Assessment of cytomegalovirus-specific cell-mediated immunity for the prediction of cytomegalovirus disease in high-risk solid-organ transplant recipients: a multicenter cohort study.

BACKGROUND: Cytomegalovirus (CMV) disease remains an important problem in solid-organ transplant recipients, with the greatest risk among donor CMV-seropositive, recipient-seronegative (D(+)/R(-)) patients. CMV-specific cell-mediated immunity may be able to predict which patients will develop CMV disease. METHODS: We prospectively included D(+)/R(-) patients who received antiviral prophylaxis. We used the Quantiferon-CMV assay to measure interferon-γ levels following in vitro stimulation with CMV antigens. The test was performed at the end of prophylaxis and 1 and 2 months later. The primary outcome was the incidence of CMV disease at 12 months after transplant. We calculated positive and negative predictive values of the assay for protection from CMV disease. RESULTS: Overall, 28 of 127 (22%) patients developed CMV disease. Of 124 evaluable patients, 31 (25%) had a positive result, 81 (65.3%) had a negative result, and 12 (9.7%) had an indeterminate result (negative mitogen and CMV antigen) with the Quantiferon-CMV assay. At 12 months, patients with a positive result had a subsequent lower incidence of CMV disease than patients with a negative and an indeterminate result (6.4% vs 22.2% vs 58.3%, respectively; P < .001). Positive and negative predictive values of the assay for protection from CMV disease were 0.90 (95% confidence interval [CI], .74-.98) and 0.27 (95% CI, .18-.37), respectively. CONCLUSIONS: This assay may be useful to predict if patients are at low, intermediate, or high risk for the development of subsequent CMV disease after prophylaxis. CLINICAL TRIALS REGISTRATION: NCT00817908.

Nonrandom Distribution of Cryptic Repeating Triplets of Purines and Pyrimidines (RNY)(n) in gp120 of HIV Type1.

Abstract We have analyzed purine (R) and pyrimidine (Y) codon patterns in variable and constant regions of HIV-1 gp120 in seven patients infected with different HIV-1 subtypes and naive to antiretroviral therapy. We have calculated the relative frequency of each in-frame codon RNY, YNR, RNR, and YNY (N=any nucleotide) in variable and constant regions of gp120, in the sequence within indels and at indels' flanking sites. Our data show that hypervariable regions V1, V2, V4, and V5 are characterized by the presence of long stretches of RNY codons constituting the majority of the sequence portion within insertions/deletions. In full-length gp120 and within inserted/deleted fragments the number of AVT (V=A, C, G) codons did not exceed 50% of the total RNY codons. RNY strings in variable regions spanned up to 21 codons and were always in frame. In contrast, RNY strings in constant regions were mostly out of frame and their length was limited to five codons. The frequency of the codon RNY was found to be significantly higher in variable regions (p<0.0001; t-test), within indels, and at indels' flanking sites (p<0.0001; χ(2) test). Analysis of the distribution of RNY strings equal to or longer than five codons in the full genome of HXB2 also shows that these sequences are mostly out of frame, unless they contain a potential N-glycosylation site or an asparagine. These data suggest that cryptic repeats of RNY may play a role in the genesis of multiple base insertions and deletions in hypervariable regions of gp120.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

Acquisitions thérapeutiques en médecine ambulatoire en 2012 [2012 literature findings in ambulatory internal medicine].

In 2012 several articles reported interesting findings for the ambulatory practice in internal general medicine. A negative rapid test for influenza does not rule out that diagnosis. A test assessing the walking speed in the elderly can help determining who would benefit from antihypertensive therapy. Antibiotic treatment has no benefit for acute uncomplicated rhinosinusitis and diverticulitis. Probiotics can reduce the risk of post-antibiotic diarrhea. Daily coffee intake could reduce mortality. Oral supplementation of calcium can be harmful to the cardiovascular system. Subclinical hyperthyroidism should be treated to prevent cardiovascular complications. Aspirin can prevent recurrences in case of a primary thromboembolic event. Local injection of corticosteroids under ultrasonographic guidance for plantar fasciitis can be a safe treatment. Ibuprofen can prevent acute mountain sickness.

Label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance.

In order to improve the efficacy and safety of treatments, drug dosage needs to be adjusted to the actual needs of each patient in a truly personalized medicine approach. Key for widespread dosage adjustment is the availability of point-of-care devices able to measure plasma drug concentration in a simple, automated, and cost-effective fashion. In the present work, we introduce and test a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup, comprised of off-the-shelf components and coupled with DNA-based aptamers specific to the antibiotic tobramycin (467 Da). The core of the T-LSPR setup are aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor. The gold NIs exhibit localized plasmon resonance in the visible range matching the sensitivity of the complementary metal oxide semiconductor (CMOS) image sensor employed as a light detector. The combination of gold NIs on the FTO substrate, causing NIs size and pattern irregularity, might reduce the overall sensitivity but confers extremely high stability in high-ionic solutions, allowing it to withstand numerous regeneration cycles without sensing losses. With this rather simple T-LSPR setup, we show real-time label-free detection of tobramycin in buffer, measuring concentrations down to 0.5 μM. We determined an affinity constant of the aptamer-tobramycin pair consistent with the value obtained using a commercial propagating-wave based SPR. Moreover, our label-free system can detect tobramycin in filtered undiluted blood serum, measuring concentrations down to 10 μM with a theoretical detection limit of 3.4 μM. While the association signal of tobramycin onto the aptamer is masked by the serum injection, the quantification of the captured tobramycin is possible during the dissociation phase and leads to a linear calibration curve for the concentrations over the tested range (10-80 μM). The plasmon shift following surface binding is calculated in terms of both plasmon peak location and hue, with the latter allowing faster data elaboration and real-time display of the results. The presented T-LSPR system shows for the first time label-free direct detection and quantification of a small molecule in the complex matrix of filtered undiluted blood serum. Its uncomplicated construction and compact size, together with the remarkable performances, represent a leap forward toward effective point-of-care devices for therapeutic drug concentration monitoring.

Multi-institutional validation of the prognostic value of Ki-67 labeling index in patients treated with radical prostatectomy.

OBJECTIVE: Several smaller single-center studies have reported a prognostic role for Ki-67 labeling index in prostate cancer. Our aim was to test whether Ki-67 is an independent prognostic marker of biochemical recurrence (BCR) in a large international cohort of patients treated with radical prostatectomy (RP). METHODS: Ki-67 immunohistochemical staining on prostatectomy specimens from 3,123 patients who underwent RP for prostate cancer was retrospectively performed. Univariable and multivariable Cox regression models were used to assess the association of Ki-67 status with BCR. RESULTS: Ki-67 positive status was observed in 762 (24.4 %) patients and was associated with lymph node involvement (LNI) (p = 0.039). Six hundred and twenty-one (19.9 %) patients experienced BCR. The estimated 3-year biochemical-free survivals were 85 % for patients with negative Ki-67 status and 82.1 % for patients with positive Ki-67 status (log-rank test, p = 0.014). In multivariable analysis that adjusted for the effects of age, preoperative PSA, RP Gleason sum, seminal vesicle invasion, extracapsular extension, positive surgical margins, lymphovascular invasion, and LNI, Ki-67 was significantly associated with BCR (HR = 1.19; p = 0.019). Subgroup analysis revealed that Ki-67 is associated with BCR in patients without LNI (p = 0.004), those with RP Gleason sum 7 (p = 0.015), and those with negative surgical margins (p = 0.047). CONCLUSION: We confirmed Ki-67 as an independent predictor of BCR after RP. Ki-67 could be particularly informative in patients with favorable pathologic characteristics to help in the clinical decision-making regarding adjuvant therapy and optimized follow-up scheduling.

Belgian experience with triple therapy with boceprevir and telaprevir in genotype 1 infected patients who inject drugs.

No data have been reported yet on treatment outcome in persons who inject drugs (PWID) infected with hepatitis C virus treated with boceprevir or telaprevir in combination with peginterferon (Peg IFN) and ribavirin (RBV). Additionally, there are concerns about the safety of boceprevir and telaprevir in some subgroups of patients with hepatitis C (HCV). In a cohort of HCV patients infected with genotype 1 in Belgium, treatment outcome of patients infected due to IV drug use was analyzed and compared with patients who have no history of substance use. The study population consisted of 179 patients: 78 PWID and 101 controls treated with boceprevir (n = 79) or telaprevir (n = 100) additional to Peg IFN and RBV; 53 (30%) had advanced disease (F3, F4) and 79 (44%) had an antiviral therapy previously. There were no significant differences in the baseline characteristics between both groups, except that PWID patients were more frequently infected with genotype 1a (67% vs 21%), were younger and were predominantly male. Psychiatric complaints during follow-up occurred more frequently in the PWID patients: 24% versus 11% (P = .02). Treatment failure for other reasons than absence of viral response was 70% and 64% in PWID and non-PWID respectively. The sustained viral response (SVR) rates were similar in both groups (71% in PWID vs 72% in non-PWID); with a non-inferiority test with -5% margin there is a difference of -1% (95% CI [-15%, 13%]) and P = 0.30. There are no reasons to exclude PWID from treatment with boceprevir, telaprevir and novel antiviral therapies. J. Med. Virol. 88:94-99, 2016. © 2015 Wiley Periodicals, Inc.

Application of intravoxel incoherent motion perfusion imaging to shoulder muscles after a lift-off test of varying duration.

Intravoxel incoherent motion (IVIM) MRI is a method to extract microvascular blood flow information out of diffusion-weighted images acquired at multiple b-values. We hypothesized that IVIM can identify the muscles selectively involved in a specific task, by measuring changes in activity-induced local muscular perfusion after exercise. We tested this hypothesis using a widely used clinical maneuver, the lift-off test, which is known to assess specifically the subscapularis muscle functional integrity. Twelve shoulders from six healthy male volunteers were imaged at 3 T, at rest, as well as after a lift-off test hold against resistance for 30 s, 1 and 2 min respectively, in three independent sessions. IVIM parameters, consisting of perfusion fraction (f), diffusion coefficient (D), pseudo-diffusion coefficient D* and blood flow-related fD*, were estimated within outlined muscles of the rotator cuff and the deltoid bundles. The mean values at rest and after the lift-off tests were compared in each muscle using a one-way ANOVA. A statistically significant increase in fD* was measured in the subscapularis, after a lift-off test of any duration, as well as in D. A fD* increase was the most marked (30 s, +103%; 1 min, +130%; 2 min, +156%) and was gradual with the duration of the test (in 10(-3) mm(2) /s: rest, 1.41 ± 0.50; 30 s, 2.86 ± 1.17; 1 min, 3.23 ± 1.22; 2 min, 3.60 ± 1.21). A significant increase in fD* and D was also visible in the posterior bundle of the deltoid. No significant change was consistently visible in the other investigated muscles of the rotator cuff and the other bundles of the deltoid. In conclusion, IVIM fD* allows the demonstration of a task-related microvascular perfusion increase after a specific task and suggests a direct relationship between microvascular perfusion and the duration of the effort. It is a promising method to investigate non-invasively skeletal muscle physiology and clinical perfusion-related muscular disorders.