Pathogen-Specific Local Immune Fingerprints Diagnose Bacterial

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Pathogen-Speciﬁc Local Immune Fingerprints Diagnose Bacterial Infection in Peritoneal Dialysis Patients

† ‡ Chan-Yu Lin,* Gareth W. Roberts, Ann Kift-Morgan,* Kieron L. Donovan,§ | Nicholas Topley, and Matthias Eberl*

*Cardiff Institute of Infection and Immunity, Cardiff University, Cardiff, Wales, United Kingdom; †Department of Nephrology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; ‡Institute of Molecular and Experimental Medicine, Cardiff University, Cardiff, Wales, United Kingdom; §Department of Nephrology and Transplant, Cardiff and Vale University Health Board, Cardiff, Wales, United Kingdom; and |Institute of Translation, Innovation, Methodology and Engagement, School of Medicine, Cardiff University, Cardiff, Wales, United Kingdom

ABSTRACT Accurate and timely diagnosis of bacterial infection is crucial for effective and targeted treatment, yet routine microbiological identification is inefficient and often delayed to an extent that makes it clinically unhelpful. The immune system is capable of a rapid, sensitive and specific detection of a broad spectrum of microbes, which has been optimized over millions of years of evolution. A patient’searlyimmuneresponse is therefore likely to provide far better insight into the true nature and severity of microbial infections than conventional tests. To assess the diagnostic potential of pathogen-specific immune responses, we characterized the local responses of 52 adult patients during episodes of acute peritoneal dialysis (PD)– associated peritonitis by multicolor flow cytometry and multiplex ELISA, and defined the immunologic signatures in relation to standard microbiological culture results and to clinical outcomes. We provide evidence that unique local “immune fingerprints” characteristic of individual organisms are evident in PD patients on the day of presentation with acute peritonitis and discriminate between culture-negative, Gram-positive, and Gram-negative episodes of infection. Those humoral and cellular parameters with the most promise for defining disease-specific immune fingerprints include the local levels of IL-1b, IL-10, IL-22, TNF-a,andCXCL10,aswellasthefrequencyoflocalgd T cells and the relative proportion of neutrophils and monocytes/macrophages among total peritoneal cells. Our data provide proof of concept for the feasibility of using immune fingerprints to inform the design of point-of-care tests that will allow rapid and accurate infection identification and facilitate targeted antibiotic prescription and improved patient management.

J Am Soc Nephrol 24: 2002–2009, 2013. doi: 10.1681/ASN.2013040332

Bacterial infections remain a leading cause of inherently slow and inefﬁcient. In many cases, no morbidity and mortality worldwide and pose a organism can be identiﬁed despite clinical signs critical challenge for public health in the 21st of infection.6,7 Even in the case of positive results, century, not the least of which is due to the neither microbiological cultures nor state-of-the- unprecedented spread of antibiotic resistance.1–5 art molecular methods such as PCR or mass Effective management of infected patients is ham- pered by the performance of standard diagnostics Received April 2, 2013. Accepted May 26, 2013. and inappropriate or delayed choice of treatments. N.T. and M.E. contributed equally to this work.

There remains a lack of appreciation of how the Published online ahead of print. Publication date available at body senses and fights different bacterial patho- www.jasn.org. gens. Diagnosis of suspected infections depends fi Correspondence: Dr.MatthiasEberl,CardiffInstituteofIn- largely on the positive identi cation of the likely fection and Immunity, Henry Wellcome Building, School of pathogen in biologic fluids, a concept that was in- Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, troduced by Robert Koch more than a century ago. Wales, United Kingdom. Email: [email protected] However, microbiological culture methods are Copyright © 2013 by the American Society of Nephrology

2002 ISSN : 1046-6673/2412-2002 JAmSocNephrol24: 2002–2009, 2013 www.jasn.org BASIC RESEARCH spectrometry–based techniques discriminate between patho- RESULTS gens causing disease, asymptomatic carriage, and sample con- taminants. Local Inflammatory Markers in Acute Peritonitis In the absence of accurate and rapid point-of-care methods Episodes of acute peritonitis in PD patients were characterized that direct therapy, especially in cases in which organism by a range of cellular and soluble biomarkers associated with virulence is a determinant of outcome, treatment of most inflammatory responses (Supplemental Table 1). This was true infections remains largely empirical.5,8–12 Ineffective and for culture-negative episodes of peritonitis as well as cases of delayed therapy can lead to chronic or recurrent infections confirmed infection by Gram-negative or Gram-positive bac- that are typically more serious and more difficult to treat. teria (Figures 1 and 2). Although peritoneal leukocytes in sta- As a result, there is a low threshold for prescribing broad- ble patients were relatively low in numbers (typically ,107 spectrum antibiotics, although many patients receiving such cells per bag) and were composed mainly of monocytes/ treatments actually may not have an infection, or could safely macrophages and T cells, acute peritonitis was dominated by be treated with better targeted drugs.13 Of note, inappropriate a massive recruitment of neutrophils, at times reaching .95% therapy and overuse of antibiotics are the main causes driv- of all peritoneal cells and .1011 cells in total. Detailed analyses ing multidrug resistance, which is one of the major global of the peritoneal leukocyte subpopulations revealed a prefer- threats for human health as identified by the World Health ential increase in Vd2+ gd T cells within the local T cell pop- Organization. ulation in acute peritonitis, whereas the percentages of CD4+ It is increasingly clear that the nature of the inflammatory and CD8+ T cells remained virtually unchanged (Supplemen- response to infection is a major determinant of outcome.14 tal Table 1). Soluble mediators increased in acute peritonitis This is especially true in vulnerable individuals such as pa- included IL-1b, IL-6, soluble IL-6 receptor (sIL-6R), IL-10, tients with acute peritoneal dialysis (PD)–associated perito- IL-22, CXCL8 (IL-8), CXCL10, TNF-a, TGF-b, and matrix nitis, in which the effect of host responses has detrimental metalloproteinase-3 (MMP-3) (Supplemental Table 1). consequences due to inflammation-related tissue damage.15,16 Key to developing better and stratified approaches to treating IL-1b and IL-10 Predict Culture-Negative Peritonitis infections is a detailed understanding of the intricate host- Wenext stratified patients into distinct subgroups according to pathogen relationships in disease, and the physiologic and the microbiological culture results provided by the routine pathophysiologic events driving early inflammatory responses diagnostic laboratory. These analyses revealed that the immune and pathogen clearance. The most meticulous cross-sectional responses were markedly different between patients with and longitudinal investigations in this respect are being made culture-negative peritonitis and those with confirmed cul- in PD patients.17–20 The peritoneal catheter in PD serves as a ture-positive infection. This latter group had significantly unique window to inflammatory scenarios that can be ob- higher numbers of all leukocyte subpopulations analyzed. served continuously in vivo. It affords noninvasive access to However, in culture-positive episodes of infection, a larger all relevant cellular components and humoral mediators in- proportion of the infiltrating immune cells were neutrophils, volved in local inflammation, as well as direct insight into whereas a smaller proportion were monocytes/macrophages, how treatment and type of infection modulate disease-specific suggesting that the ratio of neutrophils to monocytes/ pathways in a clinically relevant manner. macrophages might be of relevance for diagnostic purposes The immune system has evolved to survey the body (Supplemental Table 2). Culture-positive patients also had constantly for potentially hazardous structures.21 Different higher peritoneal levels of IL-1b,IL-2,IL-6,IL-10,IL-22,and pathogens express different molecular patterns and hence in- TNF-a (as well as sIL-6R and MMP-3 with borderline signifi- teract uniquely with different components of the immune sys- cance) (Supplemental Table 2), indicating a more severe inflam- tem. The type of infection is therefore likely to evoke distinct matory course than in patients during culture-negative peritonitis. immunologic signatures, or “immune fingerprints,” that can Given the distinct immune responses observed in the be assessed quantitatively and qualitatively.22,23 However, to different patient groups, we tested whether specific immune our knowledge, no experimental attempt has been made to fingerprints on day 1 could predict the microbiological culture systematically characterize cellular and humoral responses results. Area under the receiver operating characteristic curve to defined pathogens in a human infection scenario and to (AUROC) calculations identified a number of parameters with translate the notion of pathogen-specific immune responses discriminatory power, such as the proportion of neutrophils into the clinic to assess its diagnostic value. In this study, we among peritoneal cells as well as the levels of IL-1b, IL-10, performed a detailed immunologic and microbiological anal- TNF-a, and IL-6 (Supplemental Table 3). Levels of IL-1b and ysis of samples obtained from PD patients presenting with IL-10 gave the best Youden index (= sensitivity + specificity – acute peritonitis, and provide evidence that each bacterial in- 1) and highest overall correctness as individual parameters, fection leaves a characteristic local immune fingerprint. These for prediction of either culture-positive or culture-negative proof-of-concept findings may have applicability for rapid, peritonitis (Supplemental Table 4). A simple model for the accurate, and differentiating point-of-care diagnosis of pa- discriminatory potential between culture-negative and culture- tients with suspected infections. positive peritonitis was developed by combining biomarkers

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from patients with either culture-negative or Gram-positive peritonitis (Supplemen- tal Tables 6 and 7). Within the T cell population, Vd2+ Tcellsweresignificantly increased and expressed higher levels of the activation marker HLA-DR in Gram- negative infections. In addition, peritoneal monocytes/macrophages expressed lower levels of CD86 in Gram-negative infections compared with the rest of the patients. In- flammatory markers that significantly increased in Gram-negative infections included IL-1b, IL-10, and TNF-a (Figure 3B and Supplemental Table 7). AUROC calculations identified the combination of Vd2+ T cell frequencies and peritoneal levels of IL-10 as an excellent discriminator that predicted Gram-negative infections (Sup- plemental Table 8). This combination had the highest overall correctness, with 100% sensitivity and 93% specificity for the prediction of Gram-negative infections (Supplemental Table 9). The prognostic value of the proportion of monocytes/ macrophages among all cells (,10.7%) and Figure 1. Cellular immune biomarkers in PD patients. (A) Total number and relative their expression of CD86 (,67.9%), the + + frequency (%) of peritoneal neutrophils (CD15 ), monocytes/macrophages (CD14 ), frequency of Vd2+ TcellswithintheTcell and T cells (CD3+) in stable PD patients and on day 1 of acute peritonitis. (B) Total $ + + population ( 6.3%) and their expression number and proportion of helper T cells (CD4 ), cytotoxic T cells (CD8 ), and gd T of HLA-DR ($16.1%), and peritoneal cells (Vd2+) within the peritoneal CD3+ T cell population in stable PD patients and on IL-10 levels ($110.1 pg/ml) were all con- day 1 of acute peritonitis. Patients presenting with a cloudy bag are grouped ac- fi cording to the microbiological culture results into patients with culture-negative rmed by univariate analyses. Multivari- fi + peritonitis (n/c) or confirmed Gram-positive or Gram-negative infection. Data points ate analyses identi ed the Vd2 Tcell represent individual patients, and horizontal lines represent mean values per group. frequency as an independent prognostic biomarker for the prediction of Gram- negative peritonitis in all patients pre- according to their cut-off values (Figure 3A). When combined senting with a cloudy bag (Supplemental Table 10). together, IL-1b (,4.1 pg/ml) and IL-10 (,23.3 pg/ml) resul- + ted in 79% sensitivity and 97% specificity for the correct pre- CD4 T Cells and CXCL10 Predict Gram-Positive diction of culture-negative peritonitis (Supplemental Table 4). Infections Univariate analyses indicated that the proportion of neutro- We next attempted to identify predictors of Gram-positive phils and monocytes/macrophages among total peritoneal cells infections in an analogous way. Among all patients presenting as well as the levels of IL-1b and IL-10 had prognostic value. with acute peritonitis, patients with confirmed Gram-positive + Further multivariate analyses of these four parameters alone infections displayed larger numbers of infiltrating CD4 and + and in combination confirmed that the combination of IL-1b CD8 T cells than the rest of the patients (i.e., individuals with and IL-10 had independent prognostic significance for the pre- culture-negative or Gram-negative peritonitis) (Supplemental + diction of a culture-negative peritonitis episode on the day of Table 11). In addition, the proportion of Vd2 T cells within presentation (Supplemental Table 5). the T cell population was significantly lower in Gram-positive infections. Those inflammatory markers that significantly in- Vd2+ T Cells Predict Gram-Negative Infections creased in Gram-positive infections included IL-22 and We next tested whether pathogen-specificimmunefinger- CXCL10 (Figure 3C and Supplemental Table 12). AUROC prints existed that could predict infections by certain groups of calculations identified the combination of CXCL10 ($301.2 bacteria. PD effluent from patients with microbiologically pg/ml) and IL-22 levels ($54.3 pg/ml) and Vd2+ T cell fre- confirmed Gram-negative infections contained larger num- quencies (,2.7%) as an excellent discriminator that predicted bers of infiltrating neutrophils and consequently lower pro- Gram-positive infections (Supplemental Table 12). This com- portions of monocytes/macrophages and T cells than effluent bination also had the highest overall correctness, with 89%

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sensitivity and 67% specificity for the prediction of Gram-positive peritonitis. In addition, CXCL10 levels ($301.2 pg/ml) combined with total CD4+ T cell counts ($15.23106) had a sensitivity of only 53% yet a specificity of 95% for the prediction of Gram-positive peritonitis (Supple- mental Table 13). CD4+ Tcellcountsas well as Vd2+ T cell frequencies and CXCL10 levels had prognostic value as confirmed by univariate analyses. Multi- variate analysis of these parameters alone and in combination identified the combination of CD4+ T cell counts and CXCL10 levels as independent predictors of Gram- positive peritonitis in patients presenting with a cloudy bag (Supplemental Table 14).

Vd2+ T Cells Predict Clinical Outcome Gram-negative infections are typically associated with worse outcomes, including higher rates of technique failure and Figure 2. Peritoneal levels (in nanograms per milliliter) of IL-1b, IL-2, IL-6, sIL-6R, IL-10, mortality.24–26 Our earlier findings indi- IL-12p70, IL-17A, IL-22, CXCL8, CXCL10, IFN-g, TNF-a, GM-CSF, TGF-b1, and MMP- cated that elevated peritoneal frequencies 3 in stable PD patients and on day 1 of acute peritonitis. Patients presenting with of gd T cells within the total T cell popula- a cloudy bag are grouped according to the microbiological culture results into pa- tion on the day of presentation were asso- tients with culture-negative peritonitis (n/c) or confirmed Gram-positive or Gram- 20 negative infection. Data points represent individual patients, and horizontal lines ciated with subsequent technique failure. fi + represent mean values per group. Here, AUROC calculations identi ed Vd2 T cell frequencies $4.3% as excellent discriminator that predicted short-term technique failure within the first 1–3months (Figure 4), with an overall correctness of 94% at day 30 (4 of 48 patients with technique failure) and 77% at day 90 (9 of 48 patients) (Supplemental Tables 15–18). No other immune parameters tested, including the numbers and frequencies of neutrophils, monocytes, or CD4+ and CD8+ T cells, reached statistical significance. Taken together, these data indicate that disease- specificlocalimmunefingerprints predict not only the type of pathogen but also the associated risk of downstream complications (Table 1).

DISCUSSION

Figure 3. Examples of immunological biomarkers in patients presenting with a cloudy In a radicaldeparture from current practice, bag on day 1, depending on the microbiological culture results. (A) Discrimination our novel approach to diagnosing infec- between culture-negative and culture-positive peritonitis. (B) Discrimination between fi Gram-negative and non-Gram–negative infections. (C) Discrimination between Gram- tions exploits, for the rst time, the concept positive and non-Gram–positive infections. Data points represent individual episodes that microorganisms display distinct sets of (white, culture-negative peritonitis; blue, confirmed Gram-positive infection; red, pathogen-associated patterns and interact confirmed Gram-negative infection). Dashed lines indicate calculated cut-off values for with the immune system in a unique and positive or negative discrimination. specific manner. Conventionally, patient

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identification of low-risk patients and patients who might not require antimicrobial therapy, thereby helping to reduce unnecessary exposure to antibiotics.32,33 Our results demonstrate that designated episodes of culture-negative peritonitis can be distinguished from overt infection and have significantly different immune fingerprints that are characterized by lower numbers (and proportions) of infiltrating neutrophils and less severe inflammation, as indicated by lower peritoneal cytokine levels compared with culture-positive infections. In this respect, the cytokines IL-1b,IL-10,andTNF- a and the ratio of neutrophils to monocytes/macrophages are particularly promising candidates for a future biomarker- based diagnosis of culture-negative peritonitis. We are aware that in order to establish robust pathogen- specific immune fingerprints, accurate identification of the Figure 4. Kaplan–Meier plot showing cumulative technique causative agents is critical. Thus, for optimum stratification in survival of patients with acute peritonitis, depending on the fre- future studies and to circumvent the limitations of conven- quency of Vd2+ T cells among all peritoneal T cells on day 1. The tional culture techniques, state-of-the-art molecular methods cut-off value is determined by receiver operating characteristic such as 16S and 23S rDNA amplification, mass spectrometry, analysis, and the statistical difference between the two curves is and/or whole-sample sequencing and metagenomics might be analyzed by the log-rank test. utilized.7–11,34,35 Such approaches, however, add a further level of complication in the analysis of patient results when management is determined on the basis of symptoms, initial the data produced are inconsistent or conflicting. Conse- clinical findings, and basic laboratory markers. Yet the two quently, there is currently not sufficient evidence for recom- most widely advocated inflammatory biomarkers, C-reactive mending the routine use of molecular techniques for the protein and procalcitonin, remain of limited usefulness in the diagnosis of peritonitis.36 Our own findings suggest that there clinic because they often do not reach satisfying discriminatory is a clear microbiological and immunologic difference be- power to distinguish infectious from noninfectious inflam- tween patients with culture-negative and culture-positive mation,27,28 and have questionable relevance in patients with peritonitis, and argue against a serious bias due to potentially PD-related peritonitis.29 Our present findings demonstrate suboptimal culture techniques. It is conceivable that a pro- that different types of pathogens leave characteristic immune portion of the culture-negative episodes in this study might fingerprints early during infection that may be of diagnostic have been triggered by infectious agents (e.g., subclinical bac- and prognostic relevance, with far-reaching implications for terial or viral infection) or conversely by chemical irritation. our view of acute responses in infectious scenarios in which Because the improved outcome in that patient group does not early and accurate biomarkers are urgently needed.5,30 necessarily reflect antibiotic responsiveness, it is not possible Culture-negative peritonitis in PD patients is associated at this stage to speculate on the potential causes. This will with a fairly benign clinical course with low rates of technique require a considerably larger cohort to determine whether failure.6,31 Prediction of culture-negative episodes would thus responses to treatment in the culture-negative group can be have important implications because it might allow the further stratified and highlights the need for a more accurate

Table 1. Biomarkers of potential diagnostic and prognostic value in patients presenting with a cloudy bag on day 1, depending on the microbiological culture results and clinical outcome Culture-Negative Culture-Positive Gram-Positive Gram-Negative Technique Biomarker Peritonitis Infection Infection Infection Failure Monocytes (% of total) $19.1 ,19.1 ,10.7 CD4+ T cells (3106 cells) $15.2 Vd2+ (% of T cells) ,2.7 $6.3 $4.3 HLA-DR+ (% of gd T cells) $16.1 CD86+ (% of monocytes) ,67.9 IL-1b (pg/ml) ,4.1 $4.1 $14.0 IL-10 (pg/ml) ,23.3 $23.3 $110.1 IL-22 (pg/ml) $54.3 CXCL10 (pg/ml) $301.2 TNF-a (pg/ml) ,19.5 $19.5 $32.8

2006 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 2002–2009, 2013 www.jasn.org BASIC RESEARCH diagnostic approach to therapy including immune finger- tissue damageon thedayofpresentation. Our research highlights printing. the importance ofdetailed immunemonitoringcomprisingboth Although it is well established that patients with Gram- humoral and cellular parameters to establish accurate immune negative and Gram-positive infections present with different fingerprints. Although cellular analyses typically require trained clinical signs and outcomes,24–26 there is no rapid diagnostic laboratory staff and central equipment such as flow cytometers, test that would guide treatment and allow predictive risk mod- recent advances in microfluidics have made the development eling at the time of presentation. Here, immune fingerprints in of accurate and affordable single-use chip devices a realistic Gram-positive infections were markedly different from those option.42–44 This promising technological progress is likely to in Gram-negative infections and were indicative of a relatively allow routine monitoring of complex immune fingerprints in large underlying T cell component with higher numbers of infected patients in the near future, suggesting that rapid differ- CD4+ and CD8+ T cells and elevated levels of CXCL10, IFN-g, ential biomarker-based diagnosis and guided management of and IL-22. The chemokine CXCL10 appeared to be a partic- noninfectious and infectious syndromes at the point of care ularly good predictor of Gram-positive infections in this study, are feasible. which might have stemmed from stimulation of monocytes/ macrophages or mesothelial cells by certain microbial com- pounds such as staphylococcal a-toxin and Toll-like receptor 2 CONCISE METHODS ligands.37–39 Gram-negative infections were dominated by large num- Patients bers of infiltrating neutrophils and elevated levels of cytokines The study cohort comprised 52 adult PD patients admitted on day 1 of such as IL-1b, IL-10, and TNF-a. A proportion of these im- acute peritonitis between September 2008 and January 2012; 15 age- mune fingerprints were likely due to pronounced responses to and sex-matched stable patients with no infection in the previous bacterial LPS, which is present in the outer cell wall of Gram- 3 months were included as controls. Diagnosis of acute peritonitis was negative organisms but absent from Gram-positive organisms. based on the presence of abdominal pain and cloudy peritoneal LPS is a powerful inducer of proinflammatory cytokines and effluent with .100 white blood cells/mm3. Infections were grouped chemokines by monocytes/macrophages and neutrophils, in- into culture-negative, Gram-positive, and Gram-negative episodes, cluding IL-1b,TNF-a, and CXCL8. At the same time, LPS according to the result of the microbiological analysis of the effluent stimulation also leads to secretion or large amounts of by the routine Microbiology Laboratory, Public Health Wales; cases IL-6 and IL-10 by monocytes/macrophages (but not hu- of fungal infection (n=1) and mixed Gram-negative/Gram-positive man neutrophils).40 culture results (n=1) were excluded from this analysis. End points of Another powerful microbial factor present in the majority outcome analyses were 30th-day and 90th-day technique failure of Gram-negative bacteria (including the pathogens identified (catheter removal, transfer to hemodialysis, and/or patient death); in this study) is the isoprenoid precursor (E)-4-hydroxy-3- two patients who stopped their treatment because of noninfection- methyl-but-2-enyl pyrophosphate (HMB-PP), the natural related reasons were removed from the outcome analysis. This study activator of human Vg9/Vd2 T cells. In contrast, many wasapprovedbytheSouthEastWalesLocalEthicsCommittee Gram-positive bacteria do not produce HMB-PP and conse- (04WSE04/27), and conducted according to the principles expressed quently do not activate Vg9/Vd2 T cells.20 Despite the very in the Declaration of Helsinki. All patients provided written informed low proportion of T cells in acute Gram-negative infections, consent. Vg9/Vd2 T cells were selectively enriched among peritoneal T cells in those patients, thereby singling out Vg9/Vd2 T cells as Flow Cytometry highly sensitive and selective responder cells of diagnostic and Peritoneal cells were acquired on an eight-color FACSCanto II (BD prognostic value in acute peritonitis. This observation also has Biosciences) and analyzed with FlowJo (TreeStar), using mAbs against direct therapeutic implications because fosmidomycin, a nat- CD3 (UCHT1), CD4 (SK3), CD8 (RPA-T8), CD15 (HI98), CD69 ural antibiotic that specifically targets the HMB-PP pathway in (FN50), CD86 (2331-FUN1), HLA-DR (L234), and TCR-Vd2 (B6.1) many pathogens including multidrug-resistant Gram-negative from BD Biosciences; CD14 (61D3) from eBioscience; and TCR-Vg9 bacteria,41 possesses anti-inflammatory properties by inhibiting (Immu360) from Beckman Coulter, together with appropriate iso- Vg9/Vd2Tcell–driven immune responses to HMB-PP–producing type controls. Leukocyte populations were gated based on their bacteria.20 appearance in side scatter and forward scatter area/height, and ex- Taken together, this study provides proof of concept for clusion of live/dead staining (fixable Aqua; Invitrogen). measuring disease-specific immune responses as discriminatory markers of early infection. These responses are likely to be ELISA triggered by unique combinations of pathogen-associated Cell-free peritoneal effluents were analyzed for IL-1b, IL-2, IL-6, sIL- molecular patterns interacting with distinct aspects of the 6R, IL-10, IL-12p70, CXCL8, IFN-g,TNF-a, GM-CSF, and MMP-3 human immune system. At the same time, these results lay the on a SECTOR Imager 6000 (Meso Scale Discovery). IL-17A, IL-22, foundation for targeted therapies that modulate disease-specific and CXCL10 (R&D Systems) as well as TGF-b1 (eBioscience) were pathways to fight infection and reduce inflammation-related measured on a Dynex MRX II reader, using conventional ELISA kits.

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Statistical Analyses 7. Shafazand S, Weinacker AB: Blood cultures in the critical care unit: Statistical analyses were performed using SPSS 16.0 and GraphPad Improving utilization and yield. Chest 122: 1727–1736, 2002 Prism 4.0 software. All variables were tested for normal distributions 8. Ince J, McNally A: Development of rapid, automated diagnostics for using the Kolmogorov–Smirnov test. Differences between patient infectious disease: Advances and challenges. Expert Rev Med Devices 6: 641–651, 2009 groups were analyzed using t tests for normally distributed data or 9. Claus RA, Otto GP, Deigner HP, Bauer M: Approaching clinical reality: Mann–Whitney U tests for nonparametric data. Predictive biomark- markers for monitoring systemic inflammation and sepsis. Curr Mol ers were assessed using univariate analyses; statistically significant Med 10: 227–235, 2010 (P,0.05) variables from the univariate analysis were included in a 10. Clerc O, Greub G: Routine use of point-of-care tests: Usefulness and – multivariate analysis. Multiple logistic regression analyses were con- application in clinical microbiology. Clin Microbiol Infect 16: 1054 1061, 2010 ducted based on forward and/or backward elimination of data, as 11. Leggieri N, Rida A, François P, Schrenzel J: Molecular diagnosis of indicated in the tables. AUROC analyses were used to assess discrim- bloodstream infections: Planning to (physically) reach the bedside. Curr ination using nonparametric approaches, and to calculate cut-off Opin Infect Dis 23: 311–319, 2010 values as well as sensitivity and specificity. Cumulative survival curves 12. Chan T, Gu F: Early diagnosis of sepsis using serum biomarkers. Expert were generated using the Kaplan-Meier approach and compared us- Rev Mol Diagn 11: 487–496, 2011 fi 13. Yu VL: Guidelines for hospital-acquired pneumonia and health-care- ing log-rank tests. Differences were considered statistically signi cant fl fi , , associated pneumonia: A vulnerability, a pitfall, and a fatal aw. Lancet as indicated in the gures and tables (*P 0.05; **P 0.01; Infect Dis 11: 248–252, 2011 ***P,0.001). 14. Llewelyn MJ, Cohen J: Tracking the microbes in sepsis: Advancements in treatment bring challenges for microbial epidemiology. Clin Infect Dis 44: 1343–1348, 2007 15. Brown MC, Simpson K, Kerssens JJ, Mactier RA; Scottish Renal Regis- ACKNOWLEDGMENTS try: Peritoneal dialysis-associated peritonitis rates and outcomes in a national cohort are not improving in the post-millennium (2000-2007). – We are grateful to all patients for participating in this study. We also Perit Dial Int 31: 639 650, 2011 16. Li PK, Chow KM: Infectious complications in dialysis—epidemiology thank the staff on the B5 ward for their cooperation, and James Chess, and outcomes. Nat Rev Nephrol 8: 77–88, 2012 Chantal Colmont, Eryl Cox, Donald Fraser, David Johnson, Bernhard 17. Hurst SM, Wilkinson TS, McLoughlin RM, Jones S, Horiuchi S, Moser, Ian Weeks, and John Williams for their input. Yamamoto N, Rose-John S, Fuller GM, Topley N, Jones SA: Il-6 and its This research was supported by the National Institute for Social soluble receptor orchestrate a temporal switch in the pattern of leu- fl – Care and Health Research (NISCHR) Clinical Research Centre kocyte recruitment seen during acute in ammation. Immunity 14: 705 714, 2001 (UKCRN ID #11838, PERIT-PD), the NISCHR Academic Health 18. Roberts GW, Baird D, Gallagher K, Jones RE, Pepper CJ, Williams JD, Science Collaboration, Welsh Assembly Government (grants RFS/ Topley N: Functional effector memory T cells enrich the peritoneal H07-03-18 and RFS/HA09-009), and Baxter Healthcare (Renal Dis- cavity of patients treated with peritoneal dialysis. JAmSocNephrol20: coveries extramural grant #08AP002). 1895–1900, 2009 19. Eberl M, Roberts GW, Meuter S, Williams JD, Topley N, Moser B: A rapid crosstalk of human gammadelta T cells and monocytes drives the acute inflammation in bacterial infections. PLoS Pathog 5: e1000308, DISCLOSURES 2009 None. 20. Davey MS, Lin CY, Roberts GW, Heuston S, Brown AC, Chess JA, Toleman MA, Gahan CG, Hill C, Parish T, Williams JD, Davies SJ, Johnson DW, Topley N, Moser B, Eberl M: Human neutrophil clearance REFERENCES of bacterial pathogens triggers anti-microbial gd Tcellresponsesin early infection. PLoS Pathog 7: e1002040, 2011 21. Blander JM, Sander LE: Beyond pattern recognition: Five immune 1. Binder S, Levitt AM, Sacks JJ, Hughes JM: Emerging infectious dis- checkpoints for scaling the microbial threat. Nat Rev Immunol 12: 215– – eases: Public health issues for the 21st century. Science 284: 1311 225, 2012 1313, 1999 22. Feezor RJ, Oberholzer C, Baker HV, Novick D, Rubinstein M, Moldawer 2. Livermore DM: Has the era of untreatable infections arrived? JAnti- LL, Pribble J, Souza S, Dinarello CA, Ertel W, Oberholzer A: Molecular – microb Chemother 64[Suppl 1]: i29 i36, 2009 characterization of the acute inflammatory response to infections with — 3. Arias CA, Murray BE: Antibiotic-resistant bugs in the 21st century gram-negative versus gram-positive bacteria. Infect Immun 71: 5803– a clinical super-challenge. NEnglJMed360: 439–443, 2009 5813, 2003 4. Fauci AS, Morens DM: The perpetual challenge of infectious diseases. 23. Ramilo O, Allman W, Chung W, Mejias A, Ardura M, Glaser C, NEnglJMed366: 454–461, 2012 Wittkowski KM, Piqueras B, Banchereau J, Palucka AK, Chaussabel D: 5. Davies SC: 2011 Annual Report of the Chief Medical Officer: Infections Gene expression patterns in blood leukocytes discriminate patients and the Rise of Antimicrobial Resistance, Vol. 2, London, Department of with acute infections. Blood 109: 2066–2077, 2007 Health, 2013. Available at: https://www.gov.uk/government/publications/ 24. Abe R, Oda S, Sadahiro T, Nakamura M, Hirayama Y, Tateishi Y, chief-medical-officer-annual-report-volume-2. Accessed August 14, Shinozaki K, Hirasawa H: Gram-negative bacteremia induces greater 2013 magnitude of inflammatory response than Gram-positive bacteremia. 6. Fahim M, Hawley CM, McDonald SP, Brown FG, Rosman JB, Wiggins Crit Care 14:R27,2010 KJ, Bannister KM, Johnson DW: Culture-negative peritonitis in perito- 25. Krishnan M, Thodis E, Ikonomopoulos D, Vidgen E, Chu M, Bargman neal dialysis patients in Australia: Predictors, treatment, and outcomes JM, Vas SI, Oreopoulos DG: Predictors of outcome following bacterial in 435 cases. Am J Kidney Dis 55: 690–697, 2010 peritonitis in peritoneal dialysis. Perit Dial Int 22: 573–581, 2002

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26. Troidle L, Gorban-Brennan N, Kliger A, Finkelstein F: Differing out- kines produced by mesothelial cells: huGRO-alpha, IP-10, MCP-1 and comes of gram-positive and gram-negative peritonitis. Am J Kidney Dis RANTES. Clin Exp Immunol 112: 270–275, 1998 32: 623–628, 1998 38. Colmont CS, Raby AC, Dioszeghy V, Lebouder E, Foster TL, Jones SA, 27. Simon L, Saint-Louis P, Amre DK, Lacroix J, Gauvin F: Procalcitonin and Labéta MO, Fielding CA, Topley N: Human peritoneal mesothelial cells C-reactive protein as markers of bacterial infection in critically ill chil- respond to bacterial ligands through a specific subset of Toll-like re- dren at onset of systemic inflammatory response syndrome. Pediatr Crit ceptors. Nephrol Dial Transplant 26: 4079–4090, 2011 Care Med 9: 407–413, 2008 39. Kasraie S, Niebuhr M, Kopfnagel V, Dittrich-Breiholz O, Kracht M, 28. Tang BM, Eslick GD, Craig JC, McLean AS: Accuracy of procalcitonin Werfel T: Macrophages from patients with atopic dermatitis show a for sepsis diagnosis in critically ill patients: Systematic review and meta- reduced CXCL10 expression in response to staphylococcal a-toxin. analysis. Lancet Infect Dis 7: 210–217, 2007 Allergy 67: 41–49, 2012 29. Lam MF, Leung JC, Lam CW, Tse KC, Lo WK, Lui SL, Chan TM, Tam S, 40. Davey MS, Tamassia N, Rossato M, Bazzoni F, Calzetti F, Bruderek K, Lai KN: Procalcitonin fails to differentiate inflammatory status or predict Sironi M, Zimmer L, Bottazzi B, Mantovani A, Brandau S, Moser B, Eberl long-term outcomes in peritoneal dialysis-associated peritonitis. Perit M, Cassatella MA: Failure to detect production of IL-10 by activated – – Dial Int 28: 377–384, 2008 human neutrophils. Nat Immunol 12: 1017 1018, author reply 1018 30. Kessel AS, Sharland M: The new UK antimicrobial resistance strategy 1020, 2011 andactionplan.BMJ 346: f1601, 2013 41. Davey MS, Tyrrell JM, Howe RA, Walsh TR, Moser B, Toleman MA, 31. Chen KH, Chang CT, Weng SM, Yu CC, Fang JT, Huang JY, Yang CW, Eberl M: A promising target for treatment of multidrug-resistant bac- – Hung CC: Culture-negative peritonitis: A fifteen-year review. Ren Fail terial infections. Antimicrob Agents Chemother 55: 3635 3636, 2011 29: 177–181, 2007 42. Frankowski M, Bock N, Kummrow A, Schädel-Ebner S, Schmidt M, fl 32. Choi P, Nemati E, Banerjee A, Preston E, Levy J, Brown E: Peritoneal Tuchscheerer A, Neukammer J: A micro ow cytometer exploited for – dialysis catheter removal for acute peritonitis: A retrospective analysis the immunological differentiation of leukocytes. Cytometry A 79: 613 624, 2011 of factors associated with catheter removal and prolonged post- 43. Diaw PA, Daneau G, Coly AA, Ndiaye BP, Wade D, Camara M, Mboup operative hospitalization. AmJKidneyDis43: 103–111, 2004 S, Kestens L, Dieye TN: Multisite evaluation of a point-of-care in- 33. Rocklin MA, Teitelbaum I: Noninfectious causes of cloudy peritoneal strument for CD4(+) T-cell enumeration using venous and finger-prick dialysate. Semin Dial 14: 37–40, 2001 blood: the PIMA CD4. JAcquirImmuneDeficSyndr58: e103–e111, 34. Woo PC, Lau SK, Teng JL, Tse H, Yuen KY: Then and now: Use of 16S 2011 rDNA gene sequencing for bacterial identification and discovery of 44. Ma C, Fan R, Ahmad H, Shi Q, Comin-Anduix B, Chodon T, Koya RC, Liu novel bacteria in clinical microbiology laboratories. Clin Microbiol In- CC, Kwong GA, Radu CG, Ribas A, Heath JR: A clinical microchip for fect 14: 908–934, 2008 evaluation of single immune cells reveals high functional heterogeneity 35. Nakamura S, Nakaya T, Iida T: Metagenomic analysis of bacterial in- in phenotypically similar T cells. Nat Med 17: 738–743, 2011 fections by means of high-throughput DNA sequencing. Exp Biol Med (Maywood) 236: 968–971, 2011 36. Li PK, Szeto CC, Piraino B, Bernardini J, Figueiredo AE, Gupta A, Johnson DW, Kuijper EJ, Lye WC, Salzer W, Schaefer F, Struijk DG; International See related editorial, “Cloudy Peritoneal Dialysate: In Search of a Clear Society for Peritoneal Dialysis: Peritoneal dialysis-related infections rec- Cause?,” on pages 1929–1931. ommendations: 2010 update. Perit Dial Int 30: 393–423, 2010 37. Visser CE, Tekstra J, Brouwer-Steenbergen JJ, Tuk CW, Boorsma DM, This article contains supplemental material online at http://jasn.asnjournals. Sampat-Sardjoepersad SC, Meijer S, Krediet RT, Beelen RH: Chemo- org/lookup/suppl/doi:10.1681/ASN.2013040332/-/DCSupplemental.

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