Effects of clinically achievable pulmonary antibiotic concentrations on the recovery of bacteria: in vitro comparison of the BioFire FILMARRAY Pneumonia Panel versus conventional culture methods in bronchoalveolar lavage fluid.

Empiric antibiotics may affect bacterial pathogen recovery using conventional culture methods (CCMs), while PCR-based diagnostics are likely less affected. Herein, we conducted an in vitro study of bronchoalveolar lavage fluid (BAL) inoculated with bacteria and clinically relevant antibiotic concentrations to compare the recovery between the BioFire FILMARRAY Pneumonia Panel (Pn Panel) and CCMs. Remnant clinical BAL specimens were inoculated to ~105 cfu/mL using 12 clinical isolates. Isolates consisted of one wild-type (WT) and one or more resistant strains of: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus. Piperacillin-tazobactam, cefepime, meropenem, levofloxacin, or vancomycin was added to achieve pulmonary epithelial lining fluid peak and trough concentrations. Post-exposure cfu/mL was quantified by CCMs and simultaneously tested by the PN Panel for identification and semi-quantitative genetic copies/mL. CCM results were categorized as significant growth (SG) (≥1 × 104), no significant growth (NSG) (≥1 × 103, <1 × 104), or no growth (NG) (<1 × 103). The PN Panel accurately identified all isolates, resistance genes, and reported ≥106 genetic copies/mL regardless of antibiotic exposure. The CCM also identified all S. aureus strains exposed to vancomycin. For WT Gram-negative isolates exposed to antibiotics, SG, NSG, and NG were observed in 7/52 (13%), 18/52 (35%), and 27/52 (52%) of CCM experiments, respectively. For resistant Gram-negatives isolates, SG, NSG, and NG were observed in 62/88 (70%), 17/88 (19%), and 9/88 (10%), respectively. These in vitro data demonstrate that the PN Panel is able to identify Gram-negative pathogens in the presence of clinically significant antibiotic concentrations when CCM may not.

The Effect of Commonly Used Local Anesthetic on Bacterial Growth.

INTRODUCTION: Liposomal bupivacaine (LB) has emerged as a superior form of local anesthetic across numerous surgical subspecialties. The purpose of this study is to evaluate the ex-vivo antimicrobial effects of LB in comparison with traditional local anesthetics. METHODS: A standardized inoculum of bacteria commonly associated with surgical site infection was inoculated into a suspension of 1% lidocaine, 0.25% bupivacaine, Exparel (proprietary liposomally packaged 1.3% bupivacaine), and normal saline as a growth control. RESULTS: In all five bacteria tested, the medium inoculated with traditional local anesthetics reduced growth to a greater degree than LB-inoculated plates. Both conventional local anesthetics reduced the growth of all bacteria when compared with the control with the exception of methicillin-susceptible Staphylococcus aureus growth in bupivacaine. LB-inoculated plates had equivalent growth to the control in all plates with the exception of Escherichia coli plates which demonstrated superior growth. CONCLUSIONS: The results of this simple ex-vivo model suggest that the liposomal packaging of bupivacaine may decrease this local anesthetic's innate antibacterial properties.

ERAP1 association with ankylosing spondylitis is attributable to common genotypes rather than rare haplotype combinations.

We investigated the proposal that ankylosing spondylitis (AS) is associated with unusual ERAP1 genotypes. ERAP1 haplotypes were constructed for 213 AS cases and 46 rheumatoid arthritis controls using family data. Haplotypes were generated from five common ERAP1 single nucleotide polymorphisms (SNPs)-rs2287987 (M349V), rs30187 (K528R), rs10050860 (D575N), rs17482078 (R725Q), and rs27044 (Q730E). Haplotype frequencies were compared using Fisher's exact test. ERAP1 haplotypes imputed from the International Genetics of AS Consortium (IGAS) Immunochip study were also studied. In the family study, we identified only four common ERAP1 haplotypes ("VRNQE," "MKDRQ," "MRDRE," and "MKDRE") in both AS cases and controls apart from two rare (<0.5%) previously unreported haplotypes. There were no examples of the unusual ERAP1 haplotype combination ("*001/*005") previously reported by others in 53% of AS cases. As expected, K528-bearing haplotypes were increased in the AS family study (AS 43% vs. control 35%), due particularly to an increase in the MKDRQ haplotype (AS 35% vs. control 25%, P = 0.01). This trend was replicated in the imputed Immunochip data for the two K528-bearing haplotypes MKDRQ (AS 33% vs. controls 27%, P = 1.2 × 10-24) and MKDRE (AS 8% vs. controls 7%, P = 0.004). The ERAP1 association with AS is therefore predominantly attributable to common ERAP1 haplotypes and haplotype combinations.

The severity of ankylosing spondylitis and responses to anti-tumour necrosis factor biologics are not influenced by the tumour necrosis factor receptor polymorphism incriminated in multiple sclerosis.

Genetic polymorphism (rs1800693) of TNFRSF1A (type 1 tumour necrosis factor receptor) encodes a potentially anti-inflammatory soluble truncated form of the p55 receptor, which is associated with predisposition to multiple sclerosis but protection against ankylosing spondylitis (AS). We analysed 2917 UK Caucasian cases by linear and logistic regression for associations of rs1800693 with disease severity assessed by the Bath Ankylosing Spondylitis measures of disease activity and function (BASDAI, BAS-G and BASFI) and/or responses to anti-TNF therapy. In contrast to predictions, rs1800693 GG homozygotes actually had significantly worse BASDAI (mean 4.2, 95% CI: 4-4.5) than AA homozygotes (mean 3.8, 95% CI: 3.7-4) in both the unadjusted (difference = 0.4, p = 0.006) and adjusted analyses (difference = 0.2-0.5, p = 0.002-0.04 depending on the adjustment model). We found no evidence that rs1900693 predicted functional status (BASFI) or global disease scores (BAS-G), and it exerted no influence on either the intention to treat with or efficacy of anti-TNF treatment.

ANALYSIS OF PARS PLANA VITRECTOMY INCISIONS USING LIVE BACTERIA.

PURPOSE: To analyze small gauge pars plana vitrectomy sclerotomies using live bacteria transformed with green fluorescent protein (GFP). METHODS: Twenty-eight human cadaver eyes were specially harvested for this study. Small gauge vitrectomy was performed on each eye and the wounds were closed with various techniques (sutured, sutureless, and cauterization). Live Staphylococcus epidermidis that has been transformed with a green fluorescent protein was applied to the overlying conjunctival surface. Analysis of all vitreous samples was analyzed with confocal laser microscopy to identify the presence of bacteria. All wounds were analyzed histopathologically. RESULTS: A high concentration of bacteria was noted in 2 of 3 eyes in the sutureless, 23-G perpendicular incision group postinoculation. There were no bacteria detected in any postvitrectomy sample that were closed with cautery or a beveled incision. No bacteria were found in postvitrectomy samples of sutureless 27-G perpendicular incisions and sutureless 27-G beveled incisions. Finally, there were no bacteria detected in both eyes with 23-G perpendicular incisions that had a partial air-fill. CONCLUSION: Live bacteria can be effectively used to analyze wound integrity. Closing sclerotomy sites with cautery proved effective in a model using fresh, human cadaver eyes. 27-G perpendicular incisions may be just as competent as 27-G beveled incisions.

The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression.

OBJECTIVES: To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). METHODS: We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. RESULTS: The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10(-14)) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 'G' allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk 'A' allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). CONCLUSION: We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS.

An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation.

OBJECTIVES: To explore the functional basis for the association between ankylosing spondylitis (AS) and single-nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region. METHODS: We performed conditional analysis on genetic association data and used epigenetic data on chromatin remodelling and transcription factor (TF) binding to identify the primary AS-associated IL23R-IL12RB2 intergenic SNP. Functional effects were tested in luciferase reporter assays in HEK293T cells and allele-specific TF binding was investigated by electrophoretic mobility gel shift assays. IL23R and IL12RB2 mRNA levels in CD4+ T cells were compared between cases homozygous for the AS-risk 'A' allele and the protective 'G' allele. The proportions of interleukin (IL)-17A+ and interferon (IFN)-γ+ CD4+ T-cells were measured by fluorescence-activated cell sorting and compared between these AS-risk and protective genotypes. RESULTS: Conditional analysis identified rs11209032 as the probable causal SNP within a 1.14 kb putative enhancer between IL23R and IL12RB2. Reduced luciferase activity was seen for the risk allele (p<0.001) and reduced H3K4me1 methylation observed in CD4+ T-cells from 'A/A' homozygotes (p=0.02). The binding of nuclear extract to the risk allele was decreased ∼3.5-fold compared with the protective allele (p<0.001). The proportion of IFN-γ+ CD4+ T-cells was increased in 'A/A' homozygotes (p=0.004), but neither IL23R nor IL12RB2 mRNA was affected. CONCLUSIONS: The rs11209032 SNP downstream of IL23R forms part of an enhancer, allelic variation of which may influence Th1-cell numbers. Homozygosity for the risk 'A' allele is associated with more IFN-γ-secreting (Th1) cells. Further work is necessary to explain the mechanisms for these important observations.

Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates.

This study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with blaNDM, blaIMP, and blaOXA-48.

Bacterial Contamination in Tips of Electrocautery Devices During Total Hip Arthroplasty.

Surgical equipment can become contaminated during surgery. It is unknown if electrocautery tips can become contaminated in clean orthopedic procedures despite the produced heat. Therefore, we conducted a prospective study to address this concern. The tips from 25 primary and 25 aseptic revision THAs were collected and an additional 5 sterile tips served as negative controls. Aerobic and anaerobic cultures were incubated for a minimum of 3 days. There were 3 positive cultures (6%); one in primary THA (4%) with Lactobacillus and Enterococcus faecalis; two among revisions (8%), one with E. faecalis and another one with alpha hemolytic streptococci and coagulase negative Staphylococcus. The mean exposure time of the contaminated tips was 132.3 minutes. Patients were followed for 90 days postoperatively and none of them developed surgical site infection. This is the first study to demonstrate that electrosurgical devices can become contaminated during THA in laminar flow equipped operating rooms.

Non-telomeric epigenetic and genetic changes are associated with the inheritance of shorter telomeres in mice.

Studies using human and mouse cells have revealed some changes to non-telomeric chromatin and gene expression in response to abnormally short telomeres. To investigate this further, we studied the effect of inheriting shorter telomeres on transcription and genetic stability at non-telomeric sites in the mouse. Using multiple generations of Terc knockout mice, we show that inheriting shorter telomeres from one parent increases the likelihood of transcriptional silencing at a non-telomeric green fluorescent protein (GFP) transgene inherited from the other parent. In these cases, silencing must occur at or after zygote formation. In grand-offspring from a G3 Terc (-/-) parent, transgene expression was further reduced and associated with increased DNA methylation and, surprisingly, reduced copy number at the transgene array. In these cases, the transgene had been passed through the germline of a Terc-compromised parent, providing an opportunity for meiotic events. Furthermore, genome-wide microarray analysis of copy number variations revealed greater genetic instability in G3 Terc (-/-) mice than detected in wild-type mice of the same genetic background. Our results have implications for the molecular mechanisms underlying premature-ageing syndromes, such as dyskeratosis congenita. In autosomal-dominant dyskeratosis congenita, progressive telomere shortening is seen as it passes down the generations, and this is associated with anticipation, i.e. the disease becomes more severe earlier. The underlying mechanism is not known, but has been considered to be simply associated with decreases in telomere length. Epigenetic and/or genetic changes at non-telomeric regions could, in theory, be involved.

Mycobacterium avium complex (MAC) Immune Reconstitution Syndrome (IRIS) With Reduced Susceptibility to Ethambutol in an HIV-Infected Patient.

OBJECTIVE: To describe a case of Mycobacterium avium complex (MAC) lymphadenitis complicated by immune reconstitution syndrome (IRIS) and reduced susceptibility to ethambutol. CASE SUMMARY: A 24-year-old man was diagnosed in October 2012 with advanced HIV infection upon hospitalization for multiple opportunistic infections (OIs). Within 5 months of starting antiretroviral therapy, the patient developed significant cervical lymphadenopathy concerning for MAC/IRIS. Acid-fast bacilli were detected in the primary lymph node biopsy smear, and culture results confirmed the presence of MAC. Susceptibility testing revealed an organism susceptible to azithromycin, with an elevated minimum inhibitory concentration (MIC) to ethambutol (8 µg/mL). Currently, there is no interpretation for an ethambutol MIC of 8 µg/mL for MAC. A review of the primary literature revealed the possibility of decreased ethambutol susceptibility when the MIC is above 1 µg/mL, and therefore, therapy was replaced by rifabutin in combination with azithromycin. DISCUSSION: Current guidelines recommend a 2-drug regimen for the treatment of MAC, specifically a macrolide plus ethambutol. Guidelines also emphasize MAC susceptibility testing for macrolides only. Susceptibility results from this patient's biopsy prompted an evaluation of the effectiveness of his antimycobacterial regimen. CONCLUSIONS: Reduced ethambutol susceptibility in this patient triggered a search of the primary literature that resulted in the decision to replace ethambutol with rifabutin. Additional clinical trials are needed to define susceptibility breakpoints for ethambutol and other antimycobacterial agents used for MAC infection treatment and to direct clinical decisions when elevated MICs to primary agents are identified.

No evidence for cumulative effects in a Dnmt3b hypomorph across multiple generations.

Observations of inherited phenotypes that cannot be explained solely through genetic inheritance are increasing. Evidence points to transmission of non-DNA molecules in the gamete as mediators of the phenotypes. However, in most cases it is unclear what the molecules are, with DNA methylation, chromatin proteins, and small RNAs being the most prominent candidates. From a screen to generate novel mouse mutants of genes involved in epigenetic reprogramming, we produced a DNA methyltransferase 3b allele that is missing exon 13. Mice that are homozygous for the mutant allele have smaller stature and reduced viability, with particularly high levels of female post-natal death. Reduced DNA methylation was also detected at telocentric repeats and the X-linked Hprt gene. However, none of the abnormal phenotypes or DNA methylation changes worsened with multiple generations of homozygous mutant inbreeding. This suggests that in our model the abnormalities are reset each generation and the processes of transgenerational epigenetic reprogramming are effective in preventing their inheritance.

Directed carbapenemase testing is no longer just for Enterobacterales: cost, labor, and workflow assessment of expanding carbapenemase testing to carbapenem-resistant P. aeruginosa.

Molecular carbapenem-resistance testing, such as for the presence of carbapenemases genes, is commonly implemented for the detection of carbapenemase-producing Enterobacterales. Carbapenemase-producing P. aeruginosa is also associated with significant morbidity and mortality, although; prevalence may be underappreciated in the United States due to a lack of carbapenemase testing. The present study sought to compare hands-on time, cost and workflow implementation of carbapenemase gene testing in Enterobacterales and P. aeruginosa isolates versus sending out isolates to a public health laboratory (PHL) for testing to assess if in-house can provide actionable results. The time to carbapenemase gene results were compared. Differences in cost for infection prevention measures were extrapolated from the time of positive carbapenemase gene detection in-house versus PHL. The median time to perform carbapenemase gene testing was 7.5 min (range 5-14) versus 10 min (range 8-22) for preparation to send isolates to the PHL. In-house testing produced same day results compared with a median of 6 days (range 3-14) to receive results from PHL. Cost of in-house testing and send outs were similar ($46.92 versus $40.53, respectively). If contact precautions for patients are implemented until carbapenemase genes are ruled out, in-house testing can save an estimated $76,836.60 annually. Extension of in-house carbapenemase testing to include P. aeruginosa provides actionable results 3-14 days earlier than PHL Standard Pathway testing, facilitating guided therapeutic decisions and infection prevention measures. Supplemental phenotypic algorithms can be implemented to curb the cost of P. aeruginosa carbapenemases testing by identifying isolates most likely to harbour carbapenemases.

Reduced dosage of the modifiers of epigenetic reprogramming Dnmt1, Dnmt3L, SmcHD1 and Foxo3a has no detectable effect on mouse telomere length in vivo.

Studies carried out in cultured cells have implicated modifiers of epigenetic reprogramming in the regulation of telomere length, reporting elongation in cells that were null for DNA methyltransferase DNA methyltransferase 1 (Dnmt1), both de novo DNA methyltransferases, Dnmt3a and Dnmt3b or various histone methyltransferases. To investigate this further, we assayed telomere length in whole embryos or adult tissue from mice carrying mutations in four different modifiers of epigenetic reprogramming: Dnmt1, DNA methyltransferase 3-like, structural maintenance of chromosomes hinge domain containing 1, and forkhead box O3a. Terminal restriction fragment analysis was used to compare telomere length in homozygous mutants, heterozygous mutants and wild-type littermates. Contrary to expectation, we did not detect overall lengthening in the mutants, raising questions about the role of epigenetic processes in telomere length in vivo.

Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage.

BACKGROUND: Group A Streptococcus (GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy. METHODS: Blinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis. RESULTS: Immunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from in vivo animal models. CONCLUSION: Our study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine. TRIAL REGISTRATION: The tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.

Impact of Direct From Blood Culture Identification of Pathogens Paired With Antimicrobial Stewardship Interventions in a Pediatric Hospital.

OBJECTIVE: Identification of organisms directly from positive blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has the potential for improved clinical outcomes through earlier organism identification and shorter time to appropriate clinical intervention. The uses of this technology in pediatric patients and its impact in this patient population have not been well described. METHODS: Direct from positive blood culture organism identification via MALDI-TOF was implemented in September 2019. A quality improvement project was performed to assess its impact on admissions for contaminant blood cultures and time to effective and optimal antimicrobials and clinical decision-making. A pre- and post-implementation retrospective review for consecutive September through February time periods, was conducted on patients with positive monomicrobial blood cultures. Statistics were evaluated using Mann-Whitney U and χ2 tests. RESULTS: One hundred nineteen patients with 131 unique blood cultures (65 in pre- and 66 in post-implementation) were identified. Time to identification was shorter, median 35.4 hours (IQR, 22.7-54.3) versus 42.3 hours (IQR, 36.5-49) in post- and pre-groups, respectively (p = 0.02). Patients were less likely to be admitted for a contaminated blood culture in the post-implementation, 26% versus 11% in the pre-implementation (p = 0.03) group. In patients treated for bacteremia, there was a shorter time to optimal therapy from Gram stain reporting in the post-implementation (median 42.7 hours [IQR, 27.2-72]) versus pre-implementation (median 60.8 hours [IQR, 42.9-80.6]) (p = 0.03). CONCLUSIONS: Direct from positive blood culture identification by MALDI-TOF decreased time to effective and optimal antimicrobials and decreased unnecessary admission in pediatric patients for contaminated blood cultures.

Loss of the group A Streptococcus regulator Srv decreases biofilm formation in vivo in an otitis media model of infection.

Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the role of GAS in otitis media is underappreciated. In this study, we sought to test the hypothesis that GAS colonizes the middle ear and establishes itself in localized, three-dimensional communities representative of biofilms. To test this hypothesis, the middle ears of chinchillas were infected with either a strain of GAS capable of forming biofilms in vitro (MGAS5005) or a strain deficient in biofilm formation due to the lack of the transcriptional regulator Srv (MGAS5005 Δsrv). Infection resulted in the formation of large, macroscopic structures within the middle ears of MGAS5005- and MGAS5005 Δsrv-infected animals. Plate counts, scanning electron microscopy, LIVE/DEAD staining, and Gram staining revealed a difference in the distributions of MGAS5005 versus MGAS5005 Δsrv in the infected samples. High numbers of CFU of MGAS5005 Δsrv were isolated from the middle ear effusion, and MGAS5005 Δsrv was found randomly distributed throughout the excised macroscopic structure. In contrast, MGAS5005 was found in densely packed microcolonies indicative of biofilms within the excised material from the middle ear. CFU levels of MGAS5005 from the effusion were significantly lower than that of MGAS5005 Δsrv early during the course of infection. Allelic replacement of the chromosomally encoded streptococcal cysteine protease (speB) in the MGAS5005 Δsrv background restored biofilm formation in vivo. Interestingly, our results suggest that GAS naturally forms a biofilm during otitis media but that biofilm formation is not required to establish infection following transbullar inoculation of chinchillas.

RUNX3 and T-Bet in Immunopathogenesis of Ankylosing Spondylitis-Novel Targets for Therapy?

Susceptibility to ankylosing spondylitis (AS) is polygenic with more than 100 genes identified to date. These include HLA-B27 and the aminopeptidases (ERAP1, ERAP2, and LNPEPS), which are involved in antigen processing and presentation to T-cells, and several genes (IL23R, IL6R, STAT3, JAK2, IL1R1/2, IL12B, and IL7R) involved in IL23 driven pathways of inflammation. AS is also strongly associated with polymorphisms in two transcription factors, RUNX3 and T-bet (encoded by TBX21), which are important in T-cell development and function. The influence of these genes on the pathogenesis of AS and their potential for identifying drug targets is discussed here.

Potential role of CSF cytokine profiles in discriminating infectious from non-infectious CNS disorders.

Current laboratory testing of cerebrospinal fluid (CSF) does not consistently discriminate between different central nervous system (CNS) disease states. Rapidly distinguishing CNS infections from other brain and spinal cord disorders that share a similar clinical presentation is critical. New approaches focusing on aspects of disease biology, such as immune response profiles that can have stimulus-specific attributes, may be helpful. We undertook this preliminary proof-of-concept study using multiplex ELISA to measure CSF cytokine levels in various CNS disorders (infections, autoimmune/demyelinating diseases, lymphomas, and gliomas) to determine the potential utility of cytokine patterns in differentiating CNS infections from other CNS diseases. Both agglomerative hierarchical clustering and mixture discriminant analyses revealed grouping of CNS disease types based on cytokine expression. To further investigate the ability of CSF cytokine levels to distinguish various CNS disease states, non-parametric statistical analysis was performed. Mann-Whitney test analysis demonstrated that CNS infections are characterized by significantly higher CSF lP-10/CXCL10 levels than the pooled non-infectious CNS disorders (p = 0.0001). Within the infection group, elevated levels of MDC/CCL22 distinguished non-viral from viral infections (p = 0.0048). Each disease group of the non-infectious CNS disorders independently showed IP-10/CXCL10 levels that are significantly lower than the infection group [(autoimmune /demyelinating disorders (p = 0.0005), lymphomas (p = 0.0487), gliomas (p = 0.0294), and controls (p = 0.0001)]. Additionally, of the non-infectious diseases, gliomas can be distinguished from lymphomas by higher levels of GRO/CXCL1 (p = 0.0476), IL-7 (p = 0.0119), and IL-8 (p = 0.0460). Gliomas can also be distinguished from autoimmune/demyelinating disorders by higher levels of GRO/CXCL1 (p = 0.0044), IL-7 (p = 0.0035) and IL-8 (p = 0.0176). Elevated CSF levels of PDGF-AA distinguish lymphomas from autoimmune/demyelinating cases (p = 0.0130). Interrogation of the above comparisons using receiver operator characteristic analysis demonstrated area under the curve (AUC) values (ranging from 0.8636-1.0) that signify good to excellent utility as potential diagnostic discriminators. In conclusion, our work indicates that upon formal validation, measurement of CSF cytokine levels may have clinical utility in both identifying a CNS disorder as infectious in etiology and, furthermore, in distinguishing viral from non-viral CNS infections.