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Pathogen Concentration Combined Solid-Phase PCR on Supercritical Angle Fluorescence Microlens Array for Multiplexed Detection of Invasive Nontyphoidal Serovars.

Bloodstream infections and invasive nontyphoidal in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of serovar and in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of -spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for . and . , respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.

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The distribution of anti-Salmonella antibodies in the sera of blood donors in Yaoundé, Cameroon.

This cross-sectional study in late 1996 on a group of 230 consecutive blood donors in Yaoundé, Cameroon, found a baseline titre of 1:100 for antisalmonella O antibodies. The seroprevalence and baseline titre for antisalmonella H antibodies was much higher (1:400), suggesting that the H antibody is of limited diagnostic value in Cameroon.

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Retrospective treatment analysis of Salmonella infections.

Twenty four culture proved and nine postmortem histopathology proved cases of enteric fever were analysed retrospectively with special interest in use of various antisalmonella agents. Chloramphenicol resistance was noted in 91.7% and yet 70% of all patients received chloramphenicol alone or in combination with another antisalmonella agent. Time required for remission of fever with chloramphenical, cotrimoxazole and ciprofloxacin was 4.5, 4.1 and 6.9 days respectively. An interesting feature noted in post-mortem histopathology proved cases was enteric carditis which was documented on postmortem examination of the heart in three out of four patients who died of peripheral circulatory failure.

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Inefficient in vitro killing of virulent or nonvirulent Salmonella typhimurium by murine polymorphonuclear neutrophils.

The bactericidal capability of murine peritoneal polymorphonuclear neutrophils against virulent and nonvirulent Salmonella typhimurium was examined in an in vitro system. Although preincubation of the bacteria in specific murine antiserum elicited greater chemiluminescence from phagocytizing neutrophils than did incubation in normal murine serum, antiserum did not enhance ingestion, as less than 5% of the challenge was taken up by neutrophils under any of the conditions studied. Nonvirulent salmonellae showed a transient decrease in viable numbers early during in vitro incubation with or without intact neutrophils. Virulent salmonellae, however, were able to multiply without a lag period except when these bacteria were pretreated with antiserum and incubated in association with intact murine neutrophils. Results of these in vitro studies suggest that the murine polymorphonuclear neutrophil and antisalmonella antibody must act together to effect neutrophil-associated bactericidal activity against virulent salmonellae, and thus, that the neutrophil alone does not play a major role in the protection of unvaccinated, sensitive mice from disease caused by S. typhimurium.

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[Interaction of antibodies of different immunoglobulin classes with specific O-antigens and Salmonella haptens].

A study was made of the neutralizing properties of antisalmonella antibodies belonging to different immunoglobulin classes in respect to specific O-antigen (Lipopolysaccharide S. anatum) and haptens of salmonellae. In comparison with IgM-antibodies, IgG-antibodies were more stable bound not only with the univalent trisaccharide determinant, but also with the polysaccharide. However, in regard to the lipopolysaccharide complex the neutralizing activity of IgM- and IgG-antibodies was about the same; IgA-antibodies possessed the greatest neutralizing activity with respect to all the antigenic preparations used. The minimal neutralizing dose of the antigen and haptens increased with the reduction of the size of their molecule. A marked heterogeneity of antibodies of each of the immunoglobulin classes by their antigen-neutralizing properties was revealed in individual sera.

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Comparative clinicopathological and bacteriological studies in schistosomiasis mansoni.

FIFTY MALE Egyptian inpatients with active colonic mansoniasis were studied clinicopathologically, endoscopically, radiologically and bacteriologically. This was done to find out the pattern of colonic and urinary enterobacteria and serum antisalmonella agglutinins in various stages of mansoniasis. Cases are divided into three groups (A, B and C) according to the stage of their hepato-splenomegally. Their clinical presentations were dysentery, rectal bleeding, abdominal distension, anaemia, endocrinal changes and general weakness. Oesophageal varices were diagnosed in 19.8% of group C patients. Distal colonic polyps were noticed in 25% and 4% of group A and B cases respectively, while colonic ulcers were found in 20% of patients of either group. The total number of bilharzia ova per gram of fresh colonic biopsy was highest in group A and lowest in group C subjects. Mucosal and submucosal bilharzial granulation tissue formation together with mucosal hypertrophy were conspicuous among group A and B patients. Hematoxylin and eosin stain was of good diagnostic value and the rest of the stains used did not show any fungi or fibrinoid deposits.

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[Antisalmonella agglutinins in juvenile rheumatoid arthritis presenting with jaundice].


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Quantification of the inhibitory effect of eriochrome black and sodium nitrite on non-specific immunofluorescent staining.

To evaluate non-specific staining (NSS) of animal tissues by FITC-labelled immunoglobulins a model system was developed. HeLa cells were treated with labelled antisalmonella globulins and the fluorescence intensity of the cells was determined quantitatively by means of a fibre optic probe system. This system was used to determine the optimal treatment conditions (adequate concentrations, duration of treatment) using the two NSS-reducing agents eriochrome black and sodium nitrite. Approximately the same inhibitory effects (40-50 per cent using nitrite; about 85 per cent using eriochrome black) were obtained by conjugates of different F/P ratio values. The fibre optic probe system was also used to determine the effects of the above-mentioned agents on the NSS of liver sections. In this system, the NSS was strongly reduced by eriochrome black whereas nitrite treatment did not induce any inhibitory effect. The applicability of nitrite and eriochrome black as NSS-reducing agents was further demonstrated by the fact that they had no influence on the specific fluorescence intensity of salmonella bacteria. The effect of eriochrome black was also studied in clinical specimens infected with salmonella or tularemia bacteria.

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[Use of formalized sheep erythrocytes for the investigation of antisalmonella antibodies, with special reference to the preservability of the antigen].


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