Using the monocyte activation test as a stand-alone release test for medical devices.

Monocyte activation tests (MAT) are widely available but rarely used in place of animal-based pyrogen tests for safety assessment of medical devices. To address this issue, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods and the PETA International Science Consortium Ltd. convened a workshop at the National Institutes of Health on September 18-19, 2018. Participants included representatives from MAT testing laboratories, medical device manufacturers, the U.S. Food and Drug Administration's Center for Devices and Radiologic Health (CDRH), the U.S. Pharmacopeia, the International Organization for Standardization, and experts in the development of MAT protocols. Discussions covered industry experiences with the MAT, remaining challenges, and how CDRH's Medical Device Development Tools (MDDT) Program, which qualifies tools for use in evaluating medical devices to streamline device development and regulatory evaluation, could be a pathway to qualify the use of MAT in place of the rabbit pyrogen test and the limulus amebocyte lysate test for medical device testing. Workshop outcomes and follow-up activities are discussed.

Distribution of Roseobacter RCA and SAR11 lineages and distinct bacterial communities from the subtropics to the Southern Ocean.

We assessed the composition of the bacterioplankton in the Atlantic sector of the Southern Ocean in austral fall and winter and in New Zealand coastal waters in summer. The various water masses between the subtropics/Agulhas-Benguela boundary region and the Antarctic coastal current exhibited distinct bacterioplankton communities with the highest richness in the polar frontal region, as shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. The SAR11 clade and the Roseobacter clade-affiliated (RCA) cluster were quantified by real-time quantitative PCR. SAR11 was detected in all samples analysed from subtropical waters to the coastal current and to depths of > 1000 m. In fall and winter, this clade constituted < 3% to 48% and 4-28% of total bacterial 16S rRNA genes respectively, with highest fractions in subtropical to polar frontal regions. The RCA cluster was only present in New Zealand coastal surface waters not exceeding 17 degrees C, in the Agulhas-Benguela boundary region (visited only during the winter cruise), in subantarctic waters and in the Southern Ocean. In fall, this cluster constituted up to 36% of total bacterial 16S rRNA genes with highest fractions in the Antarctic coastal current and outnumbered the SAR11 clade at most stations in the polar frontal region and further south. In winter, the RCA cluster constituted lower proportions than the SAR11 clade and did not exceed 8% of total bacterial 16S rRNA genes. In fall, the RCA cluster exhibited significant positive correlations with latitude and ammonium concentrations and negative correlations with concentrations of nitrate, phosphate, and for near-surface samples also with chlorophyll a, biomass production of heterotrophic prokaryotes and glucose turnover rates. The findings show that the various water masses between the subtropics and the Antarctic coastal current harbour distinct bacterioplankton communities. They further indicate that the RCA cluster, despite the narrow sequence similarity of > 98% of its 16S rRNA gene, is an abundant component of the heterotrophic bacterioplankton in the Southern Ocean, in particular in its coldest regions.

Detection of pyrogens adsorbed to intraocular lenses: evaluation of limulus amoebocyte lysate and in vitro pyrogen tests.

PURPOSE: To determine the ability of the limulus amoebocyte lysate (LAL) assay and the in vitro pyrogen test (IPT) to detect pyrogens adsorbed to intraocular lenses (IOLs). SETTING: Berlin Eye Research Institute, Berlin, Germany. METHODS: Fifteen of each of the following IOLs were used: MicroSil MS 612 ASP, AcrySof SA60AT, Superflex, Sensar, XACT, and LS-106 IOLs. The challenge organism suspensions were 10(3) CFU/mL and 10(4) CFU/mL Escherichia coli, 10(3) CFU/mL and 10(4) CFU/mL Pseudomonas putida, and 10(5) CFU/mL and 10(6) CFU/mL Staphylococcus epidermidis. Two IOLs of each model were incubated at room temperature for at least 2 days in 0.6 mL of 1 of the suspensions. They were then gamma sterilized. The extract of 1 IOL was tested with the LAL assay; the other IOL was tested with the IPT. RESULTS: The LAL was negative for all incubated IOLs. The IPT was positive for all IOLs incubated in E coli and P putida suspensions, with the MicroSil MS 612 ASP, AcrySof SA60AT, XACT, and LS-106 IOLs showing a severe reaction. The Superflex and Sensar IOLs had a slight to moderate response for lower bacterial concentrations and a moderate to severe response for higher concentrations. For S epidermidis, all IOLs showed a slight IPT response except XACT IOLs, which showed a nonpyrogenic response. CONCLUSIONS: Results indicate that the LAL test may fail to detect pyrogens adsorbed to IOLs and the IPT reliably detects pyrogens with a dose-dependent response. This has relevance in the investigation of toxic anterior segment syndrome outbreaks.

Bacterial consumption of humic and non-humic low and high molecular weight DOM and the effect of solar irradiation on the turnover of labile DOM in the Southern Ocean.

The decomposition of dissolved organic matter (DOM) in pelagic ecosystems is mediated primarily by heterotrophic bacteria, but transformation by short-wave solar radiation may play an important role in surface waters, in particular when humic substances constitute a substantial fraction of the DOM pool. Most of the studies examining bacterial decomposition and photochemical transformation of DOM stem from limnetic and coastal marine systems and much less information is available from oceanic environments. To examine the bacterial decomposition of humic and non-humic DOM in the Southern Ocean we carried out microcosm experiments in which we measured bacterial growth on isolated fractions of humic and non-humic DOM of the size classes <3 kDa and >3 kDa. Experiments carried out at the Polar Front showed a preferential bacterial growth on non-humic DOM and in particular on the size fraction <3 kDa. Bacterial growth, measured as bacterial biomass production, on non-humic DOM accounted for 74% to 88% of the total growth on all four DOM fractions. In experiments in the Antarctic circumpolar current and the coastal current under pack ice, bacterial growth was 6x lower than at the Polar Front, and humic and non-humic DOM was consumed to equal amounts. The size fraction <3 kDa was always preferred. Experiments examining the effect of solar radiation on the release of dissolved amino acids (DAA) and carbohydrates (DCHO) and their subsequent bacterial utilization showed a stimulating effect on glucose uptake and the release of DAA at the Polar Front but an inhibition in the eastern Weddell Sea. Ultraviolet-B was the most effective component of the solar radiation spectrum tested. Effects of UV-B on glucose uptake and release of DAA were positively correlated with concentrations of humic-bound DAA. The data imply that at low concentrations, e.g., <100 nM (amino acid equivalent), UV-irradiation reduces, whereas at concentrations >100 nM UV-irradiation stimulates glucose uptake and release of DAA as compared to dark conditions.

Preservative-free nasal drug-delivery systems.

Unpreserved nasal sprays are the latest trend in nasal drug delivery. Different technologies are discussed here in the context of mechanically protected systems, which may provide additional benefits. New test protocols are suggested to evaluate the improved microbiological protection that can be achieved.

Influence of Myriophyllum spicatum-derived tannins on gut microbiota of its herbivore Acentria ephemerella.

The submerged living larvae of Acentria ephemerella were fed in the laboratory with either M. spicatum or Potamogeton perfoliatus, two of their host plants. Larvae exhibited a reduced growth when fed M. spicatum, a freshwater angiosperm that contains high concentrations of tannins, secondary metabolites known for their herbivore-deterrent and antimicrobial properties. In this study, we investigated the influence of food-derived tannins on gut microbiota. Bacterial densities in the guts did not differ between the food regimes, ranging from 2.8 to 13.3 x 10(6) cells per gut. Gut bacteria were characterized with cultivation techniques and subsequent identification of the strains by molecular methods. We isolated 17 bacterial strains belonging to all subdivisions, i.e., we identified alpha-, beta-, and gamma-proteobacteria, Cytophyaga/Flavobacteria (CF) and several Gram-positive bacteria. All except one Gram-positive strain were found in the guts of larvae fed with P. perfoliatus. Gram-positive bacteria and bacteria of the CF cluster were more sensitive to polyphenol-containing extracts of M. spicatum in an agar diffusion assay than strains of the alpha- or gamma-proteobacteria subdivision. Our results suggest an influence of food-derived tannins on gut microbiota in A. ephemerella.