Identification and susceptibility testing of Enterobacteriaceae and Pseudomonas aeruginosa by direct inoculation from positive BACTEC blood culture bottles into Vitek 2

Inoculation of an automated system for rapid identification (ID) and antimicrobial susceptibility testing (AST) directly from positive blood culture bottles will reduce the turnaround time of laboratory diagnosis of septicemic patients, which benefits clinical outcome and decreases patient costs. Direct test results, however, must always be confirmed by testing a pure overnight culture, which is the "gold standard." We studied the accuracy of direct testing versus repeat testing in order to investigate the possibility of refraining from repeat testing. We also assessed the clinical risk of reporting results based on direct testing only. We inoculated Vitek 2 (bioMérieux) directly from 410 positive BACTEC 9240 (BD) blood culture bottles containing gram-negative rods and studied the ID and AST results. In a comparison of direct inoculation with the standard method, a total of 344 isolates of Enterobacteriaceae and Pseudomonas aeruginosa were tested, and 93.0% were correctly identified. Of the 39 (10.2%) samples that contained bacilli not identifiable by Vitek 2, only 1 gave a conclusive, correct result. The overall MIC agreement among 312 isolates was 99.2%, with 0.8% very major and 0.02% major error rates. Of only three (polymicrobial) samples, the direct susceptibility pattern would be reported to the clinician as too sensitive. Vitek 2 results obtained from direct inoculation of blood culture bottles containing gram-negative bacilli are safe enough for immediate reporting, provided that ID and AST are consistent. Repeat testing is not necessary, unless Gram stain or overnight subculture results raise doubt about the purity of the culture.     

OH treatment of tetanus toxin reduces its susceptibility to limited proteolysis with more efficient presentation to specific T cells

At inflammatory sites, before their processing, antigens are exposed to oxygen free radicals released by activated cells. The effect of hydroxyl radicals (OH·) on the structure of a protein antigen, tetanus toxin (TT) was investigated, as well as the consequences on processing and presentation. A chemical system composed of Fe-EDTA, ascorbate and H₂O₂ was used to produce physiological amounts of OH^• radicals. TT exposed to OH· radicals presented a marked decrease of its intrinsic fluorescence with a concomitant increase of the content of bityrosine, but no fragmentation of the protein was detected by SDS-PAGE. Processing of the modified TT was analysed, by incubating TT at acidic pH with fractions enriched in plasma membranes and lysosomes obtained from a lymphoblastoid cell line (LCL). Proteolysis of OH·-treated TT was less important than proteolysis of native TT, especially upon prolonged incubations. Oxidized TT presented by LCL cells induced a greater proliferation of three different TT specific T cell clones, compared to native TT. When proteolytic digests of TT were presented by fixed LCL cells to a homologous T cell line, the proliferative response obtained in the presence of digests of OH·-treated TT was sustained, even in the case of prolonged proteolysis, whereas the response to digests of native TT fell rapidly. The relative resistance of OH·-treated TT to proteolysis appears thus responsible for its greater presentation to specific T cells, probably by protecting epitopes.     

Cytogenetic and molecular cytogenetic studies of a case of interstitial deletion of proximal 15q

A 4-month-old child with multiple anomalies was determined to have an interstitial deletion of chromosome 15, i.e., del(15) (q12q14). The deletion appears not to be a typical deletion of 15q12 such as seen in Angelman and Prader-Willi syndromes, but appears to be more distal, involving either loss of all of 15q12 and part of 15q14, or part of 15q12 and most of 15q14. In either case, 15q13 is missing. Fluorescent in situ hybridization with probes for 15 centromere (D15Z), pericentromeric satellite sequences (D15Z1), and chromosome 15 painting probes shows the deleted chromosome to involve only 15 and no other acrocentric chromosome. Hybridization with probes for the AS and PWS loci (D15S11 and GABAB3, Oncor) show both sites to be intact in the deleted 15. The case is compared with two other reports with overlapping interstitial deletions of proximal 15q, neither of which shows typical features of Angelman or Prader-Willi syndromes.     

Quantification of HIV-1 p24 by a highly improved ELISA: an alternative to HIV-1 RNA based treatment monitoring in patients from Abidjan, C?te d'Ivoire.

BACKGROUND: Quantification of HIV-1 RNA remains difficult to implement in Africa. Simple and inexpensive tests for antiretroviral treatment (ART) monitoring are needed. OBJECTIVE: To evaluate an HIV-1 p24 ELISA, which combines efficient virus disruption, heat-denaturation and signal amplification, in a West African setting. STUDY DESIGN: Eighty-six HIV-1 infected patients from Abidjan, C?te d'Ivoire, were tested for p24, HIV-1 RNA, and CD4+ count at baseline, and twice within 8 months after ART initiation. RESULTS: All patients responded to ART with a minimal HIV-1 RNA drop of 0.5 log(10) at first follow-up. Forty-one (47.7%) then rebounded >0.5 log(10) or persisted above 1000 copies/mL by week 24. The predicted baseline concentration of p24 corresponding to 100,000 copies/mL of HIV-1 RNA, above which ART is recommended, was 4546 fg/mL (95% confidence interval 3148-6566). A prediction model of virologic failure, occurring after an initial response to ART, correctly classified 84% of patients using baseline p24, p24 change on therapy, and achievement of undetectable p24 as explanatory variables. The model and further bootstrap evaluation suggested a good ability to discriminate between sustained or failing virologic response to ART. CONCLUSION: HIV-1 p24 and RNA based-ART monitoring in a low-resource country dominated by HIV-1 CRF02 AG appeared comparable.     

The mitochondrial genome can be altered or lost without lethal effect in the petite-negative yeast Debaryomyces (Schwanniomyces) occidentalis

The nature of mutations affecting several cytochrome-deficient mutants of Debaryomyces (Schwanniomyces) occidentalis has been characterized. The DR12 mutant, which is deficient in cytochrome b, and the B10Mn mutant, which is deficient in cytochromes b and a, a3, are deleted in the mitochondrial CYB and COX1 genes respectively. The B10 strain, which is partially deficient in cytochrome b, has no detectable change in its mitochondrial DNA and possibly carries nuclear lesion(s). These three mutants, unlike the rho(-) and rho degrees "petite" mutants of Saccharomyces cerevisiae, can still grow on non-fermentable substrates, due to the development of a salicylhydroxamic acid (SHAM)-sensitive alternative pathway linked to phosphorylation at site 1. A gly(-) mutant lacking mtDNA and respiratory capacity has been isolated. For the first time, it is demonstrated that mtDNA can be altered or even lost without lethal consequence in D. occidentalis, although this yeast was classified as a petite-negative species.     

Effect of articular disease and total knee arthroplasty on knee joint-position sense

Joint proprioception in the human knee has been studied using two previously described tests. Threshold of detection of slow, constant, passive motion and ability to reproduce angles to which the knee was passively placed were accurately measured. A group of postoperative total knee arthroplasty (TKA) patients were examined. All patients also had documented articular disease in the unoperated knee. Results were compared to age-matched controls. In addition, a young control group was studied for comparison to both groups. A significant difference was seen between the young control group and the older control group in both tests performed. Age-matched controls and the postoperative patients demonstrated an even greater difference. There was, however, no difference between the operated and unoperated knee among the TKA patients. It is concluded that joint proprioception declines to some degree with normal aging. A more marked decline is associated with degenerative joint disease. Total joint replacement, however, did not lead to a further decrease in sensation.     

Schizophrenia and Evoked Potentials: Maximum Amplitude, Frequency of Peaks, Variability, and Phenothiazine Effects

The purpose of this study was to compare averaged visual evoked potentials AVEPs) in normal subjects and in schizophrenics off and on phenothiazine medication. Flashes of 4 intensities were used. Ss were tested 3 times within a 1 month period. Measures of maximum amplitude (Am), frequency of peaks I FOP), and variability (V) were obtained- With increases in stimulus intensity all Ss showed increases in Ams and decreases in FOPs and V. Schizophrenics had smaller Ams. greater FOPs, and grear V than normals. Schizoprenies on phenothiazines generally had less FOPs initially and after 1 wk mi medication but not after about I month on medication. There was no consistently significant effect of phenothiazines on maximum amplitude. NO drug effect on variability was observed. Schizophrenics showed a decrease in FOPs over time while normals showed an increase. A relationship was found between variability and overall thought disturbance. Changes in clinical condition over a month were not associated with discernible AVEP changes. Relationships between Am, FOP, unit V are presented.     

Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies for inflammatory bowel disease: high prevalence in patients with celiac disease

Both celiac disease and inflammatory bowel disease (IBD) are characterized by chronic diarrhea and the presence of distinct (auto)antibodies. In the present study we wanted to determine the prevalence of serological markers for inflammatory bowel disease, i.e., perinuclear antineutrophil cytoplasmic antibodies (pANCA) and/or anti-Saccharomyces cerevisiae antibodies (ASCA), in 37 patients with biopsy-confirmed celiac disease (Marsh IIIb/c). The majority of the patients was positive for IgA (auto)antibodies typically associated with celiac disease, i.e., antiendomysium antibodies (EMA) (86.5%), antigliadin antibodies (AGA) (73%), and antirecombinant human tissue transglutaminase antibodies (rh-tTGA) (86.5%). Four patients with selective IgA deficiency could be identified by analyzing EMA, AGA, and rh-tTGA for the IgG isotype. The prevalence of pANCA and ASCA, markers that are used for IBD, was unexpectedly high in our cohort of patients with celiac disease: 8 patients were positive for pANCA (IgG) and 16 patients were positive for ASCA (IgG and/or IgA). These results indicate that the presence of pANCA or ASCA in the serum of patients with chronic diarrhea does not exclude celiac disease. A prospective study is required to determine whether pANCA and/or ASCA identify patients at risk for developing secondary autoimmune disease.     

Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils

BACKGROUND: An epidemiologic relationship between airway allergic diseases and exposure to atmospheric pollutants has been demonstrated and suggested to be one factor in the increasing prevalence of asthma. Diesel exhaust particles (DEPs) have been shown to participate in the development of allergic airway inflammation, in which the targets include macrophages, B and T cells, epithelial cells, and mast cells. In addition to the adjuvant effect of DEPs on total and allergen-specific IgE production, DEPs also act to induce chemokines and cytokines and may play a key role in primary sensitization. OBJECTIVE: DEPs have been shown to increase local IL-4-containing Kit(+) cells soon after in vivo nasal challenge. The aim of this study was to examine the effects of DEPs on human basophils, a key source of IL-4. METHODS: Peripheral blood leukocytes from allergic and control subjects were cultured in the presence of organic extracts of DEP (DEPex) with or without allergen. The cultures were analyzed for IL-4-containing cells by using multiparameter flow cytometry, IL-4 secretion with ELISA, and histamine release. RESULTS: Basophils, when exposed in vitro to DEPex, expressed IL-4 and released histamine significantly (P <.01) more than with antigen activation. DEPex did not synergize with allergen in cytokine production and histamine release. DEPex-induced basophil IL-4 expression peaked at 2 hours and persisted through 20 hours, in contrast to allergen-induced IL-4, which was transient. The effect of DEPex on basophil cytokine expression and histamine release was dose dependent and occurred with cells from both allergic and nonallergic subjects. DEPex induced IL-4 expression and histamine release in highly enriched basophil populations, suggesting it acts directly on basophils. Other peripheral blood leukocytes, including T cells, did not contribute to this cytokine expression. Preincubation with N-acetylcysteine completely abrogated DEPex-driven basophil IL-4 expression. CONCLUSIONS: Basophils are a direct target for DEPex, inducing IL-4 expression and histamine release in an IgE-allergen independent fashion. N-acetylcysteine inhibition of DEPex-driven IL-4 expression provides evidence that generation of reactive oxygen species is required for the effects observed. The capability of DEPex to activate basophils in both allergic and nonallergic subjects suggests a potential role of this pollutant in the increasing prevalence of allergic diseases.