CI, 80.5–95.9%). Accuracy was 90.0%.
RNT indicates rapid NETs test.

The rapid NETs test results for endotracheal secretions differed significantly (were less sensitive) between samples taken at the first and second occasions (all over the time frame) (P = 0.0008). There were no significant differences in CRP (P = 0.4545), procalcitonin (P = 0.7995), or leukocyte particle concentration (P = 0.1353) levels between the first and second analyses (Supplementary Material 3, Supplemental Digital Content 4, http://links.lww.com/POC/A29).

Nosocomial Urinary Tract Infection: The rapid NETs test was performed in 72 patients. The test performance of the strip test in urine samples collected from catheters was poor (sensitivity 78.6%, specificity 55.2%, negative predictive value 91.4%, and positive predictive value 29.7%).

Power Analysis

There were no available previous data and, therefore, the sample size for the present study could not be calculated. However, we showed that among the patients, 64% received empirical antibiotic therapy against nosocomial CNS infection, whereas 16% of patients had CNS infection. In 15% of cases, the diagnosis of meningitis was not verified (Supplementary Material 2, Supplemental Digital Content 3, http://links.lww.com/POC/A28). These cases were judged by the physician in charge as highly suspicious meningitis and received antibiotic therapy against meningitis. For the future studies the sample size might be calculated using the present data in order to evaluate the impact of rapid NETs test to identify the cases that should receive antibiotics. The post hoc power of the present study was 99.9% (dichotomous endpoint, 1-sample study).21

DISCUSSION

In the current study, we have evaluated the sensitivity and specificity of a rapid NETs test to distinguish the site of postsurgical infection. The rapid NETs test identified nosocomial CNS infection by analysis of CSF and was able to diagnose nosocomial pneumonia by analysis of endotracheal secretion within 1 minute with a clinically adequate power and accuracy.

Neutrophil extracellular traps are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens.15 Neutrophil extracellular traps allow neutrophils to kill extracellular pathogens while minimizing damage to the host cells. Upon in vitro activation with the pharmacological agent phorbol myristate acetate, Interleukin 8, or lipopolysaccharide, neutrophils release granule proteins and chromatin to form an extracellular fibril matrix known as NETs through an active process.15 Neutrophil extracellular traps disarm pathogens with antimicrobial proteins, such as neutrophil elastase, cathepsin G, and histones that have a high affinity for DNA.22 The rapid NETs test was designed to detect extracellular DNA.

Nosocomial bacterial meningitis may result from invasive procedures, such as craniotomy, complicated head trauma, or metastatic infection in the course of bacteremia.6 Bacterial meningitis, as a serious complication, occurs in 0.8% to 1.5% of patients who undergo craniotomy.23 The incidence of meningitis associated with internal ventricular catheters, used for the treatment of hydrocephalus, ranges from 4% to 17%.24 Although that associated with external catheters is approximately 8%,25 external lumbar catheters have been associated with meningitis rates of up to 5%.26 Meanwhile, the incidence of meningitis after head trauma is estimated to be 1.4%.27 Closed head trauma with basilar skull fracture is associated with up to 25% increased risk of infection.27 Head trauma is the most common cause of recurrent bacterial meningitis.28 Therefore, a clinical suspicion of nosocomial bacterial meningitis should prompt a diagnostic workup and antimicrobial therapy.6 The workup consists of neuroimaging, CSF analysis (cell counts, Gram staining, biochemical tests for glucose and protein, and cultures), and blood cultures.6 The diagnosis of nosocomial bacterial meningitis is based on the results of CSF cultures. However, cultures require prolonged incubation, and results may be negative in patients who have received previous antimicrobial therapy.6 Cell counts in CSF have low sensitivity and specificity and might be normal in patients in whom meningitis was confirmed by culture.8 Moreover, pleocytosis is often observed in patients without positive cultures.8 In patients subjected to neurosurgery, a lactate level of 4 mmol/L or greater in CSF showed a sensitivity of 88% and specificity of 98% to diagnose a postsurgical meningitis.29 However, in bacterial meningitis associated with CSF shunts, the sensitivity of CSF lactate level was low.24 Although elevated levels of CRP and serum procalcitonin are suggestive of bacterial infection, they do not establish the diagnosis, and the usefulness of these markers in the diagnosis of nosocomial bacterial meningitis has not been determined.11 The British Society for Antimicrobial Chemotherapy recommends empirical therapy for all patients showing signs of postoperative meningitis. Treatment should be withdrawn after 72 hours if the results of CSF cultures are negative.30 Thus, the patients in our pilot study in which CSF cultures yielded no growth, the diagnosis of meningitis was not established and this group could not be used as appositive control group. However, patients who have received previous antimicrobial therapy may require treatment despite negative culture results.6 The median antibiotic treatment duration for postneurosurgical meningitis was reported to be 17–22.5 days.31 The results of the present study showed that the rapid NETs test could rule out CNS infection with 97% negative predictive value and a significant reduction in antibiotic consumption is, therefore, expected.

Nosocomial pneumonia is the most common cause of death among nosocomial infections and is the primary cause of death in ICUs.32 Patients with risk factors or signs of clinical decompensation should have empirical therapy initiated at a lower threshold, and the therapy should be directed at a confirmed infection following positive culture results.33 Early administration of empirical antibiotic therapy is recommended in VAP. Ventilator-associated pneumonia is the main cause of antibiotic use in ICUs. The rate of VAP reaches up to 40% for brain-injured patients hospitalized in the ICU.7 Rather than using a combination of CRP, procalcitonin, or other biomarkers plus clinical criteria, the clinical practice guidelines by the Infectious Diseases Society of America and the American Thoracic Society recommend using the clinical criteria of disease alone in deciding whether or not to initiate antibiotic therapy.34 For patients with VAP, a 7-day course of antimicrobial therapy is recommended.34 The result from the present pilot study indicates that using rapid NETs test could “with a sensitivity and specificity >90%” identify the patients with VAP and therefore motivate a shorter course of antibiotic therapy using smaller spectrum antibiotics.

More than 30% of nosocomial infections comprise urinary tract infections. The origin of nosocomial bacteria is endogenous (the patient's flora) in two thirds of the cases. Patients with indwelling urinary catheters are at high risk of developing nosocomial infections, especially urinary tract infections.35 In this present study, on patients receiving antibiotic prophylaxis and febrile patients with heavy empirical antibiotic therapy, data about nosocomial urinary tract infections were not reliable and the rapid NETs test was positive in patients in which it is not possible to confirm or rule out the disease. The cutoff test used36 was not appropriate for urine samples.

Based on the clinical guidelines and due to severe complications of delayed therapy, empirical antibiotic treatment is recommended in patients who develop fever postneurosurgery. In this study, 64% of patients who underwent neurosurgery at our department received empirical therapy against nosocomial CNS infection with high doses of broad-spectrum antibiotics. Of the total 117 patients included in the study, only 8 (0.07%) patients did not receive antibiotics. However, we observed that 19 (16%) patients had a confirmed CNS infection and 32 patients (27%) had VAP. These findings indicate the value of screening tests in distinguishing the site of infection and ruling out the most severe CNS infection long before the culture results are available.

This study has limitations.

  • (A) The samples were leftover material, and the study group could not decide on sample collection. However, samples were taken to investigate the source of infection and were cultured, and information about the exact time of antibiotic administration in each case was recorded.
  • (B) It is a limitation that after case concealment, only a single observer measured the test result and documented in Excel file. However, the updated document was regularly submitted to the supervisors.
  • (C) The diagnosis of nosocomial meningitis is complicated, and it is not possible to prove that all cases included as positive controls are cases of postsurgical nosocomial meningitis. In the positive group, we have included the cases that had fever, CSF cultures yielded growth of bacteria and were judged by the physician on charge as nosocomial meningitis. As we have reported all of the cases that were collected, there are few cases that are not postoperative or nosocomial infections such as a case of toxoplasmosis. Therefore, we have called the positive control group as CNS infection.
  • (D) The present study offers no evidence that the test can result in the decrease in inappropriate use of antibiotics. Therefore, the results of this work should be confirmed in multicenter prospective studies using the results from the pilot study to calculate the patient size and power.
  • (E) The rapid test is meant to facilitate and not to replace the clinical assessment.

CONCLUSIONS

The local production of injured organ cells and activated leukocytes during an unspecific immune reaction was used to construct a test platform for locating the site of infection in febrile patients who had undergone neurosurgery. The study revealed that this rapid screening test (rapid NETs test) could identify the site of infection with clinically relevant sensitivity and specificity, indicating its potential to minimize the unnecessary use of antibiotics. If our primary results are confirmed through independent multicenter prospective studies, we will have access to a tool to guide antibiotic therapy and save resources even in non-equipped centers where the misuse of antibiotics is most rampant.

ACKNOWLEDGMENTS

The authors are grateful to Tayeb Nayeri, who hypothesized and developed the platform, to the staff at the neurosurgical ICU of Linköping University Hospital for helping with sample collection, and to Sepahdar Mansouri for the management and laboratory assistance.

REFERENCES

1. Peterson JW. Bacterial pathogenesis. In: Medical Microbiology. 4th ed.Galveston, TX: The University of Texas Medical Branch at Galveston; 1996;Chapter 7.
2. Tunkel AR, Hartman BJ, Kaplan SL, et al. Practise guidelines for the management of bacterial meningitis. Clin Infect Dis. 2004;39(9):1267–1284.
3. Infectious Diseases Society of America (IDSA). Combating antimicrobial resistance: Policy recommendations to save lives. Clin Infect Dis. 2011;52(S5):397–428.
4. Pechere JC, Hughes D, Kardas P, et al. Non-compliance with antibiotic therapy for acute community infections: a global survey. Int J Antimicrob Agents. 2007;29(3):245–253.
5. Baur D, Gladstone BP, Burkert F, et al. Effect of antibiotic stewardship on the incidence of infection and colonisation with antibiotic-resistant bacteria and Clostridium difficile infection: a systematic review and meta-analysis. Lancet Infect Dis. 2017;17(9):990–1001.
6. Vande Beek D, Drake JM, Tunkel AR. Nosocomial Bacterial Meningitis. N Engl J Med. 2010;362(2):146–154.
7. Roquilly A, Feuillet F, Seguin P, et al. Empiric antimicrobial therapy for ventilator-associated pneumonia after brain injury. Eur Respir J. 2016;47(4):1219–1228.
8. Schade RP, Schinkel J, Roelandse FW, et al. Lack of value of routine analysis of cerebrospinal fluid for prediction and diagnosis of external drainage-related bacterial meningitis. J Neurosurg. 2006;104(1):101–108.
9. Newman B, Krane EJ, Gawande R, et al. Chest CT in children: anesthesia and atelectasis. Pediatr Radiol. 2014;44:164–172.
10. Kim B-N, Choi S-I, Ryoo N-H. Three-year follow-up of an outbreak of Serratia marcescens Bacteriuria in a neurosurgical intensive care unit. J Korean Med Sci. 2006;21:973–978.
11. Nathan BR, Scheld WM. The potential roles of C-reactive protein and procalcitonin concentrations in the serum and cerebrospinal fluid in the diagnosis of bacterial meningitis. Curr Clin Top Infect Dis. 2002;22:155–165.
12. Wu C, Nakka S, Mansouri S, et al. In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments. BMC Microbiol. 2016;16(1):209.
13. Domenech M, Ramos-Sevillano E, García E, et al. Biofilm Formation Avoids Complement Immunity and Phagocytosis of Streptococcus pneumoniae. Infect Immun. 2013;81(7):2606–2615.
14. Sollberger G, Tilley DO, Zychlinsky A. Neutrophil Extracellular Traps: The Biology of Chromatin Externalization. Dev Cell. 2018;44(5):542–553.
15. Brinkmann V, Ulrike R, Christian G, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532–1535.
16. Jorch SK, Kubes P. An emerging role for neutrophil extracellular traps in noninfectious disease. Nat Med. 2017;23(3):279–287.
17. Urban CF, Ermert D, Schmid M, et al. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog. 2009;5(10):e1000639.
18. Ramezani A, Alipouratigh M, Eng L, et al. One-minute through test to distinguish lower respiratory infection by analysis of endotracheal secretions; exploring the mechanisms. BMC Res Notes. 2018;11(1):664.
19. Bergeron JA, Singer M. Metachromasy: an experimental and theoretical reevaluation. J Biophys Biochem Cytol. 1958;4(4):433–457.
20. Pham NA, Morrison A, Schwock J, et al. Quantitative image analysis of immunohistochemical stains using a CMYK color model. Diagn Pathol. 2007;2:8.
21. Rosner B. Fundamentals of Biostatistics. 7th ed. Boston: Brooks/Cole, Cengage Learning; 2011.
22. Thomas MP, Whangbo J, McCrossan GJ, et al. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins. J Immunol. 2014;192(11):5390–5397.
23. McClelland S, Hall WA. Postoperative central nervous system infection: incidence and associated factors in 2111 neurosurgical procedures. Clin Infect Dis. 2007;45(1):55–59.
24. Conen A, Walti LN, Merlo A, et al. Characteristics and treatment outcome of cerebrospinal fluid shunt–associated infections in adults: a retrospective analysis over an 11-year period. Clin Infect Dis. 2008;47(1):73–82.
25. Lozier AP, Sciacca RR, Romagnoli MF, et al. Ventriculostomy-related infections. Neurosurgery. 2002;51(1):170–181.
26. Governale LS, Fein N, Logsdon J, et al. Techniques and complications of external lumbar drainage for normal pressure hydrocephalus. Oper Neurosurg. 2008;63(4 Suppl 2):379–384.
27. Baltas I, Tsoulfa S, Sakellariou P, et al. Posttraumatic meningitis: bacteriology, hydrocephalus, and outcome. Neurosurgery. 1994;35(3):422–426.
28. Adriani KS, van de Beek D, Brouwer MC, et al. Community-acquired recurrent bacterial meningitis in adults. Clin Infect Dis. 2007;45(5):e46–e51.
29. Leib SL, Boscacci R, Gratzl O, et al. Predictive value of cerebrospinal fluid (CSF) lactate level versus CSF/blood glucose ratio for the diagnosis of bacterial meningitis following neurosurgery. Clin Infect Dis. 1999;29(1):69–74.
30. The management of neurosurgical patients with postoperative bacterial or aseptic meningitis or external ventricular drain-associated ventriculitis. Infection in Neurosurgery Working Party of the British Society for Antimicrobial Chemotherapy. Br J Neurosurg. 2000;14(1):7–12.
31. Soavi L, Rosina M, Stefini R, et al. Post-neurosurgical meningitis: management of cerebrospinal fluid drainage catheters influences the evolution of infection. Surg Neurol Int. 2016;7(Suppl 39):S927–S934.
32. Mandell GL, Douglas RG, Bennett JE, et al. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 6th ed. New York: Elsevier/Churchill Livingstone; 2005.
33. Hassinger T, Sawyer R. Should we immediately start antibiotics in every patient with a clinical suspicion of HAP/VAP?Semin Respir Crit Care Med. 2017;38(3):245–252.
34. Kalil AC, Metersky ML, Klompas M, et al. Executive summary: management of adults with hospital-acquired and ventilator-associated pneumonia: 2016 clinical practice guidelines by the Infectious Diseases Society of America and the American Thoracic Society. Clin Infect Dis. 2016;63(5):e61–e111.
35. Iacovelli V, Gaziev G, Topazio L, et al. Nosocomial urinary tract infections: a review. Urologia. 2014;81(4):222–227.
36. Ramezani A, Eng L, Turkina MV, et al. A sputum screening test to rule out pneumonia at an early stage with high negative predictive value. Point Care J -Patient Test Technol. 2018;17:101–108.
Keywords:

neutrophil extracellular trap; meningitis; neurosurgery intensive care; point of care; ventilator-associated pneumonia; screening test

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