Magnetically localized and wash-free fluorescent immuno-assay: From a research platform (MLFIA) to a multiplexed POC system (MagIA).

Sexually transmitted infections (STI) remain one of the world's public health priorities: Nearly 400 million people are infected not only in emerging, but also in western countries. HIV, HBV and HCV share common infection pathways; thus these 3 diseases are recommended to be tested at the same time. However, this combined approach is currently mainly available in laboratories, and seldomly at the Point-of-care (POC). Consequently, there is a need for a STI screening POC platform with laboratory-like performance. Such a platform should be autonomous and portable and enable multiplexed screening from capillary blood. The previously developed and introduced MLFIA (Magnetically Localized and wash-free Fluorescent Immuno-Assay) technology has the potential to address these needs, as the MLFIA 18-chamber microfluidic cartridge and the MLFIA Analyzer were previously characterized and evaluated with plasma and serum from patients infected with HIV, Hepatitis B (Hep B) or C (Hep C). Here, we present the efforts to transfer this research platform (MLFIA) to a fully integrated multi-analysis solution (MagIA). First, we present the design changes of the consumable enabling to perform multiple assays in parallel, a fast filling of the cartridge with patient samples, and a homogeneous reagent/sample incubation. Second, we describe the development a piezoelectric actuator integrated into the Analyzer: this mixing module allows for an automated, fully integrated and portable workflow, with homogeneous in-situ mixing capabilities. The obtained MagIA platform was further characterized and validated for immunoassays (LOD, cartridge stability over time), using various biological models including OVA and IgG. We discuss the performances of the MLFIA and MagIA platforms for the detection of HIV / Hep B / Hep C using results from 102 patient plasma samples. Lastly, we assessed the compatibility of the MagIA platform with veinous and capillary blood samples as a final step towards its POC validation.

Magnetically localized and wash-free fluorescence immunoassay (MLFIA): proof of concept and clinical applications.

Immunoassays are used for many applications in various markets, from clinical diagnostics to the food industry, generally relying on gold-standard ELISAs that are sensitive, robust, and cheap but also time-consuming and labour intensive. As an alternative, we propose here the magnetically localized and wash-free fluorescence immunoassay (MLFIA): a no-wash assay to directly measure a biomolecule concentration, without mixing nor washing steps. To do so, a fluorescence no-wash measurement is performed to generate a detectable signal. It consists of a differential measurement between the fluorescence of fluorophores bound to magnetic nanoparticles specifically captured by micro-magnets against the residual background fluorescence of unbound fluorophores. Targeted biomolecules (antibodies or antigens) are locally concentrated on micro-magnet lines, with the number of captured biomolecules quantitatively measured without any washing step. The performance of the MLFIA platform is assessed and its use is demonstrated with several biological models as well as clinical blood samples for HIV, HCV and HBV detection, with benchmarking to standard analyzers of healthcare laboratories. Thus, we demonstrated for the first time the versatility of the innovative MLFIA platform. We highlighted promising performances with the successful quantitative detection of various targets (antigens and antibodies), in different biological samples (serum and plasma), for different clinical tests (HCV, HBV, HIV).

Opportunistic autoimmunity secondary to cancer immunotherapy (OASI): An emerging challenge.

With "checkpoint inhibitors" targeting PD1/PD-1-ligands or CTLA-4/CD28 pathways, immunotherapy has profoundly modified therapeutic strategies in oncology. First approved in refractory metastatic neoplasms (melanoma and lung adenocarcinoma), it is now being tested broadly in other cancers and/or as adjuvant treatment. For a significant proportion of patients, immunotherapy is responsible for "immunological" events, identified as Immune-Related Adverse Events (irAEs). Owing to the increasing number of prescriptions, identification and management of specific immunological side effects is crucial and requires close collaboration between oncologists and internists and/or other organ specialists. Within irAEs, we propose to individualize the induced autoimmunity by the term "Opportunistic Autoimmunity Secondary to Cancer Immunotherapy" (OASI). The aims of this article are (1) to present the different available checkpoint inhibitors and the OASIs reported with these treatments and (2) to propose practical recommendations for diagnosis, pre-therapeutic assessment and management of OASIs. The need for predictive biomarkers of OASIs occurrence will also be discussed.

Impact of common risk factors of fibrosis progression in chronic hepatitis C.

OBJECTIVE: The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C. DESIGN: We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥ 0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥ 20 g/day for ≥ 5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR. RESULTS: Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR. CONCLUSIONS: Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.

Detection of low HCV viraemia by repeated HCV RNA testing predicts treatment failure to triple therapy with telaprevir.

BACKGROUND: Early on-treatment virological response is one of the most important predictors for sustained virological response (SVR) to treatment of chronic hepatitis C virus (HCV) genotype 1 infection with triple therapy including HCV protease inhibitors (PI). Treatment duration (24 vs. 48 weeks) is based on HCV RNA results at weeks 4 and 12 of PI therapy when HCV RNA must be 'undetectable' to allow shorter therapy. AIM: To analyse the reliability of HCV RNA measurements at key decision time points (weeks 4 and 12) and the predictive value of concordant or discordant assay results for SVR. METHODS: Weeks 4 and 12 samples of patients receiving telaprevir-containing triple therapy were initially tested with the AmpliPrep/COBAS-TaqMan_HCV-Test-v1.0 (limit of detection; LOD = 15IU/mL) and retested with the AmpliPrep/COBAS-TaqMan_HCV-Test-v2.0 (LOD = 15IU/mL) and the High_Pure/COBAS-TaqMan_HCV-Test-v2.0 (LOD = 20IU/mL). RESULTS: Concordance among the three test results in classifying samples as HCV RNA 'undetectable' or 'detectable' was only 55% at week 4, but 85% at week 12. Retesting of 'undetectable' week 4 samples with the respective other assays revealed positive HCV RNA results in 32-50%. In 30%, HCV RNA was 'undetectable' by all three tests at week 4 and all of these patients achieved SVR. In contrast, treatment failure occurred in 62% of patients with at least one 'detectable' result, including cases with one or two other 'undetectable' tests at week 4. CONCLUSIONS: A single 'undetectable' HCV RNA result at week 4 is not always associated with achieving SVR. Repeated testing in difficult-to-treat patients may identify those at risk for treatment failure.

HPV self-sampling or the Pap-smear: a randomized study among cervical screening nonattenders from lower socioeconomic groups in France.

Today in France, low attendance to cervical screening by Papanicolaou cytology (Pap-smear) is a major contributor to the 3,000 new cervical cancer cases and 1,000 deaths that occur from this disease every year. Nonattenders are mostly from lower socioeconomic groups and testing of self-obtained samples for high-risk Human Papilloma virus (HPV) types has been proposed as a method to increase screening participation in these groups. In 2011, we conducted a randomized study of women aged 35-69 from very low-income populations around Marseille who had not responded to an initial invitation for a free Pap-smear. After randomization, one group received a second invitation for a free Pap-smear and the other group was offered a free self-sampling kit for HPV testing. Participation rates were significantly different between the two groups with only 2.0% of women attending for a Pap-smear while 18.3% of women returned a self-sample for HPV testing (p ≤ 0.001). The detection rate of high-grade lesions (≥CIN2) was 0.2‰ in the Pap-smear group and 1.25‰ in the self-sampling group (p = 0.01). Offering self-sampling increased participation rates while the use of HPV testing increased the detection of cervical lesions (≥CIN2) in comparison to the group of women receiving a second invitation for a Pap-smear. However, low compliance to follow-up in the self-sampling group reduces the effectiveness of this screening approach in nonattenders women and must be carefully managed.

[Prevalence of genotype-specific HPV infection among women in France: implications for screening and vaccination]. / Prévalence des génotypes d'HPV chez les femmes en France : implications pour le dépistage et la vaccination.

OBJECTIVES: To assess human papillomavirus (HPV) prevalence and genotype distribution by age and cervical cytology/histology status among women undergoing routine gynecological examinations, and to discuss the possible impact on preventive strategies. PATIENTS AND METHODS: Liquid-based cytology (LBC) samples were tested for HPV DNA, mRNA, and HPV genotypes. Women with ASC-US+ and/or at least one positive HPV test were referred to colposcopy. Those with normal colposcopy results had biopsies taken at the 6 and 12 o'clock positions of the normal transformation zone. RESULTS: Of the 5002 women, 515 (10.3 %) were less than 25 and 4487 (89.7 %) were 25 years old or more. Overall HPV prevalence was 10.1 % to 16.1 % depending on the assay. HPV prevalence increased with the cytological and histological severity of cervical lesions. Prevalence of HPV 16/18 was 5.2 % and 2.7 % in women less than 25 and 25 years old or more, respectively. HPV 16 was the type most strongly associated with a diagnosis of CIN3+ (odds ratio=11.64 versus HPV 16 absent, P<0.001). A high proportion of high-grade cervical lesions (60.6 % of genotyping assay-positive CIN2+) were associated with HPV types 31, 33, 45, 52, or 58. DISCUSSION ET CONCLUSION: These data indicate that almost all young women could benefit from HPV prophylactic vaccination, but confirm the need for continued cervical screening and highlight the need for future vaccines to target a wider range of HPV types.

Serum bile acid levels as a predictor for the severity of liver fibrosis in patients with chronic hepatitis C.

Serum bile acids (SBAs) are commonly elevated in cholestatic liver diseases, but it is unclear if SBA levels are also elevated in noncholestatic chronic liver diseases and whether those levels correlate with disease severity. We analysed SBA levels of 135 consecutive patients with chronic hepatitis C virus infection and correlated these levels with the degree of liver fibrosis as determined by liver biopsy. In addition, we assessed the accuracy of SBA levels as a noninvasive predictor for liver fibrosis by its comparison to the patients' FibroTest scores. Two-thirds (90/135 patients, 67%) of the study patients had nonsevere liver fibrosis (Metavir F0-F2), and the others (45/135, 33%) had severe fibrosis or cirrhosis (Metavir F3-F4). The SBA levels were significantly higher in patients with severe fibrosis as compared to nonsevere fibrosis (11.46 ± 10.01 vs 6.37 ± 4.69, P < 0.0001). Furthermore, a receiver operator characteristics curve based on a model that included serum bile acids, age, body mass index, serum AST, glucose and cholesterol levels suggested that this combination reliably predicts the degree of liver fibrosis and is not inferior to the current noninvasive FibroTest score (areas under the curve of 0.837 vs 0.83, respectively, P = 0.87). We conclude that measurement of SBA levels may have a clinical role as a simple noninvasive tool to assess the severity of HCV-induced liver disease. Combined with widely available laboratory parameters, SBA levels can predict disease severity with a high degree of accuracy.

Genetic polymorphism of IL28B in hepatitis C-infected haemophilia patients in Israel.

Single-nucleotide polymorphisms (SNPs) near the IL28B gene were identified as major predictors of treatment response (sustained virologic response--SVR) and spontaneous clearance of HCV. Haemophilia patients have the highest prevalence of HCV, and are a unique target for genetic studies. The Israeli population is ethnically heterogeneous; therefore, genetic variability is anticipated. To determine the IL28B haplotypes in HCV-infected haemophilia patients and association with SVR and spontaneous viral clearance. IL28B polymorphism at SNPs rs12979860 and rs8099917 was determined in sera obtained from 130 HCV-infected haemophilia patients. The frequency of the various haplotypes was analysed according to treatment response, spontaneous HCV clearance, viral load and degree of fibrosis. The CC haplotype at SNP rs12979860 was found in 31% of patients, whereas the TT genotype at SNP rs8099917 was detected in 57% of cases. SVR was achieved in 70% of patients carrying the CC haplotype (P = 0.0196 vs. CT/TT), and 50% of the TT genotype at SNP rs8099917 (P = 0.0227 vs. TG/GG). Thirty-five percent of patients carrying the CC haplotype and 26% with the TT genotype at SNP rs8099917 showed spontaneous clearance of HCV infection (P = 0.00262 vs. CT/TT; and P = 0.00371 vs. TG/GG respectively). The C-allele frequency was exceptionally high (71%) in immigrants from the Asian republics of Russia. In HCV-infected haemophilia patients, SVR was more commonly achieved among patients who had the CC (rs12979860) or TT (rs8099917) genotype. Likewise, patients who possess harbour the CC or TT genotypes were more likely to clear HCV infection spontaneously. A unique distribution of the CC genotype was observed in some ethnic groups.

Comparison of three algorithms of non-invasive markers of fibrosis in chronic hepatitis C.

BACKGROUND: Preliminary data suggest that performance of non-invasive markers for liver fibrosis in hepatitis C may improve when combined. Three algorithms based on the combination of Fibrotest, Forns' index and AST-to-platelet ratio (APRI) have been proposed: Sequential Algorithm for Fibrosis Evaluation (SAFE biopsy); Fibropaca algorithm; Leroy algorithm. AIM: To compare three algorithms to diagnose significant fibrosis (≥ F2 by METAVIR) and cirrhosis (F4). METHODS: A total of 1013 HCV monoinfected cases undergoing liver biopsy were consecutively enrolled in seven centres. Fibrotest, APRI and Forns' index were measured at the time of liver biopsy, considered the reference standard. RESULTS: Overall, performance of combination algorithms was significantly higher than the single non-invasive methods (P < 0.0001). SAFE biopsy and Fibropaca algorithm saved a significantly higher number of liver biopsies than the single methods (P < 0.0001). For ≥ F2, Fibropaca algorithm saved more biopsies than SAFE biopsy (51.7% vs. 43.8%, P = 0.0003), but with lower accuracy (87.6% vs. 90.3%, P = 0.05). Regarding F4, the number of saved liver biopsies did not differ between SAFE biopsy and Fibropaca algorithm (79.1% vs. 76.2%, P = 0.12). However, SAFE biopsy showed a lower accuracy when compared with Fibropaca algorithm (91.2% vs. 94%, P = 0.02). As to Leroy algorithm, although it showed a good performance for ≥ F2 (93.5% accuracy), it saved less liver biopsies than SAFE biopsy and Fibropaca algorithm (29.2% vs. 43.8% and 51.7% respectively, P < 0.0001). CONCLUSIONS: SAFE biopsy and the Fibropaca algorithm have excellent performance for liver fibrosis in hepatitis C, allowing a significant reduction in the need for liver biopsies. They can be useful in clinical practice and for large-scale screening.

The impact of liver disease aetiology and the stages of hepatic fibrosis on the performance of non-invasive fibrosis biomarkers: an international study of 2411 cases.

BACKGROUND: Performance of non-invasive fibrosis biomarkers may be influenced by aetiology of chronic liver disease (CLD) and the stages of hepatic fibrosis, but large-scale studies are pending. AIM: To investigate the effect of aetiogy and stages of hepatic fibrosis on the performance of fibrosis biomarkers. METHODS: A total of 2411 patients with compensated CLD (HCV=75.1%, HBV=10.5%, NASH=7.9%, HIV/HCV=6.5%) were consecutively enrolled in 9 centres. APRI, Forns'index, Lok index, AST-to-ALT ratio, Fib-4, platelets and Fibrotest-Fibrosure were tested against liver biopsy, considered the gold standard. The effect of the stages of hepatic fibrosis to diagnose significant fibrosis and cirrhosis (≥F2 and F4 respectively) was investigated through difference between advanced and non-advanced fibrosis stages (DANA). Performance was expressed as observed area under the ROC curve (ObAUROC) and AUROC adjusted for DANA (AdjAUROC). RESULTS: Performance of APRI and Fibrotest-Fibrosure was higher than other biomarkers. In all aetiologies, AdjAUROC was higher than ObAUROC. APRI showed its best performance in HCV monoinfected cases, with an AdjAUROC of 0.77 and 0.83 for ≥F2 and F4 respectively. In HBV and non-alcoholic steatohepatitis (NASH) patients, its performance was poor (AdjAUROC <0.70). Performance of Fibrotest-Fibrosure was good in all aetiologies for both ≥F2 and F4 (AdjAUROC >0.73), except for ≥F2 in NASH (AdjAUROC = 0.64). Performance of all biomarkers was reduced in HCV cases with normal ALT. CONCLUSIONS: Aetiology is a major factor influencing the performance of liver fibrosis biomarkers. Even after correction for DANA, APRI and Fibrotest-Fibrosure exhibit the best performance. However, liver biopsy is not replaceable, especially to diagnose ≥F2 and in HCV carriers with normal ALT.

[ICD-10 adaptation of 15 Agency for Healthcare Research and Quality patient safety indicators]. / Adaptation au codage CIM-10 de 15 indicateurs de la sécurité des patients proposés par l'Agence étasunienne pour la recherche et la qualité des soins de santé (AHRQ).

BACKGROUND: In the United States, the Agency for Healthcare Research and Quality (AHRQ) has developed 20 Patient Safety Indicators (PSIs) to measure the occurrence of hospital adverse events from medico-administrative data coded according to the ninth revision of the international classification of disease (ICD-9-CM). The adaptation of these PSIs to the WHO version of ICD-10 was carried out by an international consortium. METHODS: Two independent teams transcoded ICD-9-CM diagnosis codes proposed by the AHRQ into ICD-10-WHO. Using a Delphi process, experts from six countries evaluated each code independently, stating whether it was "included", "excluded" or "uncertain". During a two-day meeting, the experts then discussed the codes that had not obtained a consensus, and the additional codes proposed. RESULTS: Fifteen PSIs were adapted. Among the 2569 proposed diagnosis codes, 1775 were unanimously adopted straightaway. The 794 remaining codes and 2541 additional codes were discussed. Three documents were prepared: (1) a list of ICD-10-WHO codes for the 15 adapted PSIs; (2) recommendations to the AHRQ for the improvement of the nosological frame and the coding of PSI with ICD-9-CM; (3) recommendations to the WHO to improve ICD-10. CONCLUSIONS: This work allows international comparisons of PSIs among the countries using ICD-10. Nevertheless, these PSIs must still be evaluated further before being broadly used.

ActiTest accuracy for the assessment of histological activity grades in patients with chronic hepatitis C, an overview using Obuchowski measure.

BACKGROUND: ActiTest (AT) is a biomarker of liver necro-inflammatory histological activity validated in patients with chronic hepatitis C (HCV). AIM: The aim was to assess the accuracy of AT in comparison with alanine aminotransferase (ALT) the standard of care. METHODS: Methods used an integrated database of individual data and the new recommended Obuchowski measures. An updated "classical" meta-analysis of AT validation studies was also performed. The main end points were the area under the ROC curves (AUROCs) for the diagnosis of each histological activity grade defined using METAVIR scoring system. To avoid repeated tests and the spectrum effect of activity grades prevalence, the comparison of AT and ALT accuracies used the Obuchowski method. RESULTS: For the individual analysis, a total of 1250 patients were included and for the meta-analysis six studies (2017 patients) were included. The overall accuracy of AT for the diagnosis of any activity grade (Obuchowski measure=0.850) was significantly higher than the accuracy of ALT (Obuchowski measure=0.837; P=0.009). The updated standard meta-analysis confirmed the accuracy of AT (p<0.0001) both in independent AUROC=0.79 (95% CI, 0.73-0.85) and in non independent studies AUROC=0.74 (95% CI, 0.67-0.81). CONCLUSIONS: The accuracy of AT for grading the necro-inflammatory activity of patients with HCV was significantly higher than ALT serum activity alone, the standard biomarker.

Fibrotest or Fibroscan for evaluation of liver fibrosis in haemophilia patients infected with hepatitis C.

Non-invasive modalities to estimate fibrosis stage are desirable in hepatitis C-infected haemophilia patients. Previous studies found a high rate of significant fibrosis both by Fibrotest (FT) and Fibroscan (FS) in these patients. To estimate liver fibrosis and to assess the concordance between FT and FS in hepatitis C-infected haemophilia patients. FT and FS were performed at different laboratories and were unaware of the results of the alternative test. Three successive liver stiffness measurements (LSM) were performed at different sites on the liver. Two-validated algorithms were used to improve evaluation of fibrosis by non-invasive methods. Fifty-seven hepatitis C-infected haemophilia patients were evaluated by FT and FS. Acquisition of LSMs was not feasible in two patients: obesity--one, surgical scars--one. Fibrosis stage > or=F2, > or =F3 or =F4 were estimated in about a half, about a third and in 15-20% of the evaluated patients by FS and FT respectively. The corresponding concordance rates and kappa score for fibrosis stage > or =F2, > or =F3 or =F4 between FT and FS were 62%, 69%, 85% and 0.24, 0.32, 0.44 respectively. Using the two aforementioned algorithms, additional 14 patients could be reliably estimated for fibrosis stage > or =F2. High proportion hepatitis C-infected haemophilia patients were estimated with significant or advanced stages of liver fibrosis using both tests. Nevertheless, the agreement between modalities was only fair and improved with more advanced stages of fibrosis. Practical algorithms for the accuracy of FT and FS may improve reliable evaluation of fibrosis in this population.