Philippe H. Lagrange

Salmonella enteritidis : AmpC plasmid-mediated inducible β-lactamase (DHA-1) with an ampR gene from Morganella morganii

ABSTRACT DHA-1, a plasmid-mediated cephalosporinase from a single clinicalSalmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable toEscherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coliJM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98.7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampCwhose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC andampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampGgene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type β-lactamase, DHA-1, probably originated from M. morganii.

Is IP-10 an accurate marker for detecting M. tuberculosis-specific response in HIV-infected persons?

Background The suboptimal sensitivity of Interferon (IFN)-γ-based in-vitro assays, especially in immunocompromised individuals, emphasizes the need for alternative markers for diagnosing tuberculosis (TB). The objective of this study was to evaluate whether interferon-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-2 and interleukin (IL)-2 can be useful biomarkers for evaluating a specific response to RD1 antigens associated to active TB disease in HIV-infected individuals. Methodology/Principal Findings The study was carried out in India, the country with the highest TB burden in the world. Sixty-six HIV-infected individuals were prospectively enrolled, 28 with active-pulmonary-TB and 38 without. The whole blood assay based on RD1-selected peptides (experimental test) and QuantiFERON-TB Gold In tube (QFT-IT) was performed. Plasma was harvested at day-1-post-culture and soluble factors were evaluated by ELISA. The results indicate that by detecting IP-10, the sensitivity of the experimental test and QFT-antigen (75% and 85.7% respectively) for active TB was higher compared to the same assays based on IFN-γ (42.9% and 60.7% respectively) and was not influenced by the ability to respond to the mitogen. By detecting IP-10, the specificity of the experimental test and QFT-antigen (57.9% and 13.2% respectively) for active TB was lower than what was reported for the same assays using IFN-γ-detection (78.9% and 68.4% respectively). On the other side, in vitro IL-2 and MCP-2 responses were not significantly associated with active TB. Conclusions HIV infection does not impair RD1-specific response detected by IP-10, while it significantly decreases IFN-γ-mediated responses. At the moment it is unclear whether higher detection is related to higher sensitivity or lower specificity of the assay. Further studies in high and low TB endemic countries are needed to elucidate this.

IP-10 response to RD1 antigens might be a useful biomarker for monitoring tuberculosis therapy

BackgroundThere is an urgent need of prognosis markers for tuberculosis (TB) to improve treatment strategies. The results of several studies show that the Interferon (IFN)-γ-specific response to the TB antigens of the QuantiFERON TB Gold (QFT-IT antigens) decreases after successful TB therapy. The objective of this study was to evaluate whether there are factors other than IFN-γ [such as IFN-γ inducible protein (IP)-10 which has also been associated with TB] in response to QFT-IT antigens that can be used as biomarkers for monitoring TB treatment.MethodsIn this exploratory study we assessed the changes in IP-10 secretion in response to QFT-IT antigens and RD1 peptides selected by computational analysis in 17 patients with active TB at the time of diagnosis and after 6 months of treatment. The IFN-γ response to QFT-IT antigens and RD1 selected peptides was evaluated as a control. A non-parametric Wilcoxon signed-rank test for paired comparisons was used to compare the continuous variables at the time of diagnosis and at therapy completion. A Chi-square test was used to compare proportions.ResultsWe did not observe significant IP-10 changes in whole blood from either NIL or QFT-IT antigen tubes, after 1-day stimulation, between baseline and therapy completion (p = 0.08 and p = 0.7 respectively). Conversely, the level of IP-10 release to RD1 selected peptides was significantly different (p = 0.006). Similar results were obtained when we detected the IFN-γ in response to the QFT-IT antigens (p = 0.06) and RD1 selected peptides (p = 0.0003). The proportion of the IP-10 responders to the QFT-IT antigens did not significantly change between baseline and therapy completion (p = 0.6), whereas it significantly changed in response to RD1 selected peptides (p = 0.002). The proportion of IFN-γ responders between baseline and therapy completion was not significant for QFT-IT antigens (p = 0.2), whereas it was significant for the RD1 selected peptides (p = 0.002), confirming previous observations.ConclusionsOur preliminary study provides an interesting hypothesis: IP-10 response to RD1 selected peptides (similar to IFN-γ) might be a useful biomarker for monitoring therapy efficacy in patients with active TB. However, further studies in larger cohorts are needed to confirm the consistency of these study results.

IFN-γ, but not IP-10, MCP-2 or IL-2 response to RD1 selected peptides associates to active tuberculosis

OBJECTIVESnTo evaluate whether in vitro response to Mycobacterium tuberculosis RD1 peptides selected by computational analysis, measured by IFN-gamma, IP-10, MCP-2 or IL-2 production, is associated with active tuberculosis (TB) in a country with a high incidence of TB.nnnMETHODSn129 individuals were prospectively enrolled, 41 with active-pulmonary TB and 88 without (household contacts and community controls). A whole blood assay based on RD1 selected peptides was performed. Soluble factors were evaluated by ELISA in plasma harvested at day1-post-culture. Enrolled individuals were also tested by QuantiFERON TB-Gold In tube (QFT-IT) and tuberculin skin tests (TST).nnnRESULTSnIFN-gamma response to RD1 selected peptides was significantly higher in active TB patients than in household contacts and community controls. IP-10 and MCP-2 response did not differ between active TB patients and household contacts, although it was higher in these groups compared to community controls; conversely IL-2 response did not differ among the three groups. When IFN-gamma response to RD1 selected peptides was scored based on receiver-operator-characteristic analysis, active TB was predicted with 68% sensitivity and 86% specificity. QFT-IT and TST showed a sensitivity for active TB of 90% and 68% and a specificity of 58% and 59%, respectively.nnnCONCLUSIONSnIFN-gamma (but not IP-10, MCP-2 and IL-2) response to RD1 selected peptides is associated with active TB with a higher specificity than QFT-IT and TST.

Point mutation in the pribnow box, the molecular basis of beta-lactamase overproduction in Klebsiella oxytoca.

Klebsiella oxytoca mutants resistant to a variety of beta-lactams were obtained in vitro on aztreonam. Constitutive beta-lactamase production was much higher in the mutants than in the susceptible strains (75-fold). The only difference observed in these mutants compared with the susceptible strains were point mutations in the Pribnow box: a transversion (G-->T) in the first base for one mutant or a transition (G-->A) in the fifth base of the -10 consensus sequence for the other three mutants. The transcriptional output of the beta-lactamase gene (blaOXY) from the mutants was significantly higher than that of the blaOXY gene from the susceptible strains.

Intermediate maturation of Mycobacterium tuberculosis LAM-activated human dendritic cells.

Contrasting observations raise the question of the role of mycobacterial derived products as compared with the whole bacterium Mycobacterium tuberculosis on maturation and function of human dendritic cells (DCs). DC‐SIGN has been identified as the key DC receptor for M.u2003tuberculosis through its interaction with the mannosylated lipoarabinomannan (ManLAM). Although ManLAM is a major mycobacterial component released from infected antigen‐presenting cells, there is no formal evidence yet for an effect of ManLAM per se on DC maturation and function. DCs activated with purified ManLAM displayed an intermediate maturation phenotype as compared with lipopolysaccharide fully matured DCs with reduced expression of MHC class I and class II molecules, CD83 and CD86 and of the chemokine receptor CCR7. They were sensitive to autologous natural killer (NK) lysis, thus behaving like immature DCs. However, ManLAM‐activated DCs lost phagocytic activity and triggered priming of naive T‐cells, confirming their intermediate maturation. Partial maturation of ManLAM‐activated DCs was overcome by triggering the CD40/CD40L pathway as a second signal, which completed maturation phenotypically and abolished autologous NK lysis susceptibility. Altogether, these data provide evidence that ManLAM may induce a partial maturation phenotype on non‐infected bystander DCs during infection suggesting that ManLAM released from infected cells might impair adaptive immune response towards M.u2003tuberculosis.

Comparison of A60 and three glycolipid antigens in an ELISA test for tuberculosis

OBJECTIVES: To compare the diagnostic usefulness in tuberculosis of the serodiagnostic enzyme-linked immunosorbent assay (ELISA) kit A60 (Anda Biologicals, Strasbourg, France) and of our domestic ELISA based on three purified cell wall glycolipid antigens. METHODS: The presence and concentrations of IgG and IgM anti-A60 antibodies and anti-LOS, anti-DAT and anti-PGLTb1 antibodies against the glycolipid antigens were determined by ELISA in 50 HIV-seronegative and 46 HIV-seropositive patients, with documented active tuberculosis. The specificity of these ELISAs was determined with use of sera from 50 healthy blood donors, 29 patients with non-mycobacterial pulmonary diseases and 24 HIV-positive patients with disseminated Mycobacterium avium infection. RESULTS: With a calculated cut-off for each antigen and immunoglobulin that gave a specificity higher than or equal to 98%, the cumulative ELISA results showed that only 36.5% of the patients with tuberculosis had a positive response in the A60 test, as compared with 84.4% who showed a response to the three glycolipid antigens (p<0.001). This striking difference persisted when the cumulative sensitivities were calculated according to the HIV status of the patients and the localization of tuberculosis. The anti-A60 antibody (IgG and IgM) levels and the degree of sensitivity of the ELISA for detection of A60 antigen were always lower in HIV-positive patients with pulmonary and extrapulmonary tuberculosis than in HIV-negative patients with tuberculosis. The sensitivity of A60 ELISA was further decreased in HIV-positive patients with low CD4+ lymphocytes counts, in contrast to the results with the three glycolipid antigens. CONCLUSIONS: These results show the limitations of the A60 ELISA, and confirm the potencies of the glycolipid antigens in serodiagnosis of tuberculosis in HIV-positive and HIV-negative patients.

Anti-PGL-Tb1 responses as an indicator of the immune restoration syndrome in HIV-TB patients.

A prospective and multi-centre study has allowed us to analyse antibody responses and Mycobacterium tuberculosis clinical isolate genotypes on 24 consecutive HIV-TB co-infected patients treated with Highly Active Antiretroviral Therapy (HAART) who either went on to develop a TB Immune Restoration Syndrome (TB-IRS), or not. Circulating free and immune-complexed antibodies against ManLAM, ESAT-6/CFP10 and PGL-Tb1 in HIV-TB co-infected patients were measured by ELISA at the initiation of anti-TB treatment, at the date of HAART initiation and thereafter. Presence of circulating B cells was also monitored by in vitro antibody production (IVAP) against ESAT-6/CFP10 and PGL-Tb1. Finally, 16 out of 24M. tuberculosis clinical isolates from patients with TB-IRS were genotyped using spoligotyping and MIRUs-VNTR typing. Eleven patients (45.8%) experienced TB-IRS (TB-IRS+). Significantly, lower anti-PGL-Tb1 antibody levels were identified in TB-IRS+ compared to TB-IRS-negative patients prior to TB-IRS development. These very low levels were neither related to CD4 counts nor with complexed antibodies. No difference in antibody levels was observed with the other tested antigens. In addition, no specific strain genotype was associated with TB-IRS. The presence of specific anti-PGL-Tb1 antibodies only in TB-IRS-negative patients represents for the first time an indicator of a potential protective response or a diagnostic biomarker for the detection of non-progression to TB-IRS in HIV-TB co-infected patients starting HAART.

A Toolbox for Tuberculosis (TB) Diagnosis: An Indian Multicentric Study (2006-2008). Evaluation of QuantiFERON-TB Gold in Tube for TB Diagnosis

Background The aim of this multicentric prospective study in India was to assess the performance of the QuantiFERON TB-Gold in tube (QFT-GIT), Tuberculin Skin Test (TST) and microbiological results as additional tools for diagnosing active tuberculosis (TB) and latent infection (LTBI) according to Human Immunodeficiency Virus (HIV) status. Methods Individuals with and without active TB and HIV infection were enrolled between 2006–2008. QFT-GIT and TST results were analyzed per se and in combination with microbiological data. Results Among the 276 individuals (96 active pulmonary TB and 180 no active TB) tested by QFT-GIT, 18 indeterminate results (6.5%) were found, more significantly numerous in the HIV-infected (15/92; 16.3%) than the HIV-uninfected (3/184; 1.6%)(p<0.0001). QFT-GIT sensitivity for active TB was 82.3% and 92.9% respectively after including or excluding indeterminate results. Clinical sensitivity was significantly lower in the HIV-infected (68.4%) than the HIV-uninfected (91.4%) patients (pu200a=u200a0.0059). LTBI was detected in 49.3% of subjects without active TB but varied according to TB exposure. When the TST and QFT-GIT were concomitantly performed, the respective sensitivity for active TB diagnosis was 95.0% and 85.0% in the HIV-uninfected (pu200a=u200a0.60), and 66.7% and 51.5% in the HIV-infected patients (pu200a=u200a0.32). QFT-GIT and TST respective specificity for active TB in the HIV-uninfected was 25.0% and 57.1% (pu200a=u200a0.028), and 64.8% and 83.3% in the HIV-infected (pu200a=u200a0.047). In those with active TB, QFT-GIT results were not associated with microbiological parameters (smear grade, liquid culture status, time-to-positivity of culture) or clinical suspicion of active TB score (provided by the clinicians at enrollment). Combining microbiological tests with both immunological tests significantly increased sensitivity for active TB diagnosis (pu200a=u200a0.0002), especially in the HIV-infected individuals (pu200a=u200a0.0016). Conclusion QFT-GIT and TST have similar diagnostic value for active TB diagnosis. In HIV-infected patients, combining microbiological tests with both immunological tests significantly increases the sensitivity for active TB diagnosis.

A toolbox for tuberculosis diagnosis: An indian multicentric study (2006-2008): Microbiological results

Background The aim of this multicentric prospective study in India was to assess the value of several microbiological tools that contribute to the diagnosis of tuberculosis (TB) according to HIV status. Methods Standard microbiological tools on individual specimens were analyzed. Results Among the 807 patients with active TB, 131 were HIV-infected, 316 HIV-uninfected and 360 had HIV-unknown status. Among the 980 non-active TB subjects, 559 were at low risk and 421 were at high risk of M. tuberculosis (Mtb) exposure. Sensitivity of smear microscopy (SM) was significantly lower in HIV-infected (42.2%) than HIV-uninfected (75.9%) (pu200a=u200a0.0001) and HIV-unknown pulmonary TB patients (61.4%) (pu200a=u200a0.004). Specificity was 94.5% in non-TB patients and 100% in health care workers (HCW) and healthy family contacts. Automated liquid culture has significantly higher diagnostic performances than solid culture, measured by sensitivity (74.7% vs. 55.9%) (pu200a=u200a0.0001) and shorter median time to detection (TTD) (12.0 vs. 34.0 days) (pu200a=u200a0.0001). Specificity was 100% in HCW and cured-TB patients, but was lower in non-TB patients (89%) due to isolation of Mycobacteria other than tuberculosis (MOTT). TTD by both methods was related to AFB score. Contamination rate was low (1.4%). AccuProbe hybridization technique detected Mtb in almost all culture-positive specimens, but MOTT were found in 4.7% with a significantly higher frequency in HIV-infected (15%) than HIV-uninfected TB patients (0.5%) (pu200a=u200a0.0007). Pre-test classification significantly increased the diagnostic value of all microbiological tests in pulmonary TB patients (p<0.0001) but to a lesser degree in extrapulmonary TB patients. Conclusions Conventional microbiological tools led to results similar to those already described in India special features for HIV-infected TB patients included lower detection by SM and culture. New microbiological assays, such as the automated liquid culture system, showed increased accuracy and speed of detection.