Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic corynebacteria

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the "gold standard" in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.     

Reversed passive latex agglutination assay for detection of toxigenic Corynebacterium diphtheriae

A reversed passive latex agglutination (RPLA) assay for determining the toxigenicity of Corynebacterium diphtheriae is presented. Rabbit antitoxin antiserum was raised by using commercially available diphtheria toxoid. This antiserum reacted with the diphtheria toxin when the culture supernatant was assayed by Western blotting, and it did not cross-react with other extracellular antigens. Affinity-purified antibodies for latex sensitization were obtained by using a Hi Trap N-hydroxysuccinimide-activated column. Demonstration of toxin in five of seven clinical isolates was in accordance with the PCR assay and the Vero cell cytotoxicity test. Culture of the bacteria for 6 h was sufficient for toxin production, and an additional 6 h was needed to observe latex agglutination. Therefore, diphtheria toxin can be detected in 12 h by this method. The lowest concentration of diphtheria toxin detectable by the RPLA assay was about 5 ng/ml. The RPLA assay can provide a convenient and reliable method for laboratories involved in the identification of toxinogenic corynebacteria.     

In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines

OBJECTIVE: Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. STUDY DESIGN: Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. RESULTS: Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. CONCLUSIONS: These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.     

Treatment of parkinsonism. Views on the recommendations issued by the Medical Products Agency

Direct detection of Escherichia coli O157 and foodborne pathogens associated with bloody diarrhea were achieved using polymerase chain reaction (PCR) after the preparation of DNA from stool specimens using the microspin technique. PCR was compared with cultivation and toxin production tests with respect to the efficiency of detection of each pathogen; E. coli O157, Vibrio parahaemolyticus, Salmonella serovar Enteritidis and Campylobacter jejuni. Detection of some or all of the above pathogens in clinical stool specimens was achieved using PCR. The minimum number of cells required for the detection of the above pathogens by PCR was 10(1) CFUs/0.5 g of stool sample. PCR was completed within 6 hr. The above pathogens were also detected in cultivation and toxin production tests. Partial purification of the template DNA using the microspin technique was essential for the elimination of PCR inhibitors from the DNA samples. This PCR method is an accurate, easy-to-read screening method for the detection of Shiga-like toxin producing E. coli O157 and enteropathogens associated with bloody diarrhea in stool specimens.     

Exfoliative toxin detection using reversed passive latex agglutination: clinical and epidemiologic applications

A rapid and simple method for detecting exfoliative toxin serotypes A and B from clinical isolates has been developed as a test kit (EXT-RPLA; Denka Seiken Co. Ltd., Niigata, Japan). This method is based on reversed passive latex agglutination. The detection limit of the EXT-RPLA observed for purified exfoliative toxin serotypes A and B was 1 ng/ml. We evaluated the clinical and epidemiologic uses of the EXT-RPLA. A total of 381 isolates of Staphylococcus aureus, 292 from various clinical specimens and 89 from the skin of dermatologic patients, were studied. The EXT-RPLA detected 19 exfoliative toxin producers, including 16 serotype A producers and 3 serotype B producers, but no double producers. The sensitivity and specificity of the EXT-RPLA were confirmed by the newborn mouse bioassay and a PCR assay for the structural genes for exfoliative toxin serotypes A and B (eta and etb, respectively). The overall positivity rate of exfoliative toxin producers was 5.0% (19 of 381), including 16 serotype A isolates and 3 serotype B isolates. Of the 89 isolates from the skin of dermatologic patients, 12 (13.5%) were positive for exfoliative toxin production. Only 2 (1.3%) of the 153 methicillin-resistant S. aureus isolates produced exfoliative toxin, while 17 (7.5%) of the 228 methicillin-sensitive isolates produced exfoliative toxin. The EXT-RPLA assay is a simple and reliable method for detecting exfoliative toxin, and we recommend its use for the rapid diagnosis of staphylococcal scalded skin syndrome. We also recommend its use for detection of this syndrome so that effective control measures can be taken against the spread of this syndrome.     

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false positive EIA results by evaluating clinical and therapeutic parameters. METHODS: 737 consecutive diarrhoeal specimens collected from patients clinically suspected of having C difficile colitis were tested for the presence of C difficile toxin by EIA for toxin A and by cytotoxicity assay. Clinical data were evaluated in all cases positive by either method. RESULTS: With the cytotoxicity assay as a gold standard, the specificity of EIA for toxin detection was 99.3% and the sensitivity was 62.2%. No false negative EIA specimens were obtained from patients already being treated for C difficile colitis. Among patients with cytotoxicity positive specimens, those with EIA positive samples had no clinical features distinguishing them from patients with EIA negative samples. CONCLUSIONS: Although specific, the new EIA method directed against toxin A lacks sensitivity compared to cytotoxicity. False negative EIA tests are not associated with concurrent treatment for C difficile colitis nor with any specific clinical features examined in our study.     

Capture EIA for determination of IgE to diphtheria and tetanus toxins in comparison to RIA

A capture IgE assay in EIA with diphtheria toxin and tetanus toxoid as model systems was developed and evaluated in relation to RIA in a DT booster study. The EIA and the RIA gave very similar results, although the EIA determined IgE over a wider range of concentrations, without interference by other subclasses. The comparison to the capture EIA indicated that the RIA gave more reliable results when a toxin as antigen. Weak correlations between local side effects and IgE responses and differences between vaccine groups were noted by RIA but not by EIA, which correlated better with the clinical findings.     

Present status and advances in detecting Shiga toxin-producing Escherichia coli O157

Currently, detection of Shiga toxin-producing Escherichia coli(STEC) in stool samples is based on the isolation method in most clinical laboratories. The procedures are as follows: i) isolation with selective agar plates, ii) biological test with differential media, iii) serological test of anti-O antisera, iv) detection of toxin or toxin gene. These procedures take 4 days, therefore more rapid method is required. In the near future, a rapid detection method that detects STEC directly from stool samples will be introduced. Polymerase-chain reaction (PCR), enzyme-linked immuno-sorbent assay (ELISA), detection of serum anti-O157 antibodies are now available in clinical laboratories. Result of PCR for detection Shiga toxin gene and serum anti-O157 antibodies are described. Fifteen stool and serum samples from patients suspected of STEC infection were examined. With the culture and PCR method, 2 patients were positive by both methods and the results were confirmed in both cases. Six patients were positive by the antibodies detection method. From these results, the PCR method using stool samples was useful as a rapid detection method in clinical laboratories. Detection of serum antibodies has been simplified and is not an expensive method. Therefore, the method is useful for clinical diagnosis of STEC infection, especially, for diagnosing HUS or after antimicrobial agents have been administered to patients.     

Methylamine stimulates the action of ricin toxin but inhibits that of diphtheria toxin

The action of ricin toxin was stimulated by addition of methylamine or some other amines, as shown by measuring the inhibition of protein synthesis of cultured cells by the toxin. Under the same conditions, however, the action of diphtheria toxin was completely inhibited by the amines. In a cell-free protein-synthesizing system, methylamine had no effect on the action of the A chain of ricin toxin and fragment A of diphtheria toxin. Studies on the interactions of 125I-labeled toxins with cells revealed that methylamine did not alter toxin-receptor bindings, but affected the entry of the toxins into the cells. Studies were also made on the effects of methylamine on the actions of two hybrid toxins, formed from a subunit of Wistaria floribunda lectin and fragment A of diphtheria toxin and the A chain of ricin toxin, respectively. Results suggested that the processes of entry of ricin toxin and diphtheria toxin, or at least parts of these processes, are different.     

Evaluation of Novapath assay for direct detection in patient stool of Vero toxin and Vero toxin-producing Escherichia coli]

We evaluated two kits which detect either Verotoxin (Novapath EHEC) or EHEC O157 (Novapath O157) directly from stool specimens and compared with the result of PCR. The tests can be completed in 3 hrs. All fecal specimens from 18 healthy volunteers showed negative results with both kits and PCR. Specimens from patients of EHEC O157 outbreak in Okayama Prefecture and asymptomatic families of patients of outbreak in Sakai City of Japan were also analyzed. Fecal specimens from which EHEC O157 was isolated showed positive results with both kits and PCR. Some specimens which were culture negative showed positive results with either Novapath EHEC or Novapath O157 or both. The sensitivity of Novapath O157 was lower than PCR to detect O157 organisms but took shorter time to get result. Novapath EHEC showed very high sensitivity in detecting Vero toxin. These results suggest that Novapath EHEC especially can be used clinically for rapid diagnosis.     

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.     

Molecular diagnosis of cat scratch disease: a two-step approach

Amplification of Bartonella henselae DNA has been proposed as a diagnostic test for cat scratch disease (CSD). The sensitivities of the following three PCR assays were compared. PCR/rRNA with universal primers amplifies part of the 16S rRNA gene, followed by hybridization with a specific B. henselae probe; PCR/CS and PCR/HSP amplify portions of the gltA and the htrA genes, respectively, each followed by restriction fragment length polymorphism analysis. The threshold of detection of B. henselae DNA in pus was 10(-4), 10(-3), and 10(-2) ng for PCR/rRNA, PCR/CS, and PCR/HSP, respectively. By these three assays, B. henselae DNA was detected in 100, 94, and 69% of 32 pus and lymph node specimens from CSD patients, respectively. The similar sensitivities of the PCR/rRNA and the PCR/CS assays for detecting B. henselae DNA in clinical specimens are in contrast to the 10-fold difference in sensitivities in favor of PCR/rRNA demonstrated with purified B. henselae DNA in sterile pus, suggesting that in the majority of cases, the bacterial load in clinical specimens is large enough to be identified by the PCR/CS assay. A two-step approach is suggested to achieve maximal sensitivity for detecting B. henselae in clinical specimens: initial testing by PCR/CS (which does not require hybridization), followed by PCR/rRNA with PCR/CS-negative specimens when CSD is strongly suspected.     

Intracellular targeting with low pH-triggered bispecific antibodies

Bispecific antibodies were designed to deliver a reversibly bound ligand into target cells and then spontaneously release it upon passage into acidified vesicles. These reagents were assembled by coupling monoclonal antibodies that recognize acid-sensitive epitopes on diphtheria toxin to cell type-specific monoclonal antibodies. The dual binding capacity of the bispecific antibodies was confirmed by delivery of 125I-diphtheria toxin to target molecules present on intact cells. Bispecific antibodies directed against transferrin receptors on human cells were loaded with toxin and tested for cytotoxicity. The mutant diphtheria toxins CRM107 and CRM45 were used since their inability to bind cell receptors renders them ordinarily nontoxic. Their full cytotoxic potential, however, was restored via bispecific antibody-mediated delivery and release within low pH intracellular vesicles. Cytotoxicity was shown to be specific by blocking receptor sites and to be acidification-dependent by protection using NH4Cl to raise endosomal pH. Kinetics for inhibition of cellular protein synthesis was identical for native diphtheria toxin and the bispecific antibody. CRM107 combination. The rate of inhibition (t1/2 = 20 min) indicated that release of CRM107 from the antibody combining site was fast, and its toxic action was unimpeded by this delivery mechanism.     

Nested PCR assay for detection of granulocytic ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

Comparison of the conformation, hydrophobicity, and model membrane interactions of diphtheria toxin to those of formaldehyde-treated toxin (diphtheria toxoid): formaldehyde stabilization of the native conformation inhibits changes that allow membrane insertion

Toxoids are inactivated protein toxins that are used in vaccines. The behavior of diphtheria toxin reacted with formaldehyde (diphtheria toxoid) was compared to that of diphtheria toxin in order to understand the nature of the changes that occur in toxoids upon protein reaction with formaldehyde. Despite the intramolecular cross-links in the toxoid, the conformations of the toxoid and the toxin were very similar in both the native and low pH-induced membrane-penetrating states as judged by fluorescence and hydrophobicity properties. However, the toxoid underwent thermal-, low-pH-, and guanidinium chloride-induced conformational changes only at more extreme conditions than needed to induce such changes in the toxin. This implies that formaldehyde modification stabilizes the native conformation relative to several conformations that involve different degrees of unfolding. The stabilization to conformational changes induced by low pH is particularly interesting because low pH induces partial unfolding of the toxin to a molten globule-like state. It was found that the toxoid only gained the ability to interact with model membrane vesicles at a lower pH than the toxin. Because low-pH-induced unfolding and membrane interaction are critical steps in the entry of diphtheria toxin into cells, the resistance of the toxoid to these changes may be linked to its lack of toxicity. The implications of these results for the construction of toxoids are discussed.     

Glycine induces a novel form of long-term potentiation in the superficial layers of the superior colliculus

Diphtheria toxin is believed to enter sensitive mammalian cells via receptor-mediated endocytosis from clathrin-coated pits, while ricin can enter via both clathrin-dependent and clathrin-independent endocytosis. The present study has confirmed this by determining the toxin sensitivity of COS-7y cells which were transiently overexpressing a trans dominant negative mutant of dynamin, a GTPase required for the budding of clathrin-coated vesicles from the plasma membrane. Cells overexpressing wild-type dynamin showed normal receptor-mediated endocytosis of transferrin and remained sensitive to both diphtheria toxin and ricin. Cells overexpressing a mutant dynamin defective in GTP binding and hydrolysis were unable to endocytose transferrin and were protected against diphtheria toxin, but they remained completely sensitive to ricin intoxication. Treating non-transfected cells or cells overexpressing mutant dynamin with nystatin caused a redistribution of the caveolae membrane marker protein VIP21-caveolin from the cell surface to intracellular locations, but did not affect their sensitivity to ricin. The redistribution of caveolin seen after nystatin treatment may reflect the disappearance of caveolae. If this is the case, caveolae are not responsible for the endocytosis of ricin. An alternative clathrin-independent route may operate for ricin, since cellular uptake, intracellular transport, and translocation into the cytosol remain unaffected when clathrin-dependent endocytosis is effectively blocked.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

The protective effect of the nerve growth factor in exposing a nerve tissue culture to diphtheria toxin

Diphtheria toxin (1.10(-1)-1.10(-6) Lf/ml) was found to inhibit neurite extension in chick embryo dorsal root ganglia in vitro. If the nerve growth factor (60 ng/ml) was added with toxin in culture media the diphtheria toxin effect was decreased and the neurite outgrowth was compared with control. Protective effect of nerve growth factor by influence of diphtheria toxin may be used in new principles of diphtheria treatment.