Differentiation of Salmonella typhi using resistotyping

The potential value of resistotyping in the investigation of strain differences is illustrated by its application to Salmonella typhi . The method, although very demanding and prone to technical difficulties, is capable of yielding information about the identity of strains. In its present form the technique was shown to be able to characterize strains in such a manner that available epidemiological information was confirmed.     

Dot immunoenzyme analysis in biological research

The modern state, general methodologic problems and the possibilities to use in biological research the dot enzyme-linked immunosorbent assay (dot-ELISA) are analysed in the review. New types of microporous polymer membranes and equipment for the application of the solid-phase reagent and performing the assay are considered. Different variants of dot-ELISA and methods for the evaluation of results obtained in the assay as well as the ways for its optimization are discussed.     

An immunoenzyme system for determining antibodies to Streptococcus pneumoniae polysaccharides in biological fluids

The enzyme immunoassay (EIA) system for the determination of antibodies to capsular polysaccharides of pneumococci, serotypes 1, 3, 6B, 8, 9N, 15F, 23F, and C-polysaccharide has been developed on the basis of poly-L-lysin-modified antigens. The use of isotype-specific conjugates in this system permits the detection of IgG and IgA antibodies in different biological fluids: blood serum, pleural fluid, saliva, milk. Samples obtained from children with pneumococcal infection and from nursing mothers have been studied. As shown in this study, the EIA system can be used for the evaluation of the dynamics of pneumococcal infection in children.     

Construction and characterization of isogenic O-antigen variants of Salmonella typhi

A 7.5 kb Kpnl-generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild-type Salmonella typhi Vi-positive strain ISP 1820 (i.e. serotype 09,12; Vi⁺ H-d), S. typhi Vi-negative strain H400 (i.e. serotype 09,12; Vi⁻; H-d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906-O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906-O4 demonstrated that these 8trains express the 04 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi⁺; H-d and the serotype of H404 is O4,12; Vi⁻; H-d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906-O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate-tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906-O4. The resistance of each strain to the bactericidal effects of guinea-pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC. The results suggest that the development of the O-antigen serotype diversity of Salmonella is probably the result of both sequence divergence and recombination     

Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress

Bacteria can, under certain conditions, enter into a cell-less state known as L-form conversion. This phenomenon is universal, but also recognized with difficultly by microbiologists. The current study addresses several aspects concerning the ability of tubercle bacilli to use L-form conversion as a unique adaptive strategy to survive and reproduce under unfavorable conditions. Nutrient starvation of M. tuberculosis in vitro followed by passages in Middlebrook 7H9 semisolid medium was used for stress induction and the selective isolation of mycobacterial L-form variants. Light and electron microscopy images evidence the peculiar characteristics of mycobacterial L-forms. For example, mycobacterial L-forms were observed to lose their acid-fastness and change their morphology. In addition, wide morphological variability, the presence of large and elementary bodies, coccoids and small granular forms, as well as the appearance of unusual modes of irregular cell division were observed. Unlike classical tubercle bacilli, L-form variants grew and developed typical "fried-egg" colonies faster. L-forms were verified as M. tuberculosis by spoligotyping. The results provide insights into the nature of L-form phenomena in M. tuberculosis and link them to the mechanisms allowing mycobacterial survival under stress.     

Use of the peroxidase method of immunoenzyme analysis for detecting Clostridium perfringens cells

The possibility of using the peroxidase method of the enzyme immunoassay for the detection of C. perfringens cells in the culture fluid of strains, as well as in affected tissues in cases of anaerobic gas gangrene infection, has been shown. This method is highly specific, convenient and takes but 1.5 hrs.     

Use of specific Bordetella pertussis antibodies in immunoenzyme analysis for detecting the pertussis antigen

The competitive EIA technique with the use of peroxidase-labeled B. pertussis antigen has been developed. The data obtained in our investigations suggest the possibility of using this technique for the detection of B. pertussis antigen in faucial smears obtained from patients.     

Selective determination of lithium in biological fluids using flow injection analysis

The use of flow injection analysis to automated extraction methods for the determination of lithium ion utilizing crown ethers or cryptands is demonstrated. The ion-pair extraction of cryptand 211, lithium, and resazurin exhibits a linear range for lithium ion of 70 ppb to 2.1 ppm. This method could tolerate up to 1000 ppm sodium ion. The chromogenic crown ether, 1-(2-hydroxy-5-nitrobenzyl)-1-aza-4,7,10-trioxacyclododecane, exhibits a linear range for lithium ion of 0.3 to 2 ppm. A sodium ion concentration of 230 ppm can be tolerated. Both extraction systems were used in the automated determination of lithium in blood serum and urine. Both methods agreed well with the known and/or atomic absorption values.     

Development of an immunoenzyme method for detecting Francisella tularensis

The conditions permitting the determination of F. tularesis cells by means of the enzyme immunoassay (EIA) in 3-5 hours have been established. Ways for enhancing the reliability of results obtained in the assay of the least possible amount of the test material have been proposed. The sensitivity and specificity of the rapid EIA technique permitting the determination of F. tularensis cells at a concentration of 20 000 cells/ml in the presence of other bacterial cells in 100-fold excess have been shown.     

Detection of microbial variants of Salmonella typhi in the bone marrow of typhoid patients and carriers

The immunofluorescence test with the use of antisera to S. typhi and its L-forms permits the detection of the infective agent in bone marrow smears. This diagnostic method is particularly important in cases of carrier state in the latent phase and in differential diagnosis. As revealed in this study, the microbial variants of S. typhi, reacting with antiserum to its L-forms, are present in the bone marrow of carriers. They are localized, as a rule, on the membranes of lymphoid and erythroid cells, which probably leads to the formation of rosettes.     

Occurrence of Salmonella typhi and Salmonella paratyphi in Jordan

In order to justify the surveillance control system and hygiene policy in Jordan, this study evaluated the occurrence of diarrhoea during the period 1988-2000, focusing on cases caused by Salmonella typhi and Salmonella paratyphi. From January 1988 to December 2000, the number of notified diarrhoeal cases by the Ministry of Health in Jordan was 1,399,563 million. Other groups of patients confined to the Governorate of Amman was diagnosed at Al-Battikhi Medical Laboratories. One-way ANOVA and Least Significant Difference (LSD) were carried out for statistical analysis. The number of reported diarrhea cases was 1,399,563, 53.0% were males, and 47.0% were females, among them, 80.3% were < 20 years and 19.7%, were > 20 years. Out of 245,255 patients tested for S. typhi and S. pararyphi, positive stool culture were 1992 (0.6%). Out of these, 960 (48.2%) were males and 1,032 (51.8%) were females (P = 0.028). The highest incidence rate (10.8) was observed in the year 1993, while the lowest incidence rate (0.9) was found in year 2000. A significant difference (P < 0.001) was found between the number of S. typhi and S. Paratyphi cases and year. The seasonal variation was also found to be significant (P < 0.0001), with the summer period showing the highest incident rate. A significant difference (P < 0.001) was observed between number of typhoid and paratyphoid cases and districts. A significance difference between number of typhoid and paratyphoid cases with age and sex. The group most affected was school age and adolescence. The demographic situation plays an important role in reporting typhoid and paratyphoid cases, where there might be an urgent indication for a better surveillance control system on water resources and disposal systems. S. typhi and S. paratyphi antibiotics resistance pattern showed they were resistant to tetracycline (56.0%, 58.0%), ampicillin (45.0%, 48.0%), trimethoprim (43.0%, 47.0%), cephtazidime (12.0%, 13.5%) chloramphenicol (6.8%, 7.2%), gentamycin (3.0%, 4.0%) neomycin (2.1. 1.8%), calvulanic acid (augmentin (1.4%, 2.2%) and norofloxacin (0.92%, 1.1%). Susceptibility to amikacin, ciprofloxacin, cetfriaxone, ofloxacine, imepenim, cefixime and cefotaxime was 100.0%. The increase in percentage of antibiotic resistant strain might indicate a need for a further prescribing policy for treatment.     

The antigenic characteristics of Salmonella typhi L forms

The study of the possibility of detecting the specific antigens of S. typhi L forms has revealed that out of three destructive methods under study (osmotic lysis, freezing-thawing, sonication) only ultrasonic disintegration has proved to be effective for S. typhi L forms. Three specific fractions capable of interacting only with specific antibodies to S. typhi L forms have been revealed in the course of chromatographic separation of the soluble antigenic complex of S. typhi stable L forms and the subsequent analysis of the fractions thus obtained in the enzyme immunoassay.     

The immunoenzyme analysis of ceruloplasmin

A highly specific and sensitive enzyme immunoassay (EIA) system, suitable for the qualitative analysis of ceruloplasmin, has been developed. The possibility of its use for the examination of children with mononucleosis and pseudotuberculosis has been studied. An increase in the concentration of ceruloplasmin has been more pronounced in infectious mononucleosis (0.506 +/- 0.026 g/l) and pseudotuberculosis (0.421 +/- 0.157 g/l). The results of EIA coincided with the data obtained by radial immunodiffusion.