Development and Validation of a Test to Monitor Endoscopic Activity in Patients With Crohn's Disease Based on Serum Levels of Proteins.

BACKGROUND & AIMS: Noninvasive tests to measure endoscopic activity in patients with Crohn's disease (CD) have limitations. We aimed to develop a test to identify patients in remission, based on endoscopic analysis, and monitor CD activity based on serum levels of proteins. METHODS: We developed a test to measure 13 proteins in blood (ANG1, ANG2, CRP, SAA1, IL7, EMMPRIN, MMP1, MMP2, MMP3, MMP9, TGFA, CEACAM1, and VCAM1), called the endoscopic healing index [EHI], using samples from 278 patients with CD from a multinational training cohort. We validated the test using 2 independent cohorts of patients with CD: 116 biologic-naive patients with early-stage CD (validation cohort 1) and 195 biologic-exposed patients with chronic CD (validation cohort 2). The ability of the test to identify patients with active disease vs patients in remission (defined as a simple endoscopic score for CD of ≤2 and ≤1 in each segment, or a total CD endoscopic index of severity score <3) was assessed by using area under receiver operating characteristic curve (AUROC) analysis. The diagnostic accuracy of the test was compared with that of measurement of serum C-reactive protein (CRP) and fecal calprotectin. RESULTS: The EHI scores range from 0 to 100 units; higher scores indicate more severe CD activity, based on endoscopy findings. The EHI identified patients in remission with an AUROC of 0.962 in validation cohort 1 (95% confidence interval, 0.942-0.982) and an AUROC of 0.693 in validation cohort 2 (95% confidence interval, 0.619-0.767), regardless of CD location or phenotype. A cutoff value of 20 points identified patients in remission with the highest level of sensitivity (97.1% in validation cohort 1 and 83.2% in validation cohort 2), with specificity values of 69.0% and 36.6%, respectively. A cutoff value of 50 points identified patients in remission with the highest level of specificity (100% in validation cohort 1 and 87.8% in validation cohort 2), with sensitivity values of 37.3% and 30.0%, respectively. The EHI identified patients in remission with a significantly higher AUROC value than the test for CRP (0.876, P < .001 in validation cohort 1 and 0.624, P = .109 in validation cohort 2). In analysis of patients with available FC measurements, the AUROC value for the EHI did not differ significantly from that of measurement of FC (AUROC, 0.950 for EHI vs AUROC, 0.923 for FC; P = .147 in validation cohort 1 and AUROC, 0.803 for EHI vs AUROC, 0.854 for FC; P = .298 in validation cohort 2). CONCLUSIONS: We developed an index called the EHI to identify patients with CD in endoscopic remission based on blood levels of 13 proteins. The EHI identified patients with resolution of endoscopic disease activity, with good overall accuracy, although with variation between the 2 cohorts assessed. The EHI AUROC values were comparable to measurement of FC and higher than measurement of serum CRP. The test might be used in practice to assess endoscopic activity in patients with CD.

Validation of Serum Test for Advanced Liver Fibrosis in Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: We analyzed markers of fibrosis in serum samples from patients with nonalcoholic fatty liver disease (NAFLD), assessed by liver biopsy. We used serum levels of markers to develop an algorithm to discriminate patients with advanced fibrosis from those with mild or moderate fibrosis and validated its performance in 2 independent cohorts of patients with NAFLD. METHODS: We performed a retrospective analysis of serum samples from 396 patients with NAFLD and different stages of fibrosis (F0-F4), collected from 2007 through 2017 on the day of liver biopsy (training cohort 1). We measured serum concentrations of alpha-2 macroglobulin (A2M), hyaluronic acid (HA), and TIMP metallopeptidase inhibitor 1 (TIMP1), and used measurements to develop an algorithm that could discriminate patients with NAFLD with advanced fibrosis (F3-F4; 24.1% of cohort) from those with mild or moderate fibrosis (F0-F2; 79.5% of cohort). We validated the algorithm using serum samples collected from a separate 396 patients from the same time period and location (validation cohort 1), as well as 244 patients with NAFLD evaluated at a separate location, from 2011 through 2017, within a median of 11 days of liver biopsy (cohort 2). RESULTS: The algorithm identified patients with advanced fibrosis vs mild or moderate fibrosis in training cohort 1 with an area under the receiver operating characteristic (AUROC) curve of 0.867 (95% CI, 0.827-0.907), 84.8% sensitivity (95% CI, 75.5%-91.0%), and 72.3% specificity (95% CI, 66.9%-77.3%), at a cutoff score of 17. The AUROC for the combined validation cohorts 1 and 2 (n=640) was 0.856 (95% CI, 0.820-0.892), identifying patients with 79.7% sensitivity (95% CI, 71.9%-86.2%) and 75.7% specificity (95% CI, 71.8%-79.4%) at the predetermined cutoff score of 17. The algorithm had negative predictive values that ranged from 92.5% to 94.7% in the validation cohorts; it correctly classified 90.0% of F0 samples, 75.0% of F1 samples, 77.4% of F3 samples, and 94.4% of F4 samples. CONCLUSION: We developed an algorithm that identifies patients with advanced fibrosis from those with mild to moderate fibrosis in patients with NAFLD with an AUROC value of approximately 0.86, based on levels of serum biomarkers. We validated the findings in 2 separate sets of patients with biopsy-proven NAFLD. The algorithm can be used non-invasively to determine risk of advanced fibrosis in patients with NAFLD.

Serum Concentrations of 7α-hydroxy-4-cholesten-3-one Are Associated With Bile Acid Diarrhea in Patients With Crohn's Disease.

BACKGROUND & AIMS: Patients with Crohn's disease (CD) often have bile acid diarrhea (BAD), due to bile acid malabsorption following ileal resection (IR). Bile acid malabsorption increases production of 7α-hydroxy-4-cholesten-3-one (C4), a bile acid precursor. We investigated relationships between serum concentrations of C4 and BAD in patients with CD. METHODS: We collected demographic data, serum samples, and information on the presence of diarrhea (>3 liquid bowel movements/day), as well as clinical, endoscopic, and histologic scores from 26 patients with CD and IR, 21 patients with CD without IR, and 37 patients with ulcerative colitis (UC). We compared serum concentrations of C4 and fibroblast growth factor 19 (FGF19) between groups. We performed area under the receiver operating characteristic curve (AUROC) analysis to identify the optimal cutoff C4 concentrations for the diagnosis of diarrhea attributable to bile acid malabsorption (BAD), defined as diarrhea and a serum concentration of FGF19 <60 pg/mL. RESULTS: Patients with UC had a median serum C4 concentration of 11.8 ng/mL, whereas patients with CD and IR with ileitis (documented endoscopically) had a median concentration of 100.0 ng/mL (P compared to UC < .0001) and patients with CD and IR without ileitis had a median concentration of 51.6 ng/mL (P compared to UC < .001). Patients with CD without IR did not have a significantly higher median concentration of C4 than patients with UC (P = .71), regardless of ileitis (P = .34). When endoscopic findings were confirmed histologically, similar results were found to analyses using endoscopic findings alone. A higher proportion of patients with active UC had diarrhea (72.0% vs 0 patients with inactive UC; P < .001), but their median concentrations of C4 did not differ significantly from that of patients with inactive UC (12.1 ng/mL vs 9.7 ng/mL; P = .3). A cutoff concentration of C4 of 48.3 ng/mL or greater identified patients with diarrhea attributable to bile acid malabsorption with 90.9% sensitivity, 84.4% specificity, and an AUROC 0.94. A significantly higher proportion of patients with concentrations of C4 above this cutoff had BAD (50.0%) than below this cutoff (1.8%) (P < .001). When we analyzed only patients with diarrhea, a C4 cutoff of 48.3 ng/mL identified those with low FGF19 concentrations (<60 pg/mL) with 91% sensitivity and 95.5% specificity (AUROC, 0.99). Above this cutoff, 83.3% of patients had a serum concentration of FGF19 <60 pg/mL compared to 4.5% below this threshold (P < .0001). C4 concentrations correlated with the number of daily bowel movements (r = 0.41; P = .004) and correlated inversely with FGF19 concentrations (r = -0.72; P<.0001). CONCLUSION: We observed significantly increased serum concentrations of C4 in patients with CD with IR, compared to patients with UC. A cutoff concentration of C4 above 48.3 ng/mL identifies patients with diarrhea likely attributable to bile acid malabsorption (BAD) with an AUROC value of 0.94. Increased serum levels of bile acid precursors identify patients with diarrhea and a low serum concentration of FGF19, and concentrations of C4 correlate with daily liquid bowel movements and correlate inversely with FGF19 concentrations. C4 may be a biomarker to identify patients with diarrhea attributable to bile acid malabsorption.

Sensitive detection of genetic variants of HIV-1 and HCV with an HIV-1/HCV assay based on transcription-mediated amplification.

This paper describes a comprehensive study of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genotype sensitivity of the transcription-mediated amplification (TMA)-based HIV-1/HCV assay, developed and manufactured by Gen-Probe Incorporated (San Diego, CA) for screening human plasma specimens in blood bank settings. The TMA HIV-1/HCV assay is a qualitative, in vitro nucleic acid testing system used for initial screening. HIV-1 and HCV discriminatory assays are used to distinguish between HIV-1 and HCV infection or co-infection. In this study, multiple unique specimens representing HCV genotypes 1-6 were tested at various dilutions. The results show that the TMA HIV-1/HCV assay and the TMA HCV discriminatory assay have similar HCV genotype sensitivity, as both assays detected all six genotypes at 100 copies/ml and nearly all replicates tested at 30 copies/ml. Similarly, numerous unique specimens representing HIV-1 group M subtypes (A-G), HIV-1 group N, and group O specimens were tested at various dilutions. The TMA HIV-1/HCV assay and the TMA HIV-1 discriminatory assay were found to have similar HIV-1 subtype sensitivity; all variants at 100 copies/ml and nearly all at 30 copies/ml were detected. These results indicate that the TMA assays meet the sensitivity requirements for blood screening in blood banks worldwide.

Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism.

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

Significant closure of the human immunodeficiency virus type 1 and hepatitis C virus preseroconversion detection windows with a transcription-mediated-amplification-driven assay.

While the present generation of serology-based assays has significantly decreased the number of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections acquired by transfusion, the possibility of infected donations escaping detection still exists. The average seronegative viremic window duration during which immunological assays are unable to detect the virus is estimated to be between 16 and 22 days for HIV-1 and approximately 70 days for HCV. Significant reduction of detection window duration was demonstrated using a nucleic acid amplification assay, the Procleix HIV-1/HCV Assay, which utilizes transcription-mediated amplification technology to simultaneously detect HIV-1 and HCV RNAs. For 26 commercially available HIV-1 seroconversion panels tested, specimens were reactive in the HIV-1/HCV assay at the same time as or earlier than in serological assays. Overall, the HIV-1/HCV assay was able to reduce the detection window duration by an average of 14 days and 6 days compared to tests relying on recognition of HIV-1 antibody and p24 antigen, respectively. For 24 commercially available HCV seroconversion panels tested, the specimens were reactive in the HIV-1/HCV assay at an earlier blood sampling date than in serological assays, reducing the detection window duration by an average of 26 days. Similar results were obtained in testing the HIV-1 and HCV seroconversion panels in the virus-specific HIV-1- and HCV-discriminatory assays, respectively. In conclusion, the HIV-1/HCV assay and corresponding discriminatory assays significantly reduced detection window durations compared to immunoassays.

HCV viral load in anti-HCV-reactive donors and infectivity for their recipients.

BACKGROUND: An attempt has been made to determine the minimum level of HCV nucleic acid in donors associated with infection of recipients. This is important for considerations about assay sensitivity, use of minipool versus single-donation testing, and continued use of serologic testing. STUDY DESIGN AND METHODS: A total of 5387 specimens from the Transfusion-Transmitted Viruses Study in the 1970s were screened for antibody to HCV (anti-HCV). The outcome in recipients of seroreactive donations was examined for viremia and seroconversion. Present techniques for both groups included third-generation EIA, RIBA, quantitative RT-PCR assay, and transcription-mediated amplification (TMA) assay. RESULTS: A total of 156 recipients of components from 180 anti-HCV-reactive donors were identified. One-hundred seven of these were HCV-naïve before transfusion and received a single, confirmed seropositive unit; 94 (88%) became infected. Eighty-five recipients had donors whose HCV RNA level was quantifiable by RT-PCR (range, 182-3,310,000 copies/mL). Eighty-three (98%) seroconverted. Of the remaining 22, a total of 10 received units positive for HCV RNA detected only by TMA; all 10 recipients seroconverted. Of the remaining 12 recipients of anti-HCV+, TMA-negative units, 1 recipient seroconverted. CONCLUSIONS: High rates of transmission were seen at all levels of viremia, and one donor transmitted with undetectable levels in the TMA assay. Current HCV RNA testing will therefore not interdict all infectious units, even with single-donation testing, and serologic screening must be continued.

Clinical specificity and sensitivity of a blood screening assay for detection of HIV-1 and HCV RNA.

BACKGROUND: An HIV-1 and HCV NAT blood screening assay (Procleix HIV-1/HCV, Gen-Probe, Inc.) simultaneously detecting HIV-1 and HCV RNA) has been implemented. Donor plasma samples reactive in the Procleix HIV-1/HCV assay are tested with the HIV-1 and HCV discriminatory assays to resolve whether HIV-1 RNA, HCV RNA, or both are present. STUDY DESIGN AND METHODS: To determine the specificity of the Procleix HIV-1/HCV assay, data were analyzed for samples from 192,288 donations, tested in 16-member pools. To determine sensitivity, data were analyzed for 2014 commercial samples known to contain HIV-1, HCV, or both, as well as 10 HIV-1 and 10 HCV commercial seroconversion panels. RESULTS: The specificity of the Procleix HIV-1/HCV assay was 99.7 percent. The HIV-1 and HCV discriminatory assays showed similar specificity. The sensitivity of the Procleix HIV-1/HCV assay was 99.9, 99.6, and 100 percent, respectively, for samples containing HIV-1, HCV, or both. The Procleix discriminatory assays were comparably sensitive. The Procleix discriminatory assays detected all tested samples of known HIV-1 subtype or HCV genotype. Procleix HIV-1/HCV testing of seroconversion panels showed that the median times to a positive reaction for HIV-1 and HCV were reduced by 3 and 25 days, respectively, compared to serologic tests. CONCLUSION: These studies support the use of the Procleix HIV-1/HCV assay for routine blood donor screening.