Antimicrobial susceptibility and molecular characterization of Mycobacterium intracellulare in China

Mycobacterium avium complex (MAC) is the most common non-tuberculosis mycobacterial pathogen isolated from respiratory samples, mainly including two species, Mycobacterium avium (M. avium) and Mycobacterium intracellulare (M. intracellulare). Although these two species belong to the same group, M. avium and M. intracellulare reveal significantly differences in pathogenicity and biology. Nevertheless, little is known regarding the drug resistant details profile of M. avium or M. intracellulare instead of MAC. Here, we examined the antimicrobial susceptibility profiles of 52 clinical M. intracellulare isolates against fourteen antimicrobial agents, which are widely selected for the treatment of nontuberculous mycobacteria (NTM) infection. The drug susceptibility test revealed that clarithromycin (47/52, 90.4%), rifampicin (41/52, 78.8%) and capreomycin (40/52, 76.9%) revealed highly antimicrobial activities against M. intracellulare isolates in vitro. Furthermore, all clarithromycin resistant isolates harbored mutations in the 23S rRNA gene, and the percentage of amikacin resistant ones with mutation in the rrs gene is 62.5% (10/16). The Hunter–Gaston Discriminatory Index (HGDI) value for the 16-loci Variable Number of Tandem Repeat (VNTR) typing of M. intracellulare isolates was 0.994, and M. intracellulare resistance to moxifloxacin was significantly more commonly found in clustered strains than in nonclustered strains (χ² = 5.551, P = 0.040). In conclusion, our data demonstrated that clarithromycin and capreomycin revealed highly antimicrobial activities against M. intracellulare isolates, and clarithromycin and amikacin resistance could be detected more readily and rapidly using molecular scanning of corresponding drug target than conventional drug susceptibility testing. We also found that infection by clustered strains was significantly associated with resistance to moxifloxacin.     

Holographic Characterization of Protein Aggregates in the Presence of Silicone Oil and Surfactants

Characterizing protein aggregates in the presence of silicone oil is a long standing challenge for the pharmaceutical industry. Silicone oil is often used as a lubricant in devices that deliver and store therapeutic protein products and has been linked to protein aggregation, which can compromise a drug’s effectiveness or cause autoimmune responses in patients. Most traditional technologies cannot quantitatively distinguish protein aggregates and silicone oil in their native formulations for sizes less than 5 μm. We use holographic video microscopy to study protein aggregation to demonstrate its capability to quantitatively distinguish protein aggregates and silicone oil in the presence of varying amounts of the surfactants SDS and polysorbate 80 in the size range of 0.5-10 μm without the need for dilution or special sample preparation. We show that SDS denatures proteins and stabilizes silicone oil. We also show that polysorbate 80 may limit protein aggregate formation if it is added to an IgG solution before introducing silicone oil.     

Application of a Best Practice Approach Using Resonant Mass Measurement for Biotherapeutic Product Characterization

Characterizing and quantifying subvisible particles in protein drug products is critical to ensuring product quality. A variety of analytical methods are used to detect and make meaningful measurements of subvisible particles. Resonant mass measurement (RMM) is a novel technology that characterizes the subvisible particle content of samples on a particle-by-particle basis. The technology presents great promise in the study of therapeutic protein products. As an emerging tool in the biopharmaceutical field, the best practices and limitations of RMM for protein products have not been well established. One key challenge of particle analysis is producing robust and reliable data, with high precision and accuracy, for particle characterization. In this study, we develop a set of possible best practices for RMM using a model protein system. We test the effects of these practices on the repeatability and reproducibility of particle measurements. Additionally, we present the data collected under a rigorously controlled set of operating conditions at 3 collaborating sites as well as a summary of the resulting optimal practices. In employing these practices, we successfully obtained improved relative standard deviation values and achieved high reproducibility and repeatability in both sizing and concentration measurement results over a broad range of sample volumes.     

Higher-Order Structure Characterization of Pharmaceutical Proteins by 2D Nuclear Magnetic Resonance Methyl Fingerprinting

A key challenge in the analytical assessment of therapeutic proteins is the comprehensive characterization of their higher-order structure (HOS). To directly assess HOS, a new type of assay is warranted. The most sensitive and detailed method for characterizing HOS is unquestionably nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy provides direct information about the HOS at an atomic level, and with modern NMR spectrometers and improved pulse sequences, this has become feasible even on unlabeled proteins. Hence, NMR spectroscopy could be a very powerful tool for control of HOS following, for example, process changes resulting in structural changes, oxidation, degradation, or chemical modifications. We present a method for characterizing the HOS of therapeutic proteins by monitoring their methyl groups using 2D H, C-correlated NMR. We use a statistical model that compares the NMR spectrum of a given sample to a reference and results in one output value describing how similar the HOS of the samples are. This makes the overall result easy to interpret even for non-NMR experts. We show that the method is applicable to proteins of varying size and complexity (here up to ～30 kDa) and that it is sufficiently sensitive for the detection of small changes in both primary and HOS.     

Human pegivirus-1 infection in kidney transplant recipients: A single-center experience

Kidney transplantation is the treatment of choice for patients with end-stage renal disease. In the posttransplant period, the induced immunosuppression leads to an increased risk of developing infectious diseases, a leading cause of death after kidney transplantation. Human pegivirus-1 (HPgV-1) is considered a nonpathogenic human virus and is highly frequent in individuals parenterally exposed, however, its impact on kidney transplantation outcome is poorly understood. Given the scarcity of epidemiological data for this infection on organ recipients in Brazil, we conducted a study in a single center for kidney transplantation in Rio de Janeiro, aiming to determine HPgV-1 prevalence and genotypic distribution. Serum samples from 61 renal recipients, followed up for the first year after transplantation, were evaluated for viral RNA and genotypes were determined by sequencing of the 5′-untranslated region. HPgV-1 RNA was detected in 36.1% (22/61) of patients. Genotype 2 was the most commonly found (80.9%), followed by genotypes 3 (9.5%), 1, and 5, in 4.8% each. Statistical comparisons did not reveal any significant impact of HPgV-1 in patient outcome. Further epidemiologic studies are needed to understand if immunosuppression may interfere in HPgV-1 persistence rates and if viremia might impact graft dysfunction rates in kidney recipients.     

Case of Desbuquois dysplasia type 1: Potentially lethal skeletal dysplasia

We report a boy with Desbuquois dysplasia type 1. He had the typical skeletal changes: a “Swedish key” appearance of the proximal femora; advanced carpal ossification and other distinctive features of the hand, including an extra‐ossification center at the base of the proximal phalanx of the index and middle fingers; dislocation of the metacarpophalangeal joint of the index finger; and bifid distal phalanx of the thumb. In addition, he presented with very severe prenatal growth failure, respiratory distress as a neonate, subsequent failure to thrive and susceptibility to airway infection, and sudden death in early childhood. Molecular analysis identified homozygous 1 bp deletion in the Calcium‐Activated Nucleotidase 1 gene (CANT1). To our knowledge, this is the first report of Desbuquois dysplasia type 1 in Japan. Our experience suggests potential lethality in the disorder.     

The effect of heating rate on the surface chemistry of NiTi

The impact of the heating rate on the Ni content at the surface of the oxide layer of biomedical NiTi is explored. Heat treatment emulating common shape-setting procedures was performed by means of conventional and inductive heating for similar annealing time and temperature, applying various heating rates from ∼0.25 K s⁻¹ to 250 K s⁻¹. A glow discharge optical emission spectroscopy method was established and employed to evaluate concentration profiles of Ni, Ti and O in the near-surface region at high resolution. The Ni content at the surface of the differently treated samples varies significantly, with maximum surface Ni concentrations of ∼20 at.% at the lowest and ∼1.5 at.% at the highest heating rate, i.e. the total amount of Ni contained in the surface region of the oxide layer decreases by >15 times. Consequently, the heating rate is a determinant for the biomedical characteristics of NiTi, especially since Ni available at the surface of the oxide layer may affect the hemocompatibility and be released promptly after surgical application of a respective implant. Furthermore, apparently contradictory results presented in the literature reporting surface Ni concentrations of ∼3 at.% to >20 at.% after heat treatment are consistently explained considering the ascertained effect of the heating rate.