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Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin-/PCR+, GDH+/toxin-/PCR-, and GDH-/toxin- samples, respectively (P 0.001). The Clarity assay may offer a single-test solution for CDI.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections that can cause significant morbidity and mortality. CDI diagnosis involves laboratory testing in conjunction with clinical assessment. The objective of this study was to assess the performance of various C. difficile tests and to compare clinical characteristics, Xpert C. difficile/Epi (PCR) cycle threshold (C(T)), and Singulex Clarity C. diff toxins A/B (Clarity) concentrations between groups with discordant test results. Unformed stool specimens from 200 hospitalized adults (100 PCR positive and 100 negative) were tested by cell cytotoxicity neutralization assay (CCNA), C. diff Quik Chek Complete (Quik Chek), Premier Toxins A and B, and Clarity. Clinical data, including CDI severity and CDI risk factors, were compared between discordant test results. Compared to CCNA, PCR had the highest sensitivity at 100% and Quik Chek had the highest specificity at 100%. Among clinical and laboratory data studied, prevalences of leukocytosis, prior antibiotic use, and hospitalizations were consistently higher across all subgroups in comparisons of toxin-positive to toxin-negative patients. Among PCR-positive samples, the median C(T) was lower in toxin-positive samples than in toxin-negative samples; however, C(T) ranges overlapped. Among Clarity-positive samples, the quantitative toxin concentration was significantly higher in toxin-positive samples than in toxin-negative samples as determined by CCNA and Quik Chek Toxin A and B. Laboratory tests for CDI vary in sensitivity and specificity. The quantitative toxin concentration may offer value in guiding CDI diagnosis and treatment. The presence of leukocytosis, prior antibiotic use, and previous hospitalizations may assist with CDI diagnosis, while other clinical parameters may not be consistently reliable.

Currently available diagnostic tests for Clostridioides difficile infection (CDI) lack specificity or sensitivity, which has led to guideline recommendations for multistep testing algorithms. Ultrasensitive assays for detection of C. difficile toxins provide measurements of disease-specific markers at very low concentrations. These assays may show improved accuracy compared to that of current testing methods and offer a potential standalone solution for CDI diagnosis, although large studies of clinical performance and accuracy are lacking.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at –80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH(+)/toxin(+), 53 GDH(−)/toxin(−), and 124 GDH(+)/toxin(−) samples, of which 39 were CCNA(+) and 85 were CCNA(−). Clarity had 96.2% negative agreement with GDH(−)/toxin(−) samples, 100% positive agreement with GDH(+)/toxin(+) samples, and 95.3% agreement with GDH(+)/toxin(−)/CCNA(−) samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH(+)/toxin(−)/CCNA(+) samples, 90.0% of GDH(+)/toxin(−)/CCNA(+) (high-positive) samples, and 31.6% of GDH(+)/toxin(−)/CCNA(+) (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT(+)/Clarity(−) and 4 NAAT(−)/Clarity(+) samples, there were 26 CCNA(−) and 4 CCNA(−) samples, respectively. CDI relapse was more common in NAAT(+)/toxin(+) patients than in NAAT(+)/toxin(−) and NAAT(−)/toxin(−) patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT(+)/toxin(−) than in NAAT(+)/toxin(+) patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

In a letter to the editor ([1][1]) in response to Sandlund and Wilcox ([2][2]), Tenover et al. suggest that on-label testing with nucleic acid amplification tests (NAATs) for the diagnosis of Clostridioides difficile infection (CDI) be discussed to put toxin testing in perspective. The authors argue

Background Clostridioides difficile infection (CDI) continues to cause significant morbidity and avoidable mortality worldwide. Results from an ultra-sensitive toxin immunoassay (Singlulex Clarity C. diff toxins A/B assay) were compared with those of various other diagnostic and reference methods/algorithms for the detection of C. difficile. Methods 293 residual clinical stool samples were tested using the Singulex assay. In total, 188 samples were tested by GDH and 239 were tested by PCR. All toxin B PCR (Serosep EntericBio C. difficile assay) positive samples (n=168) and prospectively tested GDH samples (n=97) were also tested using membrane-type toxin EIA (MT-EIA; Techlab Tox A/B Quik Cheko). Culture (alcohol shock and Brazier’s media; Oxoid) and ribotyping (capillary electrophoresis using Bidet et al. primers) information was available for 205 samples. Results The positive percent agreement (PPA) and negative percent agreement (NPA) of the Singulex Clarity C. diff toxins A/B assay compared with: GDH; toxin EIA; PCR; GDH/toxin EIA; GDH/toxin EIA/PCR; PCR/toxin EIA and culture were – 61% & 92%; 97% & 50%; 69% & 90%; 100% & 51%; 81% & 77%; 96% & 65%; and 69% AND 52% respectively. Conclusions The Singulex Clarity C. diff toxins A/B assay had high PPA compared to toxin EIA and multistep algorithms ending with toxin EIA, and high NPA compared to PCR and a multistep algorithm ending with PCR. The Singulex Clarity assay has the potential to be used as a standalone test for CDI diagnosis; additional clinical studies are required and will soon be underway.

The use of nucleic acid amplification tests (NAATs) for the diagnosis of Clostridium (Clostridioides) difficile infection (CDI) leads to overdiagnosis. To improve the clinical specificity of NAATs, there has been a recent interest in using toxin gene cycle thresholds (C(T)s) to predict the presence and absence of toxins. Although there is an association between C(T) values and fecal toxin concentrations, the predictive accuracy of the former is suboptimal for use in clinical practice. Ultrasensitive toxin immunoassays to quantify free toxins in stool offer a novel option for high-sensitivity fecal toxin detection rather than using surrogate markers for prediction.

Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity(+)/CCCNA(−)) became CCCNA(−), and 5/26 (19.2%) Clarity(+)/CCCNA(−) samples became CCCNA(+), resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) bla(OXA-48 like), 20.8% (5/24) bla(KPC), 20.8% (5/24) bla(NDM), 20.8% (5/24) bla(SME), 8.3% (2/24) bla(IMP), and 4.2% (1/24) bla(VIM). Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing bla(KPC) were 100% susceptible to ceftazidime–avibactam, meropenem–vaborbactam, and imipenem–relebactam; bla(OXA-48 like)–positive isolates were 100% susceptible to ceftazidime–avibactam; and metallo β-lactamase–positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime–avibactam, meropenem–vaborbactam, imipenem–relebactam, and ceftolozane–tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer β-lactamase inhibitors.

Background Top priorities for tuberculosis control and elimination include a simple, low-cost screening test using sputum and a non-sputum-based test in patients that do not produce sputum. The aim of this study was to evaluate the performance of a colorimetric sensor array (CSA) test, for analysis of volatile organic compounds in urine, in the diagnosis of pulmonary TB. Material and Methods Urine samples were collected from individuals suspected of having pulmonary TB in Western Kenya. Reference methods included MGIT culture and/or Xpert MTB/RIF nucleic acid amplification test on sputa. Fresh urine samples were tested with the CSA, with acid and base and without an additive. The CSA were digitally imaged, and the resulting colorimetric response patterns were used for chemometric analysis. Sensitivity, specificity, and negative (NPV) and positive predictive (PPV) values were determined for HIV-positive and HIV-negative patients. Results In HIV-negative patients, the highest accuracy was obtained in urine samples pre-treated with a base, yielding a sensitivity, specificity, PPV, and NPV of 78.3% (65/83), 69.2% (54/78), 73.0% (n/89) and 75.0% (n/72). The highest sensitivity of 79.5% was achieved using sensor data from all three test conditions at a specificity of 65.4%. In HIV-positive subjects, the sensor performance was substantially lower with sensitivity, specificity, PPV, and NPV ranging from 48.3% to 62.3%, 54.1% to 74.0%, 55.9% to 64.2%, and 60.6% to 64.9%, respectively. Conclusion The CSA fingerprint of urine headspace volatiles showed moderate accuracy in diagnosing TB in HIV-negative patients, but the sensor performance dropped substantially in HIV-coinfected patients. Further development of TB-responsive CSA indicators may improve the accuracy of CSA urine assay.

Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging as a major health threat in North America. The mechanism of resistance to carbapenems has therapeutic and public health implications. We comprehensively characterized the underlying mechanisms of carbapenem resistance in CRE isolates recovered between 2013 and 2016 at a health system in Northern California. Genotypic methods were used to detect carbapenemases and plasmid-encoded cephalosporinases, and mass spectrometry was used to quantify relative porin levels for OmpC and OmpF and their analogs. MICs for imipenem-relebactam, meropenem-vaborbactam, ceftazidime-avibactam, and ceftolozane-tazobactam were measured. Whole genome sequencing was used for strain typing. A carbapenemase gene encoding blaOXA-48 like, blaNDM, blaKPC, blaSME, blaIMP, and blaVIM was detected in 38.7% (24/62) of CRE isolates. Porin levels was down at least 2-fold in 91.9% (57/62) of isolates. Including carbapenemase genes and porin loss, the mechanism of resistance was identified in 95.2% (59/62) of CRE isolates. Of the carbapenemase gene-positive isolates, blaKPC -positive isolates were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and blaSME-positive isolates were 100% susceptible to meropenem-vaborbactam and ceftolozane-tazobactam. 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) of carbapenemase gene-negative CRE isolates were susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were genetically identical. In conclusion, at this health system in Silicon Valley, carbapenemase-producing CRE occurred sporadically and were mediated by diverse mechanisms. Nucleic acid testing for blaOXA-48 like, blaNDM, blaKPC, blaIMP, and blaVIM was sufficient to distinguish between carbapenemase-producing and non-producing CRE and accurately predicted susceptibility to ceftazidime-avibactam, meropenem-vaborbactam and imipenem-relebactam.

Aim To evaluate analytical and biological characteristics of the Singulex Clarity® cTnI assay, based upon Single Molecule Counting technology. Methods Assay's analytical sensitivity, precision, linearity, hook effect, cross-reactivity or interference by endogenous and exogenous substances, stability, 99th reference percentile [p99th] in EDTA plasma were evaluated in single or multi-site studies. Results Detection limit was 0.12 ng/L. Sensitivity was 0.14 ng/L at 20% CV (functional sensitivity) and 0.53 ng/L at 10% CV. Imprecision was 3.16%–10.0% in a multi-lot, single-site study, and 5.5%–12.0% in a single-lot, multi-site study; assay was linear from 0.08 to 25,000 ng/L. No hook effect was observed; any cross-reactivity/interference exceeded the 10%. Healthy subjects were recruited using clinical history, normal NT-proBNP and eGFR (n = 560) or plasma creatinine (n = 535) as inclusion criteria. cTnI was detectable in 96.8% of healthy subjects. The p99th were 8.01 (eGFR used) and 8.15 ng/L (plasma creatinine); both were measured with ≤5.7% CV. Median cTnI were significantly higher in older and male than in young and female subjects. Conclusions The Singulex Clarity cTnI assay show analytical features and % detection in healthy subjects that improve the corresponding values of most of the existing high-sensitivity cTnI assays.

Background Commercially available enzyme immunoassays (EIAs) for detection of norovirus antigen have poor sensitivity and are limited to use in investigations of a gastroenteritis outbreak. Hence, there remains a need for a standalone high-sensitivity assay that enables rapid and accurate detection of norovirus antigen. Methods The Singulex Clarity norovirus assay is currently in development for use on the Singulex Clarity® system (Singulex Inc., Alameda, CA, USA), a fully-automated platform powered by Single Molecule Counting technology (registered with the FDA and CE marked). The assay uses paramagnetic microparticles bound to capture antibody and a fluorescently labeled reporter antibody to detect virion capsid protein of norovirus genogroups I (GI) and II (GII) in the stool. For the development of Clarity Norovirus assay, diagnostic performance of 4 antibody pairs (as Capture and Detection reagent) were evaluated by testing 137 stool samples from patients with suspected norovirus infection. Samples were sourced from three providers: (1) 90 genotyped samples of which 75 were positive (19 different genotypes) and 15 were negative by the CDC assay, (2) 3 samples positive and 5 samples negative by the BioFire® FilmArray® Gastrointestinal Panel, and (3) 39 samples negative by a lab-developed test using Cepheid reagents (SmartCycler®). Results From all the antibody pairs tested, one of the pairs had best performance with the area under the receiver operating characteristic (AuROC) curve demonstrating a C-Statistic of 0.959 (95% CI 0.921–0.997), compared with AuROC C-statistic of 0.943 (95% CI 0.896–0.990), 0.871 (95% CI 0.807–0.936), and 0.914 (95% CI 0.863–0.964) for the three other pairs. The Clarity assay detected all 19 different genotypes tested (figures). Conclusion The ultrasensitive and rapid Clarity norovirus assay (in development) for detection of GI and GII demonstrated excellent performance with one of the antibody pairs tested and detected all 19 tested genotypes. The Clarity assay may offer a standalone solution for norovirus diagnostics. Disclosures All authors: No reported disclosures.

Background Clostridioides difficile infection (CDI) is one of the most common healthcare-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We have evaluated the clinical performance of ultrasensitive Single Molecule Counting technology for detection of C. difficile toxins A and B. Methods Stool specimens from 298 patients with suspected CDI were tested with nucleic acid amplification test (NAAT; BD MAX™ Cdiff assay or Xpert® C. difficile assay) and Singulex Clarity® C. difficile toxins A/B assay. Specimens with discordant results were tested with cell cytotoxicity neutralization assay (CCNA), and results were correlated with disease severity and outcome. Results There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity− and 4 NAAT-/Clarity+ samples, there were 26 CCNA− and 4 CCNA- samples, respectively. CDI relapse or overall death was more common in NAAT+/toxin+ patients than in NAAT+/toxin− and NAAT−/toxin− patients, and NAAT+/toxin+ patients were 3.7 times more likely to experience relapse or death (Figure 1). The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was over three times more common in NAAT+/toxin− than in NAAT+/toxin+ patients (Figure 2). Negative percent agreement between NAAT and Clarity was 98.3%, and positive percent agreement increased from 50.0% to effective 84.2% and 94.1% after CCNA testing and clinical assessment. Conclusion The Clarity assay was superior to NAATs in diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and presence of toxins was associated with disease severity and outcome. Disclosures All authors: No reported disclosures.

Growth factors are primarily responsible for the genesis, differentiation and proliferation of cells and maintenance of tissues. Given the central role of growth factors in signaling between cells in health and in disease, it is understandable that disruption of growth factor-mediated molecular signaling can cause diverse phenotypic consequences including cancer and neurological conditions. This review will focus on the specific questions of enlarged cerebral ventricles and hydrocephalus. It is also well known that angiogenic factors, such as vascular endothelial growth factor (VEGF), affect tissue permeability through activation of receptors and adhesion molecules; hence, recent studies showing elevations of this factor in pediatric hydrocephalus led to the demonstration that VEGF can induce ventriculomegaly and altered ependyma when infused in animals. In this review, we discuss recent findings implicating the involvement of biochemical and biophysical factors that can induce a VEGF-mimicking effect in communicating hydrocephalus and pay particular attention to the role of the VEGF system as a potential pharmacological target in the treatment of some cases of hydrocephalus. The source of VEGF secretion in the cerebral ventricles, in periventricular regions and during pathologic events including hydrocephalus following hypoxia and hemorrhage is sought. The review is concluded with a summary of potential non-surgical treatments in preclinical studies suggesting several molecular targets including VEGF for hydrocephalus and related neurological disorders.

Actinobaculum schaalii is a uropathogen resistant to ciprofloxacin and trimethoprim-sulfamethoxazole. It requires a long culture time and specific conditions, and is therefore easily overgrown by other bacteria and regarded as part of the normal bacterial flora. We review 17 cases of A. schaalii bacteraemia, demonstrating its invasive potential. A. schaalii should always be ruled out as causative agent in patients with urinary tract infection or urosepticaemia with treatment failure.

Background Commercially available tests for Clostridium difficile infection (CDI) make test selection by the laboratory difficult due to the following unsatisfactory characteristics: long turnaround time, poor sensitivity, and/or poor specificity. The Singulex Clarity® C. diff toxins A/B assay (in development) is a rapid and automated immunoassay for the detection of C. difficile toxins A and B in stool, with analytical limits of detection for toxins A and B at 2.0 and 0.7 pg/mL, respectively. In this multicenter study, the clinical performance of the Singulex Clarity C. diff toxins A/B assay was compared with standalone PCR, a multistep algorithm with enzyme immunoassay (EIA) and PCR, and cell cytotoxicity neutralization assay (CCNA). Methods Fresh samples from 267 subjects with suspected CDI were tested at two sites (Minneapolis Medical Research Foundation and TriCore Reference Laboratories) with the Singulex Clarity assay, PCR (Xpert®C. difficile), and EIA (C. Diff Quik Chek Complete®) for GDH and toxin testing. The performance of the assays and multistep algorithms were evaluated against CCNA (Microbiology Specialists, Inc.). Results The overall CDI prevalence was 15.7%. The Singulex Clarity C. diff toxins A/B assay had 90.5% sensitivity and 96.0% specificity, with a 98.2% negative predictive value when compared with CCNA, and the Clarity assay’s AuROC was 0.9534. PCR had 90.5% sensitivity and 91.1% specificity. Compared with CCNA, the toxin EIA had 47.6% sensitivity and 100% specificity. Testing with a multistep algorithm using EIA with discordant results reflexed to PCR resulted in 85.7% sensitivity and 94.7% specificity. Conclusion The ultrasensitive Singulex Clarity C. diff toxins A/B assay is equivalent to the sensitivity of PCR while providing higher specificity. Compared with a multistep algorithm, the Clarity assay provides higher sensitivity and specificity while providing faster time-to-result in a simpler-to-understand, one-step reporting structure, allowing for a standalone, single-step solution for detection of C. difficile toxins in patients with suspected CDI. Disclosures E. Friedland, Singulex, Inc.: Employee, Salary. A. Bartolome, Singulex, Inc.: Employee, Salary. A. Almazan, Singulex, Inc.: Employee, Salary. S. Tam, Singulex, Inc.: Employee, Salary. S. Biscocho, Singulex, Inc.: Employee, Salary. S. Abusali, Singulex, Inc.: Employee, Salary. J. Sandlund, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary.

To the Editor: Cardiac troponin I (cTnI)1 is routinely used to aid in the diagnosis of acute myocardial infarction. Few available cardiac troponin assays have adopted 99th percentiles based on patient demographics. However, there is growing evidence of age and sex differences for cTnI and cardiac troponin T (cTnT), especially when using high-sensitivity cardiac troponin assays, with sex-dependent 99th percentiles being recommended for clinical care (1–3). The Single Molecule Counting (SMCTM) cTnI test (Singulex) can measure cTnI in nearly all healthy individuals (4). We sought to establish the 99th percentile for the SMC cTnI test by deriving an objective definition of “healthy” from >100000 patient results. SMC cTnI is a laboratory-developed test (not cleared by the Food and Drug Administration). The test is a quantitative fluorescent 1-step sandwich immunoassay developed to measure human cTnI concentrations in separated EDTA plasma (5). The lower limit of quantification (LLoQ) was 0.40 ng/L (the lowest standard concentration with back-interpolated recovery bias

Background Current diagnostic methods for Helicobacter pylori infection include fecal antigen tests, 13C-urea breath test, and gastric biopsy. The breath test is limited by poor specificity and the fecal antigen tests by poor sensitivity. We have developed a prototype assay for detection of H. pylori antigen in human stool, powered by ultrasensitive Single Molecule Counting technology, and compared the analytical performance to a commercially available enzyme-linked immunoassay (EIA) antigen test. Methods The Singulex Clarity H. pylori antigen assay incubates diluted stool with capture and fluorescent-labeled detection antibodies. After incubation and wash steps, fluorescent molecules are eluted and single-molecule fluorescence measured by detected events (DE′). Analytical performance was compared with a commercial EIA (Premier Platinum HpSA® Plus, Meridian Bioscience, Inc.) using serial dilutions of H. pylori control (~37,500–1.7 ng/mL) and high positive stool (signal to noise ratio >2). Clinical performance was evaluated using two cohorts, one had 10 EIA-negative and 10 EIA-positive samples and the other 13 high positives (> 0.500 at 450/630) and 5 low positives near the EIA cutoff (0.100–0.500 at 450/630). One sample was excluded due to discordant EIA results, and three to reader flags. Results The lower limit of detection of the Clarity H. pylori assay was 1.7 ng/mL and the EIA 1,250 ng/mL (IFU: LOD 4.67 ng/mL). A high positive stool sample was detectable by the Clarity H. pylori assay diluted 1:10,000,000 and by the EIA 1:10,000. The Clarity H. pylori assay showed a 729-fold increase in lower limit of detection and 1,000-fold increase in endogenous antigen lower limit of detection compared with the EIA. Clarity signal ranged from 46–665 DE’ for EIA-negative samples and 487,484–576,747 DE’ for EIA-positive samples. Conclusion The Singulex Clarity H. pylori antigen assay may have orders of magnitude higher analytical sensitivity than the commercial EIA and demonstrated 100% positive agreement and 100% negative agreement on detection of H. pylori antigen in human stool samples. The ultrasensitive Clarity H. pylori assay has the potential for high sensitivity and specificity to improve current diagnostic options for H. pylori infection; however, additional multicenter studies are required. Disclosures All authors: No reported disclosures.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus.

Background Clostridium difficile, a spore-forming, anaerobic, Gram-positive bacterium, is the leading cause of nosocomial diarrhea. C. difficile infection (CDI) is mediated by two toxins, A (TcdA) and B (TcdB), and the role of each toxin in CDI pathogenesis remains unclear. Many assays used in CDI diagnostics, such as most NAATs and cell cytotoxicity neutralization assay (CCNA), detect presence of only tcdB or TcdB. In this study, an ultrasensitive immunoassay (UIA) powered by Single Molecule Counting technology was used for quantification of TcdA and TcdB, to assess toxin dynamics in CDI. Methods Banked samples from 46 patients with suspected CDI were tested with PCR (BD MAX™ Cdiff Assay) and CCNA, and TcdA and TcdB were quantified using the UIA (tested in triplicate). The limits of detection (LoDs) for the TcdA and TcdB assays are 0.04 and 0.12 pg/mL, respectively. Results There were 21 PCR+/CCNA+ and 25 PCR−/CCNA− samples. Both toxins were measured above LoD in all PCR+/CCNA+ samples, ranging up to 100,000 pg/mL. The average CV for the PCR+/CCNA+ samples was 9%. The median TcdA concentrations in PCR−/CCNA− and PCR+/CCNA+ samples were 0.19 pg/mL (IQR 0.12–0.67) and 3,301 pg/mL (125–8,737), respectively. The median TcdB concentrations in PCR−/CCNA− and PCR+/CCNA+ samples were 0.12 pg/mL (0.12–0.21) and 2,690 pg/mL (145–30,307), respectively. In the PCR+/CCNA+ samples, TcdA was one or more logs higher than TcdB in two samples, one or more logs lower than TcdB in six samples, and within one log of TcdB in 13 samples. In one sample (4.8% of PCR+/CCNA+ samples), TcdA was at moderately high concentration while TcdB was below a provisional cutoff, indicating that only TcdA was expressed. There was a significant correlation between TcdA and TcdB (Spearman r = 0.753). Conclusion The UIA allows for toxin quantification over a concentration range of ≥5 logs, suggesting that the quantitative TcdA and TcdB assays could be of value in CDI characterization and clinical decision making. The TcdA/TcdB ratio varied, and toxin quantification could be a useful tool in further understanding their individual roles in CDI. The TcdA concentration was not lower than TcdB (trended higher), indicating that detection of tcdB or TcdB alone may not be sufficient for accurate CDI diagnostics. Disclosures P. Katzenbach, Singulex, Inc.: Employee, Salary. G. Dave, Singulex, Inc.: Employee, Salary. A. Mukherjee, Singulex, Inc.: Employee, Salary. J. Todd, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary. J. Sandlund, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary.

Hypoxia-inducible factor (HIF)-2alpha is upregulated in hypoxia or by inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. In a number of malignancies, increased HIF-2alpha expression may indicate worse prognosis. The aim of this study was to evaluate the prognostic information of HIF-2alpha mRNA expression in renal cell carcinoma (RCC).HIF-2alpha mRNA was quantified by real time polymerase chain reaction (rt-PCR) in tumour tissue samples from 202 patients. Samples from 50 corresponding kidney cortex tissue were analysed as controls. mRNA levels were evaluated in relation to tumour cell type, TNM stage, nuclear grade and disease specific survival.The levels of HIF-2alpha mRNA were significantly higher in 168 clear cell (c)RCC than in 23 papillary (p)RCC (p0.001) or 11 chromophobe (ch)RCC (p0.006). Among cRCC there was an inverse correlation between HIF-2alpha mRNA levels and TNM stage I and II-IV tumours (p=0.01), and nuclear grade (p = 0.006). After a median follow-up time of 99 months (range 34-247), 106 patients had died of RCC. No correlation of HIF-2alpha mRNA to survival was observed. A multivariate analysis of prognostic factors in cRCC showed that TNM stage alone was an independent predictor of prognosis; HIF-2alpha mRNA levels did not add further prognostic information.The results demonstrated that HIF-2alpha mRNA levels were higher in cRCC compared to pRCC and chRCC. Furthermore, HIF-2alpha mRNA levels were inversely related to TNM stage and nuclear grade in cRCC.

Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale , which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan- Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi -specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan- Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

OBJECTIVE To evaluate the prognostic potential of endoglin (CD105) expression in human renal cell carcinoma (RCC), as endoglin is a cell membrane glycoprotein expressed in tumour-associated vascular endothelium and a marker of angiogenesis; intratumoral microvessel density assessed by endoglin staining has prognostic significance in some neoplasms. PATIENTS AND METHODS Tumour samples from 210 patients with RCC (168 conventional), diagnosed between 1982 and 1997, were assessed using the tissue microarray technique with immunohistochemical staining for endoglin. The expression of endoglin was related to clinical variables and survival. RESULTS Of the tumours, 75% expressed endoglin, and in conventional RCC the expression was inversely correlated to the Tumour-Node-Metastasis (TNM) stage (P = 0.008) and nuclear grade (P = 0.01). There was no correlation between endoglin expression and gender, age, tumour size or cell type. Patients with conventional RCC and high endoglin expression had a more favourable prognosis than those with tumours with lower expression (P = 0.04). A multivariate analysis of prognostic factors showed that TNM stage and nuclear grade were independent predictors of prognosis. Endoglin expression did not add further prognostic information. CONCLUSION These results indicate that endoglin expression is inversely related to stage and grade in RCC, and that it is associated with prognosis.

Background Assessments of the epidemiology of malaria over time are needed to understand changes in transmission and guide control and elimination strategies. Methods A longitudinal population study was established in 1985 in Nyamisati village in the Rufiji River Delta, Tanzania. A physician and research team lived in the village 1984–2000. Parasite prevalence by microscopy and two PCR methods, spleen rates and haemoglobin levels were measured in repeated cross-sectional surveys between 1985 and 2010. Passive surveillance of malaria cases was maintained until end 1999. Bed nets were distributed after the surveys 1993, 1999 and 2010. Results In 1985, overall parasite prevalence by microscopy was 70% (90% in children ages two to nine years). The prevalence decreased gradually by microscopy (38.9% 1994, 26.7% 1999) and msp2-PCR (58.7% 1994, 44.8% 1999), whereas real-time PCR prevalence remained higher throughout the 1990s (69.4% 1994, 64.8% 1999). In 2010, parasite prevalence was 17.8% by real-time PCR and 16.3% by msp2-PCR, and estimated to 4.8% by microscopy. Spleen rates in children ages two to nine years decreased earlier than parasite prevalence, from >75 to 42% in the 1980s, to nil during the 1990s. The prevalence of severe and moderate anaemia decreased from 41.1 to 13.1%. No deaths at the time of acute malaria were recorded when the research team lived in the village. Conclusions A marked decline in malaria transmission was observed over 25 years. The decrease was detected after the arrival of the research team and continued gradually both before and after distribution of bed nets. Spleen rates and microscopy identified early changes when transmission was still intense, whereas real-time PCR was a more sensitive metric when transmission was reduced. The study provides historical data on malaria within a closely monitored rural village and contributes to the understanding of changing epidemiology in sub-Saharan Africa. Electronic supplementary material The online version of this article (doi:10.1186/1475-2875-13-459) contains supplementary material, which is available to authorized users.

Hydrocephalus is a condition characterized primarily by excessive accumulation of fluid in the ventricles of the brain for which there is currently no effective pharmacological treatment. Surgery, often accompanied by complications, is the only current treatment. Extensive research in our laboratory along with work from others has suggested a link between hydrocephalus and vascular function. We hypothesized that vascular endothelial growth factor (VEGF), the major angiogenic factor, could play a role in the pathogenesis of hydrocephalus. We tested this hypothesis by examining two predictions of such a link: first, that VEGF is present in many cases of clinical hydrocephalus; and second, that exogenous VEGF in an animal model could cause ventricular enlargement and tissue changes associated with hydrocephalus. Our results support the idea that VEGF elevation can potentiate hydrocephalus. The clinical relevance of this work is that anti-angiogenic drugs may be useful in patients with hydrocephalus, either alone or in combination with the currently available surgical treatments.

Malaria predisposes children in areas where malaria is endemic to concurrent bacteremia, often with severe outcomes. The importance of bacterial coinfections in patients diagnosed with malaria in nonendemic settings has, however, not been reported. A retrospective analysis of microbiology data was performed in 755 travelers diagnosed with malaria in Sweden. Bacterial cultures from blood and other locations were correlated to clinical outcome and antibiotic treatment. Blood cultures were drawn from 417 (55%) patients (88% of whom were >15 years old), and bacterial isolates of clinical relevance ( Salmonella enterica serovar Enteritidis and Escherichia coli ) were detected in 2 patients (0.3%). Cultures from other locations (mainly urine, nasopharyngeal, and fecal samples) were obtained from 44% of the patients with 4.9% positivity. Of the 38 patients given antibiotics, 47% had neither severe malaria nor positive cultures and/or radiology signs indicative of treatment. C-reactive protein levels were associated with bacterial infections but had only a fair predictive value. Bacterial coinfections are uncommon among travelers with malaria. These data suggest a weaker association between malaria and bacteremia than previously described in endemic settings and might indicate different patient populations with different pathophysiological mechanisms and microbial environments. The study supports a restrictive antibiotic policy in returning travelers with malaria.

Glioblastomas are very difficult tumors to treat because they are highly invasive and disseminate within the normal brain, resulting in newly growing tumors. We have identified netrin-1 as a molecule that promotes glioblastoma invasiveness. As evidence, netrin-1 stimulates glioblastoma cell invasion directly through Matrigel-coated transwells, promotes tumor cell sprouting and enhances metastasis to lymph nodes in vivo. Furthermore, netrin-1 regulates angiogenesis as shown in specific angiogenesis assays such as enhanced capillary endothelial cells (EC) sprouting and by increased EC infiltration into Matrigel plugs in vivo, as does VEGF-A. This netrin-1 signaling pathway in glioblastoma cells includes activation of RhoA and cyclic AMP response element-binding protein (CREB). A novel finding is that netrin-1-induced glioblastoma invasiveness and angiogenesis are mediated by activated cathepsin B (CatB), a cysteine protease that translocates to the cell surface as an active enzyme and co-localizes with cell surface annexin A2 (ANXA2). The specific CatB inhibitor CA-074Me inhibits netrin-1-induced cell invasion, sprouting, and Matrigel plug angiogenesis. Silencing of CREB suppresses netrin-1-induced glioblastoma cell invasion, sprouting, and CatB expression. It is concluded that netrin-1 plays an important dual role in glioblastoma progression by promoting both glioblastoma cell invasiveness and angiogenesis in a RhoA-, CREB-, and CatB-dependent manner. Targeting netrin-1 pathways may be a promising strategy for brain cancer therapy.

To evaluate the relationship between osteopontin (OPN) in serum and plasma and parathyroid hormone-related protein (PTHrP) in serum, plasma and tumour tissue, and to assess the prognostic impact of OPN and PTHrP in human renal cell carcinoma (RCC).The study included 269 patients with RCC. In 189 patients, immunohistochemical (IHC) PTHrP tumour tissue expression was evaluated, and OPN and PTHrP in serum were assessed. In 80 patients, plasma OPN and PTHrP were analysed. Tumour type, TNM stage, nuclear grade and RCC-specific survival were also registered. In a sub-group, IHC expression of CD 31 was assessed. The prognostic information of the factors was analysed using uni- and multivariate analyses.The median OPN level was 2.3 times higher in plasma than in serum. Serum OPN was significantly higher in patients with papillary RCC compared to clear cell RCC and chromophobe RCC. Both serum and plasma OPN levels were positively correlated to TNM stage and nuclear grade. Multivariate analysis showed that serum and plasma OPN levels were independent prognostic factors for RCC-specific survival, along with TNM stage. Immunohistochemical expression of PTHrP associated to TNM stage but not to nuclear grade or serum OPN. Furthermore, IHC expression of PTHrP was positively correlated to serum PTHrP but inversely to tumour CD31 expression. Plasma PTHrP was increased in 20% of the patients and related to TNM stage but not to nuclear grade. Plasma OPN was significantly higher in patients with increased PTHrP levels, compared to those with normal levels.Plasma OPN levels differed between RCC types, and in clear cell RCC, both serum and plasma OPN levels were independent predictors of survival. We found no evidence for prognostic value related to circulating levels or the IHC expression of PTHrP.

This case illustrates skin lesions in a traveler staying in a hotel bed infested with tics. Although infestation of hotels with bedbugs belonging to the Cimex genus is a growing problem worldwide, tick infestation has never been reported before.

The aim of this study was to evaluate the prognostic information of soluble carbonic anhydrase (CA) IX expression in renal cell carcinoma (RCC).Serum CA IX was analysed in 361 patients. Tumour type, TNM stage, nuclear grade, and RCC-specific survival were assessed. Serum and immunohistochemical expression were compared.Median serum CA IX expression was 141 (range 2-4, 181) pg/ml. Levels were significantly higher in 287 patients with clear cell, compared to 40 papillary (p0.001) and 22 oncocytoma (p=0.002), but not to 12 chromophobe RCC (p=0.35). Serum CA IX in clear cell RCC was positively correlated to TNM stage (p=0.002). There was a positive trend between serum and immunohistochemical CA IX expression. In a multivariate analysis of clear cell RCC, TNM stage and nuclear grade were independent prognostic factors.Serum CA IX was higher in clear cell RCC compared to other RCC types. In clear cell RCC, serum CA IX correlated to TNM stage, but not survival.

Contains fulltext : 53266.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To evaluate the prognostic information of carbonic anhydrase (CA) IX expression in patients with renal cell carcinoma (RCC), as increased expression of CA IX is correlated with a worse prognosis in several malignancies. PATIENTS AND METHODS: CA IX expression was assessed in RCC tumours from 228 patients, using a tissue microarray technique on archival material. The expression was related to RCC cell type, Tumour-Node-Metastasis (TNM) stage, nuclear grade and survival. RESULTS: CA IX expression was significantly higher (P < 0.001) in 183 conventional than in 31 papillary RCC and 14 chromophobe RCC. For conventional RCC there was no correlation of CA IX expression with TNM stage or nuclear grade. To evaluate the prognostic information conventional RCC tumours were subdivided arbitrarily into three groups according to the CA IX expression, of 0-10%, 11-90% and 91-100% expression, respectively. Patients with tumours with 0-10% expression had a less favourable prognosis than those with 11-90% and 91-100% expression (P = 0.012, and 0.001), respectively. A multivariate analysis of prognostic factors for patients with conventional RCC showed that TNM stage, nuclear grade and CA IX were independent predictors of prognosis. CONCLUSION: These results show that CA IX expression is higher in conventional than other RCC cell types; furthermore, patients with conventional RCC with low CA IX expression had a less favourable prognosis.

Background The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples. Methods and Findings This study evaluated the assay’s analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51–97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163–302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs. Conclusion In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.

Intratumoural microvessel density (MVD) has prognostic significance in selected neoplasms. To evaluate the prognostic information of MVD in renal cell carcinoma (RCC) we assessed the immunohistochemical expression of CD31 in 208 tumours using the tissue microarray technique. The expression was related to tumour cell type, TNM stage, nuclear grade and survival. CD31 expression was significantly (p0.001) higher in 167 conventional RCCs (clear cell) compared to 28 papillary RCCs. In conventional RCC, CD31 expression was inversely correlated to TNM stage (p = 0.024) and nuclear grade (p = 0.010). To evaluate the impact of CD31 expression on tumour-specific survival, the conventional RCC tumours were subdivided into quartiles according to the CD31 expression. In univariate analysis, patients with tumours in the quartile with the highest expression had a more favourable prognosis (p = 0.01) compared to those with lower CD31 expression. A multivariate analysis of prognostic factors in conventional RCC showed that TNM stage and nuclear grade were independent predictors of prognosis, but CD31 expression did not add further prognostic information.