CIN2+ detection of the HPV DNA Array genotyping assay in comparison with the Cobas 4800 HPV test and cytology

BACKGROUND: HPV DNA Array is an E1-targeting PCR genotyping test, with capability of distinguishing 18 high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) and 11 low-risk HPV types (6, 11, 40, 42, 44, 54, 67, 69, 70, 85, 97). HPV DNA Array uses multiplex PCR for E1-gene sequence amplification. The amplicons are detected and genotyped by reverse hybridization to immobilized DNA probes spotted as triplets in single 96 well-plate wells and read by AID ELISPOT reader. METHODS: Aim of the study was to evaluate the clinical performance of the assay against internationally accepted and FDA approved Cobas 4800 HPV test (Roche Diagnostics). Study population comprised of 500 cervical samples. RESULTS: HPV DNA Array demonstrated a very high sensitivity of 100% for CIN2+ and 100% for CIN3+ detection, same as Cobas 4800. HPV DNA Array showed greater sensitivity for CIN2+ detection than cytology (100% vs. 13.6%). The agreement to Cobas 4800 for HPV detection, irrespective of type, was 81.4% with κ = 0.613. The agreement for HPV 16 was 92.8% (κ = 0.929), and for HPV 18 54.2% (κ = 0.681). CONCLUSION: HPV DNA Array demonstrated good clinical performance for detection of high-grade lesions, and may be considered for usage in a screening setting

Clinical performance of the HPV DNA Array genotyping assay in detection of CIN2+ lesions with BS GP5+/6+ MPG Luminex tested cervical samples

Human papillomavirus (HPV) detection is used for screening of cervical cancer and genotype-specific persistence has shown to be mandatory for dysplasia development. Aim of this study was to evaluate the clinical performance of HPV DNA Array for cervical intraepithelial neoplasia 2+ (CIN2+) lesion detection. HPV DNA Array is a polymerase chain reaction-based assay that targets E1 sequences of 29 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, and 97). The clinical evaluation was performed against the reference assay, BS-GP5+/6+ multiplex genotyping (MPG)-Luminex, with 600 cervical smear samples of a referral population. HPV DNA Array detected CIN2+ lesions with a sensitivity of 90.2%, identical to that of MPG-Luminex. Detection of CIN3+ lesions was with a sensitivity of 90.3%, as compared with 88.7% of MPG-Luminex. It demonstrated very good agreement for HPV detection, irrespective of type, of 91.5% (kappa = 0.832). HPV DNA Array is a simple and robust assay, with a short protocol of 4 hours hands-on time and automated readout by ELISpot AiDot software. It permits testing of up to 96 samples in one run and may be considered for use in organized screening programs and low resource settings

Combined antigen-specific interferon-γ and interleukin-2 release assay (FluoroSpot) for the diagnosis of Mycobacterium tuberculosis infection.

To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.Correlation of IFN-γ+ spot-forming cells in EliSpot-IGRA and FluoroSpot were R2 = 0.67 for ESAT-6 and R2 = 0.73 for CFP-10. The number of IL-2- IFN-γ+ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ+ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice

Demographic characteristics of study subjects by groups.

<p>TB = tuberculosis.</p><p>LTBI = latent infection with <i>M</i>. <i>tuberculosis</i>.</p><p>m = male.</p><p>f = female.</p><p>n = number of cases.</p><p>Demographic characteristics of study subjects by groups.</p

ESAT-6 and CFP-10- induced cytokine response in FluoroSpot.

<p>ESAT-6 induced cytokine immune response in 200.000 PBMCs/well in participants with active tuberculosis (TB, circle, n = 18), past tuberculosis (past TB, inverted triangle, n = 10), latent infection with <i>M</i>. <i>tuberculosis</i> (LTBI, square, ESAT-6-induced n = 24, CFP-10-induced n = 22), EliSpot-negative individuals (control, triangle, ESAT-6-induced n = 17, CFP-10-induced n = 19) was analysed. Groups had been defined according to the combination of their ESAT-6 and CFP-10 induced IFN-γ EliSpot-IGRA test result and clinical data. The number of IL-2⁺ (A), INF-γ⁺ (B), IL-2⁺ INF-γ^- (C), IL-2^- INF-γ⁺ (D) and IL-2⁺ INF-γ⁺ (E) spot-forming cells (SFC) were enumerated by FluoroSpot. (F) Mean proportion of ESAT-6 (top row) and CFP-10 (bottom row) -specific cytokine secreting cells for individuals with tuberculosis, past tuberculosis and LTBI are depicted as pie charts (light grey = IL-2⁺ INF-γ^-, black = IL-2^- INF-γ⁺ and dark grey = IL-2⁺ INF-γ⁺ secreting cells). Mann-Whitney U-test for non-parametric data was used for comparative analysis. A p-value of <0.05 was considered significant.</p

Flow chart of patients included in this study.

<p>TB = tuberculosis; LTBI = latent infection with <i>M</i>. <i>tuberculosis;</i> control = healthy individual with negative EliSpot-IGRA result; E = ESAT-6; C = CFP-10; dark grey = positive test result in the EliSpot-IGRA; light grey = negative test result in the EliSpot-IGRA.</p

Detection of INF-γ⁺ in EliSpot-IGRA and their concordance with INF-γ⁺ results in FluoroSpot.

<p>ESAT-6 (A) and CFP-10 (B) -induced INF-γ⁺ immune response in 200.000 PBMCs/well in participants with active tuberculosis (TB, circle, n = 18), past tuberculosis (past TB, inverted triangle, n = 10), latent infection with M. tuberculosis (LTBI, square, ESAT-6-induced n = 24, CFP-10-induced = 22) and EliSpot-negative individuals (control, triangle, ESAT-6-induced n = 17, CFP-10-induced n = 19) was analysed. LTBI and controls had been defined according to their ESAT-6 and CFP-10-induced IFN-γ EliSpot-IGRA test result and clinical data. Number of INF-γ⁺ spot-forming cells (SFC) was enumerated by EliSpot. ESAT-6 (C) and CFP-10 (D) induced- INF-γ⁺ SFC in EliSpot-IGRA (solid symbols) and FluoroSpot (open symbols) were analysed as matched pairs (connected with lines), differences were calculated using Wilcoxon signed rank test. Correlation between the number of ESAT-6 (E) and CFP-10 (F) specific INF-γ⁺ spot-forming cells (SFC) in PBMC of 69 donors detected by FluoroSpot and EliSpot-IGRA. Concordance between EliSpot-IGRA and FluoroSpot results were assessed using R² coefficient. Agreement by Bland–Altman test was expressed as mean difference (horizontal solid line) and 95% limits of agreement (dashed line) between ESAT-6 (G) and CFP-10 (H) induced- INF-γ⁺ SFC in FluoroSpot compared to EliSpot-IGRA.</p

SARS‐CoV‐2‐specific serological and functional T cell immune responses during acute and early COVID‐19 convalescence in solid organ transplant patients

International audienceThe description of protective humoral and T cell immune responses specific against SARS-CoV-2 has been reported among immunocompetent (IC) individuals developing COVID-19 infection. However, its characterization and determinants of poorer outcomes among the at-risk solid organ transplant (SOT) patient population have not been thoroughly investigated. Cytokine-producing T cell responses, such as IFN-γ, IL-2, IFN-γ/IL-2, IL-6, IL-21, and IL-5, against main immunogenic SARS-CoV-2 antigens and IgM/IgG serological immunity were tracked in SOT (n = 28) during acute infection and at two consecutive time points over the following 40 days of convalescence and were compared to matched IC (n = 16) patients admitted with similar moderate/severe COVID-19. We describe the development of a robust serological and functional T cell immune responses against SARS-CoV-2 among SOT patients, similar to IC patients during early convalescence. However, at the infection onset, SOT displayed lower IgG seroconversion rates (77% vs. 100%; p = .044), despite no differences on IgG titers, and a trend toward decreased SARS-CoV-2-reactive T cell frequencies, especially against the membrane protein (7 [0-34] vs. 113 [15-245], p = .011, 2 [0-9] vs. 45 [5-74], p = .009, and 0 [0-2] vs. 13 [1-24], p = .020, IFN-γ, IL-2, and IFN-γ/IL-2 spots, respectively). In summary, our data suggest that despite a certain initial delay, SOT population achieve comparable functional immune responses than the general population after moderate/severe COVID-19