Infusion-related reactions during Natalizumab treatment: Do we still need a post-infusion observation period?

BACKGROUND: Natalizumab (NTZ) is a humanized monoclonal antibody used in the treatment of relapsing remitting multiple sclerosis. Although NTZ is usually well-tolerated, infusion-related reactions (IRRs) may occur, and the patients have to be monitored during the infusion and for one hour afterwards. OBJECTIVE: To identify frequency and severity of IRRs during NTZ infusions and one-hour post-infusion observation period in a clinical practice setting. METHODS: Multicenter, observational study involving three Swiss (Lugano, St. Gallen and Luzern) and two Italian (Milano and Napoli) tertiary MS centers. Predisposing factors to IRRs were investigated using multivariate Cox regression models. RESULTS: A total of 11'133 infusions received by 302 MS patients were analyzed (68.9% females, median age 33.6 years, median EDSS 2.5). IRRs occurred in 24 (8%) patients during NTZ infusions and in 7 (2%) during one-hour post-infusion. Only 8 patients needed pharmacological treatment, of whom 7 during NTZ infusion. Age, sex and history of allergies were not associated with risks for IRR. The frequency of post infusion IRRs after the fifth cycle was low compared to that during the first four infusions (0.83% vs 0.06%). CONCLUSION: In our cohort, NTZ associated IRR mainly occurred during the infusion period compared to the one-hour observational period. Also, the first IRR exclusively occurred within the first 4 NTZ administrations. However, further multi-center studies with a larger sample size are needed to capture rare and serious events that could emerge during the observational period and to make clinical recommendations.

[The perspective of global eradication of poliomyelitis]. / Die Perspektive der globalen Ausrottung der Poliomyelitis.

In recent years, Germany and Switzerland have changed national policies to recommend vaccination with IPV (inactivated polio vaccine) instead of OPV (oral polio vaccine) for protection against poliomyelitis. An all IPV-schedule in routine childhood polio vaccination eliminates the - albeit minimal - risk of OPV-associated paralytic poliomyelitis. However, the impact of such a vaccination scheme on the goal to eventually eradicate poliomyelitis on a global level remains debatable. Published studies indicate that vaccine-derived poliovirus may persist in the environment for prolonged periods of time even after completion of a global eradication programme that relies on the near-exclusive use of OPV in the developing countries. Travellers vaccinated with IPV only might become silently infected with vaccine-derived virus, shedding it in large quantities. We therefore plead for a vaccination schedule that includes at least one last dose of OPV to induce strong mucosal immunity.

Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids.

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin-biotin coupling. A biotin-labeled PNA was employed in the latter approach whereas the covalent immobilization included the following steps: A maleimide group was attached to the N-terminal of the PNA using N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). To generate free thiol groups for coupling, a carboxylated dextran matrix of the sensor surface was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) and thiolated by addition of cystamine dihydrochloride followed by reduction with 1, 4-dithioerythrite (DTE). Finally, the modified PNA was coupled to the sulfhydryl groups of the activated dextran matrix. Repetitive hybridizations of a single-stranded synthetic DNA oligomer to the PNAs demonstrated the superior stability of covalent immobilization compared to noncovalent immobilization. Differentiation of point mutations in the analyte molecule was accomplished at 40 degrees C using guanidine thiocyanate concentrations of 1.5-1.7 M. In further experiments, we showed that a perfectly matched PNA allows the detection of a single-stranded DNA at a sensitivity of less than 1% in a background of single-stranded DNA having a single C to T point mutation in the region complementary to the PNA. Consequently, covalently bound PNAs provide a stable and reproducible environment for the development of mutation-specific DNA analysis assays.

Development and validation of a detection system for wild-type Vibrio cholerae in genetically modified cholera vaccine.

Orochol, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intact ctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol.

PCR-based detection of verotoxin-producing Escherichia coli (VTEC) in ground beef.

Pathogenic strains of Escherichia coli producing verotoxins (VTs) have been recognized as a cause of human disease, and rapid and sensitive detection tests are urgently needed to ensure the safety of food, especially ground beef. We applied two nested polymerase chain reaction (PCR) assays to detect the genes encoding VT1 and VT2 irrespective of the bacterial serotype. In combination with a direct sample preparation protocol, we were able to uncover the presence of about 110 CFU of verotoxinogenic E. coli (VTEC) in 10 g of ground beef. When a six-hour enrichment was included, we found the detection limit to be in the range of 1 to 10 bacterial cells per 10 g of ground beef. To evaluate our detection system, we tested 30 ground beef samples originating from butcher shops in Berne, Switzerland. One sample yielded positive PCR results for both the VT1 and VT2 genes, indicating the presence of verotoxinogenic E. coli. Finally, 20 food homogenates, shown to contain E. coli strains by standard culture, were analysed with our method, and the gene encoding VT2 was detected in one cheese sample. The results suggest that the described PCR method can serve as a valuable tool for the surveillance of VTEC contamination of foods.

Detection of Salmonella spp. in eggs: DNA analyses, culture techniques, and serology.

A polymerase chain reaction (PCR) method for direct detection of Salmonella spp. in whole-shell eggs is described. The method does not require isolating strains. Preenrichment, rapid DNA isolation, and a nested PCR system targeting the invA gene enabled detection of the genus Salmonella. The specific nested PCR product of 283 base pairs was formed from all 21 serovars, including 43 Salmonella strains tested. No PCR product was formed from 56 non-Salmonella enterobacteriacea and other bacterial strains tested. Experiments with artificially contaminated eggs showed a detection limit of about 10(3)-10(4) colony-forming units (cfu)/egg before and about 1-10 cfu/egg after preenrichment. In analysis of 180 single eggs from 4 flocks and 36 pools of 5 eggs each from another 4 flocks from the same producer, Salmonella spp. were detected in 5 of 90 eggs from 2 different flocks. Determination of anti-Salmonella antibodies in eggs yielded positive results for 2 additional and the 2 PCR-positive flocks. In contrast, classical selective culture detected Salmonella spp. in only 1 of 100 eggs in one flock when 100 eggs from each flock were analyzed.

Polymerase chain reaction-restriction fragment length polymorphism analysis: a simple method for species identification in food.

The polymerase chain reaction (PCR) technique was applied to meat species identification in marinated and heat-treated or fermented products and to the differentiation of closely related species. DNA was isolated from meat samples by using a DNA-binding resin and was subjected to PCR analysis. Primers used were complementary to conserved areas of the vertebrate mitochondrial cytochrome b (cytb) gene and yielded a 359 base-pair (bp) fragment, including a variable 307 bp region. Restriction endonuclease analysis based on sequence data of those fragments was used for differentiation among species. Restriction fragment length polymorphisms (RFLPs) were detected when pig, cattle, wild boar, buffalo, sheep, goat, horse, chicken, and turkey amplicons were cut with AluI, RsaI, TaqI, and HinfI. Analysis of sausages indicates the applicability of this approach to food products containing meat from 3 different species. The PCR-RFLP analytical method detected pork in heated meat mixtures with beef at levels below 1%, and the method was confirmed with porcine- and bovine-specific PCR assays by amplifying fragments of their growth hormone genes. Inter- and intraspecific differences of more than 22 animal species with nearly unknown cytb DNA sequences, including hoofed mammals (ungulates), and poultry were determined with PCR-RFLP typing by using 20 different endonucleases. This typing method allowed the discrimination of game meats, including stag, roe deer, chamois, moose, reindeer, kangaroo, springbok, and other antelopes in marinated and heat-treated products.

Reverse transcription PCR to detect enteroviruses in surface water.

We have developed a simple, fast, and efficient procedure to detect enteroviruses in water samples. Aliquots of water are subjected to two-step filtration, with the second filter containing a positively charged nylon membrane that holds back virus particles. Viruses thus adsorbed are directly lysed, and RNA is isolated by hybridization to specific oligonucleotides bound to magnetic beads. The solution used contains guanidine thiocyanate, which lyses virus particles, inactivates enzymes, e.g., RNases, allows mild hybridization conditions, and does not influence biotin-streptavidin interaction on magnetic beads. Detection and specific identification are accomplished by reverse transcription PCR of the highly conserved noncoding region at the 5' end of virus RNA combined with Southern hybridization. The system was tested with tap water artificially spiked with poliovirus vaccine and yielded a detection limit of 20 50% tissue culture infective doses per liter. We used the same procedure to investigate the water quality of surface water at public beaches by rivers and lakes. Of 40 samples tested, 7 were positive for enteroviruses. A comparison with enterobacterial contamination determined by PCR and classical microbiological methods in parallel showed that enteroviruses were found only in samples also positive for Escherichia coli. In conclusion, this procedure can easily be adapted to test large water samples and is simple enough to be used for routine determinations of water quality in terms of virus contamination.

Polymerase chain reaction (PCR) for detection of pathogenic microorganisms in bacteriological monitoring of dairy products.

The presence of pathogenic bacteria poses a serious problem in sustaining the safety of dairy products. Microbiological routine controls of these products make use of selective culture techniques. To detect pathogenic species, isolated colonies are characterized by specific metabolic activities and by serotyping. We present an alternative biochemical approach that does not require culture of bacteria. The total bacterial populations of food samples were isolated by centrifugation and analysed by PCRs specific for pathogenic species. A total of 90 raw milk samples and dairy products made from raw milk were screened by this method for the presence of Listeria monocytogenes, Escherichia coli, enterotoxigenic E. coli, Campylobacter jejuni and C. coli. Detection rates were 12/90 (13%) for L. monocytogenes, 41/90 (46%) for E. coli, 18/90 (20%) for enterotoxigenic E. coli producing heat-labile toxin type I or heat-stable toxin type I, and 6/90 (7%) for C. jejuni or C. coli. Except for the use of different amplification primers, this approach is identical for any bacterial species to be detected. Direct PCR analysis of food samples offers rapid screening for the presence of specific bacteria and enables selection of critical samples prior to culture.

Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes.

The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.

Reliability of PCR decontamination systems.

A major problem in the application of PCR is contamination with material amplified previously. Repeated PCRs result in the accumulation of intact and degraded amplicons and primer artifacts that can contaminate following amplification reactions. Post-PCR UV treatment and pre-PCR uracil DNA glycosylase (UDG) digestion have been recognized to efficiently inactivate or decompose intact amplification fragments. We show here that degraded amplification products and primer artifacts account for decreased sensitivity and may cause false-negative results. Our experiments indicate that partly degraded PCR products and primer artifacts containing sequences homologous to the primer oligonucleotides in the succeeding PCR reaction compete efficiently with sample DNA for the primers. The experiments done in this study may explain unexpectedly low PCR sensitivities reported in an increasing number of publications. In an attempt to solve this problem, we evaluated three post-PCR treatment methods to completely eliminate sequences competing for the amplification primers, namely, 8-methoxypsoralen (MOPS) or hydroxylamine treatment of amplified DNA and use of oligonucleotides containing 5'-ChemiClamps. However, all three methods did not sufficiently inhibit artificially produced carryover contaminations. In conclusion, false-positive results can be eliminated with UDG or UV treatment, but physical barriers are indispensable to avoid the occurrence of false-negative results.

Direct detection of Listeria monocytogenes using paramagnetic bead DNA extraction and enzymatic DNA amplification.

The potential of the polymerase chain reaction (PCR) as a tool for direct detection of Listeria monocytogenes in food and clinical samples was investigated. A specific oligonucleotide, directed against the listeriolysin 0 gene of L. monocytogenes, was coupled to paramagnetic beads and used to isolate listerial DNA from food homogenates and blood. The isolated DNA was amplified with a seminested PCR procedure, which recognized the hly gene. The detection limit for L. monocytogenes in 50 ml of a buffer solution was between 1 and 10 colony forming units (cfu). In food homogenates consisting of 10 g food and 40 ml 0.85% NaCl solution, artificially spiked with an overnight culture of L. monocytogenes, the detection limit was 1-10 cfu. The method was further evaluated by application to naturally contaminated food samples. In 250 microliters whole human blood artificially spiked with L. monocytogenes 10,000 cfu could be detected.

Detection of pork in heated meat products by the polymerase chain reaction.

A new method for the specific, sensitive, and semiquantitative detection of pork (Sus scrofa) in heat-treated meat products has been developed. DNA was isolated from meat samples by using a DNA-binding resin and subjected to polymerase chain reaction (PCR) analysis. First, oligonucleotides yielding a specific 137-base-pair (bp) fragment from eucaryotic DNA amplified from a highly conserved region of the 18-S ribosomal gene was used to assess DNA quality. Second, the presence of pork DNA was determined with specific oligonucleotides yielding a 108-bp fragment amplified from the porcine growth hormone gene. The test detected pork in fresh or heated meat mixtures of pork in beef at levels below 2%. This approach was superior to commercially available immunological tests that were not able to detect levels of pork less than 20% in cooked meat or less than 10% in fresh meat.

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction.

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32 degrees C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37 degrees C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the virF gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37 degrees C on Yersinia selective agar.

Direct polymerase chain reaction detection of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products.

A polymerase chain reaction (PCR) method designed to sensitively detect and identify Campylobacter jejuni and Campylobacter coli without the need for isolating and culturing strains is described. The intergenic sequence between the flagellin genes flaA and flaB was amplified and characterized with a triple primer or seminested primer approach. A total of 50 bacterial strains, 27 of C. jejuni and C. coli and 23 of other species, were tested, giving no false-positive or false-negative results. The detection limit as determined by ethidium bromide staining of amplification products on agarose gels was 10 bacteria or less in artificially contaminated water, milk, and soft cheese samples with the seminested primer PCR assay. As an application of the PCR system, a set of 93 samples of milk and other dairy products was screened for the presence of C. jejuni and C. coli. We identified six positive samples (6.5%), while none were found with a conventional culture method.

Polymerase chain reaction (PCR): a possible alternative to immunochemical methods assuring safety and quality of food. Detection of wheat contamination in non-wheat food products.

A rapid, sensitive and specific analysis of food samples determining wheat contamination was established using polymerase chain reaction (PCR) technology. First, primers specific for highly conserved eukaryote DNA sequences were used to prove isolated nucleic acid substrate accessibility to PCR amplification. Subsequently, a highly repetitive and specific genomic wheat DNA segment was amplified by PCR for wheat detection. This assay was tested with 35 different food samples ranging from bakery additives to heated and processed food samples. In addition, the PCR method was compared to an immunochemical assay that detected the wheat protein component gliadin. Combination of both assays allowed a detailed characterization of wheat contamination. Hence, wheat flour contamination could be distinguished from gliadin used as a carrier for spices as well as from wheat starch addition.

Comparison of "Gen-Probe" DNA probe and PCR for detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese.

A comparison was made of three approaches for the detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese after enrichment. Enrichment broths were tested by plating them onto different selective agars, by "Gen Probe" DNA hybridization and by the polymerase chain reaction (PCR). Based on two-step enrichment, all three approaches showed high specificities (90% or more) in detecting L. monocytogenes. In contrast, the sensitivity of the Gen-Probe test was low (33% or less), whereas high sensitivities were obtained with selective plating and PCR (83% or more). Based on one-step enrichment, specificities again were high for selective plating and PCR assay (100%), whereas for the Gen-Probe assay the specificity was lower (88% or more). The best sensitivities were observed with selective plating (67%) and PCR (75%). In terms of sensitivity, specificity and analysis time, PCR applied to a two-step enrichment was the most powerful assay for detecting L. monocytogenes in soft and semi-soft cheese.

Evaluation of the ligase chain reaction (LCR) for the detection of point mutations.

The ligase chain reaction (LCR) was evaluated as an amplification method for an in vivo mutation assay. Specifically, the ligase was tested for its ability to selectively amplify a DNA sequence mutated at a single base, in the presence of an excess of wild-type DNA. As a model template a 370-bp DNA fragment of the mouse Ha-ras protooncogene containing an A to T mutation at the second position of codon 61 was used. With the commercially available ligase Ampligase (Epicenter), 250 molecules of mutant fragments could be detected by an enzyme-linked immunoassay with digoxigenin marker (giving a theoretical detection limit of 1 target gene per 10(4) copies of genome). In the analysis of mixtures with corresponding wild-type DNA fragments, a 1:1 mixture resulted in a clearly stronger signal than control samples lacking wild-type and mutant DNA. However, the signal obtained from a 100-fold dilution of the mutant DNA with wild-type DNA could not be distinguished from the background noise. In this particular form, LCR lacks sufficient selectivity to be applied to an in vivo situation, where the ratio of mutant to wild-type DNA sequences might be expected to lie around 1:10(6).