Performance of two modified two-tier algorithms for the serologic diagnosis of Lyme disease.

We compared the performance of a new modified two-tier testing (MTTT) platform, the Diasorin Liaison chemiluminescent immunoassay (CLIA), to the Zeus enzyme-linked immunoassay (ELISA) MTTT and to Zeus ELISA/Viramed immunoblot standard two-tier testing (STTT) algorithm. Of 537 samples included in this study, 91 (16.9%) were positive or equivocal by one or more screening tests. Among these 91 samples, only 57 samples were concordant positive by first-tier screening tests, and only 19 of 57 were concordant by the three second-tier methods. For IgM results, positive percent agreement (PPA) was 68.1% for Diasorin versus 89.4% for Zeus compared to immunoblot. By contrast, the PPA for IgG for both Diasorin and Zeus was 100%. Using a 2-out-of-3 consensus reference standard, the PPAs for IgM were 75.6%, 97.8%, and 95.6% for Diasorin, Zeus, and immunoblot, respectively. The difference between Zeus MTTT and Diasorin MTTT for IgM detection was significant (P = 0.0094). PPA for both Diasorin and Zeus MTTT IgG assays was 100% but only 65.9% for immunoblot STTT (P = 0.0005). In total, second-tier positive IgM and/or IgG results were reported for 57 samples by Diasorin MTTT, 63 by Zeus MTTT, and 54 by Viramed STTT. While Diasorin CLIA MTTT had a much more rapid, automated, and efficient workflow, Diasorin MTTT was less sensitive for the detection of IgM than Zeus MTTT and STTT including in 5 early Lyme cases that were IgM negative but IgG positive. IMPORTANCE: The laboratory diagnosis of Lyme disease relies upon the detection of antibodies to Borrelia species. Standard two tier testing (STTT) methods rely upon immunoblots which have clinical and technical limitations. Modified two-tier testing (MTTT) methods have recently become available and are being widely adopted. There are limited independent data available assessing the performance of MTTT and STTT methods.

Clinicopathological significance of neutrophil-rich colorectal carcinoma.

AIMS: The importance of the interaction between tumour cells and neutrophils has recently begun to emerge. However, the significance of tumour-infiltrating neutrophil (TIN) in colorectal carcinomas (CRCs) remains unclear. The aim of this study was to investigate the prognostic significance of TIN in CRCs. METHODS: CRCs were evaluated for TIN and were classified as neutrophil-rich (NR), neutrophil-intermediate (NI) and neutrophil-poor (NP) based on the presence of >15, 5-15 and <5 TIN per 100 tumour cells, respectively. Various clinicopathological parameters were recorded in each case including age, gender, histological grade, tumour, node, metastasis (TNM) stage, tumour location and DNA mismatch repair (MMR) status. RESULTS: Among the 348 CRC cases reviewed, 38 cases were NR, 43 cases were NI and 267 cases were NP. High TIN was associated with higher histological grade (p=0.0222), right-sided tumour location (p=0.0025), advanced TNM stage (p=0.0346) and higher rate of MMR-deficient CRCs (p=0.0027). Patients with NR CRCs had significantly poorer 5-year recurrence-free survival comparing to patients with NI or NP CRCs on Kaplan-Meier analysis (p=0.0001) and high TIN remained an independent risk factor with multivariate analysis (p=0.0137; HR: 1.930, 95% CI: 1.144 to 3.255). NR CRCs are more commonly seen in MMR-deficient than in MMR-proficient CRCs (p=0.0006). Patients with MMR-deficient NR CRCs showed similar 5-year recurrence-free survival compared with MMR-proficient NR CRCs. CONCLUSIONS: Our findings reveal that high TIN confers poorer patient prognosis in both MMR-proficient and MMR-deficient CRCs.

Evaluation of Susceptibility and Innate Immune Response in C57BL/6 and BALB/c Mice During Candida albicans Endophthalmitis.

Purpose: Candida remains the leading cause of fungal endophthalmitis. However, the pathobiology and innate immune responses in this disease are not well characterized. Here, we developed two murine models of candida endophthalmitis and evaluated their disease susceptibility and differential immune response. Methods: Endophthalmitis was induced in C57BL/6 (B6) and BALB/c mice by intravitreal injection of Candida albicans (CA). Disease progression was monitored by slit-lamp examination and clinical scoring, followed by retinal function assessment using electroretinography (ERG). Enucleated eyes were used to estimate fungal burden and retinal tissue damage by hematoxylin and eosin and TUNEL staining. The level of inflammatory mediators were determined by quantitative Polymerase Chain Reaction (qPCR) and enzyme-linked immunosorbent assay, whereas neutrophil infiltration was assessed by flow cytometry and immunostaining. Results: Intravitreal injection of CA at 6500 colony-forming units resulted in sustained (non-resolving) ocular inflammation in both B6 and BALB/c mice as evidenced by increased levels of inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) and chemokine (CXCL2/MIP-2). In both mouse strains, fungal burden peaked at 24 to 48 hours post-infection (hpi) and decreased by 72 to 96 hpi. CA-infected eyes exhibited increased polymorphonuclear neutrophils (PMN) infiltration and retinal tissue damage. Overall retinal function declined rapidly, with a significant reduction in ERG response at 12 hpi and near-total loss by 24 hpi. Differential analyses revealed increased pathology in BALB/c versus B6 mice. Conclusions: C. albicans was able to cause endophthalmitis in mice. Although BALB/c mice were found to be more susceptible to CA endophthalmitis, both BALB/c and B6 models could be used to study fungal endophthalmitis and test therapeutic modalities.

Temporal retinal transcriptome and systems biology analysis identifies key pathways and hub genes in Staphylococcus aureus endophthalmitis.

Bacterial endophthalmitis remains a devastating inflammatory condition associated with permanent vision loss. Hence, assessing the host response in this disease may provide new targets for intervention. Using a mouse model of Staphylococcus aureus (SA) endophthalmitis and performing retinal transcriptome analysis, we discovered progressive changes in the expression of 1,234 genes. Gene ontology (GO) and pathway analyses revealed the major pathways impacted in endophthalmitis includes: metabolism, inflammatory/immune, antimicrobial, cell trafficking, and lipid biosynthesis. Among the immune/inflammation pathways, JAK/Stat and IL-17A signaling were the most significantly affected. Interactive network-based analyses identified 13 focus hub genes (IL-6, IL-1ß, CXCL2, STAT3, NUPR1, Jun, CSF1, CYR61, CEBPB, IGF-1, EGFR1, SPP1, and TGM2) within these important pathways. The expression of hub genes confirmed by qRT-PCR, ELISA (IL-6, IL-1ß, and CXCL2), and Western blot or immunostaining (CEBP, STAT3, NUPR1, and IGF1) showed strong correlation with transcriptome data. Since TLR2 plays an important role in SA endophthalmitis, counter regulation analysis of TLR2 ligand pretreated retina or the use of retinas from TLR2 knockout mice showed the down-regulation of inflammatory regulatory genes. Collectively, our study provides, for the first time, a comprehensive analysis of the transcriptomic response and identifies key pathways regulating retinal innate responses in staphylococcal endophthalmitis.