Modified oxidation-fermentation medium for detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis

A modified oxidation-fermentation medium was developed as a practical medium for highly sensitive and specific detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis. A total of 756 strains representing 17 Neisseria spp. and Branhamella catarrhalis were tested in this medium, in which the protein concentration was reduced relative to the carbohydrate concentration, phenol red was substituted for bromthymol blue at a low concentration, and the initial pH was adjusted to 7.2. Sugar utilization patterns were consistent with published results and with other cultural and biochemical characteristics for these species. The reactions obtained using this medium were qualitatively better and more reproducible than those obtained in cystine-Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) medium.     

Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains.

A regulatory gene (dtxR) responsible for iron-dependent repression of the toxin (tox) and siderophore genes in Corynebacterium diphtheriae was cloned and characterized. A DNA fragment carrying dtxR repressed expression of a tox-lacZ gene fusion in Escherichia coli DH5 alpha in a high-iron environment but not under low-iron conditions. A protein with mobility corresponding to approximately 28 to 29 kDa was identified as the product of the dtxR gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A shuttle vector designated pCM2.6 was constructed which carries the origin of replication from C. diphtheriae plasmid pNG2 and confers resistance to chloramphenicol in E. coli and C. diphtheriae. DNA fragments carrying dtxR were cloned into pCM2.6, and the hybrid shuttle plasmids were transformed by electroporation into wild-type C. diphtheriae C7(beta) and the regulatory mutant C7(beta)hm723, which produces toxin and siderophore constitutively under high-iron conditions. Expression of the cloned dtxR determinant did not affect the phenotype of C. diphtheriae C7(beta). In C. diphtheriae C7(beta)hm723, expression of cloned dtxR restored full repression of siderophore production and partial repression of diphtheria toxin production during growth in a high-iron environment.     

Differential expression of complement regulatory proteins decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 during human spermatogenesis.

We have examined the distribution of the complement (C) regulatory proteins CD59, membrane cofactor protein (MCP) and decay-accelerating factor (DAF) on mature sperm and compared expression of these proteins in parallel both during spermatogenesis and in the prostate. Enhanced immunoperoxidase staining and radioimmunoassay confirmed that C regulators are differentially expressed on sperm; CD59 was strongly expressed on the surface of acrosome intact sperm while MCP and DAF appear to be located primarily on the inner acrosomal membrane. While the MW of CD59 on sperm is typical of other systems, we confirm that in addition to a novel 40,000-46,000 MW MCP protein, sperm also express a novel 55,000 MW DAF product. Examination of normal testis by immunostaining revealed that although C regulators are differentially expressed within the germinal epithelium, all three proteins were present on the acrosomal region of condensing spermatids. We show that novel, low MW forms of MCP and DAF are expressed in normal testis membranes but are absent from testis membranes obtained from patients undergoing gender reassignment surgery in whom the germinal epithelium is diminished. Novel MW C3 convertase regulators are therefore associated with differentiating germinal epithelium. Typical CD59 components were also present on normal testis membranes confirming that CD59 is acquired during spermatogenesis. We demonstrate that the prostatic epithelium, in addition to MCP, expresses CD59 but not DAF. By comparison with CD59, therefore, our studies suggest that DAF may be acquired only in the testis. Overall, our data suggest that, on leaving the testis, sperm express the repertoire of C regulators required for protection from C during their transit through the male and female reproductive tracts.     

DNA-synthesizing cells in the blood in coeliac disease and inflammatory bowel disease.

The level of 3H-labelled thymidine ([3H]TdR) incorporation of blood cells of patients with coeliac disease was shown in two separate studies to be significantly lower than that of a normal control group. In the first study the 'background' DNA synthesis in unstimulated cultures containing a standard number of blood lymphocytes was measured on days 4, 5 and 6. In the second study a standard volume of freshly drawn whole blood was added to culture medium and the [3H]TdR incorporation measured over the first 24 hr and from 48 to 72 hr. In all cases the [3H]TdR incorporation of cells of coeliac patients on a normal or a gluten-free diet was lower than that of the control group. It is suggested that sequestration of DNA-synthesizing cells in the mucosa of the small bowel may partly explain these results. In whole-blood cultures from patients with inflammatory bowel disease in remission [3H]TdR incorporation over the first 24 hr was raised in Crohn's disease but normal in ulcerative colitis.     

Lineage extinction and replacement in dengue type 1 virus populations are due to stochastic events rather than to natural selection

Between 1996 and 1998, two clades (B and C; genotype I) of dengue virus type 1 (DENV-1) appeared in Myanmar (Burma) that were new to that location. Between 1998 and 2000, a third clade (A; genotype III) of DENV-1, which had been circulating at that locality for at least 25 years, became extinct. These changes preceded the largest outbreak of dengue recorded in Myanmar, in 2001, in which more than 95% of viruses recovered from patients were DENV-1, but where the incidence of severe disease was much less than in previous years. Phylogenetic analyses of viral genomes indicated that the two new clades of DENV-1 did not arise from the, now extinct, clade A viruses nor was the extinction of this clade due to differences in the fitness of the viral populations. Since the extinction occurred during an inter-epidemic period, we suggest that it was due to a stochastic event attributable to the low rate of virus transmission in this interval.     

Staphylococcus aureus isolates carrying Panton-Valentine leucocidin genes in England and Wales: frequency, characterization, and association with clinical disease

Staphylococcus aureus isolates carrying the genes that encode for Panton-Valentine leucocidin (PVL), a highly potent toxin, have been responsible for recent outbreaks of severe invasive disease in previously healthy children and adults in the United States of America and Europe. To determine the frequency of PVL-positive isolates sent to the Staphylococcus Reference Unit (United Kingdom) for epidemiological purposes, we tested 515 isolates of S. aureus, and 8 (1.6%) were positive for the PVL locus. A further 470 isolates were selected to explore the association of PVL-positive S. aureus with clinical disease. Of these, 23 (4.9%) were PVL positive and most were associated with skin and soft tissue infections (especially abscesses). The PVL genes were also detected in isolates responsible for community-acquired pneumonia, burn infections, bacteremia, and scalded skin syndrome. Genotyping by pulsed-field gel electrophoresis and multilocus sequence typing revealed that the PVL-positive isolates were from diverse genetic backgrounds, although one prevalent clone of 12 geographically dispersed methicillin-resistant S. aureus (MRSA) isolates was identified (ST80). All 12 isolates were stapylococcal cassette chromosome mec type IVc, had an agr3 allele, and shared a common toxin gene profile (sea-see, seg-sej, eta, etb, and tst negative but etd positive). ST80 strains with similar genetic characteristics have been responsible for community-acquired infections in France and Switzerland. The remaining PVL-positive isolates were mostly methicillin-sensitive S. aureus and belonged to 12 different sequence types, including ST22 and ST30, which are closely related to the most prevalent MRSA clones in United Kingdom hospitals, EMRSA-15 and EMRSA-16, respectively.     

Evaluation of two BBL Crystal systems for identification of some clinically important gram-negative bacteria.

The BBL Crystal system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) is a miniaturized bacterial identification method employing modified conventional and chromogenic substrates. Two products are currently available, the Rapid Stool/Enteric ID Kit and the Enteric/Nonfermenter ID Kit, each comprising thirty tests. We report an evaluation of both systems (using database version 1.1 for both) in the identification of 51 gram-negative taxa likely to be encountered commonly in the clinical laboratory. In all, 266 strains were tested in the Enteric/Nonfermenter ID Kit, and these represented 36 taxa of the family Enterobacteriaceae (188 strains), 5 oxidase-positive fermentative taxa (26 strains), and 10 nonfermentative taxa (52 strains). The majority of these same strains (203 of 266) were also tested in the Rapid Stool/Enteric ID Kit. The Enteric/Nonfermenter ID Kit performed as follows: Enterobacteriaceae, 93% correct, 6% not identified, and 1% incorrect; oxidase-positive fermenters, 88, 12, and 0%, respectively; and nonfermenters, 100% correct, although several only to the genus or group level. The Rapid Stool/Enteric ID Kit gave the following results: Enterobacteriaceae, 91% correct, 7% not identified, and 2% incorrect; oxidase-positive fermenters, 80, 13, and 7%, respectively (but results were based on only 15 strains); and nonfermenters, 100% correct (but results were based on only 11 strains). We found the systems extremely easy and rapid to use, and for the Enteric/Nonfermenter ID Kit an identification rate of 100% in 40 of 51 taxa was achieved, with corresponding figures of 29 of 39 taxa for the Rapid Stool/Enteric ID Kit.     

Complement regulatory proteins in early human fetal life: CD59, membrane co-factor protein (MCP) and decay-accelerating factor (DAF) are differentially expressed in the developing liver.

The human fetus appears to be capable of protecting itself from maternal complement (C) from an early stage in development by expressing the C regulatory proteins decay-accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 on fetally derived trophoblast at the feto-maternal interface. In this study we have examined the ontogeny of these proteins within the fetus itself and have focused on the liver which represents a major site of haemopoiesis during development. Immunostaining revealed that DAF, MCP and CD59 are all expressed from at least 6 weeks of gestation in the liver but that these proteins display distinct distribution patterns. CD59 was broadly distributed both within the epithelial and haemopoietic compartments, but expression of C3 convertase regulators was more restricted. DAF expression was limited to isolated cells within haemopoietic nests and the epithelium was DAF-negative. Although MCP expression on haemopoietic cells was also limited, by contrast with DAF the developing hepatic epithelium was strongly MCP-positive. Typical CD59 and MCP components were observed in fetal liver extracts by immunoblotting, although liver MCP components consistently migrated 4000-5000 MW ahead of those observed on placental trophoblast. Differences in the distribution of these proteins were also observed between the fetal and adult liver. In particular, by comparison with fetal hepatic epithelium, there was an apparent loss of MCP expression from adult hepatocytes. Thus, MCP appears to be developmentally regulated in the human liver and is expressed in the absence of DAF on the early hepatic epithelium. Overall, this study suggests that C regulatory proteins, and in particular CD59 and MCP, are required from the very early stages of gestation within the fetus itself.     

Test reproducibility of the API (20E), Enterotube, and Pathotec systems.

Thirty-three strains of bacteria (30 Enterobacteriaceae and one strain each of Aeromonas formicans, A. hydrophila, and Plesiomonas shigelloides) were tested three times in each of 27 conventional tests and in the API, Enterotube, and Pathotec systems. The results obtained were analysed for test reproducibility within each kit, correlation of the kit tests with the equivalent conventional media, and the identification of the strains by the kits. Difficulties in evaluation and comparison of identifications are discussed. A practical evaluation of the kits was also made.     

Comparative analysis of immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay using virus-like particles or virus-infected mouse brain antigens to detect IgM antibody in sera from patients with evident flaviviral infections

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquito-borne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 - specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain.