A need for careful evaluation of endotoxin contents in acellular pertussis-based combination vaccines

Two batches each of diphtheria-tetanus-acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from foreign markets were tested by mouse body weight decreasing (BWD) toxicity test and Limulus amaebocyte lysate (LAL) test. Three out of the four imported vaccine batches showed the levels of BWD toxicity even comparable to that of DT-whole cell pertussis vaccine. BWD toxicity test is based on endotoxin dose-dependent weight loss of mice and has been used for controlling endotoxin in DTaP. Although of the strong BWD toxicity of the imported vaccines, there was no marked difference in LAL test results between the imported vaccines and Japanese DTaP. However, one imported DTaP batch showed very strong interference with LAL activity of spiked lipopolysaccharide (LPS). The batch interfered not only with LAL activity but also with pyrogenicity and prostaglandin E₂ induction activity. However, the pyrogenicity of the spiked LPS could be recovered from the precipitated fraction of the batch by treating with phosphate buffer to suggest the possibility of recovering in vivo toxicity. As an adequate in vitro test method could not be identified for controlling the safety of the interfering batch, an appropriate in vivo test would be required for testing such vaccines.     

Assessing the Archaeoparasitological Potential of Quids As a Source Material for Immunodiagnostic Analyses

In the present study, quids from La Cueva de los Muertos Chiquitos (CMC) were subjected to ELISA tests for 2 protozoan parasites, Toxoplasma gondii (n=45) and Trypanosoma cruzi (n=43). The people who occupied CMC, the Loma San Gabriel, lived throughout much of present-day Durango and Zacatecas in Mexico. The known pathoecology of these people puts them into at-risk categories for the transmission of T. gondii and T. cruzi. Human antibodies created in response to these 2 parasites can be detected in modern saliva using ELISA kits intended for use with human serum. For these reasons, quids were reconstituted and subjected to ELISA testing. All test wells yielded negative results. These results could be a factor of improper methods because there is no precedence for this work in the existing literature. The results could equally be a simple matter of parasite absence among those people who occupied CMC. A final consideration is the taphonomy of human antibodies and whether or not ELISA is a sufficient method for recovering antibodies from archaeological contexts. An additional ELISA test targeting secretory IgA (sIgA) was conducted to further examine the failure to detect parasite-induced antibodies from quids. Herein, the methods used for quid preparation and ELISA procedures are described so that they can be further developed by future researchers. The results are discussed in light of the potential future of quid analysis.     

Co-detection of Helicobacter pylori and of its resistance to clarithromycin by PCR

Our aim was to develop a rapid molecular test based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and making it possible to detect Helicobacter pylori directly from gastric biopsy samples, and to test its susceptibility to clarithromycin. A 629-bp fragment of the 23S rRNA gene of H. pylori was amplified by PCR and the mutations responsible for clarithromycin resistance were detected with BsaI and BbsI restriction endonucleases. Thirty-five gastric samples were tested in parallel by standard microbiologic methods (culture and clarithromycin susceptibility testing with E-test strips) and by PCR-RFLP. The 10 culture-negative samples were also PCR-negative. Sixteen out of the 25 culture-positive samples (64%) were PCR-positive. RFLP analysis could be done in 12 cases and the results were in agreement with those of the E-test: susceptibility in five cases, resistance in seven (six A2144G mutations and one A2143G mutation).     

Detection of K1 antigen of Escherichia coli rods isolated from pregnant women and neonates

The K1 antigen is an important virulence determinant of Escherichia coli strains and has been shown to be associated particularly with neonatal meningitis, bacteraemia and septicaemia. Thus, its detection seems to be useful, especially in the case of E. coli strains isolated from pregnant women and newborns. In this study, the sensitivity and specificity of the latex agglutination test (Pastorex Meningitis) for identification of E. coli serogroup K1 were assessed, using PCR as the gold standard. Our results showed that consistency of results between latex agglutination test and PCR amounted to 98.5 %. Therefore, Pastorex Meningitis is a good alternative to PCR and could be used for rapid K1 antigen detection, especially in local non-specialized laboratories with limited resources where PCR assay is not applied.     

Toxoplasma gondii infection: What is the real situation?

The prevalence of chronic Toxoplasma infections reported in the literature varies enormously. We hypothesize that one factor could be due to the different methods used in the evaluation of infections. Serological evidence of Toxoplasma infections in 450 pregnant women (PW) and 300 HIV-infected patients (HIV) were investigated by the Sabin–Feldman dye test and two other commercial ELISA kits (kit1 and kit2). Anti-Toxoplasma IgG antibodies obtained from the Sabin–Feldman dye test, ELISA kit1 and ELISA kit2 in the PW subjects were 14.7%, 29.6% and 38.7%, and in the HIV subjects were 13%, 34.7% and 36.3%, respectively. So there were significant differences in the seroprevalences when different diagnostic tests were used (P < 0.05). Regarding Sabin–Feldman dye test as the gold standard for anti-Toxoplasma antibodies detection, we found that the sensitivity and specificity of the ELISA kit1 and kit2 was in the range of their specification. However as the two ELISA kits used in our study identified a much higher prevalence of Toxoplasma infections which indicated that false positive cases were being reported. Based on results obtained, it is therefore highly recommended that research workers should be aware that the reports of serological studies in terms of high positive results should be treated with some skepticism until additional precise diagnostic tools are developed.     

Oxidative Stress,Cytotoxicity and Genotoxicity in Earthworm Eisenia fetida at Different Di-n-Butyl Phthalate Exposure Levels

Recognized as ubiquitous contaminants in soil, the environmental risk of phthalic acid esters (PAEs) is of great concern recently. Effects of di-n-butyl phthalate (DnBP), an extensively used PAE compound to Eisenia fetida have been investigated in spiked natural brown yellow soil (Alfisol) for soil contact test. The toxicity of DnBP to E. fetida on the activity of superoxide dismutase (SOD) activity, peroxidase (POD), reactive oxygen species (ROS) content, and the apoptosis of coelomocytes and DNA damage at the 7th, 14th, 21st and 28th day of the incubation have been paid close attention to. In general, SOD activity and ROS content were significantly induced, opposite to total protein content and POD activity, during the toxicity test of 28 days especially under concentrations higher than 2.5 mg kg^-1. The reduction in neutral red retention (NRR) time along with the increase of dead coelomocytes as the increasing of DnBP concentrations, indicating severe damage to cell viability under varying pollutant stress during cultivation, which could also be proved by comet assay results for exerting evident DNA damage in coelomocytes. DnBP in spiked natural soil could indeed cause damage to tissues, coelomocytes and the nucleus of E. fetida. The key point of the apparent change in different indices presented around 2.5 mg DnBP kg^-1 soil, which could be recommended as the threshold of DnBP soil contamination, so that further investigation on threshold values to other soil animals or microorganisms could be discussed.     

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System,and Antibiotic Sensitivity Profiling

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.     

Additive main effects and multiplicative interaction (AMMI) analysis of genotype-location interaction in variety trials repeated over years

Genotype-location (GL) interaction effects are of special interest for breeding programmes to identify adaptation targets, adaptive traits and test sites. These effects, generally having relatively low repeatability between years, should be studied on a multiyear basis in annual crops. Their assessment by additive main effects and multiplicative interaction (AMMI) analysis is currently defined for this situation. Two procedures based on cross validations are proposed for testing the GL-interaction principal component axes, exploiting the utilities of the computer programme MATMODEL. The use of Gollob’s F test, F _GH2 test, F _R test and the heuristic criterion based on the signal-to-noise ratio is also envisaged. The consistency of results provided by the testing procedures was verified on four data sets of different cereal crops. Gollob’s test tended to be the most liberal, while the F _GH2 test appeared somewhat more liberal than the F _R test. The signal-to-noise ratio gave results consistent with the F _R test considered at a P?0.01 level of significance. These criteria disagreed in two data sets with the conclusions provided by the two cross-validation procedures which, in turn, also disagreed in one data set. Preference could be given to different testing procedures depending on the number of test years, locations and genotypes.     

山西崦山自然保护区侧柏群落优势种群种间关系分析

在样方调查的基础上,采用方差比率法、Fisher精确检验、Jaccard指数和Spearman秩相关系数研究了山西阳城崦山自然保护区侧柏群落优势种群的种间关系。结果表明：（1）群落优势种总体呈正关联,但关联性较弱,种的分布具有相对独立性。（2）Fisher精确检验与Jaccard指数结合使用可以取得较好的研究结果。（3）Jaccard指数结果表明在乔、灌、草各层中占据明显优势地位的种群其种间联结程度较大。（4）根据研究结果把侧柏群落的优势种群划分为3个生态种组：白皮松生态种组、侧柏生态种组和白莲蒿生态种组。     

Hysteresis in Audiovisual Synchrony Perception

The effect of stimulation history on the perception of a current event can yield two opposite effects, namely: adaptation or hysteresis. The perception of the current event thus goes in the opposite or in the same direction as prior stimulation, respectively. In audiovisual (AV) synchrony perception, adaptation effects have primarily been reported. Here, we tested if perceptual hysteresis could also be observed over adaptation in AV timing perception by varying different experimental conditions. Participants were asked to judge the synchrony of the last (test) stimulus of an AV sequence with either constant or gradually changing AV intervals (constant and dynamic condition, respectively). The onset timing of the test stimulus could be cued or not (prospective vs. retrospective condition, respectively). We observed hysteretic effects for AV synchrony judgments in the retrospective condition that were independent of the constant or dynamic nature of the adapted stimuli; these effects disappeared in the prospective condition. The present findings suggest that knowing when to estimate a stimulus property has a crucial impact on perceptual simultaneity judgments. Our results extend beyond AV timing perception, and have strong implications regarding the comparative study of hysteresis and adaptation phenomena.     

Photo-polymeriable chitosan derivative prepared by Michael reaction of chitosan and polyethylene glycol diacrylate (PEGDA)

N-Alkyled photo-polymeriable chitosan derivative (PEGDA-CS) was synthesized by Michael reaction of chitosan and polyethylene glycol diacrylate (PEGDA) under mild reaction conditions. The chemical structure and physical properties of PEGDA-CS were characterized by FT-IR, ¹H NMR, XRD and TG techniques. The degree of substitution (DS) of PEGDA-CS could be calculated from ¹H NMR. PEGDA-CS exhibited good solubility in distilled water. XRD analysis showed that PEGDA-CS was amorphous. TG results demonstrated that thermal stability of the derivate was lower than that of chitosan. Antimicrobial test showed that PEGDA-CS had the antimicrobial activity on Escherichia coli. It could photopolymerize under ultraviolet light with 2959 as initiator.     

Trypanosoma congolense. 3. Serological responses of experimentally infected cattle

A complement fixation (CF) test for the diagnosis of Trypanosome congolense infection in cattle was developed and compared with the indirect fluorescent antibody (IFA) test.Serological investigations were made with cattle immunized with irradiated T. congolense and challenged with untreated T. congolense. Trypanosomal antibodies were demonstrated by the CF test. The results indicated good specificity and sensitivity between the CF method and the IFA test. The CF test also afforded easier reading of results than the IFA test; however, antigen preparation was more difficult in the former.The serological responses detected by the IFA and CF test appeared to be influenced by early variations of parasitemia, although no correlation appeared to exist between titer and parasitemia at later stages of the infection. Antibodies were detected in animals which received injections of irradiated T. congolense prior to challenge with viable organisms.     

Typing of toxinogenic Microcystis from environmental samples by multiplex PCR

Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.     

Animal-based measures on fattening heavy pigs at the slaughterhouse and the association with animal welfare at the farm level: a preliminary study

Monitoring animal welfare (AW) in pig farms requires both proper indicators and a feasible approach. Animal-based measures (ABMs) are well-established AW indicators. Furthermore, AW screening at the slaughterhouses could be useful for identifying problems on farm. The aim of this study was to evaluate ABMs at the slaughterhouse and, when possible, to compare these ABMs with those collected on the farm. The study was carried out in northern Italy in a commercial abattoir and in a sample of farms. Animal-based measures were recorded on pigs from 62 batches of 54 farms, during ante-mortem (n=10 085 pigs) and post-mortem (n=7952 pigs) inspections. Sixteen of 54 farms were selected to compare ABMs collected at the slaughterhouse with ABMs collected on the farm. Overall, 2295 pigs (mean pigs examined per farm 119±45) were inspected at the slaughterhouse (group S) and 420 pigs (mean pigs per farm 26±5) on the farm (group F). Non-animal-based measures were also collected at the 16 farms. Differences between groups S and F, at the animal level, were assessed by a two-tailed paired Wilcoxon–Mann–Whitney test. Differences at the site of observation level (farm and slaughterhouse) were assessed by Fisher’s exact test using a hierarchical log-linear modelling for contingency tables. The most frequent ABMs at the slaughterhouse were manure on the body (47.7%), followed by dermatitis (28.0%), white spot (25.4%) and bursitis (24.7%). Recording ABMs at the slaughterhouse and on the farm usually yielded similar results; however, there were some exceptions. In particular, significant differences were found for non-uniformity of size (P<0.05) and dermatitis (P<0.001), which were higher at the slaughterhouse than on the farm. Results of log-linear modelling underlined the effect of the farm of origin on the percentage of pigs with bursitis, manure on the body and ear injuries that were observed at the slaughterhouse. In group S, significant associations between manure on the body and insufficient presence of clean and dry areas in the corresponding farm were found (P<0.05). Although these results should be interpreted with care due to the limited sample of farms, the slaughterhouse could be a feasible site of observation of ABMs, which could then be integrated in monitoring of AW on farm. Considering the number of slaughtered batches per farm, it would be possible to repeat assessments several times throughout the year for each farm, which could help provide an index for the continuous monitoring of AW.     

Methodological innovations for the study of irreplaceable samples reveal giardiasis in extinct animals (Nothrotherium maquinense and Palaeolama maior)

The use of diagnostic methods that prevent irreplaceable samples (from museum collections, archaeological and paleontological samples) of being consumed or that increase their yield is relevant. For museum collections, archaeological and paleontological samples it is essential to conserve samples, subsamples or portions for future research. We are addressing methods for conservation of irreplaceable samples that could be fully consumed. Innovations in methodologies that are used in studies of Paleoparasitology and Paleomicrobiology will contribute to the preservation of collections. Therefore, to the development of archaeology and paleontology in the future, we evaluated whether the discarded material of the immunochromatography test could be used for molecular diagnosis and vice versa. We used a genotyped experimental coprolite positive for Giardia duodenalis. The diagnosis was positive for giardiasis in both cases. This methodology can be corroborated with the coprolite of a Paleolama maior (extinct llama) previously diagnosed for G. duodenalis with an immunoenzymatic test. The residue of the pre-digestion step of the DNA extraction before adding Proteinase K was confirmed positive with the immunochromatographic test. Also, the DNA extraction residue from a coprolite of Nothrotherium maquinense (ground sloth) was tested positive with immunochromatographic test for G. duodenalis. These are the oldest findings for G. duodenalis confirming that this intestinal parasite occurred among Northeastern Brazilian Megafauna animals from the late Pleistocene period, correlated to human occupation. The relevance of these results will allow the study by different methodological approaches from a small amount of material, reusing discarded materials.     

Genotoxicity and antigenotoxicity of some essential oils evaluated by wing spot test of Drosophila melanogaster

Essential oils extracted from the three medicinal plants; Helichrysum italicum, Ledum groenlandicum and Ravensara aromatica, together with their mixture were tested for their genotoxic and antigenotoxic activities against urethane, a well-known promutagen. We have adopted the somatic mutations and recombination test (SMART) in the wings of Drosophila melanogaster. Three days old larvae, trans-heterozygous for two genetic markers mwh and flr, were treated by essential oil and/or urethane. A negative control corresponding to solvent was also used. Our results do not show any significant effect of the oils tested but they reduce the mutation ratio resulting from urethane. The mixture of the three oils at equal volume seems to be the most effective. The antimutagenic effect of these oils could be explained by the interaction of their constituents with cytochrome P-450 activation system leading to a reduction of the formation of the active metabolite. The effect could also be attributed to certain molecules that are involved in these oils.     

Inhibition of human placental progesterone and estrogen synthesis in early human gestation by aminoglutethimide in vivo

The inhibitory effect of d,l-aminoglutethimide (AG) on the synthesis of progesterone and estradiol in early human pregnancy (8th–12th week of gestation) was investigated in volunteers; control group (n = 11), AG group [1000 mg AG orally at test begin (n = 6)]. Venous blood samples were taken at the beginning of the test and 0.5, 1, 2, 4, 8 and 24 h thereafter. In controls, no significant changes in serum progesterone and estradiol could be observed during 24 h. In the AG group, a decrease in progesterone and estradiol could be observed within 1 h after the test began; lowest serum steroid concentrations were reached after 4 h. Relative to the initial values taken as 100%, the greatest decrease in progesterone ranged between 37 and 83%, 62 15% (x SD) (n = 6); the greatest decrease in estradiol ranged between 32 and 78%, 51 17% (x SD) (n = 6). Twenty four hours after AG treatment, both steroids reached similar concentrations to those found at test begin. No clinical signs (e.g. uterine bleeding, contractions) for the abortifacient action of AG were observed.In conclusion, a single dose of AG (1000mg given orally) cannot induce a therapeutic abortion in early pregnancy. In accordance with in vitro studies, the inhibitory effect of AG on placental progesterone formation is due to an inhibition of mitochondrial cholesterol side chain cleavage. The decrease in estradiol is thought to be related to an inhibition of placental aromatase.     

A Novel Multiplex Real-Time PCR Assay for the Concurrent Detection of Hepatitis A,B and C Viruses in Patients with Acute Hepatitis

A novel multiplex real-time PCR assay for concurrent detection of hepatitis viruses was evaluated for its clinical performance in screening patients with acute hepatitis. A total of 648 serum samples were collected from patients with acute symptoms of hepatitis. Concurrent detection of nucleic acids of HAV, HBV and HCV was performed using the Magicplex™ HepaTrio Real-time Detection test. Serum nucleic acid levels of HBV and HCV were also quantified by the Cobas® AmpliPrep/Cobas® TaqMan® (CAP/CTM) HBV and HCV tests. Patients’ medical records were also reviewed. Concordance rates between the results from the HepaTrio and the CAP/CTM tests for the detection of HBV and HCV were 94.9% (k = 0.88) and 99.2% (k = 0.98), respectively. The cycle threshold values with the HepaTrio test were also correlated well with the levels of HBV DNA (r = −0.9230) and HCV RNA (r = −0.8458). The sensitivity and specificity of the HepaTrio test were 93.8% and 98.2%, respectively, for detecting HBV infection, and 99.1% and 100.0%, respectively, for HCV infection. For the HepaTrio test, 21 (3.2%) cases were positive for both HBV and HCV. Among the positive cases, 6 (0.9%) were true coinfections. This test also detected 18 (2.8%) HAV positives. The HepaTrio test demonstrated good clinical performance and produced results that agreed well with those of the CAP/CTM assays, especially for the detection of HCV. This assay was also able to detect HAV RNA from anti-HAV IgM-positive individuals. Therefore, this new multiplex PCR assay could be useful for the concurrent detection of the three hepatitis viruses.     

Comparison of three variations of the radioallergosorbent test-inhibition assay for measuring allergenic activity of grass pollen extracts

Three variations of the radioallergosorbent (RAST)-inhibition assay (RI) have been compared for measuring the allergenic activity of grass pollen extracts. The main difference between the three assays consisted in the solid supports to which the allergens were coupled. These supports were paper disks, microcrystalline cellulose, and Matrex. It could be demonstrated that all three variations of RI yielded almost identical results as expressed by their F value (F_o). Correlations between the three methods were highly significant (P < 0.001). Three independent valid assays showed an excellent reproducibility of each test system (P < 0.01). Three different preparations of each of the supports yielded highly reproducible F_o (0.76 ± 3% SD). Evaluation of the 50% inhibition values achieved with the different assays based on protein showed differences between the three supports. Provided the same allergen extract is coupled to the different solid supports and the results are related to a reference preparation, different laboratories using different forms of RI will be able to compare their results.     

Assessment from Functional Perspectives: Using Sensorimotor Control in the Hand as an Outcome Indicator in the Surgical Treatment of Carpal Tunnel Syndrome

To investigate whether sensorimotor control of the hand could be an outcome indicator after carpal tunnel release (CTR), this work examined changes in the results of patients’ manual tactile test (MTT), pinch-holding-up activity (PHUA), two-point discrimination (2PD) and Semmes-Weinstein monofilament (SWM) tests. Participants included 30 predominantly sensory neuropathy CTS patients, as confirmed by a nerve conduction study. The MTT, precision pinch performance in PHUA and traditional sensibility (2PD and SWM) tests were used to examine different aspects of sensory status at the time-points of two weeks before operation and one month post-operation, with a single-blind design. The results showed significant improvements in the sensory function as detected by the 2PD and SWM tests (p<0.001) and sensorimotor function as detected by the MTT (p<0.001) and PHUA test (p<0.05) for patients receiving CTR. The responsiveness of the SWM, MTT and PHUA tests (effect size>0.5, p<0.01) are better than that of two-point discrimination test (effect size<0.5, p<0.001). However, pinch strength saw a decline compared to baseline with a moderate effect sizes (effect size = 0.7, p<0.001). This cohort study found that the MTT and PHUA test can both meet all the statistical criteria with regard to assessing treatment outcomes for patients with CTS. In addition, the results of this work provide clinicians with the information that the sensorimotor functions of the hands, as assessed by MTT and PHUA, are responsive to clinical changes due to CTR.