Bacterial Endotoxin Testing-Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide.

For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitability testing, quality control (QC) sample hold-time studies are requested by authorities like the Food and Drug Administration (FDA) as described in "Guidance for Industry: Pyrogen and Endotoxins Testing." It is requested to be determine whether the ability to detect endotoxins can be affected by storage and handling of the sample to be tested. To accomplish these studies, endotoxin is introduced or spiked into the undiluted product and held for a certain period of time in process-representative containers. This time period reflects procedural maximum QC sample hold time from sampling until analysis. Inadequate detection of endotoxin can be caused by adsorption of endotoxin to container surfaces or molecular masking effects, in which the binding sites on the endotoxin molecules are prevented from triggering the enzymatic cascade necessary in the assay, are obscured. The endotoxin may form macromolecular structures, such as sheets or blebs, or the binding sites may otherwise be rendered unavailable due to the sample matrix composition. In either case, the endotoxin assay may yield falsely low results if and when masking occurs. In this work, the QC sample hold times of different in-process controls within the production process of a biopharmaceutical product were analyzed. One out of eight different samples showed a strong masking of endotoxin. Analysis of the sample composition revealed that either kifunensine, mycophenolic acid (MPA), or lauryl-N, N-dimethylamine oxide (LDAO) was responsible for masking. Further analysis clearly identified LDAO as the root cause for masking. A novel one-step mechanism for LDAO-induced endotoxin masking is proposed. The principle is similar to an already-proposed two-step mechanism for endotoxin masking, but the LDAO case combines these two steps: the disturbance of the salt bridges and hydrophobic interactions with LPS in one molecule. These molecular interactions occur quickly when both endotoxin and LDAO are present in the same matrix. Thus, depending on the masking agents, low endotoxin recovery (LER) can occur regardless of the QC sample hold duration.

Molecular epidemiology of community-acquired invasive non-typhoidal Salmonella among children aged 2 29 months in rural Gambia and discovery of a new serovar, Salmonella enterica Dingiri.

Sixty-two invasive non-typhoidal Salmonella (NTS) isolates from children aged 2-29 months in rural Gambia were examined for serovar prevalence and antimicrobial susceptibility, and characterized using multilocus sequence typing (MLST) of seven genes, aroC, dnaN, hemD, hisD, purE, sucA and thrA. Salmonella enterica serovar Enteritidis was the most common serovar (80.6 %), followed by S. enterica serovar Typhimurium (8.0 %). Thirty-three per cent of the isolates were resistant to all eight antimicrobials tested, including ampicillin (74.2 %), cotrimoxazole (64.5 %) and tetracycline (63 %). A total of 40.3 % of the NTS cases had an initial clinical diagnosis of malaria, whilst 27.3 % had a diagnosis of clinical pneumonia and 18 % had a diagnosis of septicaemia. MLST of NTS resulted in ten different sequence types (STs), of which five were novel, representing five different NTS serovars. In general, STs were restricted to the same serovar. One type (ST11) encompassed 80.6 % of the NTSs. A new NTS serovar named S. enterica serovar Dingiri was discovered. S. Dingiri was isolated from a 6-month-old male with an initial clinical diagnosis of malaria but a final clinical diagnosis of anaemia and septicaemia. S. Dingiri, which possesses an antigenic formula of 17:z:1,6, was sensitive to ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, cotrimoxazole and tetracycline but resistant to gentamicin, and was ST338.

Purification and structure characterization of the active component in the pneumococcal 22F polysaccharide capsule used for adsorption in pneumococcal enzyme-linked immunosorbent assays.

Protection against pneumococcal disease is thought to be mediated primarily by antibodies that are opsonic [Musher DM, Chapman AJ, Goree A, Jonsson S, Briles D, Baughn RE. Natural and vaccine-related immunity to Streptococcus pneumoniae. J Infect Dis 1986;154(2):245-56]. Pneumococcal capsular polysaccharide (CPS) is immunogenic and induces type-specific protective immunity. For convenience, the protective capacity of serum antibodies is often evaluated by the measurement of antibody titers in an ELISA test. The pneumococcal capsular polysaccharide (CPS) used in ELISA contains several impurities; these include about 5% by weight of teicholic acid (CWPS) and the cholin binding protein, pneumococcal surface protein A (PspA) [Sorensen UB, Henrichsen J. C-polysaccharide in a pneumococcal vaccine. Acta Pathol Microbiol Immunol Scand C 1984;92(6):351-6; Yu J, Briles DE, Englund JA, Hollingshead SK, Glezen WP, Nahm MH. Immunogenic protein contaminants in pneumococcal vaccines. J Infect Dis 2003;187(6):1019-23]. All individuals have antibodies to CWPS possible as a result of early exposure to pneumococci, Streptocuccus mitis and Streptocuccus oralis [Bergstrom N, Jansson PE, Kilian M, Skov Sorensen UB. Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-polysaccharide in common with pneumococci. Eur J Biochem 2000;267(24):7147-57. [4]]. The concentration of the CWPS antibodies in non-immunized individuals often exceeds the concentration of the serotype-specific pneumococcal antibodies. Therefore, the pneumococcal ELISA requires an adsorption step to remove the unprotective CWPS antibodies [Konradsen HB, Sorensen UB, Henrichsen J. A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies. J Immunol Meth 1993;164(1):13-20. [5]; Concepcion N, Frasch CE. Evaluation of previously assigned antibody concentrations in pneumococcal polysaccharide reference serum 89SF by the method of cross-standardization. Clin Diagn Lab Immunol 1998;5(2):199-204. [6]; Kayhty H, Ahman H, Ronnberg PR, Tillikainen R, Eskola J. Pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine is immunogenic in infants and children. J Infect Dis 1995;172(5):1273-8. [7]; Koskela M. Serum antibodies to pneumococcal C polysaccharide in children: response to acute pneumococcal otitis media or to vaccination. Pediatr Infect Dis J 1987;6 (6):519-26. [8]]. Recently a new pneumococcal CPS ELISA was recommended with an extra serum absorption step with 22F CPS to remove antibodies against an extra unknown common cross-reactive component. The aim of this study was to characterize the active component in the 22F capsule. A non-capsulated pneumococci was prepared from a 22F capsulated pneumococci. The cell wall polysaccharide (CWPS2) purified from this pneumococci has a better adsorption potential than 22F capsule in the pneumococci ELISA. Structure characterization of the commercial available CWPS and CWPS2 was done by nuclear magnetic resonance (NMR). The NMR results showed that commercial CWPS had one phosporylcholine per sugar repeat while the CWPS2 had two phosporylcholine per sugar repeat explaining an immunological difference between the two variants of CWPS. In addition the LicD2 gene responsible for the attachment of the second cholin in the CWPS tetra sugar repeat was inactive in the strain used for purifying the commercial CWPS but active in the strain expressing CWPS2.

Molecular serotyping of Salmonella: identification of the phase 1 H antigen based on partial sequencing of the fliC gene.

The purpose of this study was to develop a simple and non-labour-intensive molecular method to identify the phase 1 H antigens of Salmonella. The variable region of the flagellin gene, fliC, from 96 Salmonella strains representing 51 different phase 1 H antigens was sequenced in one direction. Unique sequences were found for 45 of the 51 different antigens. We were not able to separate either H:z42 from H:d; H:g, q from H:g, m, q; H:l, w from H:Rl, z40 or H:l, (v),z13 from H:l,z,13. Several phase 2 H antigens were found to be encoded by fliC. Polymorphism, at the subspecies level, was observed in fliC of H:b, H:d, H:z10, H:z and especially H:k. By this method we were also able to confirm that one monophasic strain possesses a new antigen, H:z91. This study shows that sequence-based typing of the phase 1 H antigen of Salmonella is a good alternative to serotyping when strains are non-typable by serological methods.

Salmonella-based rodenticides and public health.

Several countries still permit strains of Salmonella enterica serotype Enteritidis, a leading cause of gastrointestinal illness in humans, to be used in rat baits. To assess the human health risk associated with such rat bait, we first reviewed historic data on health hazards associated with Ratin, a rodenticide that was used in Europe until the early 1960s. Ratin caused outbreaks of human illness, including several deaths. We then compared S. Enteritidis isolated from a current commercial product, Biorat, with S. Enteritidis from Ratin and found that the strains were both phage type 6a. Based on the similarity of the strains, currently available Salmonella-based rodenticides likely are as great a threat to public health as past strains were. Health officials should be aware that the continued use of Salmonella-based rodenticides is a risk to public health and should take appropriate measures to prevent use in their jurisdictions.